Keith Shearwin

Transcriptional interference between convergent promoters caused by elongation over the promoter

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1016/J.MOLCEL.2004.05.010

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

Publisher: Cold Spring Harbor Laboratory

Date: 27-09-2019

DOI: 10.1101/784983

Abstract: The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator binding to the phage attachment site ( att ), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA, and spreads from specific binding sites into adjacent non-specific DNA features that are shared with other RDFs. By analysing Apl’s repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

Establishing Lysogenic Transcription in the Temperate Coliphage 186

Publisher: American Society for Microbiology

Date: 04-2001

DOI: 10.1128/JB.183.7.2376-2379.2001

Abstract: A single-copy chromosomal reporter system was used to measure the intrinsic strengths and interactions between the three promoters involved in the establishment of lysogeny by coliphage 186. The maintenance lysogenic promoter p L for the immunity repressor gene c I is intrinsically ∼20-fold weaker than the lytic promoter p R . These promoters are arranged face-to-face, and transcription from p L is further weakened some 14-fold by the activity of p R . Efficient establishment of lysogeny requires the p E promoter, which lies upstream of p L and is activated by the phage CII protein to a level comparable to that of p R . Transcription of p E is less sensitive to converging p R transcription and raises c I transcription at least 55-fold. The p E promoter does not occlude p L but inhibits lytic transcription by 50%. This interference is not due to bound CII preventing elongation of the lytic transcript. The p E RNA is antisense to the anti-immune repressor gene apl , but any role of this in the establishment of lysogeny appears to be minimal.

Linkages between the Dissociation of αβ Tubulin into Subunits and Ligand Binding: The Ground State of Tubulin Is the GDP Conformation

Publisher: American Chemical Society (ACS)

Date: 02-1994

DOI: 10.1021/BI00170A006

Abstract: The effects of ligands on the dissociation of the alpha beta tubulin dimer into the two subunits were investigated using calf brain tubulin. Sedimentation equilibrium studies showed a number of linkages. In the absence of magnesium in the medium, tubulin-GTP, tubulin-GDP, and tubulin with the exchangeable site unoccupied associate with essentially the same strength (K alpha beta = 1 x 10(7) M-1). This indicates that the ground state of tubulin (i.e., in the absence of magnesium) is not affected by occupancy of the exchangeable nucleotide binding site (E site). The alpha beta association is enhanced by magnesium ions. The association of tubulin with GDP in the E site is linked to the uptake of twice as many magnesium ions as that of tubulin with GTP in the E site. This suggests that magnesium binding is linked to an E-site-related conformational change. Consideration of the linkages between the binding of magnesium ions, E-site occupancy, and tubulin conformation in terms of the model [Howard, W. D., & Timasheff, S. N. (1986) Biochemistry 25, 8292-8300] in which the tubulin alpha beta dimer exists in an equilibrium between two conformations, a microtubule-forming ("straight") state favored by GTP and a double-ring-forming ("curved") state favored by GDP, leads to the conclusion that the ground state of tubulin is the ring-forming or "curved" conformation. Thus, in the absence of magnesium, the tubulin heterodimer exists in the ring-forming conformation, whether the E site is occupied by GTP or GDP.(ABSTRACT TRUNCATED AT 250 WORDS)

APTC-C-SA01: A Novel Bacteriophage Cocktail Targeting Staphylococcus aureus and MRSA Biofilms

Publisher: MDPI AG

Date: 30-05-2022

DOI: 10.3390/IJMS23116116

Abstract: The high infection and mortality rate of methicillin-resistant Staphylococcus aureus (MRSA) necessitates the urgent development of new treatment strategies. Bacteriophages (phages) have several advantages compared to antibiotics for the treatment of multi-drug-resistant bacterial infections, and thus provide a promising alternative to antibiotics. Here, S. aureus phages were isolated from patients and environmental sources. Phages were characterized for stability, morphology and genomic sequence and their bactericidal activity against the biofilm form of methicillin-susceptible Staphylococcus aureus (MSSA) and MRSA was investigated. Four S. aureus phages were isolated and tested against 51 MSSA and MRSA clinical isolates and reference strains. The phages had a broad host range of 82–94% in idually and of % when combined and could significantly reduce the viability of S. aureus biofilms. The phages had a latent period of ≤20 min and burst size of plaque forming units (PFU)/infected cell. Transmission electron microscopy (TEM) identified phages belonging to the family of Myoviridae. Genomic sequencing indicated the lytic nature of all four phages, with no identified resistance or virulence genes. The 4 phages showed a high complementarity with 49/51 strains (96%) sensitive to at least 2/4 phages tested. Furthermore, the frequency of bacteriophage insensitive mutant (BIM) generation was lower when the phages were combined into the phage cocktail APTC-C-SA01 than for bacteria exposed to each of the phages alone. In conclusion, APTC-C-SA01, containing four lytic S. aureus phages has the potential for further development as a treatment against MSSA and MRSA infections.

Effect of Colchicine Analogues on the Dissociation of αβ Tubulin into Subunits: The Locus of Colchicine Binding

Publisher: American Chemical Society (ACS)

Date: 02-1994

DOI: 10.1021/BI00170A007

Abstract: A combination of ligand binding and sedimentation equilibrium studies was used to characterize the thermodynamic linkages between alpha beta tubulin association, nucleotide binding, and the interaction of colchicine analogues with dimeric and dissociated tubulins. The strength of binding of allocolchicine to the tubulin dimer was identical (8 x 10(5) M-1) whether the exchangeable nucleotide site (E site) was occupied by GTP or GDP. This drug bound to dimeric (alpha beta) tubulin and to one of the monomeric subunits, and the binding affinity for the dissociated state was linked to occupancy of the exchangeable nucleotide site. When the exchangeable site was occupied by GTP, the drug bound with very similar affinities to the dimeric and dissociated states of the protein. For tubulin-GDP, the binding of the drug to the dissociated state was significantly weaker (6.3 x 10(4) M-1) than to the dimeric state, suggesting the existence of an E-site-related conformational change in the dissociated state. Podophyllotoxin, which contains the A-ring portion of colchicine, bound with equal affinity to the dimeric and dissociated forms of both tubulin-GTP and tubulin-GDP, indicating that it is the C-ring portion of colchicine that is linked to the E-site-related conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)

Cro's role in the CI-Cro bistable switch is critical for λ's transition from lysogeny to lytic development

Publisher: Cold Spring Harbor Laboratory

Date: 10-2007

DOI: 10.1101/GAD.1584907

Abstract: CI represses cro Cro represses cI . This double negative feedback loop is the core of the classical CI–Cro epigenetic switch of bacteriophage λ. Despite the classical status of this switch, the role in λ development of Cro repression of the P RM promoter for CI has remained unclear. To address this, we created binding site mutations that strongly impaired Cro repression of P RM with only minimal effects on CI regulation of P RM . These mutations had little impact on λ development after infection but strongly inhibited the transition from lysogeny to the lytic pathway. We demonstrate that following inactivation of CI by ultraviolet treatment of lysogens, repression of P RM by Cro is needed to prevent synthesis of new CI that would otherwise significantly impede lytic development. Thus a bistable CI–Cro circuit reinforces the commitment to a developmental transition.

