lexA Mutants Research Articles

Pleiotropic hubs drive bacterial surface competition through parallel changes in colony composition and expansion.

Bacteria commonly adhere to surfaces where they compete for both space and resources. Despite the importance of surface growth, it remains largely elusive how bacteria evolve on surfaces. We previously performed an evolution experiment where we evolved distinct Bacilli populations under a selective regime that favored colony spreading. In just a few weeks, colonies of Bacillus subtilis showed strongly advanced expansion rates, increasing their radius 2.5-fold relative to that of the ancestor. Here, we investigate what drives their rapid evolution by performing a uniquely detailed analysis of the evolutionary changes in colony development. We find mutations in diverse global regulators, RicT, RNAse Y, and LexA, with strikingly similar pleiotropic effects: They lower the rate of sporulation and simultaneously facilitate colony expansion by either reducing extracellular polysaccharide production or by promoting filamentous growth. Combining both high-throughput flow cytometry and gene expression profiling, we show that regulatory mutations lead to highly reproducible and parallel changes in global gene expression, affecting approximately 45% of all genes. This parallelism results from the coordinated manner by which regulators change activity both during colony development-in the transition from vegetative growth to dormancy-and over evolutionary time. This coordinated activity can however also break down, leading to evolutionary divergence. Altogether, we show how global regulators function as major pleiotropic hubs that drive rapid surface adaptation by mediating parallel changes in both colony composition and expansion, thereby massively reshaping gene expression.

The influence of oxygen and oxidative stress on de novo acquisition of antibiotic resistance in E. coli and Lactobacillus lactis

BackgroundBacteria can acquire resistance through DNA mutations in response to exposure to sub-lethal concentrations of antibiotics. According to the radical-based theory, reactive oxygen species (ROS), a byproduct of the respiratory pathway, and oxidative stress caused by reactive metabolic byproducts, play a role in cell death as secondary killing mechanism. In this study we address the question whether ROS also affects development of resistance, in the conditions that the cells is not killed by the antibiotic.ResultsTo investigate whether oxygen and ROS affect de novo acquisition of antibiotic resistance, evolution of resistance due to exposure to non-lethal levels of antimicrobials was compared in E. coli wildtype and ΔoxyR strains under aerobic and anaerobic conditions. Since Lactococcus lactis (L. lactis) does not have an active electron transport chain (ETC) even in the presence of oxygen, and thus forms much less ROS, resistance development in L. lactis was used to distinguish between oxygen and ROS. The resistance acquisition in E. coli wildtype under aerobic and anaerobic conditions did not differ much. However, the aerobically grown ΔoxyR strain gained resistance faster than the wildtype or anaerobic ΔoxyR. Inducing an ETC by adding heme increased the rate at which L. lactis acquired resistance. Whole genome sequencing identified specific mutations involved in the acquisition of resistance. These mutations were specific for each antibiotic. The lexA mutation in ΔoxyR strain under aerobic conditions indicated that the SOS response was involved in resistance acquisition.ConclusionsThe concept of hormesis can explain the beneficial effects of low levels of ROS and reactive metabolic byproducts, while high levels are lethal. DNA repair and mutagenesis may therefore expedite development of resistance. Taken together, the results suggest that oxygen as such barely affects resistance development. Nevertheless, non-lethal levels of ROS stimulate de novo acquisition of antibiotic resistance.

Distinct Screening Approaches Uncover PA14_36820 and RecA as Negative Regulators of Biofilm Phenotypes in Pseudomonas aeruginosa PA14.

