ORCID Profile
0000-0002-5958-830X
Current Organisations
University of Helsinki
,
University of Sydney
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Publisher: Springer Science and Business Media LLC
Date: 08-07-2019
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: Wiley
Date: 30-09-2011
Publisher: Wiley
Date: 04-06-2009
Publisher: Frontiers Media SA
Date: 02-06-2023
DOI: 10.3389/FMICB.2023.1204979
Abstract: Bacterial cells are surrounded by a peptidoglycan (PG) wall, which is a crucial target for antibiotics. It is well known that treatment with cell wall-active antibiotics occasionally converts bacteria to a non-walled “L-form” state that requires the loss of cell wall integrity. L-forms may have an important role in antibiotic resistance and recurrent infection. Recent work has revealed that inhibition of de novo PG precursor synthesis efficiently induces the L-form conversion in a wide range of bacteria, but the molecular mechanisms remain poorly understood. Growth of walled bacteria requires the orderly expansion of the PG layer, which involves the concerted action not just of synthases but also degradative enzymes called autolysins. Most rod-shaped bacteria have two complementary systems for PG insertion, the Rod and aPBP systems. Bacillus subtilis has two major autolysins, called LytE and CwlO, which are thought to have partially redundant functions. We have dissected the functions of autolysins, relative to the Rod and aPBP systems, during the switch to L-form state. Our results suggest that when de novo PG precursor synthesis is inhibited, residual PG synthesis occurs specifically via the aPBP pathway, and that this is required for continued autolytic activity by LytE/CwlO, resulting in cell bulging and efficient L-form emergence. The failure of L-form generation in cells lacking aPBPs was rescued by enhancing the Rod system and in this case, emergence specifically required LytE but was not associated with cell bulging. Our results suggest that two distinct pathways of L-form emergence exist depending on whether PG synthesis is being supported by the aPBP or RodA PG synthases. This work provides new insights into mechanisms of L-form generation, and specialisation in the roles of essential autolysins in relation to the recently recognised dual PG synthetic systems of bacteria.
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: 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: 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: 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: Cold Spring Harbor Laboratory
Date: 03-03-2023
DOI: 10.1101/2023.03.03.530798
Abstract: Acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Despite vaccinations, the development and use of neutralizing antibodies against the viral surface spike proteins, and small molecule inhibitors targeting the viral replication machinery, COVID-19 remains a global public health crisis. Emerging mutations in the viral genome have the potential to reduce prophylactic and therapeutic efficacy of virus-directed treatments. Targeting host cell factors required for infection could, therefore, be a potential strategy to overcome this problem since mutations in the viral genome are unlikely to bypass the requirement for the targeted host factor or function. The enzymatic activity of N-myristoyltransferases (NMTs) are essential to mediate stable membrane binding and function of a erse class of cellular proteins, many of which regulate intracellular membrane trafficking. Here we report that nanomolar concentrations of the NMT inhibitor IMP-1088 inhibited SARS-CoV-2 spreading in human cells by compromising the infectivity of released viral particles, which was reduced by up to 90%. IMP-1088 also inhibited human Respiratory syncytial virus, the main cause of viral death in infants world-wide, but not the mosquito-delivered alphavirus Semliki Forest virus and the vesiculovirus Vesicular stomatitis virus. The antiviral effect of IMP-1088 against SARS-CoV-2 displayed remarkably slow reversibility, was well tolerated by cells, and is, therefore, a promising candidate for COVID-19 prophylaxis and therapy.
Publisher: Elsevier BV
Date: 07-2018
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: 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: 04-05-2006
DOI: 10.1111/J.1365-2958.2006.05184.X
Abstract: The assembly of ring-like structures, composed of FtsZ proteins (i.e. the Z ring), is the earliest and most essential process in bacterial cytokinesis. It has been shown that this process is directly regulated by the FtsZ-binding proteins, FtsA, ZapA, and EzrA, in Bacillus subtilis. In this study, protein complexes that are involved in Z-ring formation were chemically cross-linked in vivo, purified by affinity chromatography, and analysed by mass spectrometry. Analysis of the results identified YlmF as a new component of the FtsZ complex. Yeast two-hybrid analysis and fluorescence microscopy of YFP-YlmF in B. subtilis cells indicated YlmF localizes to the ision site in an FtsZ-dependent manner. A single disruption of YlmF resulted in a slight elongation of cells however, simultaneous inactivation of both YlmF and FtsA showed synthetic lethality caused by complete blockage of cell ision due to the defect in Z-ring formation. In contrast, the ftsA-null mutant phenotype, caused by inefficient Z-ring formation, could be complemented by overexpression of YlmF. These results suggest that YlmF has an overlapping function with FtsA in stimulating the formation of Z rings in B. subtilis.
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: Oxford University Press (OUP)
Date: 2007
Publisher: Wiley
Date: 02-2003
DOI: 10.1046/J.1365-2958.2003.03360.X
Abstract: A knock-out mutant of the dinR gene that encodes the SOS regulon repressor in Bacillus subtilis was constructed. The yneA, yneB and ynzC genes transcribed ergently from the dinR gene were strongly induced in mutant cells. Northern hybridization analyses revealed that these genes collectively form an operon and belong to the SOS regulon. The simultaneous deletion of dinR and yneA suppressed the filamentous phenotype of the dinR mutant. Furthermore, although yneA is suppressed in the wild-type cell in the absence of SOS induction, artificial expression of the YneA protein using an IPTG-inducible promoter resulted in cell elongation. Disruption of yneA significantly reduced cell elongation after the induction of the SOS response by mitomycin C in dinR+ cells. These results indicate that the YneA protein is responsible for cell ision suppression during the SOS response in B. subtilis. Localization of the FtsZ protein to the cell ision site was reduced in dinR-disrupted or yneA-expressing cells, further suggesting that the YneA protein suppresses cell ision through the suppression of FtsZ ring formation. Interestingly, the B. subtilis YneA protein is structurally and phylogenetically unrelated to its functional counterpart in Escherichia coli, SulA.
Publisher: Elsevier BV
Date: 02-2018
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: 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: 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: Elsevier BV
Date: 06-2015
Publisher: Wiley
Date: 11-01-2011
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: 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: Microbiology Society
Date: 04-2006
Abstract: Previous work has shown that the Bacillus subtilis EzrA protein directly inhibits FtsZ ring assembly, which is required for normal cell ision, and that loss of EzrA results in hyperstabilization of the FtsZ polymer in vivo . Here, it was found that in ezrA -disrupted cells, artificial expression of YneA, which suppresses cell ision during the SOS response, and disruption of noc ( yyaA ), which acts as an effector of nucleoid occlusion, resulted in accumulation of multiple non-constricting FtsZ rings, inhibition of cell ision, and synthetic lethality. Overexpression of the essential cell ision protein FtsL suppressed the effect of ezrA disruption. FtsL overexpression recovered the delayed FtsZ ring constriction seen in ezrA -disrupted wild-type cells. Conversely, the absence of EzrA caused lethality in cells producing a lower amount of FtsL than wild-type cells. It has previously been reported that FtsL is recruited to the ision site during the later stages of cell ision, although its exact role is currently unknown. The results of this study suggest that FtsL and EzrA synergistically regulate the FtsZ ring constriction in B. subtilis . Interestingly, FtsL overexpression also suppressed the cell ision inhibition due to YneA expression or Noc inactivation in ezrA -disrupted cells.
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.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Yoshikazu Kawai.