Insights into the Antibacterial Mechanism of Action of Chelating Agents by Selective Deprivation of Iron, Manganese, and Zinc.

Bacterial growth and proliferation can be restricted by limiting the availability of metal ions in their environment. Humans sequester iron, manganese, and zinc to help prevent infection by pathogens, a system termed nutritional immunity. Commercially used chelants have high binding affinities with a variety of metal ions, which may lead to antibacterial properties that mimic these innate immune processes. However, the modes of action of many of these chelating agents in bacterial growth inhibition and their selectivity in metal deprivation in cellulo remain ill-defined. We address this shortcoming by examining the effect of 11 chelators on Escherichia coli growth and their impact on the cellular concentration of five metals. The following four distinct effects were uncovered: (i) no apparent alteration in metal composition, (ii) depletion of manganese alongside reductions in iron and zinc levels, (iii) reduced zinc levels with a modest reduction in manganese, and (iv) reduced iron levels coupled with elevated manganese. These effects do not correlate with the absolute known chelant metal ion affinities in solution; however, for at least five chelators for which key data are available, they can be explained by differences in the relative affinity of chelants for each metal ion. The results reveal significant insights into the mechanism of growth inhibition by chelants, highlighting their potential as antibacterials and as tools to probe how bacteria tolerate selective metal deprivation. IMPORTANCE Chelating agents are widely used in industry and consumer goods to control metal availability, with bacterial growth restriction as a secondary benefit for preservation. However, the antibacterial mechanism of action of chelants is largely unknown, particularly with respect to the impact on cellular metal concentrations. The work presented here uncovers distinct metal starvation effects imposed by different chelants on the model Gram-negative bacterium Escherichia coli. The chelators were studied both individually and in pairs, with the majority producing synergistic effects in combinations that maximize antibacterial hostility. The judicious selection of chelants based on contrasting cellular effects should enable reductions in the quantities of chelant required in numerous commercial products and presents opportunities to replace problematic chemistries with biodegradable alternatives.

A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.

Antibacterial Activity of Clay Soils against Food-Borne Salmonella typhimurium and Staphylococcus aureus.

Natural clays have recently been proven to possess antibacterial properties. Effective natural antimicrobial agents are needed to combat bacterial contamination on food contact surfaces, which are increasingly more prevalent in the food chain. This study sought to determine the antibacterial activity of clays against the food-borne pathogens Salmonella typhimurium ATCC 14028 and Staphylococcus aureus ATCC 13565. Soils were processed to yield leachates and suspensions from untreated and treated clays. Soil particle size, pH, cation-exchange capacity, metal composition and mineralogy were characterized. Antibacterial screening was performed on six Malaysian soils via the disc diffusion method. In addition, a time-kill assay was conducted on selected antibacterial clays after 6 h of exposure. The screening revealed that Munchong and Carey clays significantly inhibit Salmonella typhimurium (11.00 ± 0.71 mm) and S. aureus (7.63 ± 0.48 mm), respectively. Treated Carey clay leachate and suspension completely kill Salmonella typhimurium, while S. aureus viability is reduced (2 to 3 log10). The untreated Carey and all Munchong clays proved ineffective as antibacterials. XRD analysis confirmed the presence of pyrite and magnetite. Treated Carey clays had a higher soluble metal content compared to Munchong; namely Al (92.63 ± 2.18 mg/L), Fe (65.69 ± 3.09 mg/L) and Mg (88.48 ± 2.29 mg/L). Our results suggest that metal ion toxicity is responsible for the antibacterial activity of these clays.

The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming, and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further able to bind and distort double-stranded DNA. However, Rx1 host targets that support a role for Rx1 in transcriptional reprogramming at DNA are unknown. Here, we report a functional interaction between Rx1 and NbGlk1, a Golden2-like transcription factor. Rx1 binds to NbGlk1 in vitro and in planta. NbGlk1 binds to known Golden2-like consensus DNA sequences. Rx1 reduces the binding affinity of NbGlk1 for DNA in vitro. NbGlk1 activates cellular responses to potato virus X, whereas Rx1 associates with NbGlk1 and prevents its assembly on DNA in planta unless activated by PVX. This study provides new mechanistic insight into how an NLR can coordinate an immune signaling response at DNA following pathogen perceptions.

On the Antibacterial Activity of Azacarboxylate Ligands: Lowered Metal Ion Affinities for Bis-amide Derivatives of EDTA do not mean Reduced Activity.

