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Antimicrobial resistance (AMR) is a serious global threat demanding innovations for effective control of pathogens. The bacterial SOS response, regulated by the master regulators, LexA and RecA, contributes to AMR through advantageous mutations. Targeting the LexA/RecA system with a novel inhibitor could suppress the SOS response and potentially reduce the occurrence of AMR. RecA presents a challenge as a therapeutic target due to its conserved structure and function across species, including humans. Conversely, LexA which is absent in eukaryotes, can be potentially targeted, due to its involvement in SOS response which is majorly responsible for adaptive mutagenesis and AMR. Our studies combining bioinformatic, biochemical, biophysical, molecular, and cell-based assays present a unique inhibitor of mycobacterial LexA, wherein we show that the inhibitor interacts directly with the catalytic site residues of LexA of Mycobacterium tuberculosis (Mtb), consequently hindering its cleavage, suppressing SOS response thereby reducing mutation frequency and AMR., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of the article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In 1967, in this journal, Evelyn Witkin proposed the existence of a coordinated DNA damage response in Escherichia coli , which later came to be called the "SOS response." We revisited this response using the replication inhibitor azidothymidine (AZT) and RNA-Seq analysis and identified several features. We confirm the induction of classic Save our ship (SOS) loci and identify several genes, including many of the pyrimidine pathway, that have not been previously demonstrated to be DNA damage-inducible. Despite a strong dependence on LexA, these genes lack LexA boxes and their regulation by LexA is likely to be indirect via unknown factors. We show that the transcription factor "stringent starvation protein" SspA is as important as LexA in the regulation of AZT-induced genes and that the genes activated by SspA change dramatically after AZT exposure. Our experiments identify additional LexA-independent DNA damage inducible genes, including 22 small RNA genes, some of which appear to activated by SspA. Motility and chemotaxis genes are strongly down-regulated by AZT, possibly as a result of one of more of the small RNAs or other transcription factors such as AppY and GadE, whose expression is elevated by AZT. Genes controlling the iron siderophore, enterobactin, and iron homeostasis are also strongly induced, independent of LexA. We confirm that IraD antiadaptor protein is induced independent of LexA and that a second antiadaptor, IraM is likewise strongly AZT-inducible, independent of LexA, suggesting that RpoS stabilization via these antiadaptor proteins is an integral part of replication stress tolerance., Competing Interests: Competing interests statement:The authors declare no competing interest.

In recent years, new evidence has shown that the SOS response plays an important role in the response to antimicrobials, with involvement in the generation of clinical resistance. Here we evaluate the impact of heterogeneous expression of the SOS response in clinical isolates of Escherichia coli on response to the fluoroquinolone, ciprofloxacin. In silico analysis of whole genome sequencing data showed remarkable sequence conservation of the SOS response regulators, RecA and LexA. Despite the genetic homogeneity, our results revealed a marked differential heterogeneity in SOS response activation, both at population and single-cell level, among clinical isolates of E. coli in the presence of subinhibitory concentrations of ciprofloxacin. Four main stages of SOS response activation were identified and correlated with cell filamentation. Interestingly, there was a correlation between clinical isolates with higher expression of the SOS response and further progression to resistance. This heterogeneity in response to DNA damage repair (mediated by the SOS response) and induced by antimicrobial agents could be a new factor with implications for bacterial evolution and survival contributing to the generation of antimicrobial resistance., Competing Interests: Declaration of Competing Interest On behalf of all authors, I declare that we have no commercial interests related to the submitted manuscript., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some β-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages., Competing Interests: The authors declare no conflict of interest.

The widespread use of disinfectants during the global response to the 2019 coronavirus pandemic has increased the co-occurrence of disinfection byproducts (DBPs) and antibiotic resistance genes (ARGs). Although DBPs pose major threats to public health globally, there is limited knowledge regarding their biological effects on ARGs. This study aimed to investigate the effects of two inorganic DBPs (chlorite and bromate) on the conjugative transfer of RP4 plasmid among Escherichia coli strains at environmentally relevant concentrations. Interestingly, the frequency of conjugative transfer was initially inhibited when the exposure time to chlorite or bromate was less than 24 h. However, this inhibition transformed into promotion when the exposure time was extended to 36 h. Short exposures to chlorite or bromate were shown to impede the electron transport chain, resulting in an ATP shortage and subsequently inhibiting conjugative transfer. Consequently, this stimulates the overproduction of reactive oxygen species (ROS) and activation of the SOS response. Upon prolonged exposure, the resurgent energy supply promoted conjugative transfer. These findings offer novel and valuable insights into the effects of environmentally relevant concentrations of inorganic DBPs on the conjugative transfer of ARGs, thereby providing a theoretical basis for the management of DBPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aim: Recombinase RecA and its homologs play a key role in homologous recombination DNA repair and revive stalled replication fork DNA synthesis. RecA plays an essential role in the evolution of antibiotic-resistant strains via stress-induced DNA repair mechanisms during the SOS response. Accordingly, RecA has become an attractive target to slow down antibiotic resistance rates and prevent mutations in pathogenic bacterial species., Methods and Results: We employed RecA conserved activities: DNA binding, displacement loop formation, strand exchange, ATP hydrolysis, and LexA cleavage, to elucidate the inhibitory role of gallic acid on Staphylococcus aureus RecA functions. Gallic acid inhibition of the SOS response by western blot analysis and its antibacterial activity were measured. The gallic acid inhibited all the canonical activities of S. aureus RecA protein., Conclusion: The natural phenolic compound gallic acid interferes with RecA protein DNA complex formation and inhibits activities such as displacementloop formation, strand exchange reaction, ATP hydrolysis, and coprotease activity of S. aureus. Additionally, gallic acid can obstruct ciprofloxacin-induced RecA expression and eventually confer the inhibitory role of gallic acid in the SOS survival mechanism in S. aureus., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Curcumin is a natural compound that is considered safe and may have potential health benefits; however, its poor stability and water insolubility limit its therapeutic applications. Different strategies aim to increase its water solubility. Here, we tested the compound PVP-curcumin as a photosensitizer for antimicrobial photodynamic therapy (aPDT) as well as its potential to act as an adjuvant in antibiotic drug therapy. Gram-negative E. coli K12 and Gram-positive S. capitis were subjected to aPDT using various PVP-curcumin concentrations (1-200 µg/mL) and 475 nm blue light (7.5-45 J/cm 2 ). Additionally, results were compared to aPDT using 415 nm blue light. Gene expression of recA and umuC were analyzed via RT-qPCR to assess effects on the bacterial SOS response. Further, the potentiation of Ciprofloxacin by PVP-curcumin was investigated, as well as its potential to prevent the emergence of antibiotic resistance. Both bacterial strains were efficiently reduced when irradiated with 415 nm blue light (2.2 J/cm 2 ) and 10 µg/mL curcumin. Using 475 nm blue light, bacterial reduction followed a biphasic effect with higher efficacy in S. capitis compared to E. coli K12. PVP-curcumin decreased recA expression but had limited effect regarding enhancing antibiotic treatment or impeding resistance development. PVP-curcumin demonstrated effectiveness as a photosensitizer against both Gram-positive and Gram-negative bacteria but did not modulate the bacterial SOS response.

