ORCID Profile
0000-0002-6977-9388
Current Organisations
University of Oxford
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University of Sydney
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Publisher: Springer Science and Business Media LLC
Date: 24-10-2012
DOI: 10.1038/NRMICRO2671
Abstract: The bacterial cell cycle requires the tight regulation and precise coordination of several sophisticated cellular processes. Prominent among them is the formation of the iding wall or septum, which has to take place at the right time and place to ensure equality of the progeny and integrity of the genome. Nucleoid occlusion is a defence mechanism that prevents the chromosome from being bisected and broken by the ision septum. It does so by preventing Z ring formation near the nucleoid, which also helps to determine the location of septation.
Publisher: American Society for Microbiology
Date: 02-1997
DOI: 10.1128/JB.179.3.972-975.1997
Abstract: The Bacillus subtilis spoVD gene encodes a penicillin-binding protein required for spore morphogenesis. SpoIIID is a sequence-specific DNA-binding protein that activates or represses the transcription of many different genes. We have defined the spoVD promoter region and demonstrated that it is recognized by sigmaE RNA polymerase in vitro and that SpoIIID represses spoVD transcription. Two strong SpoIIID-binding sites were mapped in the spoVD promoter region, one overlapping the -35 region and the other encompassing the -10 region and the transcriptional start site.
Publisher: Microbiology Society
Date: 10-2003
Abstract: Differentiation in the spore-forming bacterium Bacillus subtilis is governed by the sequential activation of five sporulation-specific transcription factors. The early mother-cell-specific transcription factor, σ E , directs the transcription of many genes that contribute to the formation of mature, dormant spores. In this study, DNA microarrays were used to identify genes belonging to the σ E regulon. In total, 171 genes were found to be under the control of σ E . Of these, 101 genes had not previously been described as being σ E dependent. Disruption of some of the previously unknown genes ( ydcC , yhaL , yhbH , yjaV and yqfD ) resulted in a defect in sporulation.
Publisher: American Society for Microbiology
Date: 02-1988
DOI: 10.1128/JB.170.2.796-801.1988
Abstract: We measured the synthesis of dipicolinic acid (DPA) during sporulation in spo mutants of Bacillus subtilis by a sensitive biological assay based on cross-feeding of a spoVF mutant strain and also chemically. Many spo mutations, including several that block sporulation at stage III, did not prevent synthesis of DPA but instead prevented its incorporation into the spore. In general, strains with mutations in loci that are expressed in the spore compartment synthesized DPA, whereas strains with mutations in most of the loci that are expressed in the mother-cell compartment did not. Transcription of the gerE gene, as measured by DNA-RNA hybridization, followed a dependence pattern very similar to that of DPA synthesis. However, the dependence patterns of the two operons show that at about stage IV of sporulation there is a branch in the sequence of operon expression in the mother cell. One branch leads through spoVC to synthesis of DPA synthetase, and the other leads through spoVD to expression of gerE.
Publisher: Elsevier BV
Date: 08-1993
DOI: 10.1016/0092-8674(93)90520-Z
Abstract: The establishment of compartment-specific transcription in sporulating cells of B. subtilis is governed at the level of the activity of transcription factor sigma F. Genetic experiments have suggested that SpoIIAA and SpoIIAB, the other products of the sigma F operon, are involved in regulating sigma F activity. This activity is inhibited in the pre isional cell but specifically released from inhibition in the prespore about 1.5 hr after sporulation is induced. We now show that purified SpoIIAB inhibits transcription directed by sigma F in vitro. We note that the amino acid sequence of SpoIIAB shows some similarity to a group of bacterial histidine protein kinases, and we find that SpoIIAB is indeed a protein kinase that phosphorylates SpoIIAA on a serine residue. We suggest that this phosphorylation is responsible for the compartment-specific release of sigma F activity, perhaps through the formation of a tight complex between SpoIIAB and phosphorylated SpoIIAA.
Publisher: Wiley
Date: 08-2000
Publisher: Springer Science and Business Media LLC
Date: 22-03-2017
Publisher: American Society for Microbiology
Date: 1990
DOI: 10.1128/JB.172.1.488-490.1990
Abstract: We have isolated mutations that appear to inactivate the gene (lacA) encoding an endogenous beta-galactosidase activity in Bacillus subtilis and in a closely linked negative regulatory element (lacR). Both genes map to the hisA-thrA region. The lacA mutations may help to avoid some of the problems arising from the use of the Escherichia coli lacZ gene as a reporter gene in B. subtilis.
Publisher: Wiley
Date: 2000
DOI: 10.1046/J.1365-2958.2000.01724.X
Abstract: We have characterized the role of the penicillin-binding protein PBP 2B in cell ision of Bacillus subtilis. We have shown that depletion of the protein results in an arrest in ision, but that this arrest is slow, probably because the protein is relatively stable. PBP 2B-depleted filaments contained, at about their mid-points, structures resembling partially formed septa, into which most, if not all, of the ision proteins had assembled. Although clearly deficient in wall material, membrane invagination seemed to continue, indicating that membrane and wall ingrowth can be uncoupled. At other potential ision sites along the filaments, no visible ingrowths were observed, although FtsZ rings assembled at regular intervals. Thus, PBP 2B is apparently required for both the initiation of ision and continued septal ingrowth. Immunofluorescence microscopy showed that the protein is recruited to the ision site. The pattern of localization suggested that this recruitment occurs continually during septal ingrowth. During sporulation, PBP 2B was present transiently in the asymmetrical septum of sporulating cells, and its availability may play a role in the regulation of sporulation septation.
Publisher: American Society for Microbiology
Date: 03-2003
DOI: 10.1128/MMBR.67.1.52-65.2003
Abstract: Work on two erse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell ision in a wide range of eubacteria. These proteins are directed to the ision site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents ision in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent ision in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential ision proteins assemble at the midcell ision site has been worked out for both E. coli and B. subtilis. They can be ided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the ision septum.
Publisher: Proceedings of the National Academy of Sciences
Date: 07-10-2022
Abstract: SMC complexes, loaded at ParB- parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis , ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.
Publisher: Wiley
Date: 18-05-2016
DOI: 10.1111/MMI.13393
Publisher: Elsevier BV
Date: 07-1993
Abstract: Dipicolinic acid (DPA) is a small polar molecule that accumulates to high concentrations in bacterial endospores, and is thought to play a role in spore heat resistance, or the maintenance of heat resistance. Previous work has shown that mutations in the spoVF locus of Bacillus subtilis prevent the formation of DPA, and give rise to heat-sensitive spores. Addition of exogenous DPA during spore development led to the restoration of heat resistance. This suggested that the spoVF locus encoded dipicolinic acid synthetase, the enzyme thought to catalyse the single reaction needed to synthesise DPA from dihydroxydipicolinic acid, an intermediate in the lysine biosynthetic pathway. We have now cloned and sequenced the spoVF locus of Bacillus subtilis and show that it comprises two coordinately regulated genes, now designated dpaA and dpaB. Expression of fragments of the dpa operon in Escherichia coli has shown that the two gene products together specify DPA synthetase activity. The promoter of the dpa operon, which lies just upstream of the first gene, has been identified by primer extension analysis. Sequences in this region show strong sequence similarity to several promoters recognized by the sigma K form of RNA polymerase. Transcription from this promoter was detected four hours after the onset of sporulation, at about the same time that sigma K activity is known to appear. Furthermore, transcription was abolished by mutations in a series of genes that are known to be required for the synthesis of active sigma K. These results are in accordance with previous work indicating that DPA synthetase activity was present only during the late stages of sporulation and specifically in the mother cell compartment. Transcription was enhanced by a gerE mutation, indicating that, like the previously described cotA gene, spoVF is negatively regulated by GerE. The mother-cell-specific synthesis of an enzyme responsible for a compound that accumulates to high concentrations in the prespore raises interesting questions about intercellular transport mechanisms.
Publisher: Springer Science and Business Media LLC
Date: 11-07-2023
DOI: 10.1038/S41467-023-39723-8
Abstract: Inhibition of bacterial cell wall synthesis by antibiotics such as β-lactams is thought to cause explosive lysis through loss of cell wall integrity. However, recent studies on a wide range of bacteria have suggested that these antibiotics also perturb central carbon metabolism, contributing to death via oxidative damage. Here, we genetically dissect this connection in Bacillus subtilis perturbed for cell wall synthesis, and identify key enzymatic steps in upstream and downstream pathways that stimulate the generation of reactive oxygen species through cellular respiration. Our results also reveal the critical role of iron homeostasis for the oxidative damage-mediated lethal effects. We show that protection of cells from oxygen radicals via a recently discovered siderophore-like compound uncouples changes in cell morphology normally associated with cell death, from lysis as usually judged by a phase pale microscopic appearance. Phase paling appears to be closely associated with lipid peroxidation.
Publisher: Wiley
Date: 15-04-1997
Publisher: Elsevier BV
Date: 06-2004
Publisher: Microbiology Society
Date: 11-2009
Abstract: The prokaryotic cytoskeletal protein MreB is thought to govern cell shape by positioning the cell wall synthetic apparatus at growth sites in the cell. In rod-shaped bacteria it forms helical filaments that run around the periphery of the rod during elongation. Gram-positive bacteria often contain more than one mreB gene. Bacillus subtilis has three mreB -like genes, mreB , mbl and mreBH , the first two of which have been shown to be essential under normal growth conditions. Expression of an mreB homologue from the closely related organism Bacillus licheniformis did not have any effect on cell growth or morphology. In contrast, expression of mreB from the phylogenetically more distant bacterium Clostridium perfringens produced shape defects and ultimately cell death, due to disruption of the endogenous MreB cytoskeleton. However, expression of either mreB B. licheniformis ( mreB Bl ) or mreB C. perfringens ( mreB Cp ) was sufficient to confer a rod shape to B. subtilis deleted for the three mreB isologues, supporting the idea that the three proteins have largely redundant functions in cell morphogenesis. Expression of mreBCD Bl could fully compensate for the loss of mreBCD in B. subtilis and led to the formation of rod-shaped cells. In contrast, expression of mreBCD Cp was not sufficient to confer a rod shape to B. subtilis Δ mreBCD , indicating that a complex of these three cell shape determinants is not enough for cell morphogenesis of B. subtilis .
Publisher: Wiley
Date: 22-07-2005
DOI: 10.1111/J.1365-2958.2005.04736.X
Abstract: Actin homologues of the MreB family have an important role in specifying the morphology of many non-spherical eubacteria. The mreC and mreD genes have been implicated in control of cell morphology but their precise functions are unknown. In Bacillus subtilis the MreB homologue Mbl directs helical insertion of new cell wall material in the cylindrical part of the rod-shaped cell. Depletion of either MreC or MreD abolishes the control of cell shape. In the presence of high concentrations of magnesium cells depleted of MreC or MreD can be propagated indefinitely, although they have a spheroidal shape. We show that growth of the spheroidal mutants is based on insertion of new wall material at cell ision sites and that this localized growth is dependent on cell ision. Under some conditions the MreC and MreD proteins localize in a helical configuration. This localization pattern resembles that of the helical cables of Mbl protein. These results suggest that MreC and MreD act in a morphogenic pathway that couples the helical cytosolic Mbl cables to the extracellular cell wall synthetic machinery, which is critical for cylindrical elongation of the rod-shaped cells.
Publisher: Elsevier BV
Date: 02-1999
DOI: 10.1016/S0168-9525(98)01660-6
Abstract: Recent advances have completely overturned the classical view of chromosome segregation in bacteria. Far from being a passive process involving gradual separation of the chromosomes, an active, possibly mitotic-like machinery is now known to exist. Soon after the initiation of DNA replication, the newly replicated copies of the oriC region, behaving rather like eukaryotic centromeres, move rapidly apart towards opposite poles of the cell. They then determine the positions that will be taken up by the newly formed sister nucleoids when DNA replication has been completed. Thus, the gradual expansion of the diffuse nucleoid camouflages an underlying active mechanism. Several genes involved in chromosome segregation in bacteria have now been defined their possible functions are discussed.
Publisher: Elsevier BV
Date: 11-2017
Publisher: American Society for Microbiology
Date: 12-1990
DOI: 10.1128/JB.172.12.6930-6936.1990
Abstract: During sporulation in Bacillus subtilis, expression of several prespore-specific genes is strongly dependent on the spoIIIE and spoIIIG gene products. Previous reports have also indicated a requirement for the products of the spoIIIA locus. However, we have now systematically studied six different well-defined spoIIIA mutations and find that, relative to spoIIIE and spoIIIG mutations, they have only a minor effect on the expression of two different prespore-specific genes, spoVA and sspA. Moreover, we have shown that strain IS37, which has been used as a spoIIIA mutant in several previous studies, actually contains a lesion in the spo0A gene. We conclude that spoIIIA has a relatively minor or indirect role in the regulation of prespore-specific gene expression.
Publisher: Wiley
Date: 08-1991
DOI: 10.1111/J.1365-2958.1991.TB00816.X
Abstract: We have cloned and characterized a 5 kbp region of the Bacillus subtilis chromosome and show that it contains the promoter-proximal part of the spoIIIA locus. The locus consists of a polycistronic operon containing at least three genes. We show that the operon is regulated at the transcriptional level, from a promoter that is first activated about 80 minutes after the induction of sporulation, immediately after septation. Expression of spoIIIA in different spo mutant backgrounds correlates with the ability of each strain to synthesize the sporulation-specific sigma factor, sigma E. Moreover, synthesis of sigma E in vegetative cells by use of an inducible promoter causes expression of mother-cell-specific genes spoIID, spoIIIA, and spoIIID, but not the prespore-specific genes, spoIIIG and spoVA. We suggest that sigma E may be the primary determinant of mother-cell-specific gene expression and that the SpoIIID protein exerts an additional level of regulation on spoIIIA, apparently by acting as a transcriptional repressor. Since the onset of spoIIID expression occurs about 10 minutes after that of spoIIIA, spoIIIA expression is transient. Thus spoIIIA defines a third temporal class of gene controlled by the sigma E form of RNA polymerase.
Publisher: Wiley
Date: 08-1989
DOI: 10.1111/J.1365-2958.1989.TB00255.X
Abstract: The process of spore formation in the Gram-positive bacterium Bacillus subtilis is a simple developmental system controlled by 50 or more genes. The complex pattern of regulatory interactions between these genes is beginning to be elucidated. spoVJ is a poorly characterized locus in which mutations affect spore development at a relatively late stage (Stage V). We have now cloned and physically characterized the spoVJ locus, and analysed its expression by lacZ fusion. Expression of spoVJ is temporally delayed until about two hours after the initiation of sporulation. Its expression is also spatially restricted to the mother cell compartment as such, it represents the earliest known mother-cell-specific event. Control of spoVJ transcription is complex: expression is dependent upon the products of all of the spoO genes and on some of the spoII genes but it is independent of all later genes except spoIIID. As spoIIID mutations do not affect prespore development, this gene must be an important early determinant of mother-cell-specific gene expression.
Publisher: Elsevier BV
Date: 09-1989
DOI: 10.1016/0378-1119(89)90334-X
Abstract: Zabarovsky and Allikmets [Gene 42 (1986) 119-123] have described a cloning procedure based on partial filling-in of vector and target DNA cohesive ends, which strongly enriches for recombinant molecules with single insertions. Improved Bacillus subtilis bacteriophage phi 105 vectors containing unique cloning sites for SalI have been constructed to take advantage of the partial fill-in method. The new vectors have been used to construct B. subtilis genomic libraries from which several sporulation loci have been isolated, including five not previously cloned. On inserting a promoterless lacZ gene into the cloning site, beta-galactosidase (beta Gal) was detected at a late stage in lytic phage growth, indicating that phage transcription is directed through the cloning site. When UV-irradiated cells ('maxicells') were infected with the recombinant phage containing the lacZ gene, in the presence of labelled amino acids, a protein of the expected Mr for beta Gal was visualised, in addition to the phage proteins. This system should provide a useful general approach for the identification of the products of cloned genes from B. subtilis and other Gram-positive organisms.
Publisher: American Society for Microbiology
Date: 04-1996
DOI: 10.1128/JB.178.8.2343-2350.1996
Abstract: We have cloned and sequenced the promoter-proximal region of the Bacillus subtilis operon containing the pbpB gene, encoding essential penicillin-binding protein PBP2B. The first two genes in the operon, designated yllB and yllC, are significantly similar to genes of unknown function similarly positioned upstream of pbpB in Escherichia coli. Both B. subtilis genes are shown to be nonessential. The third B. subtilis gene, yllD, is essential, as is the correspondingly positioned ftsL gene of E. coli. The predicted product of yllD is similar to FtsL in size and distribution of charged residues but is not significantly related in primary amino acid sequence. The major promoter for the cluster lies upstream of the first gene, yllB, but at least one minor promoter lies within the yllC gene. The operon is transcribed throughout growth at a low level.
Publisher: American Society for Microbiology
Date: 05-1991
DOI: 10.1128/JB.173.10.3159-3169.1991
Abstract: Electron microscopic examination of sporulating cultures of wild-type Bacillus subtilis revealed that the morphological events previously characterized as stages II and III can be ided into four substages, namely, stages IIi, IIii, IIiii, and III. The ultrastructural phenotypes of several stage II mutant strains indicate that each of the four substages has a biochemical and genetic basis. Two of the genes needed for the transition from stage II to stage III encode transcription factors sigma E and sigma F. Their roles during spore morphogenesis have been the subject of much speculation. We now show that sigma E controls genes involved in the morphological transition from stage IIi to stage IIii and then stage IIiii, while the transition to stage III may be determined by genes controlled by sigma F. The results also indicate the existence of at least two undiscovered sporulation genes involved in B. subtilis spore morphogenesis.
Publisher: The Company of Biologists
Date: 07-2018
DOI: 10.1242/JCS.221580
Publisher: Microbiology Society
Date: 2010
Abstract: Spore formation in Bacillus subtilis is a superb experimental system with which to study some of the most fundamental problems of cellular development and differentiation. Work begun in the 1980s and ongoing today has led to an impressive understanding of the temporal and spatial regulation of sporulation, and the functions of many of the several hundred genes involved. Early in sporulation the cells ide in an unusual asymmetrical manner, to produce a small prespore cell and a much larger mother cell. Aside from developmental biology, this modified ision has turned out to be a powerful system for investigation of cell cycle mechanisms, including the components of the ision machine, how the machine is correctly positioned in the cell, and how ision is coordinated with replication and segregation of the chromosome. Insights into these fundamental mechanisms have provided opportunities for the discovery and development of novel antibiotics. This review summarizes how the bacterial cell cycle field has developed over the last 20 or so years, focusing on opportunities emerging from the B. subtilis system.