Pseudomonas aeruginosa commonly infects hospitalized patients and the lungs of individuals with cystic fibrosis. This species is known for forming biofilms, which are communities of bacterial cells held together and encapsulated by a self-produced extracellular matrix. The matrix provides extra protection to the constituent cells, making P. aeruginosa infections challenging to treat. We previously identified a gene, PA14_16550, which encodes a DNA-binding TetR-type repressor and whose deletion reduced biofilm formation. Here, we assessed the transcriptional impact of the 16550 deletion and found six differentially regulated genes. Among them, our results implicated PA14_36820 as a negative regulator of biofilm matrix production, while the remaining 5 had modest effects on swarming motility. We also screened a transposon library in a biofilm-impaired ΔamrZ Δ16550 strain for restoration of matrix production. Surprisingly, we found that disruption or deletion of recA increased biofilm matrix production, both in biofilm-impaired and wild-type strains. Because RecA functions both in recombination and in the DNA damage response, we asked which function of RecA is important with respect to biofilm formation by using point mutations in recA and lexA to specifically disable each function. Our results implied that loss of either function of RecA impacts biofilm formation, suggesting that enhanced biofilm formation may be one physiological response of P. aeruginosa cells to loss of either RecA function. IMPORTANCE Pseudomonas aeruginosa is a notorious human pathogen well known for forming biofilms, communities of bacteria that protect themselves within a self-secreted matrix. Here, we sought to find genetic determinants that impacted biofilm matrix production in P. aeruginosa strains. We identified a largely uncharacterized protein (PA14_36820) and, surprisingly, RecA, a widely conserved bacterial DNA recombination and repair protein, as negatively regulating biofilm matrix production. Because RecA has two main functions, we used specific mutations to isolate each function and found that both functions influenced matrix production. Identifying negative regulators of biofilm production may suggest future strategies to reduce the formation of treatment-resistant biofilms.

Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli.

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon-insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in ΔpriB cells. Taken together, our results highlight roles for several genes in double-strand DNA break homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli.

Genotoxic hazard and oxidative stress induced by wastewater irrigated soil with special reference to pesticides and heavy metal pollution

Due to enhancement of industrial growth and urbanization, soil contamination is increasing prominently. Therefore, it is important to examine possible adverse effects of industrial waste. Soil samples were might to be polluted with several heavy-metals and pesticides. Gas chromatographic results showed occurrence of high-level of organochlorine and organophosphate pesticides in studied soil samples. Genotoxicity of soil extracts was assessed using environmental-risk assessment models. Soil samples were extracted in hexane and dichloromethane solvents and were evaluated for genotoxic potential by prokaryotic (Ames test, plasmid nicking assay and E. coli K-12 DNA repair defective mutants) and eukaryotic (Allium cepa root chromosomal aberration and Vigna radiata seed-germination test) bioassays. Strain TA98 was found the most susceptible among soil extracts. The mutagenicity of hexane soil extract from wastewater irrigation was found to be higher than that of DCM samples in terms of mutagenic index, mutagenic potential, and induction factor for Ames strains. The damage in DNA repair defective mutants of hexane extracts were found higher compared to DCM extracts at dose of 20 μl/ml of culture. Survival in polA, lexA and recA mutants were 39%, 47% and 55% while treated with hexane extract. Allium cepa test, mitotic index was decreased in dose-dependent way and various kinds of chromosomal aberrations were found. Vigna radiata seeds germination and other parameters were also affected when treated with wastewater irrigated (WWI) soil. Oxidative stress in V. radiata roots were also showed under CLS microscope. Genotoxicity of WWI soil extract was also confirmed by plasmid nicking test. Our study provides possible explanation for the assessment of potential health and environmental hazards of the industrial region.

Cytotoxic and genotoxic assessment of agricultural soils from an industrial region.

Industrial effluents contain hazardous substances that can be a serious threat to the agriculture and human health. In the present study, the cytotoxic and genotoxic impacts of agricultural soil from the industrial area of Dera Bassi (Punjab, India) have been evaluated. Assays such as defects in DNA repair in K-12 mutants of Escherichia coli and chromosomal aberrations in Allium cepa were used to estimate the acute toxicity and chromosomal mutagenesis, respectively. Atomic absorption spectrometry and GC-MS analysis revealed contamination of the soil with high concentrations of heavy metals and organic compounds, respectively. Dichloromethane extract of site I soil sample caused maximum damage to 40 μL mL-1 DNA repair defective mutants and showed 38 and 49% survival in lexA and recA mutants, respectively, which was least among all the sites. In A. cepa test, an inverse relationship between soil extract concentration and the mitotic index was observed. Exposure of growing roots of A. cepa to soil extracts induced chromosomal abnormalities and alterations in mitotic phases in root tip cells. The study concludes that agricultural sites near the industrial area were contaminated with genotoxic and mutagenic compounds. Hence, adequate measures should be taken to reduce the toxicity of industrial effluents discharged onto the agricultural fields.

The Regulation of LexA on UV-Induced SOS Response in Myxococcus xanthus Based on Transcriptome Analysis.