Although the mechanistic connections between SOS-induced mutagenesis and antibiotic resistance are well established, our current understanding of the impact of SOS response levels, recovery durations, and transcription/translation activities on mutagenesis remains relatively limited. In this study, when bacterial cells were exposed to mutagens like ultraviolet light for defined time intervals, a compelling connection between the rate of mutagenesis and the RecA-mediated SOS response levels became evident. Our observations also indicate that mutagenesis primarily occurs during the subsequent recovery phase following the removal of the mutagenic agent. When transcription/translation was inhibited or energy molecules were depleted at the onset of treatment or during the early recovery phase, there was a noticeable decrease in SOS response activation and mutagenesis. However, targeting these processes later in the recovery phase does not have the same effect in reducing mutagenesis, suggesting that the timing of inhibiting transcription/translation or depleting energy molecules is crucial for their efficacy in reducing mutagenesis. Active transcription, translation, and energy availability within the framework of SOS response and DNA repair mechanisms appear to be conserved attributes, supported by their consistent manifestation across diverse conditions, including the use of distinct mutagens such as fluoroquinolones and various bacterial strains., Competing Interests: The authors declare no conflict of interest.

Colibactin is a chemically unstable small-molecule genotoxin that is produced by several different bacteria, including members of the human gut microbiome 1,2 . Although the biological activity of colibactin has been extensively investigated in mammalian systems 3 , little is known about its effects on other microorganisms. Here we show that colibactin targets bacteria that contain prophages, and induces lytic development through the bacterial SOS response. DNA, added exogenously, protects bacteria from colibactin, as does expressing a colibactin resistance protein (ClbS) in non-colibactin-producing cells. The prophage-inducing effects that we observe apply broadly across different phage-bacteria systems and in complex communities. Finally, we identify bacteria that have colibactin resistance genes but lack colibactin biosynthetic genes. Many of these bacteria are infected with predicted prophages, and we show that the expression of their ClbS homologues provides immunity from colibactin-triggered induction. Our study reveals a mechanism by which colibactin production could affect microbiomes and highlights a role for microbial natural products in influencing population-level events such as phage outbreaks., (© 2022. The Author(s).)

Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development., (© 2022. The Author(s).)

The plasmid-mediated quinolone resistance (PMQR) genes have been shown to promote high-level bacterial resistance to fluoroquinolone antibiotics, potentially leading to clinical treatment failures. In Escherichia coli , sub-minimum inhibitory concentrations (sub-MICs) of the widely used fluoroquinolones are known to induce the SOS response. Interestingly, the expression of several PMQR qnr genes is controlled by the SOS master regulator, LexA. During the characterization of a small qnrD -plasmid carried in E. coli, we observed that the aminoglycosides become able to induce the SOS response in this species, thus leading to the elevated transcription of qnrD . Our findings show that the induction of the SOS response is due to nitric oxide (NO) accumulation in the presence of sub-MIC of aminoglycosides. We demonstrated that the NO accumulation is driven by two plasmid genes, ORF3 and ORF4, whose products act at two levels. ORF3 encodes a putative flavin adenine dinucleotide ( FAD )-binding oxidoreductase which helps NO synthesis, while ORF4 codes for a putative fumarate and nitrate reductase ( FNR )-type transcription factor, related to an O 2 -responsive regulator of hmp expression, able to repress the Hmp-mediated NO detoxification pathway of E. coli . Thus, this discovery, that other major classes of antibiotics may induce the SOS response could have worthwhile implications for antibiotic stewardship efforts in preventing the emergence of resistance., Competing Interests: AB, DS, AM, GP, ZB, TJ, MP, SG, FB, SV, SM, Cd, DM, TG No competing interests declared, (© 2022, Babosan et al.)

Antibiotics are well known drugs which, when present above certain concentrations, are able to inhibit the growth of certain bacteria. However, a growing body of evidence shows that even when present at lower doses (subMIC, for sub-minimal inhibitory concentration), unable to inhibit or affect microbial growth, antibiotics work as signaling molecules, affect gene expression and trigger important bacterial stress responses. However, how subMIC antibiotic signaling interplays with other well-known signaling networks in bacteria (and the consequences of such interplay) is not well understood. In this work, through transcriptomic and genetic approaches, we have explored how quorum-sensing (QS) proficiency of V. cholerae affects this pathogen's response to subMIC doses of the aminoglycoside tobramycin (TOB). We show that the transcriptomic signature of V. cholerae in response to subMIC TOB depends highly on the presence of QS master regulator HapR. In parallel, we show that subMIC doses of TOB are able to negatively interfere with the AI-2/LuxS QS network of V. cholerae , which seems critical for survival to aminoglycoside treatment and TOB-mediated induction of SOS response in this species. This interplay between QS and aminoglycosides suggests that targeting QS signaling may be a strategy to enhance aminoglycoside efficacy in V. cholerae .

Several classes of non-antibiotic drugs, including psychoactive drugs, proton-pump inhibitors (PPIs), non-steroidal anti-inflammatory drugs (NSAIDs), and others, appear to have strong antimicrobial properties. We considered whether psychoactive drugs induce the SOS response in E. coli bacteria and, consequently, induce Shiga toxins in Shiga-toxigenic E. coli (STEC). We measured the induction of an SOS response using a recA-lacZ   E. coli reporter strain, as RecA is an early, reliable, and quantifiable marker for activation of the SOS stress response pathway. We also measured the production and release of Shiga toxin 2 (Stx2) from a classic E. coli O157:H7 strain, derived from a food-borne outbreak due to spinach. Some, but not all, serotonin selective reuptake inhibitors (SSRIs) and antipsychotic drugs induced an SOS response. The use of SSRIs is widespread and increasing; thus, the use of these antidepressants could account for some cases of hemolytic-uremic syndrome due to STEC and is not attributable to antibiotic administration. SSRIs could have detrimental effects on the normal intestinal microbiome in humans. In addition, as SSRIs are resistant to environmental breakdown, they could have effects on microbial communities, including aquatic ecosystems, long after they have left the human body.

A broad spectrum of volatile organic compounds' (VOCs') biological activities has attracted significant scientific interest, but their mechanisms of action remain little understood. The mechanism of action of two VOCs-the cyclic monoterpenes (-)-limonene and (+)-α-pinene-on bacteria was studied in this work. We used genetically engineered Escherichia coli bioluminescent strains harboring stress-responsive promoters (responsive to oxidative stress, DNA damage, SOS response, protein damage, heatshock, membrane damage) fused to the luxCDABE genes of Photorhabdus luminescens. We showed that (-)-limonene induces the P katG and P soxS promoters due to the formation of reactive oxygen species and, as a result, causes damage to DNA (SOSresponse), proteins (heat shock), and membrane (increases its permeability). The experimental data indicate that the action of (-)-limonene at high concentrations and prolonged incubation time makes degrading processes in cells irreversible. The effect of (+)-α-pinene is much weaker: it induces only heat shock in the bacteria. Moreover, we showed for the first time that (-)-limonene completely inhibits the DnaKJE-ClpB bichaperone-dependent refolding of heat-inactivated bacterial luciferase in both E. coli wild type and mutant Δ ibpB strains. (+)-α-Pinene partially inhibits refolding only in Δ ibpB mutant strain.