Purification and self-association equilibria of the lysis-lysogeny switch proteins of coliphage 186

Publisher: Elsevier BV

Date: 05-1996

DOI: 10.1074/JBC.271.19.11525

Prophage acquisition byStaphylococcus aureuscontributes to the expansion of Staphylococcal immune evasion

Publisher: Cold Spring Harbor Laboratory

Date: 28-04-2023

DOI: 10.1101/2023.04.27.538627

Abstract: Staphylococcus aureus colonizes 30% of the human population, but only a few clones cause severe infections. S. aureus’ virulence varies and partly depends on the presence of prophages, viral DNA embedded in the S. aureus core genome, such as hlb-converting prophage (ϕSa3int). Human-adapted S. aureus often harbours a ϕSa3int group of prophages preferentially integrated into their β-hemolysin ( hlb ) gene that encodes human immune evasion cluster (IEC) genes. Exotoxins and immune modulatory molecules encoded by this prophage can inhibit human innate immunity increasing S. aureus pathogenicity. This study aims to investigate the genomic and phenotypic plasticity of S. aureus and changes in its extracellular proteome after the acquisition of ϕSa3int prophage. To achieve this, we used S. aureus strains isolated from the sinus cavities of a patient with severe chronic rhinosinusitis (CRS) at two different time points ( S. aureus SA222 and S. aureus SA333) and hybrid sequenced the strains using short-read Illumina and long-read Oxford nanopore technology. In silico analysis showed the presence of a ϕSa3int prophage in the later isolate but not in the earlier isolate while most of the core genes remained identical. Using mitomycin C, we induced the ϕSa3int prophage, and transduced it into the Sa3int-prophage-free SA222 isolate to obtain a laboratory generated ‘double lysogen’. We confirmed the successful lysogenisation with culture methods (spot assay, blood agar) and also by sequencing. We compared growth kinetics, biofilm biomass and metabolic activity between parent and the lysogen by establishing growth curves, crystal violet and resazurin assays. Exoproteins were identified and quantified using mass spectrophotometry. Integration of ϕSa3int prophage in SA222 down-regulated the beta-hemolysin expression of the lysogen . In silico analysis of the S. aureus genome confirmed the insertion of a ∼43.8 kb ϕSa3int prophage into hlb gene. Insertion of prophage DNA did not alter the growth kinetics, biofilm formation, adhesion to primary human nasal epithelial cells and the metabolic activity in a biofilm. However, the acquisition of ϕSa3int prophage significantly changed the expression of various secreted proteins, both bacterial and prophage-encoded. Altogether, thirty-eight exoproteins were significantly differentially regulated in the laboratory created lysogen, compared to its recipient strain SA222. Among these proteins, there was significant upregulation of 21 exoproteins (55.3 %) including staphylokinase (sak), SCIN (scn), and intercellular adhesion protein B (icaB) and downregulation of 17 exoproteins (44.7 %), including β-hemolysin (hlb/sph) and outer membrane porin (phoE). Most of the upregulated proteins were involved in immunomodulation that help S. aureus escape human innate immunity and help cause chronic infection. These findings may contribute to the development of novel approaches to render S. aureus susceptible to the immune response by blocking prophage-associated defence mechanisms. A ϕSa3int prophage preferentially integrates into the β-haemolysin gene ( hlb ) gene thereby disrupting the beta-hemolysin function. A ∼43.8 kb ϕSa3int prophage acquisition by S. aureus has no impact on its growth kinetics, biofilm formation and adhesion to primary human nasal epithelial cells (HNECs). The presence of a ϕSa3int group prophage likely enhances Staphylococcus aureus’ human immune evasion capability as the prophage encodes a complete set of immune evasion cluster (IEC) genes. Targeted identification of virulence factors in addition to species and strain identification may lead to better-personalized therapy as not all S. aureus carry the same virulence genes.

Promoter activation by CII, a potent transcriptional activator from bacteriophage 186

Publisher: Elsevier BV

Date: 11-2014

DOI: 10.1074/JBC.M114.608026

Hidden Secrets of Sigma54 Promoters Revealed

Publisher: Elsevier BV

Date: 12-2017

DOI: 10.1016/J.TIBS.2017.10.005

Abstract: Bacterial sigma54 (σ

Revisited gene regulation in bacteriophage λ

Publisher: Elsevier BV

Date: 04-2005

DOI: 10.1016/J.GDE.2005.02.001

Abstract: The contribution of bacteriophage lambda to gene control research is far from over. A revised model of the lambda genetic switch includes extra cooperativity through octamerization of the cI repressor protein, mediated by long-range DNA looping. Structural analysis reveals remarkably subtle transcriptional activation by cI. The action of cI, activation by cII, and aspects of antitermination by N and Q all confirm the utility and versatility of simple, weak adhesive interactions mediated by nucleic acid tethers. New genetic and quantitative analysis of the lambda gene network is challenging cherished ideas about how complex behaviours emerge from this regulatory system.

