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Objectives: Subinhibitory concentration of antibiotics in the environment is an important risk factor for the horizontal transmission of antibiotic resistance genes (ARGs). The signaling mechanism of resistance gene transmission remains unknown. The aim of this study was to investigate whether indole could be used as a molecular signal to help the spread of ARGs under the stress of subinhibitory concentrations of antibiotics. Methods: The effect of indole on conjugation frequency was investigated through a conjugation test, and its effect on the Type IV secretion system and pili gene expression of E. coli was observed. Meanwhile, we were investigating the trend of changes in indole regulatory factors ibpA, tnaA, and concentration pumps. Subsequently, we predicted the receptors that specifically bind to indole. Finally, our study focused on elucidating the regulatory mechanism of indole synthesis. Results: Conjugate frequency was significantly increased under 1/5MIC concentration cefotaxime stress. The transferred ARGs were blaCTX-M and foxA. The mobile plasmid was IncY or IncI2. Meanwhile, the concentration of endogenous indole was also significantly increased. And, surprisingly, inhibition of endogenous indole production resulted in a significant decrease in conjugate frequency. However, the conjugate frequency increased once again when the strains reacquired the exogenous indole. Furthermore, the fluctuation trends of indole‐regulated factor (ibpA, tnaA) mRNA and concentration pumps (acrEF, mtr) mRNA consistently with that of indole. Then, we found that the receptors of indole may be four targets of TCSs: CreC, PhoB, AtoC, and UhpA. More than that, when strains retrieved the exogenous indole again, the mRNA levels of T4SS (virB2, virB6, and virD4) and pppA (coding Pili precursor) genes significantly increased. This indicates that there is a close relationship between indole and conjugated channels, which are necessary for horizontal transfer of genetic material. And then, the trends of indole and tnaA mRNA were consistent with that of ibpA (one of SOS response). So, this result confirmed that indole was regulated by SOS response under subinhibitory concentrations of antibiotics. Conclusions: It is always known that subinhibitory concentrations of antibiotics stimulate an SOS response in E. coli, which helps in the horizontal spread of ARGs by modulating indole. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract The recA gene, encoding Recombinase A (RecA) is one of three Mycobacterium tuberculosis (Mtb) genes encoding an in-frame intervening protein sequence (intein) that must splice out of precursor host protein to produce functional protein. Ongoing debate about whether inteins function solely as selfish genetic elements or benefit their host cells requires understanding of interplay between inteins and their hosts. We measured environmental effects on native RecA intein splicing within Mtb using a combination of western blots and promoter reporter assays. RecA splicing was stimulated in bacteria exposed to DNA damaging agents or by treatment with copper in hypoxic, but not normoxic, conditions. Spliced RecA was processed by the Mtb proteasome, while free intein was degraded efficiently by other unknown mechanisms. Unspliced precursor protein was not observed within Mtb despite its accumulation during ectopic expression of Mtb recA within E. coli. Surprisingly, Mtb produced free N-extein in some conditions, and ectopic expression of Mtb N-extein activated LexA in E. coli. These results demonstrate that the bacterial environment greatly impacts RecA splicing in Mtb, underscoring the importance of studying intein splicing in native host environments and raising the exciting possibility of intein splicing as a novel regulatory mechanism in Mtb.

Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexAregulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains. Materials and Methods: Five clinical isolates of E. coli carrying different chromosomally-and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC-MS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software. Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins. Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance. [ABSTRACT FROM AUTHOR]

The DNA damage inducible SOS response in bacteria serves to increase survival of the species at the cost of mutagenesis. The SOS response first initiates errorfree repair followed by error-prone repair. Here, we have employed a multiomics approach to elucidate the temporal coordination of the SOS response. Escherichia coli was grown in batch cultivation in bioreactors to ensure highly controlled conditions, and a low dose of the antibiotic ciprofloxacin was used to activate the SOS response while avoiding extensive cell death. Our results show that expression of genes involved in error-free and error-prone repair were both induced shortly after DNA damage, thus, challenging the established perception that the expression of error-prone repair genes is delayed. By combining transcriptomics and a sub-proteomics approach termed signalomics, we found that the temporal segregation of error-free and error-prone repair is primarily regulated after transcription, supporting the current literature. Furthermore, the heterology index (i.e., the binding affinity of LexA to the SOS box) was correlated to the maximum increase in gene expression and not to the time of induction of SOS genes. Finally, quantification of metabolites revealed increasing pyrimidine pools as a late feature of the SOS response. Our results elucidate how the SOS response is coordinated, showing a rapid transcriptional response and temporal regulation of mutagenesis on the protein and metabolite levels. [ABSTRACT FROM AUTHOR]