A bioinformatic search for LexA boxes, combined with transcriptomic detection of loci responsive to DNA damage, identified 48 members of the SOS regulon in the genome of Salmonella enterica serovar Typhimurium. Single cell analysis using fluorescent fusions revealed that heterogeneous expression is a common trait of SOS response genes, with formation of SOS OFF and SOS ON subpopulations. Phenotypic cell variants formed in the absence of external DNA damage show gene expression patterns that are mainly determined by the position and the heterology index of the LexA box. SOS induction upon DNA damage produces SOS OFF and SOS ON subpopulations that contain live and dead cells. The nature and concentration of the DNA damaging agent and the time of exposure are major factors that influence the population structure upon SOS induction. An analogy can thus be drawn between the SOS response and other bacterial stress responses that produce phenotypic cell variants.

Horizontal gene transfer and mutation are the two major drivers of microbial evolution that enable bacteria to adapt to fluctuating environmental stressors 1 . Clustered, regularly interspaced, short palindromic repeats (CRISPR) systems use RNA-guided nucleases to direct sequence-specific destruction of the genomes of mobile genetic elements that mediate horizontal gene transfer, such as conjugative plasmids 2 and bacteriophages 3 , thus limiting the extent to which bacteria can evolve by this mechanism. A subset of CRISPR systems also exhibit non-specific degradation of DNA 4,5 ; however, whether and how this feature affects the host has not yet been examined. Here we show that the non-specific DNase activity of the staphylococcal type III-A CRISPR-Cas system increases mutations in the host and accelerates the generation of antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. These mutations require the induction of the SOS response to DNA damage and display a distinct pattern. Our results demonstrate that by differentially affecting both mechanisms that generate genetic diversity, type III-A CRISPR systems can modulate the evolution of the bacterial host.

Antimicrobial resistance is a pressing threat to global health, with multidrug-resistant pathogens becoming increasingly prevalent. The bacterial SOS pathway functions in response to DNA damage that occurs during infection, initiating several pro-survival and resistance mechanisms, such as DNA repair and hypermutation. This makes SOS pathway components potential targets that may combat drug-resistant pathogens and decrease resistance emergence. This review discusses the mechanism of the SOS pathway; the structure and function of potential targets AddAB, RecBCD, RecA and LexA; and efforts to develop selective small-molecule inhibitors of these proteins. These inhibitors may serve as valuable tools for target validation and provide the foundations for desperately needed novel antibacterial therapeutics.

Salmonella enterica subspecies enterica serovar Typhimurium ( S . Typhimurium) definitive phage type 104 (DT104), S. enterica subspecies enterica serovar Worthington ( S . Worthington) and S. bongori produce ArtA and ArtB (ArtAB) toxin homologues, which catalyse ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene ( artAB ) is encoded on prophage in DT104 and its expression is induced by mitomycin C (MTC) and hydrogen peroxide (H 2 O 2 ) that trigger the bacterial SOS response. Although the genetic regulatory mechanism associated with artAB expression is not characterized, it is thought to be associated with prophage induction, which occurs when the RecA-mediated SOS response is triggered. Here we show that subinhibitory concentration of quinolone antibiotics that are SOS-inducing agents, also induce ArtAB production in these Salmonella strains. Both MTC and fluoroquinolone antibiotics such as enrofloxacin-induced artA and recA transcription and artAB -encoding prophage (ArtAB-prophage) in DT104 and S . Worthington. However, in S. bongori , which harbours artAB genes on incomplete prophage, artA transcription was induced by MTC and enrofloxacin, but prophage induction was not observed. Taken together, these results suggest that SOS response followed by induction of artAB transcription is essential for ArtAB production. H 2 O 2 -mediated induction of ArtAB prophage and efficient production of ArtAB was observed in DT104 but not in S . Worthington and S. bongori . Therefore, induction of artAB expression with H 2 O 2 is strain-specific, and the mode of action of H 2 O 2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

DNA is exposed to the attack of several exogenous agents that modify its chemical structure, so cells must repair those changes in order to survive. Alkylating agents introduce methyl or ethyl groups in most of the cyclic or exocyclic nitrogen atoms of the ring and exocyclic oxygen available in DNA bases producing damage that can induce the SOS response in Escherichia coli and many other bacteria. Likewise, ultraviolet light produces mainly cyclobutane pyrimidine dimers that arrest the progression of the replication fork and triggers such response. The need of some enzymes (such as RecO, ExoI and RecJ) in processing injuries produced by gamma radiation prior the induction of the SOS response has been reported before. In the present work, several repair-defective strains of E. coli were treated with methyl methanesulfonate, ethyl methanesulfonate, mitomycin C or ultraviolet light. Both survival and SOS induction (by means of the Chromotest) were tested. Our results indicate that the participation of these genes depends on the type of injury caused by a genotoxin on DNA., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Increasing quantities of pharmaceutical waste in the environment have disrupted the balance of ecosystems, and may have subsequent effects on human health. Although a handful of previous studies have shown the impacts of pharmaceutically active compounds on the environment, the toxicological effects of their degradation products remain largely unknown. In the current study, the photo-degradation products of environmental ibuprofen were assessed for both ecotoxicological and human health effects using a series of in vitro assays. Here, six of the major degradation products are synthesized with high purity (>98%) and characterized with 1 HNMR, 13 CNMR, FT-IR and HRMS. To evaluate human health effects, three gut microbiota species, Lactobacillus acidophilus, Enterococcus faecalis and Escherichia coli, and two human cell lines, HEK293T and HepG2, are exposed to various concentrations of ibuprofen and its degradation products. On L. acidophilus, the ibuprofen degradation product (±)-(2R,3R)-2-(4-isobutylphenyl)-5-methylhexan-3-ol shows a greater toxic effect while ibuprofen enhances its growth at lower concentrations. At higher concentrations, ibuprofen shows at least a 2-fold higher toxicity compared to that of its degradation products. However, E. faecalis shows little or no effect upon exposure to these compounds. An induction of the SOS response in E. coli is observed but limited to only ibuprofen and 4-acetylbenzoic acid. In human cell line studies, survival of both HEK293T and HepG2 cell lines is profoundly impaired by the photo-degradation products of (±)- (2R,3R)-2-(4-isobutylphenyl)-5-methylhexan-3-ol, (±)-(2R,3S)-2-(4-isobutylphenyl)-5-methylhexan-3-ol, and (±)-1-(4-(1-hydroxy-2methylpropyl)phenyl)ethan-1-one. In this work, the bioluminescence bacterium, Aliivibrio fischeri, is used as a model to assess environmental impact. Both ibuprofen and its degradation products inhibit the growth of this gram-negative bacteria with the primary compound showing the most significant impact. Overall, our results highlight that some of the degradation products of ibuprofen can be more toxic to human kidney cell line and liver cell line than the parent compound while ibuprofen can be more toxic to human gut microbiota and A. fischeri than ibuprofen degradation products., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: LexA protein is a transcriptional repressor which regulates the expression of more than 60 genes belonging to the SOS global regulatory network activated by damages to bacterial DNA. Considering its role in bacteria, LexA represents a key target to counteract bacterial resistance: the possibility to modulate SOS response through the inhibition of LexA autoproteolysis may lead to reduced drug susceptibility and acquisition of resistance in bacteria. In our study we investigated boron-containing compounds as potential inhibitors of LexA self-cleavage., Main Methods: The inhibition of LexA self-cleavage was evaluated by following the variation of the first-order rate constant by LC-MS at several concentrations of inhibitors. In silico analysis was applied to predict the binding orientations assumed by the inhibitors in the protein active site, upon covalent binding to the catalytic Ser-119. Bacterial filamentation assay was used to confirm the ability of (3-aminophenyl)boronic acid to interfere with SOS induced activation., Key Findings: Boron-containing compounds act as inhibitors of LexA self-cleavage, as also confirmed by molecular modelling where the compounds interact with the catalytic Ser-119, via the formation of an acyl-enzyme intermediate. A new equation for the description of the inhibition potency in an autoproteolytic enzyme is also disclosed. Bacterial filamentation assays strongly support the interference of our compounds with the SOS response activation through inhibition of septum formation., Significance: The obtained results demonstrated that phenylboronic compounds could be exploited in a hit-to-lead optimization process toward effective LexA self-cleavage inhibitors. They would sustain the rehabilitation in therapy of several dismissed antibiotics., (Copyright © 2019 Elsevier Inc. All rights reserved.)