SOS response is a conserved response to DNA damage in prokaryotes and is negatively regulated by LexA protein, which recognizes specifically an "SOS-box" motif present in the promoter region of SOS genes. Myxococcus xanthus DK1622 possesses a lexA gene, and while the deletion of lexA had no significant effect on either bacterial morphology, UV-C resistance, or sporulation, it did delay growth. UV-C radiation resulted in 651 upregulated genes in M. xanthus, including the typical SOS genes lexA, recA, uvrA, recN and so on, mostly enriched in the pathways of DNA replication and repair, secondary metabolism, and signal transduction. The UV-irradiated lexA mutant also showed the induced expression of SOS genes and these SOS genes enriched into a similar pathway profile to that of wild-type strain. Without irradiation treatment, the absence of LexA enhanced the expression of 122 genes that were not enriched in any pathway. Further analysis of the promoter sequence revealed that in the 122 genes, only the promoters of recA2, lexA and an operon composed of three genes (pafB, pafC and cyaA) had SOS box sequence to which the LexA protein is bound directly. These results update our current understanding of SOS response in M. xanthus and show that UV induces more genes involved in secondary metabolism and signal transduction in addition to DNA replication and repair; and while the canonical LexA-dependent regulation on SOS response has shrunk, only 5 SOS genes are directly repressed by LexA.

In Situ Single-Molecule Dynamics of the SOS-Repressor Lexa during Antibiotic Stress

In bacteria, the key mechanism facilitating survival, mutation and adaptation upon DNA damage is the SOS response. Through autoproteolytic digestion triggered by single-stranded DNA, the repressor LexA controls over 50 SOS genes. Efforts to inhibit this response and thereby combat antibiotic resistance rely on a broad understanding of its behavior in vivo, which is still limited. Here, we use single-molecule localization microscopy to directly visualize LexA in Escherichia coli. With single-particle tracking PALM, we observe the repressor under antibiotic stress of different intensity and identify four diffusive populations. Using a set of LexA mutants, we assign operator bound, target-searching, freely diffusing and cleaved repressors. Measuring the time-evolution of these populations and developing a kinetic model, we obtain in vivo cleavage and binding rates in dependence of antibiotic dosage. A strong SOS response is observed even at low antibiotic stress. We further count molecules time-resolved in fixed cells and found that LexA is much more abundant during the late SOS response than previously thought.

Characteristics of Emergence of Mutants Resistant to Nalidixic Acid and Novobiocin in E. coli Strains with recA and lexA Mutations

The main problem of the modern medical microbiology is the widespread resistance of bacteria to many antibiotics used in therapy. Resistance to antibiotics is mainly developed due to mutations in the bacterial genome. One of the proposed mechanisms for the occurrence of mutations is the functioning of the inducible SOS response system, the proteins of which are synthesized in the cell affected by antibiotics. The RecA and LexA proteins encoded by the corresponding genes are regulators of the SOS response in bacteria. The effect of the recA13 and lexA1 mutations on bacterial resistance to nalidixic acid and novobiocin and on the rates of antibiotic resistance development was studied. The SOS response system was shown to play a minor role in the development of the bacterial resistance to quinolones and aminocoumarins, as well as in mutagenesis, during application of these antibiotics.

Mutants resistant to nalidixic acid and novobiocin. specific features of formation from E. Coli strains with RecA or LexA mutations

Mutants resistant to nalidixic acid and novobiocin. specific features of formation from E. Coli strains with RecA or LexA mutations

Systematically Altering Bacterial SOS Activity under Stress Reveals Therapeutic Strategies for Potentiating Antibiotics.