Biotin-mediated growth and gene expression in Staphylococcus aureus is highly responsive to environmental biotin

Publisher: Springer Science and Business Media LLC

Date: 05-03-2018

DOI: 10.1007/S00253-018-8866-Z

Abstract: Biotin (Vitamin B7) is a critical enzyme co-factor in metabolic pathways important for bacterial survival. Biotin is obtained either from the environment or by de novo synthesis, with some bacteria capable of both. In certain species, the bifunctional protein BirA plays a key role in biotin homeostasis as it regulates expression of biotin biosynthetic enzymes in response to biotin demand and supply. Here, we compare the effect of biotin on the growth of two bacteria that possess a bifunctional BirA, namely Escherichia coli and Staphylococcus aureus. Unlike E. coli that could fulfill its biotin requirements through de novo synthesis, S. aureus showed improved growth rates in media supplemented with 10 nM biotin. S. aureus also accumulated more radiolabeled biotin from the media highlighting its ability to efficiently scavenge exogenous material. These data are consistent with S. aureus colonizing low biotin microhabitats. We also demonstrate that the S. aureus BirA protein is a transcriptional repressor of BioY, a subunit of the biotin transporter, and an operon containing yhfT and yhfS, the products of which have a putative role in fatty acid homeostasis. Increased expression of bioY is proposed to help cue S. aureus for efficient scavenging in low biotin environments.

Cooperativity in long-range gene regulation by the λ CI repressor

Publisher: Cold Spring Harbor Laboratory

Date: 02-2004

DOI: 10.1101/GAD.1167904

Abstract: Effective repression of cI transcription from P RM by the bacteriophage λ CI repressor requires binding sites ( O L ) located 2.4 kb from the promoter. A CI tetramer bound to O L 1 .O L 2 interacts with a tetramer bound near P RM ( O R 1 .O R 2), looping the intervening DNA. We previously proposed that in this CI octamer:DNA complex, the distant O L 3 operator and the weak O R 3 operator overlapping P RM are juxtaposed so that a CI dimer at O L 3 can cooperate with a CI dimer binding to O R 3. Here we show that O L 3 is necessary for effective repression of P RM and that the repressor at O L 3 appears to interact specifically with the repressor at O R 3. The O L 3-CI- O R 3 interaction involves the same CI interface used for short-range dimer-dimer interactions and does not occur without the other four operators. The long-range interactions were incorporated into a physicochemical model, allowing estimation of the long-range interaction energies and showing the lysogenic state to be ideally poised for CI negative autoregulation. The results establish the λ system as a powerful tool for examining long-range gene regulatory interactions in vivo.

Aspects of protein–DNA interactions: a review of quantitative thermodynamic theory for modelling synthetic circuits utilising LacI and CI repressors, IPTG and the reporter gene lacZ

Publisher: Springer Science and Business Media LLC

Date: 07-11-2016

DOI: 10.1007/S12551-016-0231-9

The Colchicine-Induced GTPase Activity of Tubulin: State of the Product. Activation by Microtubule-Promoting Cosolvents

Publisher: American Chemical Society (ACS)

Date: 05-1994

DOI: 10.1021/BI00186A027

Abstract: Colchicine induces a weak assembly-independent GTPase activity in calf brain tubulin [David-Pfeuty, T., Erickson, H. P., & Pantaloni, D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5372-5376 Andreu, J. M., & Timasheff, S. N. (1981) Arch. Biochem. Biophys. 211, 151-157]. Kinetic analysis shows a turnover number of 2 x 10(-4) s-1 in 0.01 M sodium phosphate and 4 mM MgCl2, pH 7.0, with an apparent Km for GTP of 10 microM. This activity, which requires Mg2+ ions and attains a plateau at 4 mM MgCl2, is independent of pH over the pH range of 6.6-7.4. This GTPase activity was induced by all colchicine analogues that contain rings A and C (or C'), the strength varying in a manner parallel to the free energy of binding of the ligand. The specific GTPase activity was found to be independent of the tubulin-colchicine complex concentration over the range of 2-20 microM. Sedimentation velocity examination of the product of the reaction showed that GDP-tubulin-colchicine generated by hydrolysis of the E-site GTP was indistinguishable from that produced by nucleotide exchange at the site, the protein assuming the "curved" conformation in both cases. Steady-state kinetic analysis in the presence of high concentrations of microtubule-inducing cosolvent additives revealed an increase in kcat/Km of up to 1 order of magnitude that followed the order poly(ethylene glycol) 6000 (PEG-6000 > PEG-1000 = 2-methyl-2,4- pentanediol > sucrose > L-glutamate > glycerol = PEG-200 > betaine, with no apparent change in Km.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficient chromosomal-scale DNA looping in Escherichia coli using multiple DNA-looping elements

Publisher: Oxford University Press (OUP)

Date: 04-02-2017

DOI: 10.1093/NAR/GKX069

Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR

Publisher: Oxford University Press (OUP)

Date: 17-01-2011

DOI: 10.1093/NAR/GKQ1336

Instability of CII is needed for efficient switching between lytic and lysogenic development in bacteriophage 186

Publisher: Oxford University Press (OUP)

Date: 19-11-2021

DOI: 10.1093/NAR/GKAA1065

Abstract: The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.

RNA polymerase pausing at a protein roadblock can enhance transcriptional interference by promoter occlusion

Publisher: Wiley

Date: 29-03-2019

DOI: 10.1002/1873-3468.13365

Bacteriophage lambda repressor mediates the formation of a complex enhancer-like structure

Publisher: Informa UK Limited

Date: 09-2013

DOI: 10.4161/TRNS.26101

Purification of bacteriophage lambda repressor

Publisher: Elsevier BV

Date: 09-2013

DOI: 10.1016/J.PEP.2013.06.013

Effects of thermodynamic nonideality on the kinetics of ester hydrolysis by α-chymotrypsin: A model system with preexistence of the isomerization equilibrium

Publisher: Elsevier BV

Date: 10-1989

DOI: 10.1016/0003-9861(89)90414-1

Abstract: The effects of a small inert solute, sucrose, on the kinetics of hydrolysis of N-acetyl-tryptophan ethyl ester by bovine alpha-chymotrypsin have been investigated. In studies at pH 7 and 20 degrees C the presence of 0.5 M sucrose in assay mixtures caused no discernible change in kinetic parameters, a result consistent with existence of the enzyme in a single conformational state under those conditions. However, at pH 3.5 and 50 degrees C, conditions under which the enzyme comprises an equilibrium mixture of compact and expanded isomeric states, inclusion of the inert solute led to a considerable decrease in Michaelis constant (0.84 to 0.61 mM) but no significant change in maximal velocity. These results were shown to be amenable to quantitative interpretation in terms of thermodynamic nonideality effects on catalysis by an enzyme undergoing reversible isomerization in the absence of substrate. For that analysis, which required experimental estimates of the equilibrium constant for preexisting isomerization of enzyme and the activity coefficient of substrate, the magnitude of the former (0.3) was obtained by difference spectroscopy: liquid-liquid partition studies with bromobenzene as organic phase were used to determine the effect of sucrose on the activity coefficient of N-acetyltryptophan ethyl ester. Such agreement between experimental kinetic findings and theoretical predictions based on considerations of excluded volume points to the possible use of the space-filling effects of small solutes for delineating the gross extent of conformational changes associated with reversible isomerization of proteins, and hence to the potential of thermodynamic nonideality as a probe for studying protein denaturation mechanisms as well as substrate-mediated changes associated with enzyme reaction mechanisms.