Publisher: Springer Science and Business Media LLC
Date: 27-06-2016
DOI: 10.1038/NMICROBIOL.2016.91
Abstract: The peptidoglycan cell wall is a major protective external sheath in bacteria and a key target for antibiotics(1). Peptidoglycan is present in virtually all bacteria, suggesting that it was probably present in the last bacterial common ancestor(2). Cell wall expansion is orchestrated by cytoskeletal proteins related to actin (MreB) and tubulin (FtsZ)(3). FtsZ is a key essential player in a highly organized ision machine that directs an invaginating annulus of cell wall peptidoglycan. The recent discovery that cell-wall-less bacteria (L-forms) can grow and ide independently of FtsZ(4,5), provided a means of generating an ftsZ null mutant of Escherichia coli. Remarkably, we have been able to isolate variants of E. coli that lack FtsZ but are capable of efficient growth in a walled state. Genetic analysis reveals that a combination of mutations is needed for this phenotype. Importantly, the suppressive mutations lead to a major cell shape change, from the normal cylindrical shape to a branched and bulging, ramified shape, which we call 'coli-flower'. The results highlight the versatility of bacterial cells and illustrate possible evolutionary routes leading to the emergence of specialized bacteria, such as pathogenic Chlamydia or aquatic Planctomycetes, that lack FtsZ but retain the cell wall(6-8).
Publisher: Oxford University Press (OUP)
Date: 1990
Publisher: Wiley
Date: 22-12-2016
DOI: 10.1111/MMI.13286
Publisher: Wiley
Date: 03-1992
DOI: 10.1111/J.1365-2958.1992.TB01517.X
Abstract: One of the most intriguing questions posed by bacterial spore formation concerns the establishment of cell-specific gene expression in the prespore and mother cell. Recent results now suggest that sigma factors, in addition to their temporal roles in the control of gene expression, may also be the key determinants of differential gene expression during sporulation in Bacillus subtilis. The genes encoding two sporulation-specific sigma factors, sigma E and sigma F, are expressed soon after the initiation of sporulation, before the formation of the spore septum that separates the prespore and mother cell compartments. It now appears that sigma E and sigma F direct transcription only after septation and then in a specific cell type, suggesting that the segregation of the sigma activities after septation is a key event in the establishment of differential gene expression. The mechanism responsible for this segregation is complex, involving at least seven other gene products. We discuss possible models for the interactions between the sigma factors and the establishment of cell-specific transcription.
Publisher: Elsevier BV
Date: 11-2003
DOI: 10.1016/J.TCB.2003.09.005
Abstract: Actin and tubulin are the major components of the cytoskeleton that pervades the cytoplasm of all eukaryotic cells. These proteins were traditionally thought not to be present in prokaryotes, but structural and functional homologues of tubulin (FtsZ) and actin (MreB) are now known to be present virtually throughout the eubacteria and in some archae. FtsZ protein is a key player in cell ision of bacteria and some eukaryotic organelles. MreB proteins are involved in the regulation of cell shape and the segregation of some bacterial plasmids, and might have a range of other functions. Recent data demonstrate that the bacterial proteins are, like their eukaryotic counterparts, highly dynamic. Here, we review the general properties and functions of actin and tubulin homologues in bacteria, their dynamic behaviour and the implications for understanding cell ision and morphogenesis in bacteria.
Publisher: Microbiology Society
Date: 05-2020
DOI: 10.1099/MIC.0.000922
Abstract: Bacillus subtilis is the best studied model organism of the Gram-positive lineage. It is naturally transformable and has an extremely powerful genetic toolbox. It is fast growing and easy to cultivate. It is an important industrial organism, being proficient at secreting proteins and making small fine chemicals, as well as acting as a plant growth promoter. It has been an important model system for studying biofilms. Finally, it makes endospores, which have provided an exceptionally fruitful system for studying various central problems of cellular development, including the generation of asymmetry, cell fate determination and morphogenesis.
Publisher: American Society for Microbiology
Date: 03-2008
DOI: 10.1128/JB.01394-07
Abstract: The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. In Bacillus subtilis 168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis in B. subtilis : TagB, -F, -G, -H, and -O. All of these localized similarly to the inner side of the cytoplasmic membrane, in a pattern strikingly similar to that displayed by probes of nascent PG. Helix-like localization patterns are often attributable to the morphogenic cytoskeletal proteins of the MreB family. However, localization of the Tag proteins did not appear to be substantially affected by single disruption of any of the three MreB homologues of B. subtilis . Bacterial and yeast two-hybrid experiments revealed a complex network of interactions involving TagA, -B, -E, -F, -G, -H, and -O and the cell shape determinants MreC and MreD (encoded by the mreBCD operon and presumably involved in the spatial organization of PG synthesis). Taken together, our results suggest that, in B. subtilis at least, the synthesis and export of WTA precursors are mediated by a large multienzyme complex that may be associated with the PG-synthesizing machinery.
Publisher: Oxford University Press (OUP)
Date: 06-2017
Publisher: Wiley
Date: 04-2000
DOI: 10.1046/J.1365-2958.2000.01857.X
Abstract: Cell ision in most eubacteria is driven by an assembly of about eight conserved ision proteins. These proteins form a ring structure that constricts in parallel with the formation of the ision septum. Here, we show that one of the ision proteins, FtsL, is highly unstable. We also show that the protein is targeted to the ring structure and that targeting occurs in concert with the recruitment of several other membrane-associated ision proteins. FtsL stability is further reduced in the absence of DivIB protein (probably homologous to E. coli FtsQ) at high temperature, suggesting that DivIB is involved in the control of FtsL turnover. The reduced stability of FtsL may explain the temperature dependence of IB mutants, because their phenotype can be suppressed by overexpression of FtsL. The results provide new insights into the roles of the FtsL and DivIB proteins in bacterial cell ision.
Publisher: Wiley
Date: 05-2000
DOI: 10.1046/J.1365-2958.2000.01895.X
Abstract: FtsL is a small bitopic membrane protein required for vegetative cell ision and sporulation in Bacillus subtilis. We investigated its localization by fluorescence microscopy using a green fluorescent protein (GFP) fusion. GFP-FtsL was localized at mid-cell in vegetative cells and at the asymmetric septum in sporulating cells. We also show that FtsL forms a ring-like structure at the ision site and that it remains localized at mid-cell during the whole septation process. By yeast two-hybrid analysis and non-denaturing polyacrylamide gel electrophoresis (PAGE) with purified proteins, FtsL was found to interact with another membrane-bound ision protein, the FtsL-like DivIC protein.
Publisher: Microbiology Society
Date: 02-1993
DOI: 10.1099/00221287-139-2-361
Abstract: The sequence of a 4.4 kbp region of DNA from Bacillus subtilis 168, lying between sporulation genes spoVD and spoVE, has been determined as part of the B. subtilis genomic sequencing programme. The region contains three genes with high sequence similarity to the murE, mraY and murD genes of Escherichia coli. The products of these genes are likely to catalyse various steps in the formation of the precursors for peptidoglycan synthesis in B. subtilis. The regions at 133 degrees on the standard genetic map of the B. subtilis chromosome, and in the 2 min region of the E. coli genetic map, are now shown to contain a large cluster of functionally related genes. Although the linear order of the genes in the cluster is conserved, three genes that are present in the E. coli chromosome, and which are likely to be essential for peptidoglycan synthesis in both organisms, are absent from this region of the B. subtilis chromosome. In general, the B. subtilis cluster differs from that of E. coli in having more extensive intergenic regions, with less potential for translational coupling.
Publisher: Annual Reviews
Date: 12-1995
DOI: 10.1146/ANNUREV.GE.29.120195.000353
Abstract: This review addresses chromosome partitioning in Escherichia coli and Bacillus subtilis. The first part deals with events associated with completion of a round of replication to an extent that yields separable chromosomes. Events more directly involved in chromosome movement are covered in the second part. In the final section, a model for chromosome partitioning based on the information presented in the first two parts is presented.
Publisher: Wiley
Date: 18-12-2003
DOI: 10.1046/J.1365-2958.2003.03264.X
Abstract: The Bacillus subtilis soj‐spo0J locus encodes two proteins belonging to a family of proteins (the ParAB proteins) with dual roles in plasmid or chromosome segregation and transcriptional regulation. Soj protein was previously shown to be capable of abrupt subcellular relocation. The movement was highly co‐operative and at any moment, most of the Soj in any cell formed a single large ‘patch’ covering all or part of one nucleoid. Movement, and co‐operativity, in the sense of formation of a single patch, was dependent on Spo0J. Movement, but not co‐operativity, was also shown to be dependent, directly or indirectly, on FtsZ protein. We now report that the ftsZ effect arises because jumping onto a nucleoid is promoted by proximity to a cell pole. Studies of other mutants affected in cell ision suggest that the attraction to the cell pole is mediated by the ision‐site‐selection protein, MinD (which localizes at the cell poles). It does not require MinC, the main effector of the ision site selection system. A mutant form of Soj, putatively locked in the ATP form of the protein, interacts with the cell pole (dependent on MinD) but not with the nucleoid. These results identify a novel function for MinD and demonstrate an intriguing link between proteins involved in the cell ision and chromosome segregation machineries.
Publisher: Wiley
Date: 16-01-2011
DOI: 10.1111/J.1365-2958.2010.07507.X
Abstract: Control of DNA replication initiation is essential for bacterial cells to co-ordinate the faithful replication and segregation of their genetic material. The Bacillus subtilis ATPase Soj is a dynamic protein that regulates DNA replication initiation by either inhibiting or activating the DNA replication initiator protein DnaA. Here we report that the key event which switches Soj regulatory activity is a transition in its oligomeric state from a monomer to an ATP-dependent homodimer capable of DNA binding. We show that the DNA binding activity of the Soj dimer is required both for activation of DNA replication initiation and for interaction with Spo0J. Finally, we demonstrate that Spo0J inhibits Soj dimerization by stimulating Soj ATPase activity. The data provide a molecular explanation for the dichotomous regulatory activities of Soj, as well as assigning unique Soj conformations to distinct cellular localization patterns. We discuss how the regulation of Soj ATPase activity by Spo0J could be utilized to control the initiation of DNA replication during the cell cycle.
Publisher: Wiley
Date: 08-2002
DOI: 10.1046/J.1365-2958.2002.03082.X
Abstract: Starvation induces Bacillus subtilis to initiate a -simple, two-cell developmental process that begins with an asymmetric cell ision. Activation of the first compartment-specific transcription factor, sigmaF, is coupled to this morphological event. SpoIIE, a bifunctional protein, is essential for the compartment-specific activation of sigmaF and also has a morphogenic activity required for asymmetric cell ision. SpoIIE consists of three domains: a hydrophobic N-terminal domain, which targets the protein to the membrane a central domain, involved in oligomerization of SpoIIE and interaction with the cell ision protein FtsZ and a C-terminal domain comprising a PP2C protein phosphatase. Here, we report the isolation of mutations at the very beginning of the central domain of spoIIE, which are capable of activating sigmaF inde-pendently of septum formation. Purified mutant proteins showed the same phosphatase activity as the wild-type protein in vitro. The mutant proteins were fully functional in respect of their localization to sites of asymmetric septation and support of asymmetric ision. The data provide strong evidence that the phosphatase domain of SpoIIE is tightly regulated in a way that makes it respond to the formation of the asymmetric septum.
Publisher: American Society for Microbiology
Date: 26-10-2022
DOI: 10.1128/SPECTRUM.02419-22
Abstract: Bacterial L-forms require specialized culture techniques and are neither widely reported nor well understood in human infections. To date, most of the studies have been conducted on Gram-positive and stable L-form bacteria, which usually require mutagenesis or long-term passages for their generation.
Publisher: Springer Science and Business Media LLC
Date: 26-09-2019
DOI: 10.1038/S41467-019-12359-3
Abstract: Recurrent urinary tract infection (rUTI) is a major medical problem, especially in the elderly and infirm, but the nature of the reservoir of organisms responsible for survival and recolonisation after antibiotic treatment in humans is unclear. Here, we demonstrate the presence of cell-wall deficient (L-form) bacteria in fresh urine from 29 out of 30 older patients with rUTI. In urine, E. coli strains from patient s les readily transition from the walled state to L-form during challenge with a cell wall targeting antibiotic. Following antibiotic withdrawal, they then efficiently transition back to the walled state. E. coli switches between walled and L-form states in a zebrafish larva infection model. The results suggest that L-form switching is a physiologically relevant phenomenon that may contribute to the recurrence of infection in older patients with rUTI, and potentially other infections.
Publisher: Wiley
Date: 2001
DOI: 10.1046/J.1365-2958.2001.02253.X
Abstract: The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.
Publisher: Elsevier BV
Date: 12-2014
Publisher: Wiley
Date: 12-2001
DOI: 10.1046/J.1365-2958.2001.02710.X
Abstract: In rod-shaped bacteria such as Bacillus subtilis, ision site selection is mediated by MinC and MinD, which together function as a ision inhibitor. Topological specificity is imposed by DivIVA, which ensures that MinCD specifically inhibits ision close to the cell poles, while allowing ision at mid-cell. MinD plays a central role in this process, as it positions and activates MinC and is dependent on DivIVA for its own positioning at the poles. To investigate MinD activities further, we have constructed and analysed a collection of minD mutants. Mutations in the conserved ATPase motifs lead to an inactive protein, possibly unable to oligomerize, but which nevertheless retains some affinity for the cell membrane. Several mutations affecting the mid- to C-terminal parts of MinD led to a protein probably unable to interact with DivIVA, but that could still stimulate ision inhibition by MinC. These findings suggest that the ATPase activity of MinD is necessary for all its functions (possibly in part by controlling the oligomerization state of the protein). The other mutations may identify a surface of MinD involved in its interactions with DivIVA and a possible mechanism for control of MinD by DivIVA.
Publisher: Wiley
Date: 26-01-2005
DOI: 10.1111/J.1365-2958.2005.04506.X
Abstract: MreB shares a common prokaryotic ancestor with actin and is present in almost all rod-shaped bacteria. MreB proteins have been implicated in a range of important cell processes, including cell morphogenesis, chromosome segregation and cell polarity. The mreB gene frequently lies at the beginning of a cluster of genes, immediately upstream of the conserved mreC and mreD genes. RNA analysis showed that in Bacillus subtilis mreB is co-transcribed with mreC and that these genes form part of an operon under the control of a promoter(s) upstream of mreB. Construction of an in-frame deletion of mreB and its complementation by mreB(+) only, in trans, established that the gene is important for maintenance of cell width and cell viability under normal growth conditions, independent of polar effects on downstream genes. Remarkably, virtually normal growth was restored to the mreB null mutant in the presence of high concentrations of magnesium, especially when high concentrations of the osmoprotectant, sucrose were also present. Under these conditions, cells could be maintained in the complete absence of an mreB gene, with almost normal morphology. No detectable effect on chromosome segregation was evident in the mutant, nor was there an effect on the topology of nascent peptidoglycan insertion. A GFP-MreB fusion was used to look at the localization of MreB in live cells. The pattern of localization was similar to that previously described, but no tight linkage to nucleoid positioning was evident. Propagation of the mreB null mutant in the absence of magnesium and sucrose led to a progressive increase in cell width, culminating in cell lysis. Cell ision was also perturbed but this effect may be secondary to the disturbance in cell width. These results suggest that the major role of MreB in B. subtilis lies in the control of cell diameter.
Publisher: Microbiology Society
Date: 12-1992
DOI: 10.1099/00221287-138-12-2609
Abstract: The spo-87 mutation is one of two sporulation mutations originally used to define the spo0J locus of Bacillus subtilis. We now show that it blocks sporulation after completion of prespore engulfment (stage III). Surprisingly, the operon is expressed vegetatively, probably from a sigma A-dependent promoter, and its expression is shut down at the transcriptional level at about the onset of sporulation. DNA sequencing reveals that the locus defined by spo-87, which we now designate spoIIIJ, consists of a bicistronic operon. However, only the first gene is essential for sporulation the function of the second cistron is cryptic. The predicted SpoIIIJ product has an M(r) of 29,409. It probably forms a lipoprotein and is rich in basic and hydrophobic amino acids. Mutations in spoIIIJ abolish the transcription of prespore-specific genes transcribed by the sigma G form of RNA polymerase but not transcription of the spoIIIG gene encoding sigma G. The SpoIIIJ product could be involved in a signal transduction pathway coupling gene expression in the prespore to events in the mother cell, or it could be necessary for essential metabolic interactions between the two cells.
Publisher: Proceedings of the National Academy of Sciences
Date: 26-04-1994
Abstract: Soon after the initiation of sporulation, Bacillus subtilis ides asymmetrically to produce sister cells that have very different developmental fates. Recently, it has been proposed that the differential gene expression which begins soon after this ision is due to cell-specific activation of the transcription factors sigma F and sigma E in the prespore and the mother cell, respectively. We describe the use of a method for the localization of gene expression in in idual sporulating cells that lends strong support to the cell-specific localization of sigma F and sigma E activities. The dependence of sigma E activity on integrity of the gene encoding sigma F has led to the suggestion that activation of sigma F in the prespore leads to a directional signal that triggers activation of sigma E only in the mother cell. Here we show that sigma E actually specifies the fate of the mother cell in the absence of sigma E, two prespore-like cells are made. The appearance of sigma F activity at both poles of a sigma E-deficient mutant supports the idea that sigma F normally remains latent in the mother cell and that its activation depends on some morphological or physiological feature of the prespore. We present a model for the generation of asymmetry and the establishment of cell fate in B. subtilis.
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.CELL.2008.07.044
Abstract: Regulation of DNA replication and segregation is essential for all cells. Orthologs of the plasmid partitioning genes parA, parB, and parS are present in bacterial genomes throughout the prokaryotic evolutionary tree and are required for accurate chromosome segregation. However, the mechanism(s) by which parABS genes ensure proper DNA segregation have remained unclear. Here we report that the ParA ortholog in B. subtilis (Soj) controls the activity of the DNA replication initiator protein DnaA. Subcellular localization of several Soj mutants indicates that Soj acts as a spatially regulated molecular switch, capable of either inhibiting or activating DnaA. We show that the classical effect of Soj inhibiting sporulation is an indirect consequence of its action on DnaA through activation of the Sda DNA replication checkpoint. These results suggest that the pleiotropy manifested by chromosomal parABS mutations could be the indirect effects of a primary activity regulating DNA replication initiation.