EDTA is widely used as an inhibitor of bacterial growth, affecting the uptake and control of metal ions by microorganisms. We describe the synthesis and characterisation of two symmetrical bis-amide derivatives of EDTA, featuring glycyl or pyridyl substituents: AmGly2 and AmPy2 . Metal ion affinities (logK) have been evaluated for a range of metals (Mg2+ , Ca2+ , Fe3+ , Mn2+ , Zn2+ ), revealing less avid binding compared to EDTA. The solid-state structures of AmGly2 and of its Mg2+ complex have been determined crystallographically. The latter shows an unusual 7-coordinate, capped octahedral Mg2+ centre. The antibacterial activities of the two ligands and of EDTA have been evaluated against a range of health-relevant bacterial species, three Gram negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and a Gram positive (Staphylococcus aureus). The AmPy2 ligand is the only one that displays a significant inhibitory effect against K. pneumoniae, but is less effective against the other organisms. AmGly2 exhibits a more powerful inhibitory effect against E. coli at lower concentrations than EDTA (<3 mm) or AmPy2 , but loses its efficacy at higher concentrations. The growth inhibition of EDTA and AmGly2 on mutant E. coli strains with defects in outer-membrane lipopolysaccharide (LPS) structures has been assessed to provide insight into the unexpected behaviour. Taken together, the results contradict the assumption of a simple link between metal ion affinity and antimicrobial efficacy.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.

Mycobacterium tuberculosis RuvX is a Holliday junction resolvase formed by dimerisation of the monomeric YqgF nuclease domain.

The Mycobacterium tuberculosis genome possesses homologues of the ruvC and yqgF genes that encode putative Holliday junction (HJ) resolvases. However, their gene expression profiles and enzymatic properties have not been experimentally defined. Here we report that expression of ruvC and yqgF is induced in response to DNA damage. Protein-DNA interaction assays with purified M. tuberculosis RuvC (MtRuvC) and YqgF (MtRuvX) revealed that both associate preferentially with HJ DNA, albeit with differing affinities. Although both MtRuvC and MtRuvX cleaved HJ DNA in vitro, the latter displayed robust HJ resolution activity by symmetrically related, paired incisions. MtRuvX showed a higher binding affinity for the HJ structure over other branched recombination and replication intermediates. An MtRuvX(D28N) mutation, eliminating one of the highly conserved catalytic residues in this class of endonucleases, dramatically reduced its ability to cleave HJ DNA. Furthermore, a unique cysteine (C38) fulfils a crucial role in HJ cleavage, consistent with disulfide-bond mediated dimerization being essential for MtRuvX activity. In contrast, E. coli YqgF is monomeric and exhibits no branched DNA binding or cleavage activity. These results fit with a functional modification of YqgF in M. tuberculosis so that it can act as a dimeric HJ resolvase analogous to that of RuvC.

The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR.

Glycosylated Nanoparticles as Efficient Antimicrobial Delivery Agents.

Synthetic polymer nanoparticles that can be tailored through multivalent ligand display on the surface, while at the same time allowing encapsulation of desired bioactive molecules, are especially useful in providing a versatile and robust platform in the design of specific delivery vehicles for various purposes. Glycosylated nanoparticles (glyco-NPs) of a poly(n-butyl acrylate) (pBA) core and poly(N-2-(ß-d-glucosyloxy)ethyl acrylamide) (p(NßGlcEAM)) or poly(N-2-(ß-D-galactosyloxy)ethyl acrylamide) (p(NßGalEAM)) corona were prepared via nanoprecipitation in aqueous solutions of preformed amphiphilic glycopolymers. Well-defined block copolymers of (poly(pentafluorophenyl acrylate) (pPFPA) and pBA were first prepared by RAFT polymerization followed by postpolymerization functionalization with aminoethyl glycosides to yield p(NßGlcEAM-b-BA) and p(NßGalEAM-b-BA), which were then used to form glyco-NPs (glucosylated and galactosylated NPs, Glc-NPs and Gal-NPs, respectively). The glyco-NPs were characterized by dynamic light scattering (DLS) and TEM. Encapsulation and release of ampicillin, leading to nanoparticles that we have termed "glyconanobiotics", were studied. The ampicillin-loaded glyco-NPs were found to induce aggregation of Staphylococcus aureus and Escherichia coli and resulted in antibacterial activity approaching that of ampicillin itself. This glyconanobiotics strategy represents a potential new approach for the delivery of antibiotics close to the surface of bacteria by promoting bacterial aggregation. Defined release in the proximity of the bacterial envelope may thus enhance antibacterial efficiency and potentially reduce the quantities of agent required for potency.