All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated., (Copyright © 2020 American Society for Microbiology.)

Urinary tract infections are one of the most common bacterial infections and a major public health problem. The predominant causative agents are uropathogenic Escherichia coli. These strains differ from commensal E. coli by the presence of additional horizontally acquired chromosomal material, so-called pathogenicity islands, which encode traits that promote efficient bacterial colonization of the urinary tract. Uropathogenic model strain E. coli 536 possesses six archetypal pathogenicity islands. Bacteriophage-like integrases encoded by each pathogenicity island contribute to island instability. To learn more about the stability of these six islands and factors controlling their stability we constructed two chromosomal reporter systems for the measurement of island loss, as well as for the measurement of the promoter activity of the six island-associated integrase genes at the population level. We used these reporter gene modules to analyze the role of SOS response in island instability. Tests with subinhibitory concentrations of different antibiotics, including many drugs commonly used for the treatment of urinary tract infection, indicated that only SOS response-inducing antibiotics led to an increased loss of islands which was always associated with an increase in the bacterial subpopulations showing high integrase promoter activity. This suggests that island excision correlates with the expression of the cognate integrase. Our reporter modules are valuable tools to investigate the impact of various growth conditions on genome plasticity. Furthermore, a better understanding of the conditions, which affect bacterial integrase expression may open ways to specifically manipulate the genome content of bacterial pathogens by increasing pathogenicity island deletion rates in infecting or colonizing bacteria, thus leading to the attenuation of bacterial pathogens., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Shiga toxins (Stx) induce the symptoms of the life-threatening hemolytic uremic syndrome (HUS) and are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC). The bacterial SOS response is the essential signal for high level production and release of Stx1/2. To assess the potential effectiveness of different antibiotics in blocking SOS response and Stx1/2 production, we constructed a reporter gene based test system that allows for the time-resolved, simultaneous read-out of the SOS response (recAP-cfp) and Stx1 production (stx1::yfp) in EHEC O157:H7 EDL933. We find that cells exposed to inhibitory or subinhibitory concentrations of ciprofloxacin did induce the SOS response, but not when the cells were exposed to rifaximine, azithromycin, tetracycline, gentamicin or ampicillin. Cell lysis and the peak in Stx1 production were substantially delayed with respect to the peak of the SOS response. We used this feature to show that adding transcriptional or translational inhibitors can block Stx1 production even after the SOS response is fully induced. RT-qPCR based tests with other clinically relevant EHEC isolates showed similar results for both Stx1 and Stx2. These observations suggest that transcriptional and translational inhibitors may be of value in treating EHEC infections.

Adherent Invasive Escherichia coli (AIEC) strains recovered from Crohn's disease lesions survive and multiply within macrophages. A reference strain for this pathovar, AIEC LF82, forms microcolonies within phagolysosomes, an environment that prevents commensal E. coli multiplication. Little is known about the LF82 intracellular growth status, and signals leading to macrophage intra-vacuolar multiplication. We used single-cell analysis, genetic dissection and mathematical models to monitor the growth status and cell cycle regulation of intracellular LF82. We found that within macrophages, bacteria may replicate or undergo non-growing phenotypic switches. This switch results from stringent response firing immediately after uptake by macrophages or at later stages, following genotoxic damage and SOS induction during intracellular replication. Importantly, non-growers resist treatment with various antibiotics. Thus, intracellular challenges induce AIEC LF82 phenotypic heterogeneity and non-growing bacteria that could provide a reservoir for antibiotic-tolerant bacteria responsible for relapsing infections., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Gaëlle Demarre was employed by Inovarion at the time of the study.