The bacterial SOS response is a DNA damage repair network that is strongly implicated in both survival and acquired drug resistance under antimicrobial stress. The two SOS regulators, LexA and RecA, have therefore emerged as potential targets for adjuvant therapies aimed at combating resistance, although many open questions remain. For example, it is not well understood whether SOS hyperactivation is a viable therapeutic approach or whether LexA or RecA is a better target. Furthermore, it is important to determine which antimicrobials could serve as the best treatment partners with SOS-targeting adjuvants. Here we derived Escherichia coli strains that have mutations in either lexA or recA genes in order to cover the full spectrum of possible SOS activity levels. We then systematically analyzed a wide range of antimicrobials by comparing the mean inhibitory concentrations (MICs) and induced mutation rates for each drug-strain combination. We first show that significant changes in MICs are largely confined to DNA-damaging antibiotics, with strains containing a constitutively repressed SOS response impacted to a greater extent than hyperactivated strains. Second, antibiotic-induced mutation rates were suppressed when SOS activity was reduced, and this trend was observed across a wider spectrum of antibiotics. Finally, perturbing either LexA or RecA proved to be equally viable strategies for targeting the SOS response. Our work provides support for multiple adjuvant strategies, while also suggesting that the combination of an SOS inhibitor with a DNA-damaging antibiotic could offer the best potential for lowering MICs and decreasing acquired drug resistance. IMPORTANCE Our antibiotic arsenal is becoming depleted, in part, because bacteria have the ability to rapidly adapt and acquire resistance to our best agents. The SOS pathway, a widely conserved DNA damage stress response in bacteria, is activated by many antibiotics and has been shown to play central role in promoting survival and the evolution of resistance under antibiotic stress. As a result, targeting the SOS response has been proposed as an adjuvant strategy to revitalize our current antibiotic arsenal. However, the optimal molecular targets and partner antibiotics for such an approach remain unclear. In this study, focusing on the two key regulators of the SOS response, LexA and RecA, we provide the first comprehensive assessment of how to target the SOS response in order to increase bacterial susceptibility and reduce mutagenesis under antibiotic treatment.

The SOS Response Master Regulator LexA Is Associated with Sporulation, Motility and Biofilm Formation in Clostridium difficile.

The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.

Genotoxicity assessments of alluvial soil irrigated with wastewater from a pesticide manufacturing industry.

In this study, organochlorine pesticides (OCP) and heavy metals were analyzed from wastewater- and groundwater- irrigated soils (control samples) by gas chromatography (GC) and atomic absorption spectrophotometry (AAS), respectively. Gas chromatographic analysis revealed the presence of high concentration of pesticides in soil irrigated with wastewater (WWS). These concentrations were far above the maximum residue permissible limits indicating that alluvial soils have high binding capacity of OCP. AAS analyses revealed higher concentration of heavy metals in WWS as compared to groundwater (GWS). Also, the DNA repair (SOS)-defective Escherichia coli K-12 mutant assay and the bacteriophage lambda system were employed to estimate the genotoxicity of soils. Therefore, soil samples were extracted by hexane, acetonitrile, methanol, chloroform, and acetone. Both bioassays revealed that hexane-extracted soils from WWS were most genotoxic. A maximum survival of 15.2% and decline of colony-forming units (CFUs) was observed in polA mutants of DNA repair-defective E. coli K-12 strains when hexane was used as solvent. However, the damage of polA (-) mutants triggered by acetonitrile, methanol, chloroform, and acetone extracts was 80.0, 69.8, 65.0, and 60.7%, respectively. These results were also confirmed by the bacteriophage λ test system as hexane extracts of WWS exhibited a maximum decline of plaque-forming units for lexA mutants of E. coli K-12 pointing to an elevated genotoxic potential. The lowest survival was observed for lexA (12%) treated with hexane extracts while the percentage of survival was 25, 49.2, 55, and 78% with acetonitrile, methanol, chloroform, and acetone, respectively, after 6 h of treatment. Thus, our results suggest that agricultural soils irrigated with wastewater from pesticide industries have a notably high genotoxic potential.

Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains under aerobic and anaerobic conditions

During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-damaging agents and these agents can induce several defence mechanisms. Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species (ROS) generated by chemical and physical agents or by metabolism. In this work, we have evaluated whether the interaction with an abiotic surface by mutants derived from E. coli K-12 deficient in some enzymes that are part of BER causes DNA damage and associated filamentation. Moreover, we studied the role of endonuclease V (nfi gene; 1506 mutant strain) in biofilm formation. Endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions. We verified that endonuclease V is involved in biofilm formation. Our results showed more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) than in the wild-type strain (AB1157). By contrast, the mutant nfi did not present filamentation in biofilm, although its wild-type strain (1466) showed rare filaments in biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of SOS induction measured by the SOS chromotest. However, biofilm formation depended on the ability of the bacteria to induce the SOS response since the mutant lexA Ind(-) did not induce the SOS response and did not form any biofilm. Oxygen tension was an important factor for the interaction of the BER mutants, since these mutants exhibited decreased quantitative adherence under anaerobic conditions. However, our results showed that the presence or absence of oxygen did not affect the viability of BW9091 and BW535 strains. The nfi mutant and its wild-type did not exhibit decreased biofilm formation under anaerobic conditions. Scanning electron microscopy was also performed on the E. coli K-12 strains that had adhered to the glass, and we observed the presence of a structure similar to an extracellular matrix that depended on the oxygen tension. In conclusion, it was proven that bacterial interaction with abiotic surfaces can lead to SOS induction and associated filamentation. Moreover, we verified that endonuclease V is involved in biofilm formation.