Publisher: Elsevier BV
Date: 03-2001
DOI: 10.1016/S0092-8674(01)00287-2
Abstract: In the absence of an overt cytoskeleton, the external cell wall of bacteria has traditionally been assumed to be the primary determinant of cell shape. In the Gram-positive bacterium Bacillus subtilis, two related genes, mreB and mbl, were shown to be required for different aspects of cell morphogenesis. Subcellular localization of the MreB and Mbl proteins revealed that each forms a distinct kind of filamentous helical structure lying close to the cell surface. The distribution of the proteins in different species of bacteria, and the similarity of their sequence to eukaryotic actins, suggest that the MreB-like proteins have a cytoskeletal, actin-like role in bacterial cell morphogenesis.
Publisher: Elsevier BV
Date: 09-2006
DOI: 10.1016/J.DEVCEL.2006.07.017
Abstract: MreB proteins are bacterial actin homologs involved in cell morphogenesis and various other cellular processes. However, the effector proteins used by MreBs remain largely unknown. Bacillus subtilis has three MreB isoforms. Mbl and possibly MreB have previously been shown to be implicated in cell wall synthesis. We have now found that the third isoform, MreBH, colocalizes with the two other MreB isoforms in B. subtilis and also has an important role in cell morphogenesis. MreBH can physically interact with a cell wall hydrolase, LytE, and is required for its helical pattern of extracellular localization. Moreover, lytE and mreBH mutants exhibit similar cell-wall-related defects. We propose that controlled elongation of rod-shaped B. subtilis depends on the coordination of cell wall synthesis and hydrolysis in helical tracts defined by MreB proteins. Our data also suggest that physical interactions with intracellular actin bundles can influence the later localization pattern of extracellular effectors.
Publisher: Wiley
Date: 02-09-1985
Publisher: Elsevier BV
Date: 06-2003
DOI: 10.1016/S0092-8674(03)00421-5
Abstract: Cell shape in most eubacteria is maintained by a tough external peptidoglycan cell wall. Recently, cell shape determining proteins of the MreB family were shown to form helical, actin-like cables in the cell. We used a fluorescent derivative of the antibiotic vancomycin as a probe for nascent peptidoglycan synthesis in unfixed cells of various Gram-positive bacteria. In the rod-shaped bacterium B. subtilis, synthesis of the cylindrical part of the cell wall occurs in a helical pattern governed by an MreB homolog, Mbl. However, a few rod-shaped bacteria have no MreB system. Here, a rod-like shape can be achieved by a completely different mechanism based on use of polar growth zones derived from the ision machinery. These results provide insights into the erse molecular strategies used by bacteria to control their cellular morphology, as well as suggesting ways in which these strategies may impact on growth rates and cell envelope structure.
Publisher: Wiley
Date: 13-09-2007
Publisher: American Society for Microbiology
Date: 07-11-2018
Publisher: Springer Science and Business Media LLC
Date: 09-2009
DOI: 10.1038/NRMICRO2198
Abstract: Bacterial cell ision is orchestrated by a tubulin homologue, FtsZ, which polymerizes to form a ring-like structure that is both a scaffold for the assembly of the bacterial cytokinetic machinery and, at least in part, a source of the energy for constriction. FtsZ assembly is tightly regulated, and a erse repertoire of accessory proteins contributes to the formation of a functional ision machine that is responsive to cell cycle status and environmental stress. In this Review, we describe the interaction of these proteins with FtsZ and discuss recent advances in our understanding of Z ring assembly.
Publisher: Microbiology Society
Date: 12-1993
DOI: 10.1099/00221287-139-12-3197
Abstract: The spoIIAB gene of Bacillus subtilis encodes an inhibitor of sigma F, a transcription factor that plays a crucial role in the establishment of prespore-specific gene expression during sporulation. The SpoIIAB protein can probably also inhibit a closely related sigma factor sigma G, which determines the later phase of prespore-specific transcription. We have isolated two new missense mutations in the spoIIAB gene. spoIIAB28 behaves like the previously described spoIIAB1 mutation, in that it mainly affects the activity of sigma G. In contrast, the spoIIAB22 mutation seems to be impaired mainly in its ability to inhibit sigma F. All three missense mutations are clustered in the N-terminal coding region of spoIIAB, suggesting that this region of the protein interacts with the sigma factors. The extreme N-terminal part of SpoIIAB may be specifically concerned with the regulation of sigma G activity.
Publisher: Wiley
Date: 02-11-2006
DOI: 10.1111/J.1365-2958.2006.05485.X
Abstract: The bacterial actin homologue MreB forms helical filaments in the cytoplasm of rod-shaped bacteria where it helps maintain the shape of the cell. MreB is co-transcribed with mreC that encodes a bitopic membrane protein with a major periplasmic domain. Like MreB, MreC is localized in a helical pattern and might be involved in the spatial organization of the peptidoglycan synthesis machinery. Here, we present the structure of the major, periplasmic part of MreC from Listeria monocytogenes at 2.5 A resolution. MreC forms a dimer through an intimate contact along an N-terminal alpha-helix that connects the transmembrane region with two C-terminal beta-domains. The translational relationship between the molecules enables, in principle, filament formation. One of the beta-domains shows structural similarity to the chymotrypsin family of proteins and possesses a highly conserved Thr Ser dipeptide. Unexpectedly, mutagenesis studies show that the dipeptide is dispensable for maintaining cell shape and viability in both Escherichia coil and Bacillus subtilis. Bacterial two-hybrid experiments reveal that MreC Interacts with high-molecular-weight penicillin-binding proteins (PBPs), rather than with low-molecular-weight endo- and carboxypeptidases, indicating that MreC might act as a scaffold to which the murein synthases are recruited in order to spatially organize the synthesis of new cell wall material. Deletion analyses indicate which domains of B. subtilis MreC are required for interaction with MreD as well as with the PBPs.
Publisher: Cold Spring Harbor Laboratory
Date: 05-1998
Abstract: The spoIIE gene is essential for the compartment-specific activation of transcription factor sigmaF during sporulation in Bacillus subtilis. SpoIIE is a membrane protein that is targeted to the potential sites of asymmetric septation near each pole of the sporulating cell. The cytoplasmic carboxy-terminal domain of SpoIIE contains a serine phosphatase that triggers the release of sigmaF in the prespore compartment after septation. To understand how septum-located SpoIIE is activated selectively in the prespore, we examined the distribution of a SpoIIE-GFP fusion protein. We show that the polar bands of SpoIIE protein actually form sequentially and that the most prominent band develops at the pole where the prespore forms. We also show that the protein is sequestered to the prespore side of the asymmetric septum. Sequestration of SpoIIE into the prespore compartment provides a mechanism that could explain the cell specificity of sigmaF activation.
Publisher: Wiley
Date: 11-08-2003
DOI: 10.1046/J.1365-2958.2003.03643.X
Abstract: Sporulating cells of Bacillus subtilis undergo a highly polarized cell ision and possess a specialized mechanism to move the oriC region of the chromosome close to the cell pole before septation. DivIVA protein, which localizes to the cell pole, and the Soj and Spo0J proteins, which associate with the chromosome, are part of the mechanism that delivers the chromosome to the cell pole. A sporulation-specific protein, RacA, encodes a third DNA-binding protein, which acts in conjunction with Soj and Spo0J to effect efficient polar chromosome segregation. IVA mutants and soj racA double mutants have an unexpected phenotype in which specific markers to the left and right of oriC can be captured in the prespore compartment but the central oriC region is efficiently excluded. This 'residual' trapping requires Spo0J protein. We suggest that the Soj RacA DivIVA system is required to extract the oriC region from its position determined by the vegetative chromosome segregation machinery and anchor it to the cell pole.
Publisher: Wiley
Date: 16-12-2004
DOI: 10.1046/J.1365-2958.2003.03854.X
Abstract: Bacterial cell shape is determined by a rigid external cell wall. In most non-coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl. To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin-binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis. The localization of these fusions was studied in a wild-type background as well as in strains deficient in FtsZ, MreB or Mbl. PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum. All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum. This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall. The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth. Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP-PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl.
Publisher: Wiley
Date: 04-2001
DOI: 10.1046/J.1365-2958.2001.02356.X
Abstract: The actin-like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell ision. Here, we show that Bacillus subtilis FtsA is targeted to ision sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential ision sites at both poles of the cell, but asymmetric ision occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the ision site. Therefore, FtsA may have a direct structural role in cell ision. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP-binding and ATP-hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell ision in B. subtilis (and possibly in most eubacteria).
Publisher: Wiley
Date: 10-1996
DOI: 10.1046/J.1365-2443.1996.750275.X
Abstract: Differential gene expression during sporulation in the prespore and mother cell of Bacillus subtilis is dependent on the correct timing and localization of the activity of specific transcription (sigma) factors. The first sigma factor activated is sigmaF, which directs gene expression specifically in the prespore compartment. Release of sigmaF activity is tightly controlled through a series of complex interactions involving an anti-sigma factor, SpoIIAB, an anti-anti-sigma factor SpoIIAA and a phosphoprotein phosphatase SpoIIE. In vitro studies have shown that SpoIIAB binds to sigmaF, preventing transcription of the sigmaF regulon, and that it can also phosphorylate SpoIIAA, thereby inactivating it. However, non-phosphorylated SpoIIAA can displace sigmaF from SpoIIAB. The SpoIIE phosphatase provides a means of reactivating SpoIIAA-P. We have directly determined the cellular distributions of sigmaF, SpoIIAB, SpoIIAA-P and SpoIIAA during sporulation, using recently developed immunofluorescence methods. While sigmaF activity is restricted to the prespore, the protein is present in both compartments. As development proceeds the sigmaF signal disappears. The anti-sigma factor SpoIIAB is also distributed throughout both cells and rapidly disappears from both cellular compartments soon after sigmaF becomes active. Disappearance of SpoIIAB seems to be closely associated with the activation of the second prespore-specific sigma factor sigmaF. The distribution of phosphorylated SpoIIAA closely mimics that of SpoIIAB, being non-compartmentalized and disappearing soon after sigmaF activation occurs. Significantly, the active, non-phosphorylated form of the anti-anti-sigma factor, SpoIIAA, accumulates in the prespore just before sigmaF becomes active. These results support the hypothesis that the accumulation of SpoIIAA within the prespore is the single most important requirement for activation of sigmaF.
Publisher: Springer Science and Business Media LLC
Date: 12-01-2015
DOI: 10.1038/NRMICRO3398
Abstract: Work over the past decade has highlighted the pivotal role of the actin-like MreB family of proteins in the determination and maintenance of rod cell shape in bacteria. Early images of MreB localization revealed long helical filaments, which were suggestive of a direct role in governing cell wall architecture. However, several more recent, higher-resolution studies have questioned the existence or importance of the helical structures. In this Opinion article, I navigate a path through these conflicting reports, revive the helix model and summarize the key questions that remain to be answered.
Publisher: Wiley
Date: 08-1996
DOI: 10.1111/J.1365-2958.1996.TB02559.X
Abstract: During sporulation in Bacillus subtilis a small prespore cell is formed by an asymmetric cell ision. Pre-spore chromosome partitioning occurs by a specialised mechanism in which septation precedes chromosome movement. We show that the spo0J gene is needed to specify the orientation of the chromosome at the time of polar ision and to impose directionality on the subsequent transport of the remainder of the chromosome through the septum. Both phenotypes may arise by disruption of a centromere-like apparatus that anchors the or/C region of the prespore chromosome in the pole of the cell.
Publisher: Wiley
Date: 24-11-2008
DOI: 10.1111/J.1365-2958.2008.06501.X
Abstract: Cell ision in bacteria is governed by a complex cytokinetic machinery in which the key player is a tubulin homologue, FtsZ. Most rod-shaped bacteria ide precisely at mid-cell between segregated sister chromosomes. Selection of the correct site for cell ision is thought to be determined by two negative regulatory systems: the nucleoid occlusion system, which prevents ision in the vicinity of the chromosomes, and the Min system, which prevents inappropriate ision at the cell poles. In Bacillus subtilis recruitment of the ision inhibitor MinCD to cell poles depends on DivIVA, and these proteins were thought to be sufficient for Min function. We have now identified a novel component of the ision-site selection system, MinJ, which bridges DivIVA and MinD. minJ mutants are impaired in ision because MinCD activity is no longer restricted to cell poles. Although MinCD was thought to act specifically on FtsZ assembly, analysis of minJ and IVA mutants showed that their block in ision occurs downstream of FtsZ. The results support a model in which the main function of the Min system lies in allowing only a single round of ision per cell cycle, and that MinCD acts at multiple levels to prevent inappropriate ision.
Publisher: American Society for Microbiology
Date: 15-12-2013
DOI: 10.1128/JB.00507-13
Abstract: Bacterial cell ision is a highly coordinated process that begins with the polymerization of the tubulin-like protein FtsZ at midcell. FtsZ polymerization is regulated by a set of conserved cell ision proteins, including ZapA. However, a zapA mutation does not result in a clear phenotype in Bacillus subtilis . In this study, we used a synthetic-lethal screen to find genes that become essential when ZapA is mutated. Three transposon insertions were found in yvcL . The deletion of yvcL in a wild-type background had only a mild effect on growth, but a yvcL zapA double mutant is very filamentous and sick. This filamentation is caused by a strong reduction in FtsZ-ring assembly, suggesting that YvcL is involved in an early stage of cell ision. YvcL is 25% identical and 50% similar to the Streptomyces coelicolor transcription factor WhiA, which induces ftsZ and is required for septation of aerial hyphae during sporulation. Using green fluorescent protein fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP-on-chip assay gave no indication that YvcL functions as a transcription factor. To gain more insight into the function of YvcL, we searched for suppressors of the filamentous phenotype of a yvcL zapA double mutant. Transposon insertions in gtaB and pgcA restored normal cell ision of the double mutant. The corresponding proteins have been implicated in the metabolic sensing of cell ision. We conclude that YvcL (WhiA) is involved in cell ision in B. subtilis through an as-yet-unknown mechanism.
Publisher: Elsevier BV
Date: 11-1999
DOI: 10.1016/S1097-2765(00)80378-0
Abstract: The Spo0J and Soj proteins of B. subtilis belong to a widespread family of bacterial proteins required for accurate segregation of plasmids and chromosomes. Spo0J binds to several sites around the oriC region of the chromosome, which are organized into compact foci that may play a centromere-like role in active chromosome segregation. We now show that Soj has a role in organization or compaction of Spo0J-oriC complexes and possibly other regions of the nucleoid. This activity is accompanied by a dynamic localization pattern in which Soj protein undergoes assembly and disassembly into large nucleoid-associated patches on a timescale of minutes. The dynamic behavior of Soj, like its previously described transcriptional repression activity, is controlled by Spo0J. These interactions may constitute a checkpoint coupling developmental transcription to cell cycle progression.
Publisher: Wiley
Date: 20-03-2008
DOI: 10.1111/J.1365-2958.2008.06210.X
Abstract: The characteristic shape of bacterial cells is mainly determined by the cell wall, the synthesis of which is orchestrated by penicillin-binding proteins (PBPs). Rod-shaped bacteria have two distinct modes of cell wall synthesis, involved in cell elongation and cell ision, which are believed to employ different sets of PBPs. A long-held question has been how these different modes of growth are co-ordinated in space and time. We have now identified the cell ision protein, EzrA, and a newly discovered protein, GpsB, as key players in the elongation- ision cycle of Bacillus subtilis. Mutations in these genes have a synthetic phenotype with defects in both cell ision and cell elongation. They also have an unusual bulging phenotype apparently due to a failure in properly completing cell pole maturation. We show that these phenotypes are tightly associated with disturbed localization of the major transglycosylase/transpeptidase of the cell, PBP1. EzrA and GpsB have partially differentiated roles in the localization cycle of PBP1, with EzrA mainly promoting the recruitment of PBP1 to ision sites, and GpsB facilitating its removal from the cell pole, after the completion of pole maturation.
Publisher: Wiley
Date: 05-1993
DOI: 10.1111/J.1365-2958.1993.TB01639.X
Abstract: We have established a time course for the early morphological events of sporulation in Bacillus subtilis and related this to changes in gene expression, particularly those occurring in the prespore compartment. We have also systematically studied the effects of mutations in various regulatory (spo) genes on prespore-specific gene expression. On the basis of these results, and those of other laboratories, at least four distinct temporal classes of prespore-specific gene expression can now be distinguished. The first class begins within 15 min of the formation of the spore septum, and requires the sigma F form of RNA polymerase. The second class, also directed by RNA polymerase containing sigma F, begins soon after the completion of prespore engulfment, and depends on an intercellular signal from the mother cell. This transcription results in synthesis of sigma G. However, sigma G activity, directing the third class of gene expression, appears only about 30 min later and is dependent on the completion of prespore engulfment and on further interactions with the mother cell. The fourth class of gene expression has been described. The results demonstrate that the prespore programme of gene expression incorporates a series of control points modulated by information from the mother cell and on progress through the morphogenetic process.
Publisher: Wiley
Date: 04-1990
DOI: 10.1111/J.1365-2958.1990.TB00622.X
Abstract: The gene spoIIID, which is essential for spore formation in Bacillus subtilis, was cloned and sequenced. It consists of one open reading frame which would encode a 93-amino-acid protein with a classic helix-turn-helix motif, characteristic of sequence-specific DNA-binding proteins. SpoIIID protein is a previously identified transcription factor, capable of altering the specificity of RNA polymerase containing sigma K in vitro (Kroos et al., 1989). The spoIIID83 mutation (by which the locus was originally identified), was sequenced and found to be a single base substitution in the ribosome binding site upstream of the spoIIID open reading frame. A transcriptional fusion to lacZ was constructed and used to examine the regulation of spoIIID. Expression of spoIIID occurred only during sporulation, beginning 1.5 to 2 hours after the initiation of sporulation. The dependence of spoIIID expression on other spo loci suggests that it is mother-cell-specific, and that it is transcribed by sigma E-containing RNA polymerase.
Publisher: Wiley
Date: 04-2006
Abstract: Construction and microscopic imaging of protein fusions to green fluorescent protein (GFP) have revolutionised our understanding of bacterial structure and function. We have undertaken a systematic study of the localisation of over 100 Bacillus subtilis proteins, following the development of high-throughput construction and analysis procedures. We focused on proteins linked in various ways to the DNA replication machinery, as well as on proteins exemplifying a range of other cellular functions and structures. The results validate the approach as a way of obtaining systematic protein localisation information. They also provide a range of novel biological insights, particularly through the identification of a number of proteins not previously known to be associated with the DNA replication factory.