Bacterial RecA plays an important role in the evaluation of antibiotic resistance via stress-induced DNA repair mechanism; SOS response. Accordingly, RecA became an important therapeutic target against antimicrobial resistance. Small molecule inhibitors of RecA may prevent adaptation of antibiotic resistance mutations and the emergence of antimicrobial resistance. In our study, we observed that phenolic compound p-Coumaric acid as potent RecA inhibitor. It inhibited RecA driven biochemical activities in vitro such as ssDNA binding, strand exchange, ATP hydrolysis and RecA coprotease activity of E. coli and L. monocytogenes RecA proteins. The mechanism underlying such inhibitory action of p-Coumaric acid involves its ability to interfere with the DNA binding domain of RecA protein. p-Coumaric acid also potentiates the activity of ciprofloxacin by inhibiting drastic cell survival of L. monocytogenes as well as filamentation process; the bacteria defensive mechanism in response to DNA damage. Additionally, it also blocked the ciprofloxacin induced RecA expression leading to suppression of SOS response in L. monocytogenes. These findings revealed that p-Coumaric acid is a potent RecA inhibitor, and can be used as an adjuvant to the existing antibiotics which not only enhance the shelf-life but also slow down the emergence of antibiotic resistance in bacteria., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Escherichia coli cells have been observed earlier to display caspase-3-like protease activity (CLP) and undergo programmed cell death (PCD) when exposed to gamma rays. The presence of an irreversible caspase-3 inhibitor (Ac-DEVD-CMK) during irradiation was observed to increase cell survival. Since radiation is known to induce SOS response, the effect of a caspase-3 inhibitor on SOS response was studied in E. coli. UV, a well-known SOS inducer, was used in the current study. Cell filamentation in E. coli upon UV exposure was found to be inhibited by ninefold in the presence of a caspase-3 inhibitor. CLP activity was found to increase twofold in UV-exposed cells than in control (non-treated) cells. Further, bright fluorescing filaments were observed in UV-exposed E. coli cells treated with FITC-DEVD-FMK, a fluorescent dye tagged with an irreversible caspase-3 inhibitor (DEVD-FMK), indicating the presence of active CLP in these cells. Unlike caspase-3 inhibitor, a serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), was not found to improve cell survival after UV treatment. Additionally, a SOS reporter system known as SIVET (selectable in vivo expression technology) assay was performed to reconfirm the inhibition of SOS induction in the presence of caspase-3 inhibitor. SIVET assay is used to quantify cells in which the SOS response has been induced leading to a scorable permanent selectable change in the cell. The SIVET induction frequency (calculated as the ratio of SIVET-induced cells to total viable cells) increased around tenfold in UV-exposed cultures. The induction frequency was found to decrease significantly to 51 from 80% in the cells pre-incubated with caspase-3 inhibitor. On the contrary, caspase-3 inhibitor failed to improve cell survival of E. coli ΔrecA and E. coli DM49 (SOS non-inducible) cells post UV treatment. Summing together, the results indicated a possible linkage of SOS response and the PCD process in E. coli. The findings also indicated that functional SOS pathway is required for CLP-like activity; however, the exact mechanism remains to be elucidated.

Background: Fluoroquinolones such as ciprofloxacin induce the mutagenic SOS response and increase the levels of intracellular reactive oxygen species (ROS). Both the SOS response and ROS increase bacterial mutagenesis, fuelling the emergence of resistant mutants during antibiotic treatment. Recently, there has been growing interest in developing new drugs able to diminish the mutagenic effect of antibiotics by modulating ROS production and the SOS response., Objectives: To test whether physiological concentrations of N-acetylcysteine, a clinically safe antioxidant drug currently used in human therapy, is able to reduce ROS production, SOS induction and mutagenesis in ciprofloxacin-treated bacteria without affecting antibiotic activity., Methods: The Escherichia coli strain IBDS1 and its isogenic mutant deprived of SOS mutagenesis (TLS-) were treated with different concentrations of ciprofloxacin, N-acetylcysteine or both drugs in combination. Relevant parameters such as MICs, growth rates, ROS production, SOS induction, filamentation and antibiotic-induced mutation rates were evaluated., Results: Treatment with N-acetylcysteine reduced intracellular ROS levels (by ∼40%), as well as SOS induction (by up to 75%) and bacterial filamentation caused by subinhibitory concentrations of ciprofloxacin, without affecting ciprofloxacin antibacterial activity. Remarkably, N-acetylcysteine completely abolished SOS-mediated mutagenesis., Conclusions: Collectively, our data strongly support the notion that ROS are a key factor in antibiotic-induced SOS mutagenesis and open the possibility of using N-acetylcysteine in combination with antibiotic therapy to hinder the development of antibiotic resistance., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Antibiotic resistance is considered a global threat to public health. Adaptive resistance mutations and the acquisition of resistance genes by horizontal gene transfer are known to be facilitated by the RecA-dependent SOS response during antibiotic treatment, making RecA inhibitors promising agents for the prevention of antibiotic resistance. However, the impact of RecA inactivation on antibiotic sensitivities remains unclear. Therefore, in this study, we performed high-throughput screening to determine the minimum inhibitory concentrations (MICs) of 217 chemicals, including both antibiotics and toxic chemicals of unknown drug action, in the wild-type MDS42 and the ΔrecA mutant strains of Escherichia coli. The ΔrecA mutant showed increased sensitivity to DNA-damaging agents, DNA replication inhibitors, and chromate stress, as well as to other chemicals, such as S-(2-aminoethyl)-L-cysteine, L-histidine, ruthenium red, D-penicillamine, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), cerulenin, and L-cysteine. Microarray analysis showed further that the ΔrecA mutant had lower expressions of glnK, nac, and glnLG, which encode nitrogen assimilation regulators, as well as amtB, which encodes an ammonium transporter, compared with the wild type. These findings suggest that the ΔrecA mutation affects not only the SOS response but also amino acid metabolism.

Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations by triggering transient differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS). Cipro-induced DNA breaks activate the Escherichia coli SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is limited to a cell subpopulation in which electron transfer together with SOS induce ROS, which activate the sigma-S (σ S ) general-stress response, which allows mutagenic DNA-break repair. When sorted, this small σ S -response-"on" subpopulation produces most antibiotic cross-resistant mutants. A U.S. Food and Drug Administration (FDA)-approved drug prevents σ S induction, specifically inhibiting antibiotic-promoted mutagenesis. Further, SOS-inhibited cell division, which causes multi-chromosome cells, promotes mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a "gambler" cell subpopulation promote resistance evolution without risking most cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The occurrence of damage on bacterial DNA (mediated by antibiotics, for example) is intimately associated with the activation of the SOS system. This pathway is related to the development of mutations that might result in the acquisition and spread of resistance and virulence factors. The inhibition of the SOS response has been highlighted as an emerging resource, in order to reduce the emergence of drug resistance and tolerance. Herein, we evaluated the ability of betulinic acid (BA), a plant-derived triterpenoid, to reduce the activation of the SOS response and its associated phenotypic alterations, induced by ciprofloxacin in Staphylococcus aureus . BA did not show antimicrobial activity against S. aureus (MIC > 5000 µg/mL), however, it (at 100 and 200 µg/mL) was able to reduce the expression of recA induced by ciprofloxacin. This effect was accompanied by an enhancement of the ciprofloxacin antimicrobial action and reduction of S. aureus cell volume (as seen by flow cytometry and fluorescence microscopy). BA could also increase the hyperpolarization of the S. aureus membrane, related to the ciprofloxacin action. Furthermore, BA inhibited the progress of tolerance and the mutagenesis induced by this drug. Taken together, these findings indicate that the betulinic acid is a promising lead molecule in the development helper drugs. These compounds may be able to reduce the S. aureus mutagenicity associated with antibiotic therapies.