In vitro analysis of the phytotoxic and genotoxic potential of Aligarh wastewater and Mathura refinery wastewater

Present report deals with the phytotoxicity and genotoxicity of Mathura refinery wastewater and Aligarh wastewater of Northern India. The IC50 value in Allium cepa root growth inhibition test was recorded to be 0.14X and 0.10X for Mathura refinery and Aligarh industrial wastewaters, respectively. Significant decline in the survival of various Escherichia coli K12 DNA repair defective mutants was observed when the tester strains were exposed to the aforementioned samples. The order of sensitivity was invariably as: AB1157 (wild type) < AB2494 (lexA mutant) < AB2463 (recA mutant) < AB2480 (uvrA recA double mutant). These results suggested a significant amount of DNA damage within the bacterial cells exposed to test wastewaters. A. cepa genotoxicity test also demonstrated a considerable amount of chromosomal damage of A. cepa brought about by the test samples. The aberration index (A.I.) for Aligarh wastewater and refinery wastewater was recorded to be 11.2% and 14.7%, respectively, whereas the aquaguard mineral water serving as negative control displayed the A.I. value to be 2.6%. Interestingly, genotoxicity of both industrial wastewaters was reduced to a remarkable extent in presence of mannitol, the hydroxyl radical scavenger. Present study clearly indicated a distinct pattern of the chromosomal aberrations showing predominantly stickiness and stray chromosomes in case of AWW while clumping and stickiness in case of RWW, thereby affirming the genotoxicity of both test waters.

Opposing effects of aminocoumarins and fluoroquinolones on the SOS response and adaptability in Staphylococcus aureus

RecA is the key enzyme involved in DNA repair, recombination and induction of the SOS response and is central to the development of antibiotic resistance. Here we assessed the interaction of two different gyrase inhibitors, ciprofloxacin (a fluoroquinolone) and novobiocin (an aminocoumarin), on RecA activity and the SOS response in Staphylococcus aureus. The influence of different gyrase inhibitors on the SOS response of S. aureus (including recA and lexA mutants) was analysed by northern blot analysis, real-time RT-PCR, western blot analysis and promoter activity assays. Recombination as well as mutation frequencies were determined for the different antibiotic combinations. We verified that ciprofloxacin leads to RecA activation and therefore induction of the SOS response. In contrast, novobiocin treatment resulted in an inhibition of recA transcription independent of LexA. When novobiocin and ciprofloxacin were added simultaneously, recA was reduced to the same level as with novobiocin alone. In combination, novobiocin also partially reduces the ciprofloxacin-mediated induction of the LexA target gene umuC (error-prone polymerase). Apart from reducing recA and umuC expression, novobiocin also inhibited the frequency of recombination, mutation and the formation of non-haemolytic variants. In summary, aminocoumarins inhibit recA expression in S. aureus and probably delay the process of developing antibiotic resistance and gene transfer. A clinical re-evaluation of these compounds as well as designing more applicable derivatives should be considered.

Effect of Translesion DNA Polymerases, Endonucleases and RpoS on Mutation Rates inSalmonella typhimurium