Clonetegration Using OSIP Plasmids: One-Step DNA Assembly and Site-Specific Genomic Integration in Bacteria

Publisher: Springer New York

Date: 27-09-2016

DOI: 10.1007/978-1-4939-6343-0_11

Abstract: Clonetegration is a method for site-specific insertion of DNA into prokaryotic chromosomes, based on bacteriophage integrases. The method combines DNA cloning/assembly and chromosomal integration into a single step, providing a simple and rapid strategy for inserting DNA sequences into bacterial chromosomes.

Programmable DNA looping using engineered bivalent dCas9 complexes

Publisher: Springer Science and Business Media LLC

Date: 20-11-2017

DOI: 10.1038/S41467-017-01873-X

Abstract: DNA looping is a ubiquitous and critical feature of gene regulation. Although DNA looping can be efficiently detected, tools to readily manipulate DNA looping are limited. Here we develop CRISPR-based DNA looping reagents for creation of programmable DNA loops. Cleavage-defective Cas9 proteins of different specificity are linked by heterodimerization or translational fusion to create bivalent complexes able to link two separate DNA regions. After model-directed optimization, the reagents are validated using a quantitative DNA looping assay in E. coli . Looping efficiency is ~15% for a 4.7 kb loop, but is significantly improved by loop multiplexing with additional guides. Bivalent dCas9 complexes are also used to activate endogenous norVW genes by rewiring chromosomal DNA to bring distal enhancer elements to the gene promoters. Such reagents should allow manipulation of DNA looping in a variety of cell types, aiding understanding of endogenous loops and enabling creation of new regulatory connections.

Simplified heavy-atom derivatization of protein structures via co-crystallization with the MAD tetragon tetrabromoterephthalic acid

Publisher: International Union of Crystallography (IUCr)

Date: 28-04-2021

DOI: 10.1107/S2053230X21004052

Abstract: The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound. Incorporating B4C into the crystal lattice using co-crystallization, the crystal structure of hen egg-white lysozyme was solved using MAD phasing. The strong anomalous signal generated by its four Br atoms coupled with its compatibility with commonly used crystallization reagents render B4C an effective experimental phasing compound that can be used to overcome the phase problem.

The helix-turn-helix motif of the coliphage 186 immunity repressor binds to two distinct recognition sequences

Publisher: Elsevier BV

Date: 02-2002

DOI: 10.1074/JBC.M107740200

Road rules for traffic on DNA—systematic analysis of transcriptional roadblockingin vivo

Publisher: Oxford University Press (OUP)

Date: 17-07-2014

DOI: 10.1093/NAR/GKU627

The pIT5 Plasmid Series, an Improved Toolkit for Repeated Genome Integration in E. coli

Publisher: American Chemical Society (ACS)

Date: 30-06-2021

DOI: 10.1021/ACSSYNBIO.1C00215

Abstract: We describe a new set of tools for inserting DNA into the bacterial chromosome. The system uses site-specific recombination reactions carried out by bacteriophage integrases to integrate plasmids at up to eight phage attachment sites in

Prophages encoding human immune evasion cluster genes are enriched in Staphylococcus aureus isolated from chronic rhinosinusitis patients with nasal polyps

Publisher: Microbiology Society

Date: 15-12-2021

DOI: 10.1099/MGEN.0.000726

Abstract: Prophages affect bacterial fitness on multiple levels. These include bacterial infectivity, toxin secretion, virulence regulation, surface modification, immune stimulation and evasion and microbiome competition. Lysogenic conversion arms bacteria with novel accessory functions thereby increasing bacterial fitness, host adaptation and persistence, and antibiotic resistance. These properties allow the bacteria to occupy a niche long term and can contribute to chronic infections and inflammation such as chronic rhinosinusitis (CRS). In this study, we aimed to identify and characterize prophages present in Staphylococcus aureus from patients suffering from CRS in relation to CRS disease phenotype and severity. Prophage regions were identified using PHASTER. Various in silico tools like ResFinder and VF Analyzer were used to detect virulence genes and antibiotic resistance genes respectively. Progressive MAUVE and maximum likelihood were used for multiple sequence alignment and phylogenetics of prophages respectively. Disease severity of CRS patients was measured using computed tomography Lund–Mackay scores. Fifty-eight S. aureus clinical isolates (CIs) were obtained from 28 CRS patients without nasal polyp (CRSsNP) and 30 CRS patients with nasal polyp (CRSwNP). All CIs carried at least one prophage (average=3.6) and prophages contributed up to 7.7 % of the bacterial genome. Phage integrase genes were found in 55/58 (~95 %) S. aureus strains and 97/211 (~46 %) prophages. Prophages belonging to Sa3int integrase group (phiNM3, JS01, phiN315) (39/97, 40%) and Sa2int (phi2958PVL) (14/97, 14%) were the most prevalent prophages and harboured multiple virulence genes such as sak, scn, chp, luk E/D, sea . Intact prophages were more frequently identified in CRSwNP than in CRSsNP ( P =0.0021). Intact prophages belonging to the Sa3int group were more frequent in CRSwNP than in CRSsNP ( P =0.0008) and intact phiNM3 were exclusively found in CRSwNP patients ( P =0.007). Our results expand the knowledge of prophages in S. aureus isolated from CRS patients and their possible role in disease development. These findings provide a platform for future investigations into potential tripartite associations between bacteria-prophage-human immune system, S. aureus evolution and CRS disease pathophysiology.

Advanced resistance studies identify two discrete mechanisms in Staphylococcus aureus to overcome antibacterial compounds that target biotin protein ligase

Publisher: MDPI AG

Date: 06-04-2020

DOI: 10.3390/ANTIBIOTICS9040165

Abstract: Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound ( −9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

Characterizing Transcriptional Interference between Converging Genes in Bacteria

Publisher: American Chemical Society (ACS)

Date: 05-02-2019

DOI: 10.1021/ACSSYNBIO.8B00477

Abstract: Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two.