Drug tolerance in mycobacterial pathogens is a global concern. Fluoroquinolone (FQ) treatment is widely used for induction of persisters in bacteria. Although FQs that target DNA gyrase are currently used as second-line anti-tuberculosis (TB) drugs, little is known about their impact on Mycobacterium tuberculosis (Mtb) persister formation. Here we explored the CRISPRi-based genetic repression for better understanding the effect of DNA gyrase depletion on Mtb physiology and response to anti-TB drugs. We find that suppression of DNA gyrase drastically affects intra- and extracellular growth of Mtb. Interestingly, gyrase depletion in Mtb leads to activation of RecA/LexA-mediated SOS response and drug tolerance via induction of persister subpopulation. Chemical inhibition of RecA in gyrase-depleted bacteria results in reversion of persister phenotype and better killing by antibiotics. This study provides evidence that inhibition of SOS response can be advantageous in improving the efficacy of anti-TB drugs and shortening the duration of current TB treatment.

Bacterial persisters are able to tolerate high levels of antibiotics and give rise to new populations. Persister tolerance is generally attributed to minimally active cellular processes that prevent antibiotic-induced damage, which has led to the supposition that persister offspring give rise to antibiotic-resistant mutants at comparable rates to normal cells. Using time-lapse microscopy to monitor Escherichia coli populations following ofloxacin treatment, we find that persisters filament extensively and induce impressive SOS responses before returning to a normal appearance. Further, populations derived from fluoroquinolone persisters contain significantly greater quantities of antibiotic-resistant mutants than those from untreated controls. We confirm that resistance is heritable and that the enhancement requires RecA, SOS induction, an opportunity to recover from treatment, and the involvement of error-prone DNA polymerase V (UmuDC). These findings show that fluoroquinolones damage DNA in persisters and that the ensuing SOS response accelerates the development of antibiotic resistance from these survivors.

The ability of fermentates of two potential probiotic strains, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, to lower the SOS response in bacteria was evaluated using Escherichia coli-based Lux biosensors (pRecA-lux) and the tested bacilli fermentates obtained through solid-state fermentation. The SOS response was stimulated by the addition of ciprofloxacine. Preparations of both Bacillus fermentates demonstrated SOS-inhibitory activity (up to 54.21%). The strain КATMIRA1933 was characterized by higher SOS-inhibitory activity. The active components of the fermentates were stable against heating, proteinase, and RNase action.

Periplanetasin-2 is a 15-mer antimicrobial peptide (AMP), derived from the American cockroach Periplaneta americana. This novel AMP exhibits potent antibacterial effect against several pathogenic bacteria including Escherichia coli. Distinct from the targeting cell membrane, which is the general antibacterial mechanism of AMP, periplanetasin-2 exerts its antibacterial activity via apoptosis-like death, which is physiologically and mechanistically similar to eukaryotic apoptosis. E. coli cells treated with periplanetasin-2 showed features of apoptosis in a concentration-dependent manner, such as membrane depolarization, DNA fragmentation, caspase-like protein activation, and phosphatidylserine externalization. These physiological changes were attenuated by pretreatment with the reactive oxygen species (ROS) scavenger, which demonstrates that periplanetasin-2 induced apoptosis-like death in E. coli by generating ROS. In addition, periplantasin-2-induced apoptotic death was affected by SOS response components. In the absence of RecA, an essential protein for SOS response, apoptosis did not occur and the antibacterial activity of periplanetasin-2 was decreased. In contrast, deletion of the SOS gene dinF caused higher ROS accumulation and apoptotic features were detected. Collectively, these results indicate that the antibacterial mechanism of periplanetasin-2 is ROS-induced apoptosis-like death, which requires RecA for proceeding it, and the role of DinF is assumed to contribute to the ROS defense SOS response.

Antimicrobial peptides are one of the promising substitutes for conventional antibiotics. To examine their potential usefulness for controlling bacterial infections, the present study aimed to determine the induction of the self-destruction of Escherichia coli mediated by bac8c, which was modified from template peptide bactenecin. The potential of bac8c (RIWVIWRR) was reflected by the change in physiological following exposure, which led to the accumulation of reactive oxygen species (ROS) with an imbalance in the antioxidant system and damage to the intracellular lipids. In addition, bac8c-mediated death exhibited typical features to bacterial apoptosis-like death (ALD). The pathway was not enhanced in the absence of autocleavage of RecA and LexA, the SOS response proteins. We observed that elevated levels of intracellular ROS induced ALD, but this effect was insufficient before preceding RecA activation and LexA autocleavage. Furthermore, dinF, which encodes a member of the toxic compound extrusion (MATE) family, was evaluated to examine the role of the ALD pathway. dinF protein was required to accelerate the ALD-mediated bac8c in combination with RecA. Taken together, bac8c-mediated cell death underlies the severe SOS response, leading to RecA activation, LexA proteolysis, and dinF overexpression. Since then, overproduction of intracellular ROS facilitated the cell death., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Cells contain a variety of proteins, including those forming a cellular defense system against oxidative stress and DNA damage. In this study, we examined the antibacterial effect of Tat (47-58) peptide, a cell-penetrating peptide, derived from human immunodeficiency virus-1 by focusing on the glutathione and SOS response. First, total glutathione levels were measured to reveal the cellular defense capacity against oxidative stress. Tat (47-58) decreased total glutathione and generated excessive reactive oxygen species, leading to oxidative damage. Second, the expression of RecA protein, which activates the SOS response system in bacterial cells, was detected. Tat (47-58) induced the expression of RecA protein by damaging chromatin and DNA; these actions were confirmed by DAPI and TUNEL staining. Moreover, membrane depolarization and phosphatidylserine exposure, regarded as apoptotic markers, were observed in Tat (47-58)-treated cells. In conclusion, the bactericidal action of Tat (47-58) attenuated the cellular defense systems, including the antioxidant defense system and DNA repair system, and induced apoptosis-like death of Escherichia coli., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The SOS response is a conserved response to DNA damage that is found in Gram-negative and Gram-positive bacteria. When DNA damage is sustained and severe, activation of error-prone DNA polymerases can induce a higher mutation rate than is normally observed, which is called the SOS mutator phenotype or hypermutation. We previously showed that zinc blocked the hypermutation response induced by quinolone antibiotics and mitomycin C in Escherichia coli and Klebsiella pneumoniae . In this study, we demonstrate that zinc blocks the SOS-induced development of chloramphenicol resistance in Enterobacter cloacae . Zinc also blocked the transfer of an extended spectrum beta-lactamase (ESBL) gene from Enterobacter to a susceptible E. coli strain. A zinc ionophore, zinc pyrithione, was ~100-fold more potent than zinc salts in inhibition of ciprofloxacin-induced hypermutation in E. cloacae . Other divalent metals, such as iron and manganese, failed to inhibit these responses. Electrophoretic mobility shift assays (EMSAs) revealed that zinc, but not iron or manganese, blocked the ability of the E. coli RecA protein to bind to single-stranded DNA, an important early step in the recognition of DNA damage in enteric bacteria. This suggests a mechanism for zinc's inhibitory effects on bacterial SOS responses, including hypermutation.