Preparation of an Antibacterial Poly(ionic liquid) Graft Copolymer of Hydroxyethyl Cellulose.

Poly(ionic liquid)s (P(IL)s) of different degrees of polymerization (10, 50, and 100) were prepared via RAFT polymerization using an alkyne-terminated xanthate as transfer agent, with a monomer conversion of up to ∼80% and a DM of 1.5 for P(IL)100. Subsequently, P(IL) chains were coupled to (15)N-labeled azido-functionalized hydroxyethyl cellulose (HEC), forming graft copolymers of HEC with different chain length and graft densities, which were characterized using ((13)C and (15)N) CP-MAS NMR and FT-IR spectroscopies. The antibacterial activities of HEC-g-P(IL)s were tested against Escherichia coli and Staphylococcus aureus and were comparable to ampicillin, a well-known antibiotic, demonstrating efficient activity of the graft copolymers against bacteria. Moreover, HEC-g-P(IL)s were slightly more effective against E. coli than S. aureus. A decrease in graft density of P(IL)10 on the HEC backbone decreased the activity of the graft copolymers against both bacteria. These findings suggest that HEC-g-P(IL) could find applications as an antiseptic compound, for example, in paint formulation.

Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry.

Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively-charged residues in the outer sides of the junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild-type, while retaining the ability to bind X junctions. Crucially, these two variants cleaved Holliday junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts.

Antibacterial mechanism of Malaysian Carey clay against food-borne Staphylococcus aureus.

Depending on their chemical structure and geochemistry, clay minerals can display potent antibacterial properties against a range of bacterial pathogens. Malaysian Carey clay was evaluated for its antibacterial activity against food-borne Staphylococcus aureus ATCC 13565 strains. The minimum inhibitory concentration (MIC) and minimum bactericidal activity (MBC) of both Carey clay leachates and suspension were 125 mg/mL and 250 mg/mL, respectively. Time-kill assay revealed that 2x MIC and 4x MIC Carey clay in both leachate and suspension forms resulted in complete killing of S. aureus. Antibacterial mechanism was investigated through imaging of bacterial morphology using TEM and determination of reactive oxygen species (ROS) using NBT assay. Imaging of bacterial morphology using TEM showed abnormalities, including disrupted cell walls following exposure to Carey clay, and the antibacterial activity was associated with generation of ROS. Our study suggests that Carey clay displays promising functionality as a natural antibacterial agent in the food industry.

A critical role for iron and zinc homeostatic systems in the evolutionary adaptation of Escherichia coli to metal restriction.

Host nutritional immunity utilizes metal deprivation to help prevent microbial infection. To investigate bacterial adaptation to such restrictive conditions, we conducted experimental evolution with two metal sequestering agents. Ethylenediaminetetraacetic acid (EDTA) and diethylenetriamine pentamethylene phosphonic acid (DTPMP) were selected as ligands because they differentially affect cellular levels of iron, manganese and zinc in Escherichia coli. Mutants of E. coli strain BW25113 were isolated after cultivation at sub-minimum inhibitory concentration (MIC) chelant levels and genetic changes potentially responsible for tolerance were identified by whole-genome sequencing. In EDTA-selected strains, mutations in the promoter region of yeiR resulted in elevated gene expression. The yeiR product, a zinc-specific metallochaperone, was confirmed to be primarily responsible for EDTA resistance. Similarly, in two of the DTPMP-selected strains, a promoter mutation increased expression of the fepA-entD operon, which encodes components of the ferric-enterobactin uptake pathway. However, in this case improved DTPMP tolerance was only detectable following overexpression of FepA or EntD in trans. Additional mutations in the cadC gene product, an acid-response regulator, preserved the neutrality of the growth medium by constitutively activating expression of the cadAB regulon. This study uncovers specific resistance mechanisms for zinc and iron starvation that could emerge by selection against host nutritional immunity or competition with heterologous metallophores. It also provides insight into the specific metals affected by these two widely used chelators critical for their antibacterial mode of action.

Effect of natural antibacterial clays against single biofilm formation by Staphylococcus aureus and Salmonella Typhimurium bacteria on a stainless-steel surface.