Efficient O-demethylation of lignin monoaromatics using the peroxygenase activity of cytochrome P450 enzymes

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D2CC04698A

Abstract: Selective O -demethylation of the lignin monoaromatics, syringol and guaiacol, using the peroxygenase activity of two distinct cytochrome P450 enzymes.

Functional Alignment of Regulatory Networks: A Study of Temperate Phages

Publisher: Public Library of Science (PLoS)

Date: 23-12-2005

DOI: 10.1371/JOURNAL.PCBI.0010074

When push comes to shove - RNA polymerase and DNA-bound protein roadblocks

Publisher: Springer Science and Business Media LLC

Date: 06-2023

DOI: 10.1007/S12551-023-01064-7

Abstract: In recent years, transcriptional roadblocking has emerged as a crucial regulatory mechanism in gene expression, whereby other DNA-bound obstacles can block the progression of transcribing RNA polymerase (RNAP), leading to RNAP pausing and ultimately dissociation from the DNA template. In this review, we discuss the mechanisms by which transcriptional roadblocks can impede RNAP progression, as well as how RNAP can overcome these obstacles to continue transcription. We examine different DNA-binding proteins involved in transcriptional roadblocking and their biophysical properties that determine their effectiveness in blocking RNAP progression. The catalytically dead CRISPR-Cas (dCas) protein is used as an ex le of an engineered programmable roadblock, and the current literature in understanding the polarity of dCas roadblocking is also discussed. Finally, we delve into a stochastic model of transcriptional roadblocking and highlight the importance of transcription factor binding kinetics and its resistance to dislodgement by an elongating RNAP in determining the strength of a roadblock.

Repurposing site-specific recombinases for synthetic biology

Publisher: Future Medicine Ltd

Date: 11-2013

DOI: 10.2217/FMB.13.119

Effect of calcium ion on the dimerization of α-chymotrypsin

Publisher: Elsevier BV

Date: 03-1990

DOI: 10.1016/0167-4838(90)90022-8

Abstract: Results of a sedimentation equilibrium study of the inhibitory effect of calcium ion on the dimerization of alpha-chymotrypsin (pH 3.9, I 0.2) are used to establish that the phenomenon does not reflect increased electrostatic repulsion between Ca2(+)-saturated enzyme molecules, but rather the displacement of the monomer-dimer equilibrium by the specific, weak interaction of metal ion with a single site on monomeric enzyme.

A Mathematical Model for Transcriptional Interference by RNA Polymerase Traffic in Escherichia coli

Publisher: Elsevier BV

Date: 03-2000

DOI: 10.1016/J.JMB.2004.11.075

Abstract: Interactions between RNA polymerases (RNAP) resulting from tandem or convergent arrangements of promoters can cause transcriptional interference, often with important consequences for gene expression. However, it is not known what factors determine the magnitude of interference and which mechanisms are likely to predominate in any situation. We therefore developed a mathematical model incorporating three mechanisms of transcriptional interference in bacteria: occlusion (in which passing RNAPs block access to the promoter), collisions between elongating RNAPs, and "sitting duck" interference (in which RNAP complexes waiting to fire at the promoter are removed by passing RNAP). The predictions of the model are in good agreement with a recent quantitative in vivo study of convergent promoters in E.coli. Our analysis predicts that strong occlusion requires the interfering promoter to be very strong. Collisions can also produce strong interference but only if the interfering promoter is very strong or if the convergent promoters are far apart (>200 bp). For moderate strength interfering promoters and short inter-promoter distances, strong interference is dependent on the sitting duck mechanism. Sitting duck interference is dependent on the relative strengths of the two promoters. However, it is also dependent on the "aspect ratio" (the relative rates of RNAP binding and firing) of the sensitive promoter, allowing promoters of equal strength to have very different sensitivities to transcriptional interference. The model provides a framework for using transcriptional interference to investigate various dynamic processes on DNA in vivo.

Positive and Negative Control of Enhancer-Promoter Interactions by Other DNA Loops Generates Specificity and Tunability

Publisher: Elsevier BV

Date: 02-2019

DOI: 10.1016/J.CELREP.2019.02.002

Abstract: Enhancers are ubiquitous and critical gene-regulatory elements. However, quantitative understanding of the role of DNA looping in the regulation of enhancer action and specificity is limited. We used the Escherichia coli NtrC enhancer-σ54 promoter system as an in vivo model, finding that NtrC activation is highly sensitive to the enhancer-promoter (E-P) distance in the 300-6,000 bp range. DNA loops formed by Lac repressor were able to strongly regulate enhancer action either positively or negatively, recapitulating promoter targeting and insulation. A single LacI loop combining targeting and insulation produced a strong shift in specificity for enhancer choice between two σ54 promoters. A combined kinetic-thermodynamic model was used to quantify the effect of DNA-looping interactions on promoter activity and revealed that sensitivity to E-P distance and to control by other loops is itself dependent on enhancer and promoter parameters that may be subject to regulation.

Combining random microseed matrix screening and the magic triangle for the efficient structure solution of a potential lysin from bacteriophage P68

Publisher: International Union of Crystallography (IUCr)

Date: 07-2019

DOI: 10.1107/S2059798319009008

Abstract: Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.

Transcriptional interference - A crash course

Publisher: Elsevier BV

Date: 06-2005

DOI: 10.1016/J.TIG.2005.04.009

The Structural Basis of Cooperative Regulation at an Alternate Genetic Switch

Publisher: Elsevier BV

Date: 03-2006

DOI: 10.1016/J.MOLCEL.2006.01.019

Abstract: Bacteriophage lambda is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of lambda and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 A resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 A resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and lambda repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.

Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor

Publisher: Oxford University Press (OUP)

Date: 24-04-2013

DOI: 10.1093/NAR/GKT298

Mechanisms of biotin-regulated gene expression in microbes

Publisher: Elsevier BV

Date: 03-2016

DOI: 10.1016/J.SYNBIO.2016.01.005

RNA polymerase efficiently transcribes through DNA‐scaffolded, cooperative bacteriophage repressor complexes

Publisher: Wiley

Date: 22-07-2022

DOI: 10.1002/1873-3468.14447

Abstract: DNA can act as a scaffold for the cooperative binding of protein oligomers. For ex le, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA with specific binding sites, while phage λ CI repressor dimers bind to two well‐separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by Escherichia coli RNA polymerase (RNAP) through these protein complexes. 186 CI, or λ CI, bound along unlooped DNA negligibly interfered with transcription by RNAP. Wrapped and looped topologies induced by these scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with high affinity, these repressors are not effective roadblocks to transcription.