Publisher: Wiley
Date: 29-08-2017
DOI: 10.1111/MMI.13765
Publisher: Oxford University Press (OUP)
Date: 1993
Publisher: American Society for Microbiology
Date: 15-01-2000
DOI: 10.1128/JB.182.2.418-424.2000
Abstract: Differential gene expression during Bacillus subtilis sporulation is controlled by sigma factors and other regulatory effectors. The first compartmentalized sigma factor, ς F , is active specifically in the prespore compartment. During our screening for new chromosome segregation mutants using a ς F -dependent gpr-lacZ reporter as a probe, we identified a new gene ( ywfN ) required for maximal expression of the reporter and named it rsfA . The product of rsfA has features of gene regulatory proteins, and the protein colocalizes with DNA. The expression of rsfA is under the control of both ς F and ς G . Null mutations in rsfA have different effects on the expression of ς F -dependent genes, suggesting that the RsfA protein is a regulator of transcription that fine-tunes gene expression in the prespore.
Publisher: Wiley
Date: 09-1999
DOI: 10.1046/J.1365-2958.1999.01543.X
Abstract: Early in the process of spore formation in Bacillus subtilis, asymmetric cell ision produces a large mother cell and a much smaller prespore. Differentiation of the prespore is initiated by activation of an RNA polymerase sigma factor, sigmaF, specifically in that cell. sigmaF is controlled by a regulatory cascade involving an anti-sigma factor, SpoIIAB, an anti-anti-sigma factor, SpoIIAA, and a membrane-bound phosphatase, SpoIIE, which converts the inactive, phosphorylated form of SpoIIAA back to the active form. SpoIIE is required for proper asymmetric ision and much of the protein is sequestered into the prespore during septation. Importantly, activation of sigmaF is dependent on formation of the asymmetric septum. We have now characterized this morphological checkpoint in detail, using strains affected in cell ision and/or spoIIE function. Surprisingly, we found that significant dephosphorylation of SpoIIAA occurred even in the absence of septation. This shows that the SpoIIE phosphatase is at least partially active independent of the morphological event and also that cells can tolerate significant levels of unphosphorylated SpoIIAA without activating sigmaF. We also describe a spoIIE mutant in which the checkpoint is bypassed, probably by an increase in the dephosphorylation of SpoIIAA. Taken together, the results support the idea that sequestration of SpoIIE protein into the prespore plays an important role in the control of sigmaF activation and in coupling this activation to septation.
Publisher: Elsevier BV
Date: 12-2003
Publisher: Microbiology Society
Date: 06-2005
Abstract: A key event in cytokinesis in bacteria is the assembly of the essential ision protein FtsZ into ring-like structures at the nascent ision site. FtsZ is the prokaryotic homologue of tubulin, and is found in nearly all bacteria. In vitro , FtsZ polymerizes in the presence of GTP to form higher-ordered polymers. FtsZ consists of two domains, with the GTP-binding site located in the N-terminal domain. The less-conserved C-terminal domain contains residues important for GTP hydrolysis, but its overall function is still unclear. This paper reports the development of a simple strategy to generate mutations in the essential ision gene ftsZ . Nine novel and viable ftsZ mutants of Bacillus subtilis are described. Eight of the mutations would affect the C-terminus of FtsZ. The collection of mutants exhibits a range of morphological phenotypes, ranging from normal to highly filamentous cells some produce minicells, or ide in a twisted configuration one mutation has a temperature-sensitive effect specifically impairing sporulation. The sites of the amino acid changes generated by the mutations could be informative about FtsZ function and its protein–protein interactions.
Publisher: Oxford University Press (OUP)
Date: 11-2004
DOI: 10.1016/J.FEMSLE.2004.09.038
Abstract: DivIVA is involved in placement of the ision septum and chromosome segregation in Bacillus subtilis and it plays important roles in cell ision or morphogenesis in erse Gram-positive bacteria. In Staphylococcus aureus, DivIVA is localized at the ision septum, but it does not colocalize with the chromosomal origin of replication, as labeled with SpoOJ protein. Unexpectedly, a IVA null mutant is not impaired in growth, nor is it affected in chromosome segregation or cell morphology.
Publisher: American Chemical Society (ACS)
Date: 29-11-2017
DOI: 10.1021/ACSCHEMBIO.7B00733
Abstract: Antibiotics that interfere with the bacterial cytoplasmic membrane have long-term potential for the treatment of infectious diseases as this mode of action is anticipated to result in low resistance frequency. Vancoresmycin is an understudied natural product antibiotic consisting of a terminal tetramic acid moiety fused to a linear, highly oxygenated, stereochemically complex polyketide chain. Vancoresmycin shows minimum inhibitory concentrations (MICs) from 0.125 to 2 μg/mL against a range of clinically relevant, antibiotic-resistant Gram-positive bacteria. Through a comprehensive mode-of-action study, utilizing Bacillus subtilis reporter strains, DiSC
Publisher: Wiley
Date: 15-02-2000
Publisher: American Society for Microbiology
Date: 15-01-2007
DOI: 10.1128/JB.01381-06
Abstract: To address the need for new antibacterials, a number of bacterial genomes have been systematically disrupted to identify essential genes. Such programs have focused on the disruption of single genes and may have missed functions encoded by gene pairs or multiple genes. In this work, we hypothesized that we could predict the identity of pairs of proteins within one organism that have the same function. We identified 135 putative protein pairs in Bacillus subtilis and attempted to disrupt the genes forming these, singly and then in pairs. The single gene disruptions revealed new genes that could not be disrupted in idually and other genes required for growth in minimal medium or for sporulation. The pairwise disruptions revealed seven pairs of proteins that are likely to have the same function, as the presence of one protein can compensate for the absence of the other. Six of these pairs are essential for bacterial viability and in four cases show a pattern of species conservation appropriate for potential antibacterial development. This work highlights the importance of combinatorial studies in understanding gene duplication and identifying functional redundancy.
Publisher: Wiley
Date: 29-09-2005
Publisher: Springer Science and Business Media LLC
Date: 09-11-2017
DOI: 10.1038/S41467-017-01596-Z
Abstract: The central player in bacterial cell ision, FtsZ, is essential in almost all organisms in which it has been tested, with the most notable exception being Streptomyces . Streptomycetes differ from many bacteria in growing from the cell tip and undergoing branching, similar to filamentous fungi. Here we show that limited cell damage, either mechanical or enzymatic, leads to near complete destruction of mycelial microcolonies of a Streptomyces venezuelae ftsZ mutant. This result is consistent with a lack of ftsZ -dependent cross-walls and may be inconsistent with a recently proposed role for membrane structures in the proliferation of ftsZ mutants in other Streptomyces species. Rare surviving fragments of mycelium, usually around branches, appear to be the preferred sites of resealing. Restoration of growth in hyphal fragments of both wild-type and ftsZ mutant hyphae can occur at multiple sites, via branch-like outgrowths containing DivIVA protein at their tips. Thus, our results highlight branching as a means of FtsZ-independent cell proliferation.
Publisher: The Royal Society
Date: 29-04-1996
Abstract: Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell ision. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (σ), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called σ F , the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue SpoIIAA then remains free to inhibit σ F . In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and σ F is set free. Release of σ F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/A DP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by σ F . First it regulates σ F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.
Publisher: Microbiology Society
Date: 10-2006
Abstract: Systematic inactivation of Bacillus subtilis genes has previously revealed that 271 are indispensable for growth. In the present study, 11 of these ( yacA , ydiB , ydiC , ykqC , ylaN , yloQ , ymdA , yneS , yqeI , yqjK and ywlC ) were identified as genes encoding proteins of unknown function. By analysing the effects of protein depletion, and examining the subcellular localization of these proteins, a start has been made in elucidating their functions. It was found that four of these genes ( ydiB , yloQ , yqeI and ywlC ) were not required for B. subtilis viability. Analysis of the localization of YkqC suggests that it co-localizes with ribosomes, and it is proposed that it is involved in processing either rRNA or specific mRNAs when they are associated with the ribosome. The results suggest that other novel essential proteins may be involved in lipid synthesis and control of cell wall synthesis.
Publisher: Cold Spring Harbor Laboratory
Date: 11-02-2021
DOI: 10.1101/2021.02.11.430593
Abstract: ATP and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis , Noc nucleates on 16-bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell ision machinery. By in vitro reconstitution, we show that (i) CTP is required for Noc to form the NBS -dependent nucleoprotein complex, and (ii) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.
Publisher: American Society for Microbiology
Date: 12-1993
DOI: 10.1128/JB.175.23.7604-7616.1993
Abstract: The pbpB gene, which encodes penicillin-binding protein (PBP) 2B of Bacillus subtilis, has been cloned, sequenced, mapped, and mutagenized. The sequence of PBP 2B places it among the class B high-molecular-weight PBPs. It appears to contain three functional domains: an N-terminal domain homologous to the corresponding domain of other class B PBPs, a penicillin-binding domain, and a lengthy carboxy extension. The PBP has a noncleaved signal sequence at its N terminus that presumably serves as its anchor in the cell membrane. Previous studies led to the hypothesis that PBP 2B is required for both vegetative cell ision and sporulation septation. Its sequence, map site, and mutant phenotype support this hypothesis. PBP 2B is homologous to PBP 3, the cell ision protein encoded by pbpB of Escherichia coli. Moreover, both pbpB genes are located in the same relative position within a cluster of cell ision and cell wall genes on their respective chromosomes. However, immediately adjacent to the B. subtilis pbpB gene is spoVD, which appears to be a sporulation-specific homolog of pbpB. Inactivation of SpoVD blocked synthesis of the cortical peptidoglycan in the spore, whereas carboxy truncation of PBP 2B caused cells to grow as filaments. Thus, it appears that a gene duplication has occurred in B. subtilis and that one PBP has evolved to serve a common role in septation during both vegetative growth and sporulation, whereas the other PBP serves a specialized role in sporulation.
Publisher: eLife Sciences Publications, Ltd
Date: 30-10-2014
DOI: 10.7554/ELIFE.04629
Abstract: The peptidoglycan cell wall is a defining structural feature of the bacterial kingdom. Curiously, some bacteria have the ability to switch to a wall-free or ‘L-form’ state. Although known for decades, the general properties of L-forms are poorly understood, largely due to the lack of systematic analysis of L-forms in the molecular biology era. Here we show that inhibition of peptidoglycan precursor synthesis promotes the generation of L-forms from both Gram-positive and Gram-negative bacteria. We show that the L-forms generated have in common a mechanism of proliferation involving membrane blebbing and tubulation, which is dependent on an altered rate of membrane synthesis. Crucially, this mode of proliferation is independent of the essential FtsZ based ision machinery. Our results suggest that the L-form mode of proliferation is conserved across the bacterial kingdom, reinforcing the idea that it could have been used in primitive cells, and opening up its use in the generation of synthetic cells.
Publisher: Wiley
Date: 24-07-2009
Publisher: Annual Reviews
Date: 08-09-2017
DOI: 10.1146/ANNUREV-MICRO-102215-095630
Abstract: The bacterial cytoplasmic membrane is composed of roughly equal proportions of lipids and proteins. The main lipid components are phospholipids, which vary in acyl chain length, saturation, and branching and carry head groups that vary in size and charge. Phospholipid variants determine membrane properties such as fluidity and charge that in turn modulate interactions with membrane-associated proteins. We summarize recent advances in understanding bacterial membrane structure and function, focusing particularly on the possible existence and significance of specialized membrane domains. We review the role of membrane curvature as a spatial cue for recruitment and regulation of proteins involved in morphogenic functions, especially elongation and ision. Finally, we examine the role of the membrane, especially regulation of synthesis and fluid properties, in the life cycle of cell wall–deficient L-form bacteria.
Publisher: Wiley
Date: 05-2901
DOI: 10.1111/J.1365-2958.1991.TB01887.X
Abstract: After the initiation of spore formation in Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e. the prespore and the mother cell. The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non-sporulating cells on the basis of its resistance properties. We have used a procedure originally developed to label the terminus region of the B. subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation. We have purified prespore DNA and used strand-specific probes to measure the radioactivity incorporated. The results show that the sister chromosomes segregate at random into the prespore. This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation.
Publisher: American Society for Microbiology
Date: 10-2000
DOI: 10.1128/JB.182.19.5572-5579.2000
Abstract: The ftsL gene is required for the initiation of cell ision in a broad range of bacteria. Bacillus subtilis ftsL encodes a 13-kDa protein with a membrane-spanning domain near its N terminus. The external C-terminal domain has features of an α-helical leucine zipper, which is likely to be involved in the heterodimerization with another ision protein, DivIC. To determine what residues are important for FtsL function, we used both random and site-directed mutagenesis. Unexpectedly, all chemically induced mutations fell into two clear classes, those either weakening the ribosome-binding site or producing a stop codon. It appears that the random mutagenesis was efficient, so many missense mutations must have been generated but with no phenotypic effect. Substitutions affecting hydrophobic residues in the putative coiled-coil domain, introduced by site-directed mutagenesis, also gave no observable phenotype except for insertion of a helix-breaking proline residue, which destroyed FtsL function. ftsL homologues cloned from three erse Bacillus species, Bacillus licheniformis , Bacillus badius , and Bacillus circulans , could complement an ftsL null mutation in B. subtilis , even though up to 66% of the amino acid residues of the predicted proteins were different from B. subtilis FtsL. However, the ftsL gene from Staphylococcus aureus (whose product has 73% of its amino acids different from those of the B. subtilis ftsL product) was not functional. We conclude that FtsL is a highly malleable protein that can accommodate a large number of sequence changes without loss of function.
Publisher: Elsevier BV
Date: 02-2018
Publisher: Wiley
Date: 08-2002
DOI: 10.1093/EMBOJ/CDF393
Publisher: Wiley
Date: 09-1997
DOI: 10.1111/J.1365-2958.1997.MMI530.X
Abstract: We have developed methods for labelling regions of the Bacillus subtilis chromosome with the nucleotide analogue 5-bromodeoxyuridine (BrdU) and for subcellular visualization of the labelled DNA. Examination of oriC-labelled chromosomes in outgrowing spores has provided direct evidence for active segregation of sister chromosomes. Co-immunodetection of Spo0J and BrdU-labelled DNA has directly confirmed the expected close association between this chromosome partitioning protein and the oriC region of the chromosome. The results provide further support for the notion that bacterial cells use an active mitotic-like mechanism to segregate their chromosomes.
Publisher: Wiley
Date: 25-11-2005
DOI: 10.1111/J.1365-2958.2004.04420.X
Abstract: Staphylococcus aureus penicillin-binding protein PBP2 is an enzyme involved in the last stages of peptidoglycan assembly and is an important player in the mechanism of methicillin resistance of this pathogen. PBP2 localized to the ision site but its recruitment to the forming ision septum was prevented after acylation by oxacillin. The presence of the antibiotic did not affect FtsZ ring maintenance nor the localization of externalized peptidoglycan precursors. Delocalization of PBP2 was also observed when its pentapeptide substrate was eliminated by addition of d-cycloserine or blocked by addition of vancomycin. Taken together these observations suggest that PBP2 is recruited to the ision site by binding to its substrate, which is localized at that place. In methicillin-resistant S. aureus, addition of oxacillin does not result in delocalization of PBP2 indicating that acylated PBP2 can be maintained in place by functional PBP2A, the central element of this resistance mechanism.
Publisher: Cold Spring Harbor Laboratory
Date: 05-1997
Abstract: The Bacillus subtilis spo0J gene is required for accurate chromosome partitioning during growth and sporulation. We have characterized the subcellular localization of Spo0J protein by immunofluorescence and, in living cells, by use of a spo0J-gfp fusion. We show that the Spo0J protein forms discrete stable foci usually located close to the cell poles. The foci replicate in concert with the initiation of new rounds of DNA replication, after which the daughter foci migrate apart inside the cell. This migration is independent of cell length extension, and presumably serves to direct the daughter chromosomes toward opposite poles of the cell, ready for ision. During sporulation, the foci move to the extreme poles of the cell, where they function to position the oriC region of the chromosome ready for polar septation. These observations provide strong evidence for the existence of a dynamic, mitotic-like apparatus responsible for chromosome partitioning in bacteria.
Publisher: Proceedings of the National Academy of Sciences
Date: 11-08-2009
Abstract: Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.
Publisher: Wiley
Date: 19-02-2009
Publisher: American Society for Microbiology
Date: 15-01-2003
DOI: 10.1128/JB.185.2.693-697.2003
Abstract: DivIVA is involved in Bacillus subtilis cell ision and is located at the cell poles. Previous experiments suggested that the cell ision proteins FtsZ and PBP 2B are required for polar targeting of DivIVA. By using outgrowing spores, we show that DivIVA accumulates at the cell poles independent of the presence of FtsZ or PBP 2B.
Publisher: Cold Spring Harbor Laboratory
Date: 11-1998
Abstract: Cell ision in rod-shaped bacteria is initiated by formation of a ring of the tubulin-like protein FtsZ at mid-cell. Division site selection is controlled by a conserved ision inhibitor MinCD, which prevents aberrant ision at the cell poles. The Bacillus subtilis DivIVA protein controls the topological specificity of MinCD action. Here we show that DivIVA is targeted to ision sites late in their assembly, after some MinCD-sensitive step requiring FtsZ and other ision proteins has been passed. DivIVA then recruits MinD to the ision sites preventing another ision from taking place near the newly formed cell poles. Sequestration of MinD to the poles also releases the next mid-cell sites for ision. Remarkably, this mechanism of DivIVA action is completely different from that of the equivalent protein MinE of Escherichia coli , even though both systems operate via the same ision inhibitor MinCD.
Publisher: Elsevier BV
Date: 2003
Publisher: American Society for Microbiology
Date: 03-3009
DOI: 10.1128/JB.01497-08
Abstract: Bacterial actin homologues are thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. They are also thought to control other functions, including cell polarity and chromosome segregation in various organisms. Bacillus subtilis and many other gram-positive bacteria have three actin isoforms, MreB, Mbl, and MreBH, which colocalize in helical structures that span the length of the cell, close to the inner surface of the cytoplasmic membrane. Deletion of the mbl gene has previously been reported to produce viable, although poorly growing, mutant cells. We now show that under normal conditions Δmbl cells are nonviable but suppressors allowing growth readily accumulate. In the presence of high concentrations of Mg 2+ , viable, nonsuppressed mutants can be obtained. A screen for suppressor mutations revealed that deletion of rsgI restores Mg 2+ -independent growth of the mbl mutant. Recent work has shown that rsgI deletion leads to upregulation of the alternative sigma factor σ I . The basis of suppression is not yet clear, but it is independent of the Mg 2+ effect. We found that the construction of a triple mutant lacking all three actin homologues became possible in the rsgI background. Triple mutant cells are spherical, but no significant defect in chromosome segregation was detected.