Staphylococcus aureus and Salmonella Typhimurium have a propensity to develop biofilms on food contact surfaces, such as stainless-steel, that persist despite rigorous cleaning and sanitizing procedures. Since both bacterial species pose a significant public health risk within the food chain, improved anti-biofilm measures are needed. This study examined the potential of clays as antibacterial and anti-biofilm agents against these two pathogens on appropriate contact surfaces. Natural soil was processed to yield leachates and suspensions of both untreated and treated clays. Soil particle size, pH, cation-exchange capacity, and metal ions were characterized to assess their importance in bacterial killing. Initial antibacterial screening was performed on nine distinct types of natural Malaysian soil using a disk diffusion assay. Untreated leachate from Kuala Gula and Kuala Kangsar clays were found to inhibit S. aureus (7.75 ± 0.25 mm) and Salmonella Typhimurium (11.85 ± 1.63 mm), respectively. The treated Kuala Gula suspension (50.0 and 25.0 %) reduced S. aureus biofilms by 4.4 and 4.2 log at 24 and 6 h, respectively, while treated Kuala Kangsar suspension (12.5 %) by a 4.16 log reduction at 6 h. Although less effective, the treated Kuala Gula leachate (50.0 %) was effective in removing Salmonella Typhimurium biofilm with a decrease of >3 log in 24 h. In contrast to Kuala Kangsar clays, the treated Kuala Gula clays contained a much higher soluble metal content, especially Al (301.05 ± 0.45 ppm), Fe (691.83 ± 4.80 ppm) and Mg (88.44 ± 0.47 ppm). Elimination of S. aureus biofilms correlated with the presence of Fe, Cu, Pb, Ni, Mn and Zn irrespective of the pH of the leachate. Our findings demonstrate that a treated suspension is the most effective for eradication of S. aureus biofilms with a potential as a sanitizer-tolerant, natural antibacterial against biofilms for applications in the food industry.

The antibacterial activity of a photoactivatable diarylacetylene against Gram-positive bacteria.

The emergence of antibiotic resistance is a growing threat to human health, and therefore, alternatives to existing compounds are urgently needed. In this context, a novel fluorescent photoactivatable diarylacetylene has been identified and characterised for its antibacterial activity, which preferentially eliminates Gram-positive over Gram-negative bacteria. Experiments confirmed that the Gram-negative lipopolysaccharide-rich outer surface is responsible for tolerance, as strains with reduced outer membrane integrity showed increased susceptibility. Additionally, bacteria deficient in oxidative damage repair pathways also displayed enhanced sensitivity, confirming that reactive oxygen species production is the mechanism of antibacterial activity. This new diarylacetylene shows promise as an antibacterial agent against Gram-positive bacteria that can be activated in situ, potentially for the treatment of skin infections.

The C-terminus of the phage λ Orf recombinase is involved in DNA binding.

Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA. To help identify interacting domains we analysed Orf and SSB proteins carrying mutations or truncations in the C-terminal region. A cluster of acidic residues at the carboxy-terminus of SSB is known to attract multiple protein partners to assist in DNA replication and repair. In this case an alternative domain must be utilized since Orf association with SSB was unaffected by an SSB113 point mutant (P176S) or removal of the last ten residues (ΔC10). Structurally the Orf C-terminus consists of a helix with a flexible tail that protrudes from each side of the dimer and could serve as a binding site for either SSB or DNA. Eliminating the six residue flexible tail (ΔC6) or the entire helix (ΔC19) had no significant impact on the Orf-SSB interaction. However, the OrfΔC6 protein exhibited reduced DNA binding, a feature shared by single amino acid substitutions within (W141F) or adjacent (R140A) to this region. The OrfΔC19 mutant bound poorly to DNA and secondary structure analysis in solution revealed that this truncation induces protein misfolding and aggregation. The results show that the carboxy-terminus of Orf is involved in nucleic acid recognition and also plays an unexpected role in maintaining structural integrity.

Bioinspired and eco-friendly high efficacy cinnamaldehyde antibacterial surfaces.

Antimicrobial essential oils are incorporated into mussel-inspired and natural plant polyphenol coatings as part of a single-step fabrication process. Polydopamine-cinnamaldehyde, polyethyleneimine-cinnamaldehyde, and tannic acid-cinnamaldehyde coatings exhibit strong antibacterial activities against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus (with the polydopamine- and tannic acid-based systems displaying log10 Reduction = 8). Cinnamaldehyde impregnation into porous non-woven polypropylene cloth, polytetrafluoroethylene membrane, and knitted cotton cloth also gives rise to high levels of antibacterial activity (log10 Reduction = 8). No loss in antibacterial efficacy is observed for non-woven polypropylene cloth impregnated with cinnamaldehyde over 17 recycle tests.