Linkage between Ligand Binding and Control of Tubulin Conformation

Publisher: American Chemical Society (ACS)

Date: 09-1992

DOI: 10.1021/BI00149A044

Abstract: The effect of both antimitotic drugs and nucleotide analogues on the magnesium-induced self-association of purified tubulin into 42S double rings has been examined by sedimentation velocity. In the absence of magnesium, all complexes sedimented as the 5.8S species. The binding of colchicine to tubulin led to a small but consistent (-0.1 to -0.2 kcal/mol) enhancement in the self-association of tubulin alpha-beta dimers. In the absence of nucleotide at the exchangeable site, tubulin retained a weak ability (K2 = 7.5 x 10(3) M-1) to self-associate, which was unchanged by the addition of guanosine or GMP. Analogues with altered P-O-P bonds (GMPPCP, GMPPNP) did not support ring formation at the protein concentrations examined, although GMPPCP supported microtubule assembly. When the exchangeable site was occupied by nucleotides altered on the gamma-phosphate (GTP gamma S, GTP gamma F), rings were formed tubulin-GTP gamma F formed rings to an extent slightly greater than did tubulin-GTP, and tubulin-GTP gamma S to about the same extent as tubulin-GDP. Both of these analogues are inhibitors of microtubule assembly. These results are consistent with a model [Melki, R., Carlier, M.-F., Pantaloni, D., & Timasheff, S. N. (1989) Biochemistry 28, 9143-9152] in which an equilibrium exists between straight (microtubule-forming) and curved (ring-forming) conformations of tubulin. Furthermore, the present results indicate that the "switch" which controls the nature of the final polymeric product via free energy linkages is the occupancy of the gamma-phosphate binding locus of the exchangeable site by a properly coordinated metal-nucleotide complex.

The role of repressor kinetics in relief of transcriptional interference between convergent promoters

Publisher: Oxford University Press (OUP)

Date: 04-07-2016

DOI: 10.1093/NAR/GKW600

Modelling Transcriptional Interference and DNA Looping in Gene Regulation

Publisher: Elsevier BV

Date: 06-2007

DOI: 10.1016/J.JMB.2007.04.041

Abstract: We describe a hybrid statistical mechanical and dynamical approach for modelling the formation of closed, open and elongating complexes of RNA polymerase, the interactions of these polymerases to produce transcriptional interference, and the regulation of these processes by a DNA-binding and DNA-looping regulatory protein. As a model system, we have used bacteriophage 186, for which genetic, biochemical and structural studies have suggested that the CI repressor binds as a 14-mer to form alternative DNA-looped complexes, and activates lysogenic transcription indirectly by relieving transcriptional interference caused by the convergent lytic promoter. The modelling showed that the original mechanisms proposed to explain this relief of transcriptional interference are not consistent with the available in vivo reporter data. However, a good fit to the reporter data was given by a revised model that incorporates a novel predicted regulatory mechanism: that RNA polymerase bound at the lysogenic promoter protects itself from transcriptional interference by recruiting CI to the lytic promoter. This mechanism and various estimates of in vivo biochemical parameters for the 186 CI system should be testable. Our results demonstrate the power of mathematical modelling for the extraction of detailed biochemical information from in vivo data.

Thermodynamic nonideality as a probe of reversible protein unfolding effected by variations in pH and temperature: Studies of ribonuclease

Publisher: Elsevier BV

Date: 11-1990

DOI: 10.1016/0003-9861(90)90120-N

Abstract: Thermodynamic nonideality arising from the space-filling effect of added sucrose is employed to confirm that the reversible unfolding of ribonuclease A effected by acid may be described as an equilibrium between native and unfolded states of the enzyme. However, the extent of the volume change is far too small for the larger isomer to be the fully expanded state, a result signifying that the acid-mediated unfolding of ribonuclease does not conform with the two-state equilibrium model of protein denaturation. Although the thermal denaturation of ribonuclease A is characterized by a larger increase in volume, quantitative reappraisal of published results on the effects of glycerol on this transition at pH 2.8 (Gekko, K., and Timasheff, S. N., 1981 Biochemistry 20, 4677-4686) leads to an estimated volume increase that is much smaller than that inferred from hydrodynamic studies--a disparity attributed to the dual actions of glycerol as a space-filling solute and as a ligand that binds preferentially to the thermally unfolded form of the enzyme. Even in this unfavorable circumstance the fact that glycerol exerts a net excluded volume effect at least confirms that the thermal unfolding of ribonuclease A is an equilibrium transition between two discrete states. The strengths and limitations of using thermodynamic nonideality as a probe of the two-state equilibrium model of protein denaturation are discussed in the light of these findings.

Overcoming bacteriophage insensitivity in Staphylococcus aureus using clindamycin and azithromycinat subinhibitory concentrations

Publisher: Wiley

Date: 16-05-2021

DOI: 10.1111/ALL.14883

Abstract: Staphylococcus aureus is a pathogen of major concern in both acute infections and chronic conditions such as chronic rhinosinusitis (CRS). Bacteriophage (phage) therapy has recently regained interest for its potential to treat infections caused by antibiotic resistant strains including Methicillin Resistant Staphylococcus aureus (MRSA). However, bacteria can adapt and become resistant to phages. The aim of this study is to determine the potential for antibiotics to overcome phage resistance. The susceptibility of S. aureus clinical isolates (CIs) to phages J‐Sa36, Sa83 and Sa87 alone or in combination with protein synthesis inhibitor (PSI) antibiotics clindamycin, azithromycin and erythromycin was assessed using plaque spot assays, minimum inhibitory concentration (MIC) assays, double layer spot assays and resazurin assays. The safety and efficacy of subinhibitory PSI antibiotics in combination with phage was tested in a Sprague Dawley rat model of sinusitis infected with a phage resistant S. aureus CI. All three antibiotics at subinhibitory concentrations showed synergy when combined with all 3 phages against S. aureus CIs in planktonic and biofilm form and could sensitize phage‐resistant S. aureus to promote phage infection. The combination of topical subinhibitory clindamycin or azithromycin and phage was safe and could eradicate S . aureus sinonasal biofilms in vivo . Subinhibitory concentrations of PSI antibiotics could sensitize phage‐resistant S . aureus and MRSA strains to phages in vitro and in vivo . This data supports the potential use of phage‐PSI antibiotic combination therapies, in particular for difficult‐to‐treat infections with phage‐resistant S . aureus and MRSA strains.