The adaptive survival mechanisms of bacterial pathogens under host-induced stress are crucial for understanding pathogenesis. Recently, Uppalapati et al. revealed a unique dual function of the Gifsy-1 prophage terminase in Salmonella enterica: it acts as a transfer ribonuclease (tRNase) under oxidative stress. The Gifsy-1 prophage terminase targets and fragments tRNALeu to halt translation and temporarily impairs bacterial growth when exposed to high levels of ROS generated by the host immune cells. This response not only preserves genomic integrity by facilitating DNA repair but also inhibits prophage mobilization, thereby aiding in bacterial survival within vertebrate hosts. This study highlights a novel intersection between phage biology and bacterial adaptive strategies.

The adaptation of Salmonella enterica serovar Typhimurium to stress conditions involves expression of genes within the regulon of the alternative sigma factor RpoN (σ54). RpoN-dependent transcription requires an activated bacterial enhancer binding protein (bEBP) that hydrolyzes ATP to remodel the RpoN-holoenzyme-promoter complex for transcription initiation. The bEBP RtcR in S. Typhimurium strain 14028s is activated by genotoxic stress to direct RpoN-dependent expression of the RNA repair operon rsr-yrlBA-rtcBA. The molecular signal for RtcR activation is an oligoribonucleotide with a 3'-terminal 2',3'-cyclic phosphate. We show in S. Typhimurium 14028s that the molecular signal is not a direct product of nucleic acid damage, but signal generation is dependent on a RecA-controlled SOS-response pathway, specifically, induction of prophage Gifsy-1. A genome-wide mutant screen and utilization of Gifsy prophage-cured strains indicated that the nucleoid-associated protein Fis and the Gifsy-1 prophage significantly impact RtcR activation. Directed-deletion analysis and genetic mapping by transduction demonstrated that a three-gene region (STM14_3218-3220) in Gifsy-1, which is variable between S. Typhimurium strains, is required for RtcR activation in strain 14028s and that the absence of STM14_3218-3220 in the Gifsy-1 prophages of S. Typhimurium strains LT2 and 4/74, which renders these strains unable to activate RtcR during genotoxic stress, can be rescued by complementation in cis by the region encompassing STM14_3218-3220. Thus, even though RtcR and the RNA repair operon are highly conserved in Salmonella enterica serovars, RtcR-dependent expression of the RNA repair operon in S. Typhimurium is controlled by a variable region of a prophage present in only some strains. IMPORTANCE The transcriptional activator RtcR and the RNA repair proteins whose expression it regulates, RtcA and RtcB, are widely conserved in Proteobacteria. In Salmonella Typhimurium 14028s, genotoxic stress activates RtcR to direct RpoN-dependent expression of the rsr-yrlBA-rtcBA operon. This work identifies key elements of a RecA-dependent pathway that generates the signal for RtcR activation in strain 14028s. This signaling pathway requires the presence of a specific region within the prophage Gifsy-1, yet this region is absent in most other wild-type Salmonella strains. Thus, we show that the activity of a widely conserved regulatory protein can be controlled by prophages with narrow phylogenetic distributions. This work highlights an underappreciated phenomenon where bacterial physiological functions are altered due to genetic rearrangement of prophages.