Citric acid and EGCG at their minimum inhibitory concentrations were tested in this study. Logarithmic phase cells of Escherichia coli O157:H7 (ATCC 43895) were exposed to EGCG and citric acid respectively. The results of RT-real time PCR showed that both EGCG and citric acid increased stx2 and oxyR expression and decreased stx1, recA and Q expression. The result of Western blotting for RecA protein further indicated that both EGCG and citric acid decreased RecA production. Both EGCG and citric acid increased the level of intracellular reactive oxygen species and H 2 O 2 production and decreased superoxide dismutase activity. Therefore, EGCG and citric acid might induce stx2 production by increasing oxidative stress response and inhibit stx1 production by suppressing SOS response. In our study, the differential effects of the two antimicrobials were observed. EGCG reduced ompC and rpoS expression. However, citric acid caused an increase in ompC and rpoS expression. Membrane permeability is associated with toxin release. Citric acid increased the outer membrane permeability of E. coli O157:H7. However, the outer membrane of E. coli O157:H7 remained unaffected by EGCG., Significance and Impact of the Study: Shiga toxins are the major virulence factors of Escherichia coli O157:H7. The use of antimicrobials triggering Shiga toxin production is controversial. (-)-epigallocatechin-3-gallate (EGCG) citric acid are often used singly or in combination to prevent micro-organisms in some food products. This study evaluated toxin induction in E. coli O157:H7 in response to EGCG and citric acid and investigated the potential mechanism of action. The findings may contribute to the proper use of EGCG and citric acid as antimicrobials., (© 2018 The Society for Applied Microbiology.)

Evolutionary processes are driven by diverse molecular mechanisms that act in the creation and prevention of mutations. It remains unclear how these mechanisms are regulated because limitations of existing mutation assays have precluded measuring how mutation rates vary over time in single cells. Toward this goal, I detected nascent DNA mismatches as a proxy for mutagenesis and simultaneously followed gene expression dynamics in single Escherichia coli cells using microfluidics. This general microscopy-based approach revealed the real-time dynamics of mutagenesis in response to DNA alkylation damage and antibiotic treatments. It also enabled relating the creation of DNA mismatches to the chronology of the underlying molecular processes. By avoiding population averaging, I discovered cell-to-cell variation in mutagenesis that correlated with heterogeneity in the expression of alternative responses to DNA damage. Pulses of mutagenesis are shown to arise from transient DNA repair deficiency. Constitutive expression of DNA repair pathways and induction of damage tolerance by the SOS response compensate for delays in the activation of inducible DNA repair mechanisms, together providing robustness against the toxic and mutagenic effects of DNA alkylation damage., Competing Interests: The author declares no conflict of interest.

Background: The SOS response is an almost ubiquitous response of cells to genotoxic stresses. The full complement of genes in the SOS regulon for Vibrio species has only been addressed through bioinformatic analyses predicting LexA binding box consensus and in vitro validation. Here, we perform whole transcriptome sequencing from Vibrio cholerae treated with mitomycin C as an SOS inducer to characterize the SOS regulon and other pathways affected by this treatment., Results: Comprehensive transcriptional profiling allowed us to define the full landscape of promoters and transcripts active in V. cholerae. We performed extensive transcription start site (TSS) mapping as well as detection/quantification of the coding and non-coding RNA (ncRNA) repertoire in strain N16961. To improve TSS detection, we developed a new technique to treat RNA extracted from cells grown in various conditions. This allowed for identification of 3078 TSSs with an average 5'UTR of 116 nucleotides, and peak distribution between 16 and 64 nucleotides; as well as 629 ncRNAs. Mitomycin C treatment induced transcription of 737 genes and 28 ncRNAs at least 2 fold, while it repressed 231 genes and 17 ncRNAs. Data analysis revealed that in addition to the core genes known to integrate the SOS regulon, several metabolic pathways were induced. This study allowed for expansion of the Vibrio SOS regulon, as twelve genes (ubiEJB, tatABC, smpA, cep, VC0091, VC1190, VC1369-1370) were found to be co-induced with their adjacent canonical SOS regulon gene(s), through transcriptional read-through. Characterization of UV and mitomycin C susceptibility for mutants of these newly identified SOS regulon genes and other highly induced genes and ncRNAs confirmed their role in DNA damage rescue and protection., Conclusions: We show that genotoxic stress induces a pervasive transcriptional response, affecting almost 20% of the V. cholerae genes. We also demonstrate that the SOS regulon is larger than previously known, and its syntenic organization is conserved among Vibrio species. Furthermore, this specific co-localization is found in other γ-proteobacteria for genes recN-smpA and rmuC-tatABC, suggesting SOS regulon conservation in this phylum. Finally, we comment on the limitations of widespread NGS approaches for identification of all RNA species in bacteria.

It has been demonstrated that deuterium oxide enhances the SOS response of Escherichia coli cells induced by chemical genotoxicants and mutagens. This demonstrates that the heavy nonradioactive hydrogen isotope deuterium can be considered to be a comutagen.

The RecA/LexA axis of the bacterial DNA damage (SOS) response is a promising, yet nontraditional, drug target. The SOS response is initiated upon genotoxic stress, when RecA, a DNA damage sensor, induces LexA, the SOS repressor, to undergo autoproteolysis, thereby derepressing downstream genes that can mediate DNA repair and accelerate mutagenesis. As genetic inhibition of the SOS response sensitizes bacteria to DNA damaging antibiotics and decreases acquired resistance, inhibitors of the RecA/LexA axis could potentiate our current antibiotic arsenal. Compounds targeting RecA, which has many mammalian homologues, have been reported; however, small-molecules targeting LexA autoproteolysis, a reaction unique to the prokaryotic SOS response, have remained elusive. Here, we describe the logistics and accomplishments of an academic-industry partnership formed to pursue inhibitors against the RecA/LexA axis. A novel fluorescence polarization assay reporting on RecA-induced self-cleavage of LexA enabled the screening of 1.8 million compounds. Follow-up studies on select leads show distinct activity patterns in orthogonal assays, including several with activity in cell-based assays reporting on SOS activation. Mechanistic assays demonstrate that we have identified first-in-class small molecules that specifically target the LexA autoproteolysis step in SOS activation. Our efforts establish a realistic example for navigating academic-industry partnerships in pursuit of anti-infective drugs and offer starting points for dedicated lead optimization of SOS inhibitors that could act as adjuvants for current antibiotics.

Natural peptides with antimicrobial activity are extremely diverse, and peptide synthesis technologies make it possible to significantly improve their properties for specific tasks. Here, we investigate the biological properties of the natural peptide indolicidin and the indolicidin-derived novel synthetic peptide In-58. In-58 was generated by replacing all tryptophan residues on phenylalanine in D-configuration; the α-amino group in the main chain also was modified by unsaturated fatty acid. Compared with indolicidin, In-58 is more bactericidal, more resistant to proteinase K, and less toxic to mammalian cells. Using molecular physics approaches, we characterized the action of In-58 on bacterial cells at the cellular level. Also, we have found that studied peptides damage bacterial membranes. Using the Escherichia coli luminescent biosensor strain MG1655 (pcolD'::lux), we investigated the action of indolicidin and In-58 at the subcellular level. At subinhibitory concentrations, indolicidin and In-58 induced an SOS response. Our data suggest that indolicidin damages the DNA, but bacterial membrane perturbation is its principal mode of action. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.)