Native mass spectrometry identifies an alternative DNA-binding pathway for BirA from Staphylococcus aureus

Publisher: Springer Science and Business Media LLC

Date: 26-02-2019

DOI: 10.1038/S41598-019-39398-6

Abstract: An adequate supply of biotin is vital for the survival and pathogenesis of Staphylococcus aureus . The key protein responsible for maintaining biotin homeostasis in bacteria is the biotin retention protein A (BirA, also known as biotin protein ligase). BirA is a bi-functional protein that serves both as a ligase to catalyse the biotinylation of important metabolic enzymes, as well as a transcriptional repressor that regulates biotin biosynthesis, biotin transport and fatty acid elongation. The mechanism of BirA regulated transcription has been extensively characterized in Escherichia coli , but less so in other bacteria. Biotin-induced homodimerization of E. coli BirA ( Ec BirA) is a necessary prerequisite for stable DNA binding and transcriptional repression. Here, we employ a combination of native mass spectrometry, in vivo gene expression assays, site-directed mutagenesis and electrophoretic mobility shift assays to elucidate the DNA binding pathway for S. aureus BirA ( Sa BirA). We identify a mechanism that differs from that of Ec BirA, wherein Sa BirA is competent to bind DNA as a monomer both in the presence and absence of biotin and/or MgATP, allowing homodimerization on the DNA. Bioinformatic analysis demonstrated the Sa BirA sequence used here is highly conserved amongst other S. aureus strains, implying this DNA-binding mechanism is widely employed.

Directing traffic on DNA—How transcription factors relieve or induce transcriptional interference

Publisher: Informa UK Limited

Date: 03-2017

DOI: 10.1080/21541264.2017.1285851

Enhancer-like long-range transcriptional activation by λ CI-mediated DNA looping

Publisher: Proceedings of the National Academy of Sciences

Date: 04-02-2013

DOI: 10.1073/PNAS.1221322110

Abstract: How distant enhancer elements regulate the assembly of a transcription complex at a promoter remains poorly understood. Here, we use long-range gene regulation by the bacteriophage λ CI protein as a powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed by CI bridging its binding sites at OR and OL , is known already to enhance repression at the lysogenic promoter PRM , located at OR . Here, we show that CI looping also activates PRM by allowing the C-terminal domain of the α subunit of the RNA polymerase bound at PRM to contact a DNA site adjacent to the distal CI sites at OL . Our results establish OL as a multifaceted enhancer element, able to activate transcription from long distances independently of orientation and position. We develop a physicochemical model of our in vivo data and use it to show that the observed activation is consistent with a simple recruitment mechanism, where the α–C-terminal domain to DNA contact need only provide ∼2.7 kcal/mol of additional binding energy for RNA polymerase. Structural modeling of this complete enhancer–promoter complex reveals how the contact is achieved and regulated, and suggests that distal enhancer elements, once appropriately positioned at the promoter, can function in essentially the same way as proximal promoter elements.

One-Step Cloning and Chromosomal Integration of DNA

Publisher: American Chemical Society (ACS)

Date: 20-05-2013

DOI: 10.1021/SB400021J

Abstract: We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids.

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Publisher: MyJove Corporation

Date: 16-01-2021

DOI: 10.3791/61894

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

Publisher: Oxford University Press (OUP)

Date: 13-08-2020

DOI: 10.1093/NAR/GKAA655

Abstract: The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

Thermodynamic nonideality in macromolecular solutions: Evaluation of parameters for the prediction of covolume effects

Publisher: Wiley

Date: 06-1990

DOI: 10.1111/J.1432-1033.1990.TB15605.X

Abstract: Second virial coefficients and hence covolumes for self-interaction of five proteins, viz. ribonuclease, ovalbumin, bovine serum albumin, catalase and alpha-crystallin, have been determined by analyzing the concentration dependence of the partition coefficient obtained from frontal chromatographic studies on either Fractogel TSK HW55 or porous glass beads. The resulting estimates of the effective radii essentially duplicate their Stokes counterparts and thereby provide further justification for assuming the approximate identity of the thermodynamic and hydrodynamic radii of hydrated globular proteins. Gel chromatographic evaluation of second virial coefficients for protein/dextran systems has led to elimination of the sphere/sphere model as a valid thermodynamic description of the space-filling effects in protein olymer mixtures, since it does not predict the observed independence of covolume, expressed per unit mass of polymer, upon size of the polymer. This requirement is met by the sphere/rod model [Edmond, E. & Ogston, A. G. (1968) Biochem. J. 109, 569-576] and also by the sphere/flexible-segment model [Hermans, J. (1982) J. Chem. Phys. 77, 2193-2203]. Furthermore, similar studies of the effect of solute radius on covolume for interaction with dextran T70 attest to the adequacy of either model for predicting the thermodynamic nonideality arising from the inclusion of dextrans in protein solutions, and also provide the relevant calibration of the model.

Slow growth leads to a switch

Publisher: Springer Science and Business Media LLC

Date: 11-2009

DOI: 10.1038/NCHEMBIO.248

The tum protein of coliphage 186 is an antirepressor

Publisher: Elsevier BV

Date: 03-1998

DOI: 10.1074/JBC.273.10.5708

The loopometer: a quantitative in vivo assay for DNA-looping proteins

Publisher: Oxford University Press (OUP)

Date: 28-01-2021

DOI: 10.1093/NAR/GKAA1284

Abstract: Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this ‘loopometer’ assay to measure DNA looping for a variety of bacterial and phage proteins.

Transcriptional interference by RNA polymerase pausing and dislodgement of transcription factors

Publisher: Informa UK Limited

Date: 2011

DOI: 10.4161/TRNS.2.1.13511

Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells

Publisher: Proceedings of the National Academy of Sciences

Date: 06-10-2014

DOI: 10.1073/PNAS.1410764111

Abstract: Genes are frequently regulated by interactions between proteins that bind to the DNA near the gene and proteins that bind to DNA sites located far away, with the intervening DNA looped out. In eukaryotic genomes, genes and their distant sites are intermingled in complex ways and it is not understood how the correct connections are formed. Using two pairs of DNA-looping sites in bacterial cells, we tested the idea that one DNA loop can either assist or interfere with the formation of another DNA loop. By measuring the strength of these interactions between loops, we showed that this mechanism is capable of directing a distant site to the correct gene and preventing it contacting the wrong gene.