Our laboratory recently reported that induction of the SOS response, triggered by SOS-inducing drugs, was accompanied by a large release of DNA from enteric bacteria. The SOS response release had not previously been reported to include release of extracellular DNA from bacterial cells. We followed up on those observations in this current study and found that not just double-stranded DNA was being released, but also single-stranded DNA, RNA, and protein. SOS-inducing drugs also triggered formation of biofilm at the air–fluid interface on glass, and the biofilms contained DNA. We extended our study to test whether inhibitors of the SOS response would block DNA release and found that SOS inhibitors, including zinc salts, nitric oxide donors, and dequalinium, inhibited SOS-induced DNA release. The understanding that SOS-induced DNA release is associated with formation of biofilms increases our appreciation of the role of the SOS response in pathogenesis, as well as in emergence of new antibiotic resistance. Our findings with SOS inhibitors also suggest that regimens might be devised that could block the deleterious effects of the SOS response, at least temporarily, when this is desired. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) has been recognized as one of the most important crises affecting global human health in the 21st century. Tigecycline is one of the last resort antibiotics for treating severe infections caused by multi-drug resistant Enterobacteriaceae. However, the mobile resistance gene tet(X4), which could mediate high-level tigecycline resistance, was discovered in 2019. The outer membrane vesicle (OMV) has been recognized as a new route for horizontal gene transfer; antimicrobial resistant bacteria also have the ability to secret OMVs, while little is known about the impact of antibiotics on the secretion and characteristics of OMVs from tigecycline resistant bacteria till now. This study aimed to investigate the effects of antibiotics on the production and traits of a tigecycline resistant Escherichia coli strain of 47EC. The results showed that sub-inhibitory (1/2 MIC or 1/4 MIC) concentrations of gentamicin, meropenem, ceftazidime, chloramphenicol, tigecycline, ciprofloxacin, polymycin, rifaximin and mitomycin C could significantly increase the secretion of OMVs (0.713 ± 0.05~6.333 ± 0.15 mg/mL) from E. coli 47EC compared to the respective untreated control (0.709 ± 0.03 mg/mL). In addition, the particle sizes of OMVs were generally larger, and the zeta potential were lower in the antibiotics-treated groups than those of the antibiotic-free group. The copy numbers of the tigecycline resistance gene of tet(X4) in the OMVs of most antimicrobial-treated groups were higher than that of the control group. Moreover, transcriptome analysis on ciprofloxacin-treated E. coli 47EC indicated that the SOS response and prophage activation might participate in the ciprofloxacin-induced OMV formation. In conclusion, the clinical application of antibiotics in treating bacterial infections, especially multi-drug resistant bacteria, might lead to the increased secretion of bacterial OMVs and the enrichment of antimicrobial-resistant genes in the OMVs. [ABSTRACT FROM AUTHOR]

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response. [ABSTRACT FROM AUTHOR]

Bacteria have evolved numerous regulatory pathways to survive in changing environments. The SOS response is an inducible DNA damage repair system that plays an indispensable role in bacterial adaptation and pathogenesis. Here we report a discovery of the previously uncharacterized protein Lmo0946 as an SOS response interfering factor (Sif) in the human pathogen Listeria monocytogenes. Functional genetic studies demonstrated that sif is indispensable for normal growth of L. monocytogenes in stress-free as well as multi-stress conditions, and sif contributes to susceptibility to β-lactam antibiotics, biofilm formation and virulence. Absence of Sif promoted the SOS response and elevated expression of mobilome genes accompanied by mobilization of the A118 prophage and ICELm-1 mobile genetic elements (MGEs). These changes were found to be associated with decreased expression of general stress response genes from the σB regulon as well as virulence genes, including the PrfA regulon. Together, this study uncovers an unexpected role of a previously uncharacterized factor, Sif, as an inhibitor of the SOS response in L. monocytogenes. [ABSTRACT FROM AUTHOR]

Introduction/objectiveSuppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains.Materials and methodsFive clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC–MS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software.ResultsThe proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins.ConclusionHigh genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance.

It was shown for the first time that preliminary deuteration of Esherichia coli bacteria with deuterium oxide (D2O) at concentrations from 5 to 10% in a medium leads to an enhancement of the SOS response in the biosensors E. coli MG1655 (pColD::lux) and E. coli MG1655 (pRecA::lux) induced by UV at a dose of 12 J/m2. The biosensors used in this work contain hybrid plasmids carrying the luxCDABE operon of the Photorhabdus luminescens photobacterium, placed under the control of the cda (colD) and recA gene promoters. The induction of genotoxic factors in biosensors indicates the activation of the corresponding E. coli SOS system of gene expression. A simultaneous study of the intensity of luminescence and the cell viability of the E. coli MG1655 biosensor (pCol-lux) bacteria showed that UV irradiation at a dose of 12 J/m2 increases the intensity of luminescence from 2111.1 in a medium without D2O to 5030.3 RLU for 107 viable cells in a medium with D2O. The intensity of luminescence of the biosensor was 2.5 times higher in the medium with deuterium than without it. [ABSTRACT FROM AUTHOR]