Publisher: Springer Science and Business Media LLC
Date: 18-08-2020
DOI: 10.1038/S41467-020-17988-7
Abstract: Many bacteria can form wall-deficient variants, or L-forms, that ide by a simple mechanism that does not require the FtsZ-based cell ision machinery. Here, we use microfluidic systems to probe the growth, chromosome cycle and ision mechanism of Bacillus subtilis L-forms. We find that forcing cells into a narrow linear configuration greatly improves the efficiency of cell growth and chromosome segregation. This reinforces the view that L-form ision is driven by an excess accumulation of surface area over volume. Cell geometry also plays a dominant role in controlling the relative positions and movement of segregating chromosomes. Furthermore, the presence of the nucleoid appears to influence ision both via a cell volume effect and by nucleoid occlusion, even in the absence of FtsZ. Our results emphasise the importance of geometric effects for a range of crucial cell functions, and are of relevance for efforts to develop artificial or minimal cell systems.
Publisher: The Royal Society
Date: 22-05-1991
Abstract: Sigma factors (sigma) are transcription factors that operate global switches in gene expression in prokaryotes. They work by directing core RNA polymerase to specific cis-acting promoter sequences each sigma has a cognate class of promoters with specific sequence characteristics. In Bacillus subtilis four different sigma factors have been implicated in the regulation of gene expression during spore formation, which is a simple differentiation system involving two cell types. In this review I show how the modern developmental system may have arisen from a primitive organism that used only two sigma factors, by a series of steps involving gene duplication and ergence. The increasing sophistication of eukaryotic developmental systems may reflect similar evolutionary processes.
Publisher: Elsevier BV
Date: 02-2005
DOI: 10.1016/0378-1119(93)90223-P
Abstract: A novel expression system based on the Bacillus subtilis bacteriophage phi 105 has been developed to permit the high-level synthesis and secretion of beta-lactamase I (BlaI) from Bacillus cereus. Shotgun insertion of a promoterless lacZ gene into the phage genome permitted the identification of a clone producing large amounts of beta-galactosidase (beta Gal), indicating the transcription of the reporter gene from a strong phage promoter. The insertion also blocked lysis of the host cell. Although the insertion in the original prophage was complex, plasmid vectors and prophage derivatives have been developed to facilitate the replacement of lacZ with other genes for expression. The new prophages contain two additional mutations: an ind mutation, which greatly enhances the normally poor transformability of phi 105 lysogens, and a cts mutation, which allows thermo-induction of phage development and protein production. Induction of a derivative prophage containing the blaI gene from B. cereus resulted in the production of up to 500 micrograms of secreted BlaI per ml of culture supernatant.
Publisher: Springer Science and Business Media LLC
Date: 08-07-2019
Publisher: Wiley
Date: 27-04-2007
DOI: 10.1002/PROT.21377
Abstract: The crystal structure of a conserved leucine rich protein, YlaN, from Staphylococcus aureus has been determined by X-ray crystallography to 2.3 A resolution. Whilst the precise function of S. aureus YlaN is unknown its homologue in B. subtilis has been shown to be essential for cell survival and is thought to be involved in controlling cell shape. The structure of S. aureus YlaN provides the first view of its protein family, which reveals that it is a novel homodimer whose subunit architecture is comprised of an antiparallel 3 helix bundle reminiscent of the helical arrangements seen in leucine zipper proteins. Analysis of the pattern of sequence conservation on the structure has led to the identification of two connected solvent exposed patches of conserved residues in each subunit located at one end of but on opposite faces of the molecule. We suggest that YlaN has a binding role in the cell rather than a catalytic function and a search for its ligand is underway to accelerate its exploitation as a target for antibiotic discovery.
Publisher: Microbiology Society
Date: 07-2004
Abstract: At the onset of sporulation in Bacillus subtilis , an asymmetric cell ision gives rise to two unequal-sized compartments with distinct developmental fates. The smaller compartment, or prespore, becomes the spore, whilst the larger compartment, or mother cell, eventually lyses after contributing to spore maturation. The fate of each compartment is determined by differential gene expression, controlled by the activation of four compartment-specific σ -factors. The expression and activity of all four σ -factors are tightly regulated to ensure the correct sequence of morphological events. Prespore-specific genes are transcribed by two σ -factors, σ F followed by σ G . The gene encoding σ G ( sigG ) is transcribed by σ F , but also requires the activity of one of the mother-cell-specific σ -factors, σ E , for its expression. The minimal promoter required for dependence on σ E was found to stretch to just upstream of the −35 site. Analysis of mutant sigG promoters generated by site-directed mutagenesis and sigG promoters from other species suggests the presence of a binding site for a transcriptional repressor within the sigG promoter region. Replacement of the wild-type promoter with σ E -independent promoters resulted in impairment of sporulation. These data support the idea that σ E activity is required for the transcription of sigG .
Publisher: American Society for Microbiology
Date: 03-1991
DOI: 10.1128/JB.173.6.1911-1919.1991
Abstract: Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins.
Publisher: Elsevier BV
Date: 04-2014
Publisher: Elsevier BV
Date: 02-2013
DOI: 10.1016/J.CELL.2013.01.043
Abstract: The peptidoglycan cell wall is a hallmark of the bacterial subkingdom. Surprisingly, many modern bacteria retain the ability to switch into a wall-free state called the L-form. L-form proliferation is remarkable in being independent of the normally essential FtsZ-based ision machinery and in occurring by membrane blebbing and tubulation. We show that mutations leading to excess membrane synthesis are sufficient to drive L-form ision in Bacillus subtilis. Artificially increasing the cell surface area to volume ratio in wild-type protoplasts generates similar shape changes and cell ision. Our findings show that simple biophysical processes could have supported efficient cell proliferation during the evolution of early cells and provide an extant biological model for studying this problem.
Publisher: American Society for Microbiology
Date: 07-1995
DOI: 10.1128/JB.177.14.3923-3931.1995
Abstract: To elucidate the process of asymmetric ision during sporulation of Bacillus subtilis, we have measured changes in cell cycle parameters during the transition from vegetative growth to sporulation. Because the propensity of B. subtilis to grow in chains of cells precludes the use of automated cell-scanning devices, we have developed a fluorescence microscopic method for analyzing cell cycle parameters in in idual cells. From the results obtained, and measurements of DNA replication fork elongation rates and the escape time of sporulation from the inhibition of DNA replication, we have derived a detailed time scale for the early morphological events of sporulation which is mainly consistent with the cell cycle changes expected following nutritional downshift. The previously postulated sensitive stage in the DNA replication cycle, beyond which the cell is unable to sporulate without a new cell cycle, could represent a point in the ision cycle at which the starved cell cannot avoid attaining the initiation mass for DNA replication and thus embarking on another round of the cell cycle. The final cell cycle event, formation of the asymmetric spore septum, occurs at about the time in the cell cycle at which the uninduced cell would have ided centrally, in keeping with the view that spore septation is a modified version of vegetative ision.
Publisher: American Society for Microbiology
Date: 1992
DOI: 10.1128/JB.174.2.586-594.1992
Abstract: Mutations in the spoIVA locus of Bacillus subtilis abolish cortex synthesis and interfere with the synthesis and assembly of the spore coat. We have characterized the cloned spoIVA locus in terms of its physical structure and regulation during sporulation. The locus contains a single gene capable of encoding an acidic protein of 492 amino acids (molecular weight, 55,174). The gene is transcribed from a sigma E-dependent promoter soon after the formation of the spore septum. A genetic test indicated that expression of spoIVA is only necessary in the mother cell compartment for the formation of a mature spore. This, together with the phenotypic properties of spoIVA mutations, would be in accord with the hypothesis that sigma E is only active after septation and in the mother cell compartment.
Publisher: Elsevier BV
Date: 12-2001
DOI: 10.1016/S1369-5274(01)00266-1
Abstract: The early stages of sporulation in Bacillus subtilis incorporate a modified, highly asymmetric cell ision. It is now clear that most, if not all, of the components of the vegetative ision machinery are used also for asymmetric ision. However, the machinery for chromosome segregation may differ significantly between vegetative growth and sporulation. Several interesting checkpoint mechanisms couple cell cycle events to gene expression early in sporulation. This review summarises important advances in the understanding of chromosome segregation and cell ision at the onset of sporulation in B.subtilis in the past three years.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 22-04-1994
Abstract: Sporulation in Bacillus subtilis begins with an asymmetric cell ision, producing a smaller prespore and a larger mother cell, both of which contain intact copies of the chromosome. The spoIIIE gene is required for chromosome segregation into the prespore compartment. The effects of the spoIIIE36 mutation on sigma F-dependent transcription are an indirect consequence of the failure of certain genes to enter the cellular compartment in which their transcription factor has become active. SpoIIIE may also be required to prevent sigma F from becoming active in the mother cell.
Publisher: Springer Science and Business Media LLC
Date: 11-2003
DOI: 10.1038/NRMICRO750
Abstract: Spore formation in bacteria poses a number of biological problems of fundamental significance. Asymmetric cell ision at the onset of sporulation is a powerful model for studying basic cell-cycle problems, including chromosome segregation and septum formation. Sporulation is one of the best understood ex les of cellular development and differentiation. Fascinating problems posed by sporulation include the temporal and spatial control of gene expression, intercellular communication and various aspects of cell morphogenesis.
Publisher: Elsevier BV
Date: 02-2014
Publisher: Wiley
Date: 07-1999
DOI: 10.1046/J.1365-2958.1999.01450.X
Abstract: Bacterial cell ision commences with the assembly of the tubulin-like protein, FtsZ, at midcell to form a ring. Division site selection in rod-shaped bacteria is mediated by MinC and MinD, which form a ision inhibitor. Bacillus subtilis DivIVA protein ensures that MinCD specifically inhibits ision close to the cell poles, while allowing ision at midcell. We have examined the localization of MinC protein and show that it is targeted to midcell and retained at the mature cell poles. This localization is reminiscent of the pattern previously described for MinD. Localization of MinC requires both early (FtsZ) and late (PbpB) ision proteins, and it is completely dependent on MinD. The effects of a IVA mutation on localization of MinC now suggest that the main role of DivIVA is to retain MinCD at the cell poles after ision, rather than recruitment to nascent ision sites. By overexpressing minC or minD, we show that both proteins are required to block ision, but that only MinD needs to be in excess of wild-type levels. The results suggest a mechanism whereby MinD is required both to pilot MinC to the cell poles and to constitute a functional ision inhibitor.
Publisher: Public Library of Science (PLoS)
Date: 16-03-2010
Publisher: American Society for Microbiology
Date: 08-2004
DOI: 10.1128/JB.186.15.5153-5156.2004
Abstract: Bacillus subtilis penicillin-binding protein PBP1 has been implicated in cell ision. We show here that a PBP1 knockout strain is affected in the formation of the asymmetric sporulation septum and that green fluorescent protein-PBP1 localizes to the sporulation septum. Localization of PBP1 to the vegetative septum is dependent on various cell ision proteins. This study proves that PBP1 forms part of the B. subtilis cell ision machinery.
Publisher: Wiley
Date: 24-05-2013
Abstract: Cell ision in bacteria is carried out by a set of conserved proteins that all have to function at the correct place and time. A cell cycle-dependent transcriptional programme drives cell ision in bacteria such as Caulobacter crescentus. Whether such a programme exists in the Gram-positive model organism Bacillus subtilis is unknown. Here, we investigate the role of gene transcription as a potential regulatory mechanism for control of ision in B. subtilis. Transcriptional GFP fusions in combination with flow cytometry demonstrated a constitutive promoter activity, independent of growth rate, of nine tested cell ision genes. These measurements were verified by quantitative real-time reverse-transcription PCR (qrtPCR). Time-lapse fluorescence microscopy was performed on a set of selected reporter strains to test transcriptional regulation during the cell cycle. Interestingly, although the average fluorescence remained constant during cell-cycle progression, in idual cells demonstrated a roughly twofold higher promoter activity at the end of the cell cycle. This cell cycle-dependent increased promoter activity can be partly explained by the doubled promoter copy number after DNA replication. Our results indicate that the transcriptional activity of promoters for cell ision genes remains constant regardless of growth rate and cell-cycle state, suggesting that regulation of cell ision in B. subtilis predominantly takes place at the post-translational level.
Publisher: American Society for Microbiology
Date: 12-1990
DOI: 10.1128/JB.172.12.6937-6941.1990
Abstract: Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two sister cells, the prespore and the mother cell. Many of the genes that regulate sporulation (spo genes) are thought to be expressed differentially. However, direct demonstration of differential gene expression, by fractionation of prespore and mother cell proteins, is possible only at a relatively late stage of development. H. De Lencastre and P. J. Piggot (J. Gen. Microbiol. 114:377-389, 1979) have described a genetic method for determining the cellular location of the requirement for spo gene expression. Here we describe a similar method based on the use of integrational plasmids that can insertionally inactivate any given spo gene. Loss of the integrated plasmid by homologous recombination leads to the restoration of spo gene function. If this occurs just before sporulation begins, the phenotypes of the progeny of heat-resistant spores should depend on whether the gene is required in the prespore or the mother cell. Thus, we show that for known prespore-specific genes, such as spoIIIG and spoVA, only phenotypically Spo+ progeny that have lost the integrated plasmid are produced. In contrast, for mother-cell-specific genes, such as spoIIIC and spoVJ, a substantial proportion of the progeny are asporogenous, having retained the integrated plasmid. On the basis of our results, the spoIID and spoIIIA genes, which are expressed soon after ision, appear to be required only in the mother cell compartment.
Publisher: Microbiology Society
Date: 03-1998
DOI: 10.1099/00221287-144-3-801
Abstract: The sequence of a 28 kbp segment of DNA surrounding the spoVM gene of Bacillus subtilis 168 (lying at approximately 145 on the standard genetic map) has been determined. The region contains 27 ORFs, a number of which have predicted products significantly similar to proteins in sequence databases, particularly to proteins involved in macromolecular synthesis of nucleic acids, proteins and phospholipids. A pair of closely linked genes encode a likely serine protein phosphatase and a serine protein kinase, respectively. Such proteins play important regulatory roles in eukaryotic cells but are rare in prokaryotes.
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.MOLCEL.2011.02.024
Abstract: The sliding cl is an essential component of the replisome required for processivity of DNA synthesis and several other aspects of chromosome metabolism. However, the in vivo dynamics of the cl are poorly understood. We have used various biochemical and cell biological methods to study the dynamics of cl association with the replisome in Bacillus subtilis cells. We find that cl s form large assemblies on DNA, called "cl zones." Loading depends on DnaG primase and is probably driven by Okazaki fragment initiation on the lagging strand. Unloading, which is probably regulated, only occurs after many cl s have accumulated on the DNA. On/off cycling allows chromosomal zones of about 200 accumulated cl s to follow the replisome. Since we also show that cl zones recruit proteins bearing a cl -binding sequence to replication foci, the results highlight the cl as a central organizer in the structure and function of replication foci.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 19-09-2008
Abstract: FtsZ is an essential bacterial guanosine triphosphatase and homolog of mammalian β-tubulin that polymerizes and assembles into a ring to initiate cell ision. We have created a class of small synthetic antibacterials, exemplified by PC190723, which inhibits FtsZ and prevents cell ision. PC190723 has potent and selective in vitro bactericidal activity against staphylococci, including methicillin- and multi-drugâresistant Staphylococcus aureus . The putative inhibitor-binding site of PC190723 was mapped to a region of FtsZ that is analogous to the Taxol-binding site of tubulin. PC190723 was efficacious in an in vivo model of infection, curing mice infected with a lethal dose of S. aureus . The data validate FtsZ as a target for antibacterial intervention and identify PC190723 as suitable for optimization into a new anti-staphylococcal therapy.
Publisher: Cold Spring Harbor Laboratory
Date: 11-1994
Abstract: Genetic experiments have suggested that σ F , the first compartment-specific transcription factor in sporulating B. subtilis , is regulated by an anti-σ factor SpoIIAB and an anti-anti-σ factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of σ F -directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA–phosphate, and SpoIIAB is immediately released but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA–phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-σ F activity, whereas SpoIIAAS58A, which cannot be phosphorylated, makes complexes with SpoIIAB in the presence of ADP or ATP and has constitutive anti-anti-σ F activity both in vivo and in vitro. It seems likely that the alternative reactions of SpoIIAA and SpoIIAB, involving ADP or ATP, regulate the anti-anti-σ capacity of SpoIIAA and hence the activity of σ F .
Publisher: Springer Science and Business Media LLC
Date: 18-05-2020
Publisher: Wiley
Date: 17-11-2005
DOI: 10.1111/J.1365-2958.2004.04399.X
Abstract: The initiation of the developmental process of sporulation in the rod-shaped bacterium Bacillus subtilis involves the activation of the Spo0A response regulator. Spo0A then drives the switch in the site of ision septum formation from midcell to a polar position. Activated Spo0A is required for the transcription of key sporulation loci such as spoIIG, which are negatively regulated by the Soj protein. The transcriptional repressing activity of Soj is antagonized by Spo0J, and both proteins belong to the well-conserved Par family of partitioning proteins. Soj has been shown to jump from nucleoid to nucleoid via the cell pole. The dynamic behaviour of Soj is somehow controlled by Spo0J, which prevents the static association of Soj with the nucleoid, and presumably its transcriptional repression activity. Soj in turn is required for the proper condensation of Spo0J foci around the oriC region. The asymmetric partitioning of the sporangial cell requires DivIB and other proteins involved in vegetative (medial) ision. We describe an allele of the cell ision gene IB ( IB80) that reduces the cellular levels of DivIB, and affects nucleoid structure and segregation in growing cells, yet has no major impact on cell ision. In IB80 cells Spo0J foci are not correctly condensed and Soj associates statically with the nucleoid. The IB80 allele prevents transcription of spoIIG, and arrests sporulation prior to the formation of the asymmetric ision septum. The defect in Spo0A-dependent gene expression, and the Spo- phenotype can be suppressed by expression of IB in trans or by deletion of the soj-spo0J locus. However, deletion of the spo0J-soj region does not restore the normal cellular levels of DivIB. Therefore, the reduced levels of DivIB in the IB80 mutant are sufficient for efficient cell ision, but not to sustain a second, earlier function of DivIB related to the activity of the Spo0J/Soj system of chromosome segregation.
Publisher: Wiley
Date: 29-11-2011
DOI: 10.1111/J.1365-2958.2011.07920.X
Abstract: L-forms are variants of common bacteria that can grow and proliferate without a cell wall. Little is known about their molecular cell biology but they undergo a remarkable mode of proliferation that is independent of the normally essential FtsZ-dependent ision machinery. We have isolated a strain of Bacillus subtilis that can quickly and quantitatively convert from the walled to the L-form state. Analysis of the transition process identified an unexpected 'escape' step needed for L-form emergence from the rod. Mutations in two different genes, walR and sepF, contribute to the high frequency of escape: walR, a transcriptional regulator involved in cell wall homeostasis and sepF, required for accurate and efficient cell ision. Time-lapse imaging shows that the mutations act by facilitating the release of the L-form from its walled parent cell but that they act in different ways. The walR mutation renders the activity of the protein partially constitutive, inappropriately upregulating the activity of autolytic enzymes that weaken the cell wall. The sepF mutation probably works by perturbing the formation of a properly constructed ision septum, generating a mechanical breach in the wall. The new strain provides a powerful experimental system for studying the genetics and cell biology of L-forms.