Analysis of Infection Time Courses Shows CII Levels Determine the Frequency of Lysogeny in Phage 186

Publisher: MDPI AG

Date: 29-09-2021

DOI: 10.3390/PH14100998

Abstract: Engineered phage with properties optimised for the treatment of bacterial infections hold great promise, but require careful characterisation by a number of approaches. Phage–bacteria infection time courses, where populations of bacteriophage and bacteria are mixed and followed over many infection cycles, can be used to deduce properties of phage infection at the in idual cell level. Here, we apply this approach to analysis of infection of Escherichia coli by the temperate bacteriophage 186 and explore which properties of the infection process can be reliably inferred. By applying established modelling methods to such data, we extract the frequency at which phage 186 chooses the lysogenic pathway after infection, and show that lysogenisation increases in a graded manner with increased expression of the lysogenic establishment factor CII. The data also suggest that, like phage λ, the rate of lysogeny of phage 186 increases with multiple infections.

Substrate as a source of thermodynamic nonideality in enzyme kinetic studies: Invertase-catalyzed hydrolysis of sucrose

Publisher: Elsevier BV

Date: 02-1988

DOI: 10.1016/0003-9861(88)90478-X

Abstract: Expressions for the effects of thermodynamic nonideality arising from the use of high concentrations of small substrate in enzyme kinetic studies are derived. Their application to experimental results for the hydrolysis of sucrose by yeast invertase (pH 4.9, 37 degrees C) signifies that the progressive decrease in initial velocity at high sucrose concentration is consistent with the occurrence of isomeric expansion during the transition of an enzyme-substrate complex to its activated state. Ultracentrifuge studies on the yeast enzyme preparation are then used to establish the physical acceptability of the volume change required to account for the kinetic effects in these terms: the postulated expansion of 1.3 liter/mol would represent a mere 0.16% increase in hydrated volume (or a corresponding increase in extent of asymmetry). Finally, although originally interpreted to signify an effect of sucrose on water concentration, published results for the invertase-sucrose system [J. M. Nelson and M. P. Schubert (1928) J. Amer. Chem. Soc. 50, 2188-2193] also find a rational explanation in terms of the present analysis based on effects of thermodynamic nonideality in enzyme kinetic studies.

Allowance for thermodynamic nonideality and Donnan effects in binding studies. Activity coefficients of charged ligands in the presence of albumin

Publisher: Elsevier BV

Date: 08-1990

DOI: 10.1016/0301-4622(90)80029-7

Abstract: A combination of ultrafiltration with either equilibrium dialysis or frontal gel chromatography has been used to evaluate the effects of thermodynamic nonideality in mixtures of bovine serum albumin and charged ligands. Studies with methyl orange, chlorpromazine and chromate as ligand all demonstrated inadequacy of the Donnan effect for description of the difference between the concentrations of free ligand in a mixture and the protein-free phase with which it is in dialysis equilibrium. On the basis of a quantitative relationship derived for the situation in which Donnan and thermodynamic nonideality effects both operate, values of the second virial coefficient for albumin and ligand have been determined. For albumin and either methyl orange or chlorpromazine the magnitude of this second virial coefficient has been rationalized on the statistical-mechanical basis of excluded volume. For the albumin-chromate system, however, the thermodynamic nonideality was manifested as a negative deviation from Raoult's Law, in keeping with the classical behaviour of electrolyte ions. From the viewpoint of the characterization of ligand binding a unique feature of the ultrafiltration/gel chromatography and ultrafiltration/equilibrium dialysis methods is their ability to define not only the binding function but also the activity coefficient of ligand for a given acceptor-ligand mixture. Consequently, irrespective of whether the ligand is charged or uncharged, the intrinsic binding constant that is determined is the thermodynamic parameter instead of the apparent value that is obtained from methods based on assumed thermodynamic ideality.

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors

Publisher: Proceedings of the National Academy of Sciences

Date: 16-12-2013

DOI: 10.1073/PNAS.1317817111

Abstract: Proteins bound to DNA often interact with proteins bound elsewhere on the same DNA to regulate gene expression. The intervening DNA tethers the proteins near each other, making their interaction efficient and specific, but the importance of this tethering effect is poorly understood at large DNA separations. We quantitated tethering inside bacterial cells, using two different proteins at separations up to 10,000 bp, to show that tethering is strong enough to drive efficient interactions over these distances. The same interactions were ∼10-fold weaker outside cells, implying that cellular factors enhance tethering. However, tethering was lost at a DNA separation of 500,000 bp inside bacteria, indicating special mechanisms inside eukaryotic cells to provide efficient and specific interactions over such distances.

Potent Transcriptional Interference by Pausing of RNA Polymerases over a Downstream Promoter

Publisher: Elsevier BV

Date: 06-2009

DOI: 10.1016/J.MOLCEL.2009.04.018

Transcription disrupts DNA-scaffolded bacteriophage repressor complexes

Publisher: Cold Spring Harbor Laboratory

Date: 30-10-2021

DOI: 10.1101/2021.10.29.466366

Abstract: DNA can act as a scaffold for the cooperative binding of protein oligomers. For ex le, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA carrying specific binding sites, while the phage λ CI repressor binds in units of dimers to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by E. coli RNA polymerase through these protein complexes. 186 CI or λ CI bound along unlooped DNA negligibly interfered with transcription by RNAP. More complex topologies induced by scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with rather high affinity, these repressors are not effective roadblocks to transcription.

University Of Adelaide

Location: Australia

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Discovery Projects - Grant ID: DP150103009

Start Date: 2015

End Date: 12-2018

Amount: $241,600.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110101470

Start Date: 2011

End Date: 12-2014

Amount: $300,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0665185

Start Date: 2006

End Date: 12-2009

Amount: $376,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP230103062

Start Date: 2023

End Date: 12-2025

Amount: $522,724.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP160101450

Start Date: 2016

End Date: 02-2020

Amount: $410,100.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110100824

Start Date: 2011

End Date: 12-2014

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989336

Start Date: 08-2009

End Date: 12-2010

Amount: $560,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882382

Start Date: 08-2008

End Date: 08-2008

Amount: $245,000.00

Funder: Australian Research Council