Expansion microscopy (ExM) is a sample preparation technique which allows to achieve improved visualization of biological structures based on the physical expansion of the sample. This method is used in combination with traditional light microscopy and allows to achieve visualization of biological structures with higher resolution, without the use of complex technical devices typical for super-resolution microscopy. Unlike the methods of super-resolution microscopy, expansion microscopy does not make it possible to overcome the diffraction limit; however, the observed effect can be considered equal to an increase in the spatial resolution. The relative simplicity of the method and low requirements for the microscope used, have made expansion microscopy a fairly popular method to visualize various biological structures recently. This paper describes the use of expansion microscopy to visualize DNA and structures formed by the FtsZ protein in Escherichia coli cells during the SOS response. The results of the work confirm the previously obtained data that the FtsZ protein in cells in the state of the SOS response is unevenly distributed. The protocol used in this work for visualization of E. coli cells using the expansion microscopy method can be used in the future to study the internal structures of other cells, both bacterial and eukaryotic. [ABSTRACT FROM AUTHOR]

Cross-interface spread and aggregation of antibiotic resistance genes (ARGs) at human-animal-environment has become a major problem in public health and environmental ecosystems, ARGs and mobile genetic elements (MGEs) carrying ARGs have been recognized as emerging environmental contaminants. Heavy metal-mediated horizontal transfer of ARGs increases the potential risk of ARG enrichment in indigenous microorganisms. Currently, the study of horizontal transfer of ARGs mediated by heavy metals and other emerging contaminants has become a hot research topic in the field of environment and its related disciplines. The review outlines the effects of heavy metals as co-selectants on the conjugation of ARGs in the environment, focusing on the mechanisms associated with the increased conjugation of ARGs induced by heavy metals from a molecular biological perspective. The heavy metal-mediated conjugation of ARGs was the main reason for the increased abundance of ARGs, and the heavy metal-induced changes in conjugation frequency were mainly related to their concentrations, which is consistent with the hormesis. Related regulatory mechanisms include regulation of reactive oxygen species (ROS) production and initiation of SOS responses, enhancement of cell membrane permeability, and effects on conjugation transfer-related gene expression and ATP synthesis. Elucidation of the mechanisms that enhance the horizontal transfer of ARGs in contaminated environments with heavy metals (or compound heavy metals) and antibiotics under different environmental conditions is a key direction for future research. It can provide some reference for the study of heavy metal-mediated environmental behavior of ARGs. [ABSTRACT FROM AUTHOR]

The DNA damage inducible SOS response in bacteria serves to increase survival of the species at the cost of mutagenesis. The SOS response first initiates error-free repair followed by error-prone repair. Here, we have employed a multi-omics approach to elucidate the temporal coordination of the SOS response. Escherichia coli was grown in batch cultivation in bioreactors to ensure highly controlled conditions, and a low dose of the antibiotic ciprofloxacin was used to activate the SOS response while avoiding extensive cell death. Our results show that expression of genes involved in error-free and error-prone repair were both induced shortly after DNA damage, thus, challenging the established perception that the expression of error-prone repair genes is delayed. By combining transcriptomics and a sub-proteomics approach termed signalomics, we found that the temporal segregation of error-free and error-prone repair is primarily regulated after transcription, supporting the current literature. Furthermore, the heterology index (i.e., the binding affinity of LexA to the SOS box) was correlated to the maximum increase in gene expression and not to the time of induction of SOS genes. Finally, quantification of metabolites revealed increasing pyrimidine pools as a late feature of the SOS response. Our results elucidate how the SOS response is coordinated, showing a rapid transcriptional response and temporal regulation of mutagenesis on the protein and metabolite levels.

As an effective non-thermal sterilization method, ultrasound remains at the level of passive bacterial death despite the initial understanding of its sterilization mechanism. Here, we present the perspective that bacteria can choose to actively enter an apoptosis-like death state in response to external ultrasonic stress. In this study, Vibrio parahaemolyticus exhibited apoptotic markers such as phosphatidylserine ectropion and activated caspases when subjected to ultrasound stress. Additionally, the accumulation of reactive oxygen species (ROS) and enhanced calcium signaling were observed. Further transcriptomic analysis was conducted to investigate the regulatory mechanism of the SOS response in Vibrio parahaemolyticus during an apoptosis-like state. The results showed that the genes encoding the citrate cycle were down-regulated in Vibrio parahaemolyticus cells adapted to ultrasonic stress, leading to an apoptosis-like state and a decrease in production capacity and ability to catabolize carbon dioxide. Furthermore, the level of oxidized glutathione increased, suggesting that the bacteria were engaged in various anti-oxidative stress responses, ultimately leading to apoptosis. Moreover, the ultrasound field activated the regulatory factor CsrA, which facilitates stress survival as cells transition from rapid growth to an apoptotic state through a stringent response and catabolic inhibition system. Parallel reaction monitoring (PRM) revealed that the expression of certain key SOS proteins in Vibrio parahaemolyticus was up-regulated following ultrasound treatment, resulting in a gradual adaptation of the cells to external stress and ultimately leading to active cell death. In conclusion, the biological lethal effect of ultrasound treatment is not solely a mechanical cell necrosis process as traditionally viewed, but also a programmed cell death process regulated by cellular adaptation. This enriched the biological effect pathway of ultrasound sterilization.