Fluoroquinolone antibiotics are prescribed for the treatment of Salmonella enterica infections, but resistance to this family of antibiotics is growing. Here we report that loss of the global regulatory protein cyclic AMP (cAMP) receptor protein (CRP) or its allosteric effector, cAMP, reduces susceptibility to fluoroquinolones. A Δ crp mutation was synergistic with the primary fluoroquinolone resistance allele gyrA83 , thus able to contribute to clinically relevant resistance. Decreased susceptibility to fluoroquinolones could be partly explained by decreased expression of the outer membrane porin genes ompA and ompF with a concomitant increase in the expression of the ciprofloxacin resistance efflux pump gene acrB in Δ crp cells. Expression of gyrAB , which encode the DNA supercoiling enzyme GyrAB, which is blocked by fluoroquinolones, and expression of topA , which encodes the dominant supercoiling-relaxing enzyme topoisomerase I, were unchanged in Δ crp cells. Yet Δ crp cells maintained a more relaxed state of DNA supercoiling, correlating with an observed increase in topoisomerase IV ( parCE ) expression. Surprisingly, the Δ crp mutation had the unanticipated effect of enhancing fitness in the presence of fluoroquinolone antibiotics, which can be explained by the observation that exposure of Δ crp cells to ciprofloxacin had the counterintuitive effect of restoring wild-type levels of DNA supercoiling. Consistent with this, Δ crp cells did not become elongated or induce the SOS response when challenged with ciprofloxacin. These findings implicate the combined action of multiple drug resistance mechanisms in Δ crp cells: reduced permeability and elevated efflux of fluoroquinolones coupled with a relaxed DNA supercoiling state that buffers cells against GyrAB inhibition by fluoroquinolones., (Copyright © 2017 American Society for Microbiology.)

The RecX protein, a very active natural RecA protein inhibitor, can completely disassemble RecA filaments at nanomolar concentrations that are two to three orders of magnitude lower than that of RecA protein. Based on the structure of RecX protein complex with the presynaptic RecA filament, we designed a short first in class α-helical peptide that both inhibits RecA protein activities in vitro and blocks the bacterial SOS-response in vivo. The peptide was designed using SEQOPT, a novel method for global sequence optimization of protein α-helices. SEQOPT produces artificial peptide sequences containing only 20 natural amino acids with the maximum possible conformational stability at a given pH, ionic strength, temperature, peptide solubility. It also accounts for restrictions due to known amino acid residues involved in stabilization of protein complexes under consideration. The results indicate that a few key intermolecular interactions inside the RecA protein presynaptic complex are enough to reproduce the main features of the RecX protein mechanism of action. Since the SOS-response provides a major mechanism of bacterial adaptation to antibiotics, these results open new ways for the development of antibiotic co-therapy that would not cause bacterial resistance., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The widespread use of organic ultraviolet (UV) filters in personal care products raises concerns about their potentially hazardous effects on human and ecosystem health. In this study, the toxicities of four commonly used benzophenones (BPs) UV filters including benzophenone (BP), 2-Hydroxybenzophenone (2HB), 2-Hydroxy-4-methoxybenzophenone (BP3), and 2-Hydroxy-4-methoxybenzophenone-5-sulfonicacid (BP4) in water were assayed in vitro using Vibrio fischeri, SOS/umu assay, and yeast estrogen screen (YES) assay, as well as in vivo using zebrafish larvae. The results showed that the luminescent bacteria toxicity, expressed as logEC 50 , increased with the lipophilicity (logKow) of BPs UV filters. Especially, since 2HB, BP3 and BP4 had different substituent groups, namely -OH, -OCH 3 and -SO 3 H, respectively, these substituent functional groups had a major contribution to the lipophilicity and acute toxicity of these BPs. Similar tendency was observed for the genotoxicity, expressed as the value of induction ratio=1.5. Moreover, all the target BPs UV filters showed estrogenic activity, but no significant influences of lipophilicity on the estrogenicity were observed, with BP3 having the weakest estrogenic efficiency in vitro. Although BP3 displayed no noticeable adverse effects in any in vitro assays, multiple hormonal activities were observed in zebrafish larvae including estrogenicity, anti-estrogenicity and anti-androgenicity by regulating the expression of target genes. The results indicated potential hazardous effects of BPs UV filters and the importance of the combination of toxicological evaluation methods including in vitro and in vivo assays., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown., Purpose: Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens., Methods: The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated., Results: Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC 50 ) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein., Conclusion: In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Zinc inhibits the virulence of diarrheagenic E. coli by inducing the envelope stress response and inhibiting the SOS response. The SOS response is triggered by damage to bacterial DNA. In Shiga-toxigenic E. coli, the SOS response strongly induces the production of Shiga toxins (Stx) and of the bacteriophages that encode the Stx genes. In E. coli, induction of the SOS response is accompanied by a higher mutation rate, called the mutator response, caused by a shift to error-prone DNA polymerases when DNA damage is too severe to be repaired by canonical DNA polymerases. Since zinc inhibited the other aspects of the SOS response, we hypothesized that zinc would also inhibit the mutator response, also known as hypermutation. We explored various different experimental paradigms to induce hypermutation triggered by the SOS response, and found that hypermutation was induced not just by classical inducers such as mitomycin C and the quinolone antibiotics, but also by antiviral drugs such as zidovudine and anti-cancer drugs such as 5-fluorouracil, 6-mercaptopurine, and azacytidine. Zinc salts inhibited the SOS response and the hypermutator phenomenon in E. coli as well as in Klebsiella pneumoniae, and was more effective in inhibiting the SOS response than other metals. We then attempted to determine the mechanism by which zinc, applied externally in the medium, inhibits hypermutation. Our results show that zinc interferes with the actions of RecA, and protects LexA from RecA-mediated cleavage, an early step in initiation of the SOS response. The SOS response may play a role in the development of antibiotic resistance and the effect of zinc suggests ways to prevent it.

Aberrant DNA replication is a major source of the mutations and chromosomal rearrangements associated with pathological disorders. In bacteria, several different DNA lesions are repaired by homologous recombination, a process that involves sister chromatid pairing. Previous work in Escherichia coli has demonstrated that sister chromatid interactions (SCIs) mediated by topological links termed precatenanes, are controlled by topoisomerase IV. In the present work, we demonstrate that during the repair of mitomycin C-induced lesions, topological links are rapidly substituted by an SOS-induced sister chromatid cohesion process involving the RecN protein. The loss of SCIs and viability defects observed in the absence of RecN were compensated by alterations in topoisomerase IV, suggesting that the main role of RecN during DNA repair is to promote contacts between sister chromatids. RecN also modulates whole chromosome organization and RecA dynamics suggesting that SCIs significantly contribute to the repair of DNA double-strand breaks (DSBs).