Publisher: Informa UK Limited
Date: 14-12-2016
DOI: 10.1080/14786419.2016.1263854
Abstract: The actinomycete DEM20745, collected from non-rhizosphere soil adjacent to Paraserianthes falactaria trees (Cangkringan, Indonesia), is an efficient producer of the anticancer ansamycin polyketide 17-O-demethyl-geldanamycin (17-O-DMG), a biosynthetic precursor of the Hsp90 inhibitor geldanamycin (GDM). In DEM20745, 17-O-DMG is the major ansamycin product observed reaching a maximum titre of 17 mg/L in the fermentation broth. 17-O-DMG has the potential to be a key starting material for the semi-synthesis of GDM analogues for use in anticancer therapy. Thus, this preferential biosynthesis of 17-O-DMG facilitates easy access to this important molecule and provides further insight in the biosynthesis of the geldanamycins.
Publisher: Wiley
Date: 04-06-2009
Publisher: EMBO
Date: 07-01-2015
Publisher: The Royal Society
Date: 05-11-2016
Abstract: The peptidoglycan cell wall is widely conserved across the bacterial domain, suggesting that it appeared early in the evolution of bacteria. It is normally essential but under certain conditions wall-deficient or ‘L-form’ bacteria can be isolated. In Bacillus subtilis this normally requires two genetic changes. The first, exemplified by mutations shutting down wall precursor synthesis, works by increasing membrane synthesis. This promotes the unusual form of proliferation used by L-forms, involving a range of relatively disorganized membrane blebbing or vesiculation events. The secondary class of mutations probably work by relieving oxidative stress that L-forms may incur due to their unbalanced metabolism. Repression or inhibition of cell wall precursor synthesis can stimulate the L-form transition in a wide range of bacteria, of both Gram-positive and -negative lineages. L-forms are completely resistant to most antibiotics working specifically on cell wall synthesis, such as penicillins and cephalosporins, consistent with the many reports of their involvement in various chronic diseases. They are potentially important in biotechnology, because lack of a wall can be advantageous in a range of production or strain improvement applications. Finally, L-forms provide an interesting model system for studying early steps in the evolution of cellular life. This article is part of the themed issue ‘The new bacteriology’.
Publisher: Wiley
Date: 04-1996
DOI: 10.1111/J.1365-2958.1996.TB02482.X
Abstract: Several crucial genes required for bacterial ision lie close together in a region called the dcw cluster. Within the cluster, gene expression is subject to complex transcriptional regulation, which serves to adjust the cell cycle in response to growth rate. The pivotally important FtsZ protein, which is needed to initiate ision, is now known to interact with many other components of the ision machinery in Escherichia coli. Some biochemical properties of FtsZ, and of another ision protein called FtsA, suggest that they are similar to the eukaryotic proteins tubulin and actin respectively. Cell ision needs to be closely co-ordinated with chromosome partitioning. The mechanism of partitioning is poorly understood, though several genes involved in this process, including several muk genes, have been identified. The min genes may participate in both septum positioning and chromosome partitioning. Coupled transcription and translation of membrane-associated proteins might also be important for partitioning. In the event of a failure in the normal partitioning process, Bacillus subtilis, at least, has a mechanism for removing a bisected nucleoid from the ision septum.
Publisher: Microbiology Society
Date: 12-1996
DOI: 10.1099/13500872-142-12-3445
Abstract: Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types called the prespore and the mother cell. The process is induced by nutrient deprivation and culminates with the formation of a mature spore, which is released by lysis of the mother cell. We have studied commitment to sporulation with several different assays. The results indicate that commitment occurs soon after the formation of the asymmetrically positioned ision septum that separates the prespore and the mother cell. This is earlier than the previously postulated point of commitment, prespore engulfment by the mother cell. Commitment coincides approximately with activation of the early prespore- and mother-cell-specific sigma factors, sigma(E) and sigma(F).
Publisher: Wiley
Date: 06-1998
DOI: 10.1046/J.1365-2958.1998.00857.X
Abstract: The Spo0J protein of Bacillus subtilis is required for normal chromosome segregation and forms discrete subcellular assemblies closely associated with the oriC region of the chromosome. Here we show that duplication of Spo0J foci occurs early in the DNA replication cycle and that this requires the initiation of DNA replication at oriC but not elongation beyond the nearby STer sites. Soon after duplication, sister oriC/Spo0J foci move rapidly apart to achieve a fixed separation of about 0.7 microm, reminiscent of the segregation of eukaryotic chromosomes on the mitotic spindle. The magnitude of the fixed separation distance may explain how chromosome segregation is kept in close register with cell growth and the initiation mass for DNA replication. It could also explain how segregation can proceed accurately in the absence of cell ision. The kinetics of focal separation suggest that one role of Spo0J protein may be to facilitate formation of separate sister oriC complexes that can be segregated.
Publisher: Springer Science and Business Media LLC
Date: 07-2001
DOI: 10.1038/35080005
Publisher: The Royal Society
Date: 29-03-2005
Abstract: Bacterial cells are much smaller and have a much simpler overall structure and organization than eukaryotes. Several prominent differences in cell organization are relevant to the mechanisms of chromosome segregation, particularly the lack of an overt chromosome condensation/decondensation cycle and the lack of a microtubule-based spindle. Although bacterial chromosomes have a rather dispersed appearance, they nevertheless have an underlying high level of spatial organization. During the DNA replication cycle, early replicated ( oriC ) regions are localized towards the cell poles, whereas the late replicated terminus ( terC ) region is medially located. This spatial organization is thought to be driven by an active segregation mechanism that separates the sister chromosomes continuously as replication proceeds. Comparisons of various well-characterized bacteria suggest that the mechanisms of chromosome segregation are likely to be erse, and that in many bacteria, multiple overlapping mechanisms may contribute to efficient segregation. One system in which the molecular mechanisms of chromosome segregation are beginning to be elucidated is that of sporulating cells of Bacillus subtilis . The key components of this system have been identified, and their functions are understood, in outline. Although this system appears to be specialized, most of the functions are conserved widely throughout the bacteria.
Publisher: Elsevier BV
Date: 02-1995
DOI: 10.1016/0378-1119(94)00874-R
Abstract: A defective prophage vector, phi 105MU331, for high-level protein overproduction in Bacillus subtilis, was derived by random insertion of a lacZ reporter gene. The site of insertion not only provided efficient inducible transcription of heterologous genes, but also prevented lysis of the host cell. The region of the insertion in phi 105MU331 lies close to the right cohesive end of phi 105. DNA sequence analysis revealed that this region of phi 105 somewhat resembles the lysis cassette of various phages, including lambda. The site of insertion lies in a possible 'holin' gene, which could explain the block in host cell lysis. Dual promoters apparently responsible for the strong inducible transcription lie in an untranslated region just upstream from the putative holin gene. This region is probably equivalent to the site of the major late promoter and antiterminator of the lambdoid phages. The sequence features could, thus, account for the useful properties of the phi 105MU331 vector system.
Publisher: Elsevier BV
Date: 10-2018
Publisher: Wiley
Date: 08-2006
DOI: 10.1111/J.1365-2958.2006.05316.X
Abstract: Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.
Publisher: Wiley
Date: 04-1991
DOI: 10.1111/J.1365-2958.1991.TB00750.X
Abstract: Many differentiation processes in both prokaryotes and eukaryotes begin with an asymmetric ision, producing 'daughter' cells that differ in size and developmental fate. This is particularly obvious in the well-studied prokaryotic life cycles of Caulobacter and Bacillus. In no system, however, is the mechanism of asymmetric ision understood. Here I propose a model for the mechanism of asymmetric ision during sporulation in Bacillus subtilis. The model explains both the timing and asymmetric localization of spore-septum formation. It also explains the morphological phenotypes of various asporogenous (spo) mutants.
Publisher: Public Library of Science (PLoS)
Date: 25-07-2022
DOI: 10.1371/JOURNAL.PNTD.0010128
Abstract: Mycetoma is a neglected tropical chronic granulomatous inflammatory disease of the skin and subcutaneous tissues. More than 70 species with a broad taxonomic ersity have been implicated as agents of mycetoma. Understanding the full range of causative organisms and their antibiotic sensitivity profiles are essential for the appropriate treatment of infections. The present study focuses on the analysis of full genome sequences and antibiotic inhibitory concentration profiles of actinomycetoma strains from patients seen at the Mycetoma Research Centre in Sudan with a view to developing rapid diagnostic tests. Seventeen pathogenic isolates obtained by surgical biopsies were sequenced using MinION and Illumina methods, and their antibiotic inhibitory concentration profiles determined. The results highlight an unexpected ersity of actinomycetoma causing pathogens, including three Streptomyces isolates assigned to species not previously associated with human actinomycetoma and one new Streptomyces species. Thus, current approaches for clinical and histopathological classification of mycetoma may need to be updated. The standard treatment for actinomycetoma is a combination of sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Most tested isolates had a high IC (inhibitory concentration) to sulfamethoxazole/trimethoprim or to amoxicillin alone. However, the addition of the β-lactamase inhibitor clavulanic acid to amoxicillin increased susceptibility, particularly for Streptomyces somaliensis and Streptomyces sudanensis . Actinomadura madurae isolates appear to have a particularly high IC under laboratory conditions, suggesting that alternative agents, such as amikacin, could be considered for more effective treatment. The results obtained will inform future diagnostic methods for the identification of actinomycetoma and treatment.
Publisher: Elsevier BV
Date: 07-2001
DOI: 10.1016/S1534-5807(01)00014-4
Abstract: The MinCDE system regulates the position of the ision plane in rod-shaped bacteria. New results from Escherichia coli provide insight into how this operates by showing that MinE stimulates the ATPase activity of MinD.
Publisher: Wiley
Date: 09-1989
DOI: 10.1111/J.1365-2958.1989.TB00275.X
Abstract: The spoIIIG gene encodes a sigma factor that determines prespore-specific gene expression during sporulation in Bacillus subtilis. Correct spatial and temporal expression of the spoIIIG gene depends on a number of other sporulation (spo) genes, but only one of these genes, spoIIIE, has a specific effect on spoIIIG expression and not on gene expression in the other differentiating cell, the mother cell. However, the spoIIIE gene is expressed predominantly before differentiation begins. Thus, its product must play an important role in sensing or determining the spatial localization of prespore-specific gene expression in this system.
Publisher: Springer Science and Business Media LLC
Date: 03-2003
DOI: 10.1038/NCB0303-175
Publisher: Elsevier BV
Date: 10-1995
DOI: 10.1016/0378-1119(95)00467-K
Abstract: Sequence similarity analysis has revealed that orf2, in the cell ision 135-137 degrees region of the Bacillus subtilis (Bs) chromosome, is the probable homolog of Escherichia coli murB (encoding a reductase involved in peptidoglycan synthesis). The amino-acid sequences of the two protein products show 24% identity (47% overall similarity), with several regions of higher similarity which may represent functional domains of the proteins. Attempts to insertionally inactivate orf2 were unsuccessful, strongly suggesting that it is an essential Bs gene. A small gene found in the same region as orf2, sbp (encoding the 'small basic protein'), was shown to be non-essential in Bs.
Publisher: Springer Science and Business Media LLC
Date: 06-1988
DOI: 10.1038/333399A0
Abstract: Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming covalent DNA adducts. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. Here, a comparison of the toxicity of ellipticine to human breast adenocarcinoma MCF-7 cells, leukemia HL-60 and CCRF-CEM cells, neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cells and U87MG glioblastoma cells and mechanisms of its action to these cells were evaluated. Treatment of all cells tested with ellipticine resulted in inhibition of cell growth and proliferation. This effect was associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP and peroxidase enzymes, in MCF-7, HL-60, CCRF-CEM, UKF-NB-3, UKF-NB-4 and U87MG cells, but not in neuroblastoma UKF-NB-3 cells. Therefore, DNA adduct formation in most cancer cell lines tested in this comparative study might be the predominant cause of their sensitivity to ellipticine treatment, whereas other mechanisms of ellipticine action also contribute to its cytotoxicity to neuroblastoma UKF-NB-3 cells.
Publisher: Springer Science and Business Media LLC
Date: 16-09-2022
DOI: 10.1038/S41598-022-18726-3
Abstract: A genomic and bioactivity informed analysis of the metabolome of the extremophile Amycolatopsis sp. DEM30355 has allowed for the discovery and isolation of the polyketide antibiotic tatiomicin. Identification of the biosynthetic gene cluster was confirmed by heterologous expression in Streptomyces coelicolor M1152. Structural elucidation, including absolute stereochemical assignment, was performed using complementary crystallographic, spectroscopic and computational methods. Tatiomicin shows antibiotic activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Cytological profiling experiments suggest a putative antibiotic mode-of-action, involving membrane depolarisation and chromosomal decondensation of the target bacteria.
Publisher: Elsevier BV
Date: 06-2015
Publisher: Research Square Platform LLC
Date: 08-11-2022
DOI: 10.21203/RS.3.RS-2150762/V1
Abstract: Cell wall synthesis is an essential function for the growth of almost all bacteria, and a major target for our most effective antibiotics. Inhibition of cell wall synthesis by antibiotics such as β-lactams is thought to cause explosive lysis through loss of cell wall integrity. However, recent studies on a wide range of bacteria have suggested that these antibiotics also perturb central carbon metabolism, contributing to death via oxidative damage. We have genetically dissected this connection in Bacillus subtilis and identified key enzymatic steps in upstream and downstream pathways that stimulate the generation of reactive oxygen species (ROS) through cellular respiration. Our results also reveal the critical role of iron homeostasis for the ROS-mediated lethal effects, through iron-dependent enhancement of lipid peroxidation when cell wall synthesis is perturbed. Unexpectedly, we found that protection of cells from ROS via a recently discovered siderophore-like compound uncoupled changes in cell morphology normally associated with cell death, from lysis as usually judged by a phase pale microscopic appearance. Phase paling appears to be specifically associated with lipid peroxidation, presumably the result of membrane destabilization. Finally, we provide a new view of the differentiated roles of the widely conserved Rod and aPBP mechanisms of bacterial cell wall synthesis.
Publisher: Wiley
Date: 10-2009
DOI: 10.1111/J.1365-2958.2009.06872.X
Abstract: The molecular mechanisms underlying cell growth, cell ision and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zinc-inducible system for the fine control of gfp-fusion gene expression and for protein depletion experiments in S. pneumoniae. Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.
Publisher: Portland Press Ltd.
Date: 13-04-2017
DOI: 10.1042/BST20160435
Abstract: The peptidoglycan (PG) cell wall is a defining feature of the bacteria. It emerged very early in evolution and must have contributed significantly to the success of these organisms. The wall features prominently in our thinking about bacterial cell function, and its synthesis involves the action of several dozen proteins that are normally essential for viability. Surprisingly, it turns out to be relatively simple to generate bacterial genetic variants called L-forms that completely lack PG. They grow robustly provided that lack of the cell wall is compensated for by an osmoprotective growth medium. Although their existence has been noted and studied on and off for many decades, it is only recently that modern molecular and cellular methods have been applied to L-forms. We used Bacillus subtilis as an experimental model to understand the molecular basis for the L-form switch. Key findings included the discovery that L-forms use an unusual blebbing, or tubulation and scission mechanism to proliferate. This mechanism is completely independent of the normal FtsZ-based ision machinery and seems to require only an increased rate of membrane synthesis, leading to an increased surface area-to-volume ratio. Antibiotics that block cell wall precursor synthesis, such as phosphomycin, efficiently induce the L-form switch without the need for genetic change. The same antibiotics turned out to induce a similar L-form switch in a wide range of bacteria, including Escherichia coli, in which we showed that proliferation was again FtsZ-independent. Aside from further basic science, future work on L-forms is likely to focus on their possible role in chronic or recurrent infections, their use as a model in studies of the origins of life, and possibly, biotechnological applications.
Publisher: Wiley
Date: 02-1996
DOI: 10.1046/J.1365-2958.1996.434964.X
Abstract: Sigma-factor F (sigmaF) is a key transcription factor that initiates prespore development in Bacillus subtilis. Its activity is controlled by an anti-sigma factor, SpoIIAB, which is also a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA. We have examined our earlier prediction that SpoIIAA must undergo a major change in its properties when phosphorylated. Upon gel filtration in the presence of ADP, SpoIIAA-P was eluted from a Superdex column much later than SpoIIAB, whereas SpoIIAA was coeluted with SpoIIAB, indicating the formation of a protein rotein complex. The complex contained ADP, and had two monomers of SpoIIAA to each SpoIIAB dimer. Its dissociation constant was 13 mu M. Gel permeation on high-performance liquid chromatography (HPLC) suggested an apparent molecular mass for SpoIIAA-P which was much higher (23.5 kDa) than that of SpoIIAA (15.8 kDa), but Ferguson plots showed that SpoIIAA-P was not a phosphorylated dimer of SpoIIAA. Our tentative conclusion, that SpoIIAA and SpoIIAA-P differ markedly in conformation, was confirmed by the results of partial digestion with chymotrypsin.
Publisher: Wiley
Date: 20-09-2006
DOI: 10.1111/J.1365-2958.2006.05402.X
Abstract: The small bitopic ision protein FtsL is an essential part of the ision machinery ( isome) in most eubacteria. In Bacillus subtilis FtsL is a highly unstable protein and the turnover has been implicated in regulation of ision in response to DNA damage. N-terminal deletions and a domain swap experiment identified the short cytoplasmic domain of FtsL as being required for instability. We then identified a zinc metalloprotease, YluC, required for turnover, and likely sequence motifs involved in substrate recognition. YluC belongs to the site-2-protease (S2P) family of proteases involved in regulated intramembrane proteolysis (RIP), which plays a role in erse regulatory phenomena from bacteria to man. The yluC mutant, and strains with N-terminal truncations of ftsL have a short cell phenotype, indicating that that FtsL is normally rate-limiting for ision. Coexpression experiments of FtsL and YluC in Escherichia coli corroborated a model in which FtsL is directly cleaved by the membrane metalloprotease. The results shed new light on the regulation of cell ision in B. subtilis and identify a novel class of targets for RIP.