Conservation of RecG activity from pathogens to hyperthermophiles.

Maintaining the integrity of the genome is essential for the survival of all organisms. RecG helicase plays an important part in this process in Escherichia coli, promoting recombination and DNA repair, and providing ways to rescue stalled replication forks by way of a Holliday junction intermediate. We purified RecG proteins from three other species: two Gram-positive mesophiles, Bacillus subtilis and Streptococcus pneumoniae, and one extreme thermophile, Aquifex aeolicus. All three proteins bind and unwind replication fork and Holliday junction DNA molecules with efficiencies similar to the E. coli protein. Proteins from the Gram-positive species promote DNA repair in E. coli, indicating either that RecG acts alone or that any necessary protein-protein interactions are conserved. The S. pneumoniae RecG reduces plasmid copy number when expressed in E. coli, indicating that like the E. coli protein it unwinds plasmid R loop structures used to prime replication. This effect is not seen with B. subtilis RecG; the protein either lacks R loop unwinding activity or is compromised by having insufficient ATP. The A. aeolicus protein unwinds DNA well at 60 degrees C but is less efficient at 37 degrees C, explaining its inability to function in E. coli at this temperature. The N-terminal extension present in this protein was investigated and found to be dispensable for activity and thermo-stability. The results presented suggest that the role of RecG in DNA replication and repair is likely to be conserved throughout all bacteria, which underlines the importance of this protein in genome duplication and cell survival.

VceR regulates the vceCAB drug efflux pump operon of Vibrio cholerae by alternating between mutually exclusive conformations that bind either drugs or promoter DNA.

VceR, a member of the TetR family of transcriptional regulators, is a repressor of the vceCAB operon, which encodes a multidrug efflux pump in Vibrio cholerae. VceR binds to a 28 bp inverted-repeat within the vceR-vceC intergenic region and is dissociated from this site with CCCP, a pump substrate. The rate of the CCCP-induced conformational change in VceR was determined by stopped-flow fluorescence spectroscopy, revealing a highly co-operative process that occurs with a Hill coefficient of approximately 4. The apparent affinity for CCCP decreased in a linear manner with increasing concentrations of DNA, indicative of competition between the CCCP and DNA for binding to VceR. These data are consistent with an equilibrium between mutually exclusive conformations that are supported by the binding of DNA and CCCP to the N and C termini of VceR, respectively. Size-exclusion chromatography and dynamic light-scattering studies indicate that VceR exists predominantly as a dimer; however, a pair of dimers binds to the DNA. In order to account for the fact that VceR is a dimer in the absence of DNA but binds CCCP with a Hill co-efficient of 4, implying that it has at least four binding-sites, we propose that the VceR monomer possesses a pair of binding sites that can be simultaneously occupied by CCCP. Using a gene-reporter system and stopped-flow spectroscopy, we established that the equilibrium between free VceR and VceR-CCCP plays a critical role in controlling expression of the pump. The co-operative transition between these states allows the repressor to respond to relatively small changes in drug concentration. Thus, repression and induction can be readily switched about a critical drug concentration which will prove toxic to the cell.

The RuvAB branch migration translocase and RecU Holliday junction resolvase are required for double-stranded DNA break repair in Bacillus subtilis.

In models of Escherichia coli recombination and DNA repair, the RuvABC complex directs the branch migration and resolution of Holliday junction DNA. To probe the validity of the E. coli paradigm, we examined the impact of mutations in DeltaruvAB and DeltarecU (a ruvC functional analog) on DNA repair. Under standard transformation conditions we failed to construct DeltaruvAB DeltarecG, DeltarecU DeltaruvAB, DeltarecU DeltarecG, or DeltarecU DeltarecJ strains. However, DeltaruvAB could be combined with addAB (recBCD), recF, recH, DeltarecS, DeltarecQ, and DeltarecJ mutations. The DeltaruvAB and DeltarecU mutations rendered cells extremely sensitive to DNA-damaging agents, although less sensitive than a DeltarecA strain. When damaged cells were analyzed, we found that RecU was recruited to defined double-stranded DNA breaks (DSBs) and colocalized with RecN. RecU localized to these centers at a later time point during DSB repair, and formation was dependent on RuvAB. In addition, expression of RecU in an E. coli ruvC mutant restored full resistance to UV light only when the ruvAB genes were present. The results demonstrate that, as with E. coli RuvABC, RuvAB targets RecU to recombination intermediates and that all three proteins are required for repair of DSBs arising from lesions in chromosomal DNA.