It has been suggested that bacteria have evolved mechanisms to increase their mutation rate in response to various stresses and that the translesion DNA polymerase Pol IV under control of the LexA regulon and the alternative sigma factor RpoS are involved in regulating this mutagenesis. Here we examined in Salmonella enterica serovar Typhimurium LT2 the rates for four different types of mutations (rifampicin, nalidixic acid, and chlorate resistance and Lac(+) reversion) during various growth conditions and with different levels of four translesion DNA polymerases (Pol II, Pol IV, Pol V, and SamAB) and RpoS. Constitutive derepression of the LexA regulon by a lexA(def) mutation had no effect on Lac(+) reversion rates but increased the other three mutation rates up to 11-fold, and the contribution of the translesion DNA polymerases to this mutagenesis varied with the type of mutation examined. The increase in mutation rates in the lexA(def) mutant required the presence of the LexA-controlled UvrB protein and endonucleases UvrC and Cho. With regard to the potential involvement of RpoS in mutagenesis, neither an increase in RpoS levels conferred by artificial overexpression from a plasmid nor long-term stationary phase incubation or slow growth caused an increase in any of the four mutation rates measured, alone or in combination with overexpression of the translesion DNA polymerases. In conclusion, mutation rates are remarkably robust and no combination of growth conditions, induction of translesion DNA polymerases by inactivation of LexA, or increased RpoS expression could confer an increase in mutation rates higher than the moderate increase caused by derepression of the LexA regulon alone.

Differences in stress response after UVC or UVA irradiation in Vibrio parahaemolyticus

The SOS response is a global regulatory network for repairing DNA damage induced by various environmental stresses such as UV irradiation. The Escherichia coli SOS response has been extensively studied. However, there are no reports on the SOS response in Vibrio parahaemolyticus. In this study, we examined the SOS response in V. parahaemolyticus and compared the differential expression of genes induced by UVC and UVA irradiation. In UVC-exposed wild-type cells, expression of several DNA repair genes was increased. However, expression of these genes was not increased in ΔrecA or lexA mutants. Cell filamentation was observed in wild-type cells, but not in ΔrecA and lexA mutant cells. Sensitivity to UVC was significantly increased in ΔrecA, lexA mutant and Δlon strains compared with wild type. In the case of UVA irradiation, LexA-controlled DNA repair genes were minimally induced and cell filamentation was not observed. Sensitivity to UVA was the same in the mutant and wild-type strains. These findings suggest that there is a RecA-LexA-mediated SOS response in V. parahaemolyticus, and that this response is important to UVC tolerance but does not contribute to UVA tolerance.

Antibacterial effects of silver ions on growth of gram-negative bacteria and biofilm formation

The average minimal inhibiting concentrations (MICs) of AgNO3 for the gram-negative bacteria Escherichia coli K12, Serratia proteamaculans 94, and Serratia liquefaciens MG1 were found to be 0.075–0.3 μg/ml and, for Pseudomonas aeruginosa PAO1 and P. chlororaphis 449, this concentration was 0.15–0.3 μg/ml. The formation of a biofilm by Escherichia coli AB1157 and S. proteamaculans 94 was completely inhibited by 0.3 μg/ml of AgNO3 and, in the case of Pseudomonas aeruginosa PAO1, biofilm formation was inhibited by 0.6 μg/ml of AgNO3. Mutations in E. coli genes that encode global regulators of gene expression, such as sigma S and sigma N subunits of RNA polymerase, the catabolite repression protein CRP, and the Lon protease, had no marked effect on the sensitivity of cells to silver. Wild-type E. coli strains and strains deficient in excision repair (uvrA and uvrB), SOS repair, and recombination (recA, lexA, recBC, and recF mutants) did not differ in their sensitivity to silver. This suggests that the sensitivity of the bacteria to the silver was not associated with DNA lesions that could be repaired by these repair and recombination systems. E. coli mutant strains deficient in OmpF or OmpC porins were three to four times more resistant to silver ions than the wild-type strain. Experiments with the pME6863 plasmid, which bears the gene of N-acyl-homoserine lactonase AiiA, demonstrated that the quorum sensing (QS) regulation did not participate in controlling the sensitivity of S. proteamaculans 94 and P. chlororaphis 449 to silver. The same conclusion was drawn from a comparison of AgNO3 MICs of the S. liquefaciens wild-type strain and a mutant strain deficient in QS.

A Genetic Screen for Isolating “Lariat” Peptide Inhibitors of Protein Function

Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. We developed a genetic screen for isolating "lariat" peptides that function as trans dominant inhibitors of protein function. A lariat consists of a lactone-cyclized peptide with a covalently attached transcription activation domain, which allows combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay. We isolated lariats against the bacterial repressor protein LexA. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolysis make bacteria more sensitive to, and inhibit resistance against, cytotoxic reagents. We showed that an anti-LexA lariat blocked LexA autoproteolysis and potentiated the antimicrobial activity of mitomycin C.