Publisher: Cold Spring Harbor Laboratory
Date: 14-08-2009
DOI: 10.1101/GAD.528209
Abstract: Coordination of DNA replication with cellular development is a crucial problem in most living organisms. Bacillus subtilis cells switch from vegetative growth to sporulation when starved. Sporulation normally occurs in cells that have stopped replicating DNA and have two completed chromosomes: one destined for the prespore and the other for the mother cell. It has long been recognized that there is a sensitive period in the cell cycle during which the initiation of spore development can be triggered, presumably to allow for the generation of exactly two complete chromosomes. However, the mechanism responsible for this has remained unclear. Here we show that the sda gene, previously identified as a checkpoint factor preventing sporulation in response to DNA damage, exerts cell cycle control over the initiation of sporulation. Expression of sda occurs in a pulsatile manner, with a burst of expression each cell cycle at the onset of DNA replication. Up-regulation of the intrinsically unstable Sda protein, which is dependent on the active form of the DNA replication initiator protein, DnaA, transiently inhibits the initiation of sporulation. This regulation avoids the generation of spore formers with replicating chromosomes, which would result in diploid or polyploid spores that we show have reduced viability.
Publisher: Wiley
Date: 11-01-2011
Publisher: Wiley
Date: 04-08-2013
DOI: 10.1111/MMI.12335
Publisher: Wiley
Date: 28-05-2009
Publisher: Wiley
Date: 12-12-2005
DOI: 10.1111/J.1365-2958.2005.04987.X
Abstract: Cell ision in nearly all bacteria is initiated by polymerization of the conserved tubulin-like protein FtsZ into a ring-like structure at midcell. This Z-ring functions as a scaffold for a group of conserved proteins that execute the synthesis of the ision septum (the isome). Here we describe the identification of a new cell ision protein in Bacillus subtilis. This protein is conserved in Gram positive bacteria, and because it has a role in septum development, we termed it SepF. sepF mutants are viable but have a cell ision defect, in which septa are formed slowly and with a severely abnormal morphology. Yeast two-hybrid analysis showed that SepF can interact with itself and with FtsZ. Accordingly, fluorescence microscopy showed that SepF accumulates at the site of cell ision, and this localization depends on the presence of FtsZ. Combination of mutations in sepF and ezrA, encoding another Z-ring interacting protein, had a synthetic lethal ision effect. We conclude that SepF is a new member of the Gram positive isome, required for proper execution of septum synthesis.
Publisher: Wiley
Date: 07-1998
DOI: 10.1046/J.1365-2958.1998.00954.X
Abstract: We have identified the Bacillus subtilis homologue of the essential cell ision gene, ftsL, of Escherichia coli. Repression of ftsL in a strain engineered to carry a conditional promoter results in cell filamentation, with a near immediate arrest of cell ision. The filaments show no sign of invagination, indicating that ision is blocked at an early stage. FtsL is also shown to be required for septation during sporulation, and depletion of FtsL blocks the activation but not the synthesis of the prespore-specific sigma factor, sigmaF. Immunofluorescence microscopy shows that depletion of FtsL has little or no effect on FtsZ ring formation, but the assembly of other ision proteins, DivIB and DivIC, at the site of ision is prevented. Repression of FtsL also results in a rapid loss of DivIC protein, indicating that DivIC stability is dependent on the presence of FtsL, in turn suggesting that FtsL is intrinsically unstable. The instability of one or more components of the ision apparatus may be important for the cyclic assembly/disassembly of the ision apparatus.
Publisher: Elsevier BV
Date: 05-2009
DOI: 10.1016/J.CELL.2009.02.035
Abstract: Proper segregation of DNA replication products is essential in all cells. In Bacillus subtilis, two protein complexes have been implicated in this process: the ParAB homologs, Soj and Spo0J, and the bacterial Smc/ScpAB complex, also called condensin. Here we demonstrate that Smc is highly enriched in the region around the origin of replication, specifically near parS sites to which Spo0J binds and at highly transcribed genes. Furthermore, we find that efficient recruitment of Smc to a large region around the origin of replication depends on the presence of Spo0J. We show that Spo0J performs two independent functions: regulation of initiation of DNA replication via Soj and promotion of chromosome segregation by Smc recruitment. Our results demonstrate a direct functional interaction between two widely conserved systems involved in chromosome replication and segregation.
Publisher: Wiley
Date: 08-2009
DOI: 10.1111/J.1365-2958.2009.06805.X
Abstract: MreB proteins are bacterial actin homologues thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. Many bacteria, particularly Gram-positives, have more than one MreB isoform. Bacillus subtilis has three, MreB, Mbl and MreBH, which colocalize in a single helical structure. We now show that the helical pattern of peptidoglycan (PG) synthesis in the cylindrical part of the rod-shaped cell is governed by the redundant action of the three MreB isoforms. Single mutants for any one of mreB isoforms can still incorporate PG in a helical pattern and generate a rod shape. However, after depletion of MreB in an mbl mutant (or depletion of all three isoforms) lateral wall PG synthesis was impaired and the cells became spherical and lytic. Overexpression of any one of the MreB isoforms overcame the lethality as well as the defects in lateral PG synthesis and cell shape. Furthermore, MreB and Mbl can associate with the peptidoglycan biosynthetic machinery independently. However, no single MreB isoform was able to support normal growth under various stress conditions, suggesting that the multiple isoforms are used to allow cells to maintain proper growth and morphogenesis under changing and sometimes adverse conditions.
Publisher: Wiley
Date: 02-1998
DOI: 10.1046/J.1365-2958.1998.00724.X
Abstract: Soon after the onset of sporulation in Bacillus subtilis, asymmetric cell ision occurs to generate the differentiating prespore and mother cell types. Formation of the septum close to the cell pole initially bisects the nucleoid destined for the prespore, trapping only about one-third of the DNA in the small compartment. The remaining part of the chromosome is then transported through the septum. spoIIIE mutant cells fail to transfer the DNA and arrest with only partially segregated prespore chromosomes. Previous work has shown that the orientation of the chromosome at the time of septation is not random. Here, we use both physical and genetic methods to characterize the trapped DNA. The results show that the chromosome has a very specific orientation at the time of septation, consistent with the action of a centromere-like sequence near oriC. They also demonstrate that the chromosome is folded, or otherwise organized, in a highly ordered manner.
Publisher: American Society for Microbiology
Date: 15-02-2002
DOI: 10.1128/JB.184.4.1102-1111.2002
Abstract: We have characterized the yyaA gene of Bacillus subtilis , located near the origin of chromosome replication ( oriC ). Its protein product is similar to the Spo0J protein, which belongs to the ParB family of chromosome- and plasmid-partitioning proteins. Insertional inactivation of the yyaA gene had no apparent effect on chromosome organization and partitioning during vegetative growth or sporulation. Subcellular localization of YyaA by immunofluorescence microscopy indicated that it colocalizes with the nucleoid, and gel retardation studies confirmed that YyaA binds relatively nonspecifically to DNA. Overexpression of yyaA caused a sporulation defect characterized by the formation of multiple septa within the cell. This phenotype indicates that YyaA may have a regulatory role at the onset of sporulation.
Publisher: Cold Spring Harbor Laboratory
Date: 04-1996
DOI: 10.1101/GAD.10.7.794
Abstract: During sporulation in Bacillus subtilis an asymmetric cell ision gives rise to unequal progeny called the prepore and the mother cell. Gene expression in the prespore is initiated by cell-specific activation of the transcription factor sigma(F). Three proteins participate in the regulation of sigma(F) activity. The first, SpoIIAB, is an inhibitor of sigma(F), that is, an anti-sigma factor. SpoIIAB is also a protein kinase that catalyzes phosphorylation of the second regulatory protein SpoIIAA (the anti-anti-sigma factor), and thus inactivates it. A third protein, SpoIIE, was shown recently to be able to dephosphorylate SpoIIAA-P in vitro. Here we show that SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate. First, we confirm by the use of in vivo experiments that it regulates the release of sigma(F) activity by dephosphorylating SpoIIAA-P. Second, we show that SpoIIE is needed for normal formation of the asymmetric septum that separates the prespore from the mother cell. Combination of these two functions in a single polypeptide may serve to couple the release of the cell-specific transcription factors with the formation of the differentiating cells.
Publisher: Proceedings of the National Academy of Sciences
Date: 12-09-1995
Abstract: Mutations in the spoIIIE gene prevent proper partitioning of one chromosome into the developing prespore during sporulation but have no overt effect on partitioning in vegetatively iding cells. However, the expression of spoIIIE in vegetative cells and the occurrence of genes closely related to spoIIIE in a range of nonsporulating eubacteria suggested a more general function for the protein. Here we show that SpoIIIE protein is needed for optimal chromosome partitioning in vegetative cells of Bacillus subtilis when the normal tight coordination between septation and nucleoid partitioning is perturbed or when septum positioning is altered. A functional SpoIIIE protein allows cells to recover from a state in which their chromosome has been trapped by a closing septum. By analogy to its function during sporulation, we suggest that SpoIIIE facilitates partitioning by actively translocating the chromosome out of the septum. In addition to enhancing the fidelity of nucleoid partitioning, SpoIIIE also seems to be required for maximal resistance to antibiotics that interfere with DNA metabolism. The results have important implications for our understanding of the functions of genes involved in the primary partitioning machinery in bacteria and of how septum placement is controlled.
Publisher: Springer Science and Business Media LLC
Date: 20-11-2019
DOI: 10.1038/S41467-019-13256-5
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Publisher: American Chemical Society (ACS)
Date: 23-02-2007
DOI: 10.1021/NL0700853
Abstract: The clinically important vancomycin antibiotic inhibits the growth of pathogens such as Staphylococcus aureus by blocking cell wall synthesis through specific recognition of nascent peptidoglycan terminating in D-Ala-D-Ala. Here, we demonstrate the ability of single-molecule atomic force microscopy with antibiotic-modified tips to measure the specific binding forces of vancomycin and to map in idual ligands on living bacteria. The single-molecule approach presented here provides new opportunities for understanding the binding mechanisms of antibiotics and for exploring the architecture of bacterial cell walls.
Publisher: Microbiology Society
Date: 09-2009
Abstract: In bacteria, DNA replication initiation is tightly regulated in order to coordinate chromosome replication with cell growth. In Escherichia coli , positive factors and negative regulatory mechanisms playing important roles in the strict control of DNA replication initiation have been reported. However, it remains unclear how bacterial cells recognize the right time for replication initiation during the cell cycle. In the Gram-positive bacterium Bacillus subtilis , much less is known about the regulation of replication initiation, specifically, regarding negative control mechanisms which ensure replication initiation only once per cell cycle. Here we report that replication initiation was greatly enhanced in strains that had the origin of replication ( oriC ) relocated to various loci on the chromosome. When oriC was relocated to new loci further than 250 kb counterclockwise from the native locus, replication initiation became asynchronous and earlier than in the wild-type cells. In two oriC -relocated strains ( oriC at argG or pnbA , 25 ° or 30 ° on the 36 ° chromosome map, respectively), DnaA levels were higher than in the wild-type but not enough to cause earlier initiation of replication. Our results suggest that the initiation capacity of replication is accumulated well before the actual time of initiation, and its release may be suppressed by a unique DNA structure formed near the native oriC locus.
Publisher: Wiley
Date: 30-11-1987
DOI: 10.1016/0014-5793(87)80465-9
Abstract: The sigma-subunit of RNA polymerase is responsible for promoter recognition in prokaryotes [(1969) Nature 221, 43-46]. Alterations in the sigma-subunit are thought to be involved in controlling 'global' changes in gene expression, such as those involved in differentiation in the spore-forming bacterium Bacillus subtilis [(1981) Cell 25, 582-584]. Stragier et al. [(1985) FEBS Lett. 195, 3-11] have proposed that sigma-factors are composed of two domains: a C-terminal domain involved in promoter recognition and an N-terminal domain involved in interactions with RNA polymerase. We have sequenced another developmental gene from B. subtilis, spoIIIC, and the strong homology of its predicted product suggests that it too may be a sigma-factor. However, the spoIIIC product is small and lacks completely the conserved N-terminal domain of the sigma-subunits. I propose that the product of the spoIIIC gene may carry out the DNA-recognition functions of a sigma-factor but that it probably requires an auxiliary factor to interact with core RNA polymerase.
Publisher: Proceedings of the National Academy of Sciences
Date: 11-06-2002
Abstract: A protein-interaction network centered on the replication machinery of Bacillus subtilis was generated by genome-wide two-hybrid screens and systematic specificity assays. The network consists of 91 specific interactions linking 69 proteins. Over one fourth of the interactions take place between homologues of proteins known to interact in other organisms, indicating the high biological significance of the other interactions we report. These interactions provide insights on the relations of DNA replication with recombination and repair, membrane-bound protein complexes, and signaling pathways. They also lead to the biological role of unknown proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control. Thus, our interaction map provides a valuable tool for the discovery of aspects of bacterial DNA replication.
Publisher: Wiley
Date: 06-10-2003
DOI: 10.1046/J.1365-2958.2003.03719.X
Abstract: We have developed several new fluorescent staining procedures that enabled us to study the synthesis of cell wall material in the spherical Gram-positive bacterium Staphylococcus aureus. The results obtained support previous proposals that these cells synthesize new wall material specifically at cell ision sites, in the form of a flat circular plate that is subsequently cleaved and remodelled to produce the new hemispherical poles of the daughter cells. We have shown that formation of the septal peptidoglycan is dependent on the key cell ision protein FtsZ, which recruits penicillin-binding protein (PBP) 2. Unexpectedly, in FtsZ-depleted cells, the cell wall synthetic machinery becomes dispersed and new wall material is made in dispersed patches over the entire surface of the cells, which increase in volume by up to eightfold before lysing. The results have implications for understanding the nature of S. aureus morphogenesis and for inhibitors of cell ision proteins as drug targets.
Publisher: Springer Science and Business Media LLC
Date: 02-2009
DOI: 10.1038/NATURE07742
Abstract: The cell wall is an essential structure for virtually all bacteria, forming a tough outer shell that protects the cell from damage and osmotic lysis. It is the target of our best antibiotics. L-form strains are wall-deficient derivatives of common bacteria that have been studied for decades. However, they are difficult to generate and typically require growth for many generations on osmotically protective media with antibiotics or enzymes that kill walled forms. Despite their potential importance for understanding antibiotic resistance and pathogenesis, little is known about their basic cell biology or their means of propagation. We have developed a controllable system for generating L-forms in the highly tractable model bacterium Bacillus subtilis. Here, using genome sequencing, we identify a single point mutation that predisposes cells to grow without a wall. We show that propagation of L-forms does not require the normal FtsZ-dependent ision machine but occurs by a remarkable extrusion-resolution mechanism. This novel form of propagation provides insights into how early forms of cellular life may have proliferated.
Publisher: American Chemical Society (ACS)
Date: 14-12-2020
DOI: 10.26434/CHEMRXIV.13286078.V1
Abstract: The application of genomic techniques to the investigation of understudied species of actinobacteria provides an expedited route to the discovery of new bioactive natural products. We report the isolation of the antibiotic polyketide tatiomicin, through a genomics and bioactivity informed analysis of the metabolome of the extremophile Amycolatopsis sp. DEM30355. Structural elucidation including absolute stereochemical assignment was performed using complementary crystallographic, spectroscopic and computational methods. Tatiomicin shows antibiotic activity against Gram-positive bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA).
Publisher: Wiley
Date: 08-1994
DOI: 10.1111/J.1365-2958.1994.TB00459.X
Abstract: A rapid and sensitive method for detection of cell- and compartment-specific gene expression in in idual cells of both Gram-negative and Gram-positive microorganisms is described. The method combines the use of gene fusions to lacZ, and a fluorogenic beta-galactosidase substrate, fluorescein-di-(beta-D-galactopyranoside), with digitized video microscopy. All of the reporter constructs tested were successfully detected. Secondary staining of the cells with a nucleic acid-specific dye, propidium iodide, allowed cells devoid of nucleic acid to be identified, while cell nucleoid shape and the morphological stage of development could be correlated with the location of beta-galactosidase activity. The double-staining procedure was used to show that gene expression can be induced in non-culturable cells of Salmonella enteritidis produced by carbon/nitrogen starvation. The resolution was sufficient to distinguish between cells at different morphological stages of sporulation in Bacillus subtilis. This highly sensitive and rapid method may have many other applications in basic and applied microbiology.
Publisher: Springer Science and Business Media LLC
Date: 02-2008
DOI: 10.1038/451900A
Publisher: American Association for the Advancement of Science (AAAS)
Date: 23-11-2001
Abstract: DNA replication in bacteria is carried out by a multiprotein complex, which is thought to contain only one essential DNA polymerase, specified by the dnaE gene in Escherichia coli and the polC gene in Bacillus subtilis . Bacillus subtilis genome analysis has revealed another DNA polymerase gene, dnaE BS , which is homologous to dnaE . We show that, in B. subtilis , dnaE BS is essential for cell viability and for the elongation step of DNA replication, as is polC, and we conclude that there are two different essential DNA polymerases at the replication fork of B. subtilis , as was previously observed in eukaryotes. dnaE BS appears to be involved in the synthesis of the lagging DNA strand and to be associated with the replication factory, which suggests that two different polymerases carry out synthesis of the two DNA strands in B. subtilis and in many other bacteria that contain both polC and dnaE genes.
Publisher: The Company of Biologists
Date: 2017
DOI: 10.1242/JCS.194571
Abstract: This study was designed to identify bioactive compounds that alter the cellular shape of the fission yeast Schizosaccharomyces pombe, by affecting functions involved in the cell cycle or cell morphogenesis. We used a multidrug sensitive fission yeast strain, SAK950 to screen a library of 657 actinomycete bacteria and identified 242 strains that induced eight different major shape phenotypes in S. pombe. These include the typical cell cycle related phenotype, Elongated cells and cell morphology related phenotype Rounded cells. As a proof of principle we purified four of these activities, one of which is a novel compound and three that are the previously known compounds, Leptomycin B, Streptonigrin and Cycloheximide. In this study we have also shown novel effects for two of these compounds, Leptomycin B and Cycloheximide. The identification of these four compounds and the explanation of the S. pombe phenotypes in terms of their known, or predicted bioactivities, confirm the effectiveness of this approach.
Publisher: Proceedings of the National Academy of Sciences
Date: 07-04-2003
Abstract: To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ≈4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and ision, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, ision, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden–Meyerhof–Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.
Publisher: Elsevier BV
Date: 1996
DOI: 10.1016/0168-9525(96)81386-2
Abstract: On starvation, the soil bacterium Bacillus subtilis stops iding and initiates sporulation, a simple developmental process involving the differentiation of two cell types. Sporulation begins with a reorganization of the cell cycle, to produce cells with the size and chromosome content appropriate for the developmental process. The central ision that would normally occur, to produce a pair of identical daughter cells, is blocked and the cell ides asymmetrically to produce a small, polar prespore cell and a much larger mother cell. The developmental fates of the two cells are dictated by the localized activation of cell-specific transcription factors, which are controlled by mechanisms that respond to the cellular asymmetry.
Publisher: American Society for Microbiology
Date: 08-2003
DOI: 10.1128/JB.185.15.4615-4619.2003
Abstract: The activation of σ G , a transcription factor, in Bacillus subtilis is coupled to the completion of engulfment during sporulation. SpoIIAB, an anti-sigma factor involved in regulation of σ F , is also shown to form a complex with σ G in vitro. SpoIIAA, the corresponding anti-anti-sigma factor, can disrupt the SpoIIAB:σ G complex, releasing free σ G . The data suggest the existence of an as-yet-unknown mechanism to keep σ G inactive prior to engulfment.
Publisher: Wiley
Date: 08-2001
DOI: 10.1046/J.1365-2958.2001.02551.X
Abstract: Spo0J and Soj belong to the ParB/ParA family of proteins involved in chromosome and plasmid segregation in bacteria. In Bacillus subtilis, Spo0J protein binds to several specific sites, parS, located on both sides of the origin of DNA replication, oriC, and apparently self-associates to form large discrete foci visible by fluorescence microscopy. Soj protein forms large 'patches' probably associated with the nucleoid, which can undergo dynamic, co-operative jumping from nucleoid to nucleoid in the presence of Spo0J. Patches of Soj protein somehow help to bring about the condensation of Spo0J foci. Soj is also a negative regulator of transcription. In the absence of Spo0J, Soj is statically distributed on each of the nucleoids in the cell and blocks the transcription of several sporulation genes. To analyse the functional interaction between Spo0J and Soj further, we have constructed and studied a collection of spo0J mutants. Most of the mutants completely prevent Spo0J from interacting with DNA. One mutation impairs the formation of compact Spo0J foci and simultaneously results in loss of Soj movement. We also isolated one spo0J mutant, in which the frequency of Soj internucleoid oscillation is highly increased. Both mutations affecting the interaction with Soj lie in the N-terminal coding part of spo0J, whereas the substitutions affecting DNA binding lie in the mid- to C-terminal coding region.
Publisher: Frontiers Media SA
Date: 13-10-2022
DOI: 10.3389/FMICB.2022.1004737
Abstract: Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a protein machine called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg 2+ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening & 2,000 extracts from a erse collection of actinobacteria, we identified a compound, mirubactin C, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed using synthetic mirubactin C. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin C rescuing activity is not due simply to iron sequestration. The results support an emerging view that the functions of bacterial siderophores extend well beyond simply iron binding and uptake.
Publisher: Wiley
Date: 03-1991
DOI: 10.1111/J.1365-2958.1991.TB00746.X
Abstract: Sporulation in Bacillus subtilis is a simple developmental system in which a single cell undergoes differentiation to two 'sister' cells, namely the prespore and the sporangium. Prespore-specific gene expression is largely dependent on the synthesis of a transcription factor, sigma G. Transcription of spolllG, the gene encoding sigma G, is under precise temporal and spatial control, requiring the products of at least eight genes that are expressed in the pre- isional cell. Here we show that the product of one of these genes, another sigma factor, sigma F, is by itself sufficient to direct transcription of spolllG in non-sporulating cells. The results indicate that the cell-specificity of prespore gene expression is determined by a mechanism that exerts temporal and spatial control over the activity of sigma F.
Publisher: American Chemical Society (ACS)
Date: 21-10-2022
Publisher: Public Library of Science (PLoS)
Date: 15-10-2020
Publisher: Elsevier BV
Date: 1994
DOI: 10.1016/S0022-2836(05)80027-0
Abstract: The Bacillus subtilis spoVD gene has been cloned and sequenced. It encodes a 71,262 Da protein with extensive sequence similarity to penicillin-binding proteins from various organisms. The context of this gene in the B. subtilis chromosome, immediately upstream of the mur operon, suggests that it is related to the pbpB gene of Escherichia coli, which is involved in the synthesis of septal peptidoglycan during cell ision. Expression of spoVD in E. coli leads to the synthesis of a membrane-associated protein of the size expected for SpoVD, which can bind labelled penicillin. However, insertional disruption of the spoVD gene has no effect on vegetative growth or ision: a second pbp-like gene immediately upstream of spoVD is probably the functional homologue of E. coli pbpB. spoVD seems instead to have a specialized role in the morphogenesis of the spore cortex, which is a modified form of peptidoglycan. spoVD transcription appears to occur from a promoter recognized by the sigma E form of RNA polymerase. Analysis of the expression of a spoVD'-lacZ reporter gene supports this notion and indicates that a second level of negative regulation is dependent on the SpoIIID protein. SpoVD synthesis probably occurs only in the mother cell since both sigma E and SpoIIID are thought to be specific to this cell type. Such localization of SpoVD synthesis was supported by the results of a genetic test showing that expression of spoVD only in the mother cell is sufficient for spore formation. The results support the proposition that spore cortex formation is determined primarily by the mother cell.
Publisher: Wiley
Date: 06-1991
DOI: 10.1111/J.1365-2958.1991.TB00783.X
Abstract: The spoVJ gene of Bacillus subtilis encodes a 36 kDa protein and is expressed only in the mother cell. spoVJ has an interesting pattern of regulation during sporulation because it is expressed from sequentially activated promoters. These promoters, designated P1 and P2, are under the control of different sigma factors, sigma E and sigma K, which become active at separate times during sporulation. Removal of promoter P1, leaving promoter P2 active, resulted in about a 30-minute delay in the formation of heat-resistant spores and demonstrated that the expression of spoVJ from both promoters is essential for normal sporulation. A comparison is made between the sequences of the spoVJ promoters and the promoters of other genes dependent upon sigma E and sigma K.
Publisher: Wiley
Date: 30-09-2011
Publisher: Springer International Publishing
Date: 2017
Publisher: Microbiology Society
Date: 07-1995
Publisher: American Society for Microbiology
Date: 11-2006
DOI: 10.1128/JB.01031-06
Abstract: About 11 essential proteins assemble into a ring structure at the surface of the cell to bring about cytokinesis in bacteria. Several of these proteins have their major domains located outside the membrane, forming an assembly that we call the outer ring (OR). Previous work on ision in Bacillus subtilis has shown that four of the OR proteins—FtsL, DivIC, DivIB, and PBP 2B—are interdependent for assembly. This contrasts with the mainly linear pathway for the equivalent proteins in Escherichia coli . Here we show that the interdependent nature of the B. subtilis pathway could be due to effects on FtsL and DivIC stability and that DivIB is an important player in regulating this turnover. Two-hybrid approaches suggest that a multiplicity of protein-protein interactions contribute to the assembly of the OR. DivIC is unusual in interacting strongly only with FtsL. We propose a model for the formation of the OR through the mutual association of the membrane proteins directed by the cytosolic inner-ring proteins.
Publisher: American Society for Microbiology
Date: 09-1997
DOI: 10.1128/JB.179.17.5636-5638.1997
Abstract: We have isolated transposon insertions in the lacA gene encoding an endogenous beta-galactosidase of Bacillus subtilis. Upstream of the putative operon containing lacA is a negative regulator, lacR, which encodes a product related to a family of regulators that includes the lactose repressor, lacI, of Escherichia coli. New strains with insertions in the lacA gene should be of use in studies using lacZ fusions in B. subtilis.
Publisher: Wiley
Date: 27-01-2012
DOI: 10.1038/EMBOJ.2012.6
Publisher: Microbiology Society
Date: 04-1996
DOI: 10.1099/00221287-142-4-733
Abstract: Wild-type and mutant forms of the gene encoding green fluorescent protein (GFP) from Aequorea victoria have been introduced into Bacillus subtilis as translational fusions to the prespore-specific and mother-cell-specific genes dacF and spoIVA . In both cases, the protein was readily detected by fluorescence microscopy, and its synthesis was correctly localized. The S65T substitution, which improves the quantum yield and rate of development of fluorescence, also produced a spectral shift that allowed the protein to be colocalized with DNA, after staining with 4′,6-diamidino-2-phenylindole. Three different translational fusions to the N-terminal region of GFP all produced active protein. Moreover, a full-length spoIVA-GFP fusion showed proper targeting to the surface of the spore, albeit at low temperature and in the presence of wild-type spoIVA protein. A mutation in the gfp gene which changes the light emitted by the protein from green to blue was found not to be useful because of the intrinsic autofluorescence of B. subtilis in the blue part of the spectrum.
Publisher: Wiley
Date: 06-1997
DOI: 10.1046/J.1365-2958.1997.3811764.X
Abstract: The Bacillus subtilis IVA gene, first defined by a mutation giving rise to anucleate minicells, has been cloned and characterized. Depletion of DivIVA leads to inhibition of the initiation of cell ision. The residual isions that do occur are abnormally placed and sometimes misorientated relative to the long axis of the cell. The DivIVA phenotype can be suppressed by disruption of the MinCD ision inhibitor, suggesting that DivIVA controls the topological specificity of MinCD action and thus septum positioning. A DivIVA-GFP fusion targets to new and used sites of cell ision, consistent with it having a direct role in topological specification.
Publisher: Elsevier BV
Date: 11-1992
DOI: 10.1016/0378-1119(92)90172-L
Abstract: We have developed a novel expression vector based on the bacteriophage phi 105, and employed it for the production of mutant beta-lactamases in Bacillus subtilis. Expression of the beta-lactamase-encoding gene was low when cloned into the prophage under the control of its own promoter. However, expression was considerably elevated when the gene was inserted into the phage genome in the same orientation as phage transcription. A defective phi 105 vector was constructed with a deletion removing a region needed for cell lysis, and with a mutation in the immunity repressor, rendering it temperature sensitive. Production of beta-lactamase could then be induced by a shift in temperature and without concomitant cell lysis, facilitating purification of the protein from the culture supernatant. This phage has considerable potential for development as a vector for controllable production of heterologous proteins in B. subtilis.
Publisher: Wiley
Date: 15-02-2011
DOI: 10.1111/J.1365-2958.2011.07559.X
Abstract: Cell ision in almost all bacteria is orchestrated by the essential tubulin homologue FtsZ, which assembles into a ring-like structure and acts as a scaffold for the ision machinery. Division was recently validated as an important target for antibiotics by the demonstration that low-molecular-weight inhibitors of FtsZ, called benzamides, can cure mice infected with Staphylococcus aureus. In treated cells of Bacillus subtilis we show that FtsZ assembles into foci throughout the cell, including abnormal locations at the cell poles and over the nucleoid. These foci are not inactive aggregates because they remain dynamic, turning over almost as rapidly as untreated polymers. Remarkably, although ision is completely blocked, the foci efficiently recruit ision proteins that normally co-assemble with FtsZ. However, they show no affinity for components of the Min or Nucleoid occlusion systems. In vitro, the benzamides strongly promote the polymerization of FtsZ, into hyperstable polymers, which are highly curved. Importantly, even at low concentrations, benzamides transform the structure of the Z ring, resulting in abnormal helical cell ision events. We propose that benzamides act principally by promoting an FtsZ protomer conformation that is incompatible with a higher-order level of assembly needed to make a ision ring.
Publisher: American Chemical Society (ACS)
Date: 14-12-2020
DOI: 10.26434/CHEMRXIV.13286078
Abstract: The application of genomic techniques to the investigation of understudied species of actinobacteria provides an expedited route to the discovery of new bioactive natural products. We report the isolation of the antibiotic polyketide tatiomicin, through a genomics and bioactivity informed analysis of the metabolome of the extremophile i Amycolatopsis /i sp. DEM30355. Structural elucidation including absolute stereochemical assignment was performed using complementary crystallographic, spectroscopic and computational methods. Tatiomicin shows antibiotic activity against Gram-positive bacteria, including Methicillin-resistant i Staphylococcus aureus /i (MRSA).
Publisher: Wiley
Date: 18-02-2009
DOI: 10.1111/J.1365-2958.2009.06601.X
Abstract: The bacterial actin homologue MreB plays a key role in cell morphogenesis. In Bacillus subtilis MreB is essential under normal growth conditions and mreB mutants are defective in the control of cell diameter. However, the precise role of MreB is still unclear. Analysis of the lethal phenotypic consequences of mreB disruption revealed an unusual bulging phenotype that precedes cell death. A similar phenotype was seen in wild-type cells at very low Mg(2+) concentrations. We found that inactivation of the major bi-functional penicillin-binding protein (PBP) PBP1 of B. subtilis restored the viability of an mreB null mutant as well as preventing bulging in both mutant and wild-type backgrounds. Bulging was associated with delocalization of PBP1. We show that the normal pattern of localization of PBP1 is dependent on MreB and that the proteins can physically interact using in vivo pull-down and bacterial two-hybrid approaches. Interactions between MreB and several other PBPs were also detected. Our results suggest that MreB filaments associate directly with the peptidoglycan biosynthetic machinery in B. subtilis as part of the mechanism that brings about controlled cell elongation.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 03-11-2000
DOI: 10.1126/SCIENCE.290.5493.995
Abstract: The SpoIIIE protein of Bacillus subtilis is required for chromosome segregation during spore formation. The COOH-terminal cytoplasmic part of SpoIIIE was shown to be a DNA-dependent adenosine triphosphatase (ATPase) capable of tracking along DNA in the presence of ATP, and the NH 2 -terminal part of the protein was found to mediate its localization to the ision septum. Thus, during sporulation, SpoIIIE appears to act as a DNA pump that actively moves one of the replicated pair of chromosomes into the prespore. The presence of SpoIIIE homologs in a broad range of bacteria suggests that this mechanism for active transport of DNA may be widespread.
Publisher: Cold Spring Harbor Laboratory
Date: 07-2001
DOI: 10.1101/GAD.197501
Abstract: DivIVA is a coiled-coil, tropomyosin-like protein of Gram-positive bacteria. Previous work showed that this protein is targeted to ision sites and retained at the cell poles after ision. In vegetative cells, DivIVA sequesters the MinCD ision inhibitor to the cell poles, thereby helping to direct cell ision to the correct midcell site. We now show that DivIVA has a second, quite separate role in sporulating cells of Bacillus subtilis . It again acts at the cell pole but in this case interacts with the chromosome segregation machinery to help position the oriC region of the chromosome at the cell pole, in preparation for polar ision. We isolated mutations in IVA that separate the protein's role in sporulation from its vegetative function in cell ision. DivIVA therefore appears to be a bifunctional protein with distinct roles in ision-site selection and chromosome segregation.
Publisher: Springer Science and Business Media LLC
Date: 12-1987
DOI: 10.1007/BF00325704
Abstract: Here we provide a genome-wide, high-resolution map of the phylogenetic origin of the genome of most extant laboratory mouse inbred strains. Our analysis is based on the genotypes of wild-caught mice from three subspecies of Mus musculus. We show that classical laboratory strains are derived from a few fancy mice with limited haplotype ersity. Their genomes are overwhelmingly Mus musculus domesticus in origin, and the remainder is mostly of Japanese origin. We generated genome-wide haplotype maps based on identity by descent from fancy mice and show that classical inbred strains have limited and non-randomly distributed genetic ersity. In contrast, wild-derived laboratory strains represent a broad s ling of ersity within M. musculus. Intersubspecific introgression is pervasive in these strains, and contamination by laboratory stocks has played a role in this process. The subspecific origin, haplotype ersity and identity by descent maps can be visualized using the Mouse Phylogeny Viewer (see URLs).
Publisher: American Society for Microbiology
Date: 23-09-2020
DOI: 10.1128/JB.00296-20
Abstract: Endospore formation in Firmicutes bacteria provides one of the most highly resistant life forms on earth. During the early stages of endospore formation, the cell cycle is reorganized so that exactly two fully replicated chromosomes are generated, before the cell ides asymmetrically to generate the prespore and mother cell compartments that are critical for the developmental process. Decades ago, it was discovered that just prior to asymmetrical ision the two chromosomes enter an unusual elongated configuration called the axial filament. This paper provides new insights into the nature of the axial filament structure and suggests that cohesion of the normally separated sister chromosome termini plays an important role in axial filament formation.
Publisher: American Society for Microbiology
Date: 03-1988
DOI: 10.1128/JB.170.3.1162-1167.1988
Abstract: The spoIIIC locus of Bacillus subtilis has been cloned from the lambda library of Ferrari et al. (E. Ferrari, D. J. HEnner, and J. A. Hoch, J. Bacteriol. 146:430-432, 1981) by using as an assay transformation of the mutant allele spoIIIC94 to the wild type. Regulation of the spoIIIC locus was studied by hybridization of cloned spoIIIC DNA to RNA pulse-labeled at various times during growth and sporulation. The relative rate of transcription of the spoIIIC locus was highest 3 h after the end of growth. The DNA sequence of the spoIIIC transcription unit indicated the coding capacity for a small protein (138 amino acids) having significant similarity with one domain of RNA polymerase sigma factors. Interruption of this coding sequence by an insertion mutation caused cells to become Spo-.
Publisher: Cold Spring Harbor Laboratory
Date: 24-04-2022
DOI: 10.1101/2022.04.24.489322
Abstract: Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a complex protein machinery called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg 2+ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening ,000 extracts from a erse collection of actinobacteria, we identified a new compound, mirubactin B, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed by synthesising mirubactin B. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin B rescuing activity is not due simply to iron sequestration. The results demonstrate a novel antibacterial compound and add to growing evidence that bacterial siderophores have a range of activities beyond simple iron sequestration.
Publisher: Elsevier BV
Date: 12-2005
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.CELREP.2012.03.008
Abstract: The cell wall is a defining structural feature of the bacterial subkingdom. However, most bacteria are capable of mutating into a cell-wall-deficient "L-form" state, requiring remarkable physiological and structural adaptations. L-forms proliferate by an unusual membrane deformation and scission process that is independent of the conserved and normally essential FtsZ based ision machinery, and which may provide a model for the replication of primitive cells. Candidate gene screening revealed no requirement for the cytoskeletal systems that might actively drive membrane deformation or scission. Instead, we uncovered a crucial role for branched-chain fatty acid (BCFA) synthesis. BCFA-deficient mutants grow and undergo pulsating shape changes, but membrane scission fails, abolishing the separation of progeny cells. The failure in scission is associated with a reduction in membrane fluidity. The results identify a step in L-form proliferation and demonstrate that purely biophysical processes may have been sufficient for proliferation of primitive cells.
Publisher: Wiley
Date: 13-06-2019
DOI: 10.1002/MBO3.876
Publisher: American Chemical Society (ACS)
Date: 11-04-2017
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
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End Date: 2017
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Funder: European Commission
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Funder: International Human Frontier Science Program Organization
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Funder: Australian Research Council
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Funder: Medical Research Council
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