Bacteria have evolved numerous regulatory pathways to survive in changing environments. The SOS response is an inducible DNA damage repair system that plays an indispensable role in bacterial adaptation and pathogenesis. Here we report a discovery of the previously uncharacterized protein Lmo0946 as an SOS response interfering factor (Sif) in the human pathogen Listeria monocytogenes. Functional genetic studies demonstrated that sif is indispensable for normal growth of L. monocytogenes in stress-free as well as multi-stress conditions, and sif contributes to susceptibility to β-lactam antibiotics, biofilm formation and virulence. Absence of Sif promoted the SOS response and elevated expression of mobilome genes accompanied by mobilization of the A118 prophage and ICELm-1 mobile genetic elements (MGEs). These changes were found to be associated with decreased expression of general stress response genes from the σB regulon as well as virulence genes, including the PrfA regulon. Together, this study uncovers an unexpected role of a previously uncharacterized factor, Sif, as an inhibitor of the SOS response in L. monocytogenes.

Kai Tang, Heng Zhao Fujian Provincial Key Laboratory of Innate Immune Biology, Fujian Normal University, Fujian, People’s Republic of ChinaCorrespondence: Heng Zhao, Fujian Provincial Key Laboratory of Innate Immune Biology, Fujian Normal University, Fujian, People’s Republic of China, Tel +86-17689970104, Email QBX20200151@yjs.fjnu.edu.cnAbstract: The clinical application of quinolone antibiotics is particularly extensive. In addition to their high efficiency in infectious diseases, the treatment process brings multiple hidden dangers or side effects. In this regard, drug resistance becomes a major challenge and is almost unavoidable in the clinical application of quinolones. Both genetic and phenotypic variations contribute to bacterial survival resistance under antibiotic therapy. This review is focusing on the drug discovery history, compound structure, and bactericidal mechanism of quinolone antibiotics. Recent studies bring a more in-depth insight into the research progress of quinolone antibiotics in the causes of death, drug resistance formation, and closely related SOS response after disease treatment at this stage. Combined with the latest clinical studies, we summarize the clinical application of quinolone antibiotics and further lay a theoretical foundation for the mechanism study of resistant or sensitive bacteria in response to quinolone treatment.Keywords: quinolone antibiotics, disease treatment, SOS response, lethality, bacteria

Antimicrobial resistance (AMR) has been recognized as one of the most important crises affecting global human health in the 21st century. Tigecycline is one of the last resort antibiotics for treating severe infections caused by multi-drug resistant Enterobacteriaceae. However, the mobile resistance gene tet(X4), which could mediate high-level tigecycline resistance, was discovered in 2019. The outer membrane vesicle (OMV) has been recognized as a new route for horizontal gene transfer; antimicrobial resistant bacteria also have the ability to secret OMVs, while little is known about the impact of antibiotics on the secretion and characteristics of OMVs from tigecycline resistant bacteria till now. This study aimed to investigate the effects of antibiotics on the production and traits of a tigecycline resistant Escherichia coli strain of 47EC. The results showed that sub-inhibitory (1/2 MIC or 1/4 MIC) concentrations of gentamicin, meropenem, ceftazidime, chloramphenicol, tigecycline, ciprofloxacin, polymycin, rifaximin and mitomycin C could significantly increase the secretion of OMVs (0.713 ± 0.05~6.333 ± 0.15 mg/mL) from E. coli 47EC compared to the respective untreated control (0.709 ± 0.03 mg/mL). In addition, the particle sizes of OMVs were generally larger, and the zeta potential were lower in the antibiotics-treated groups than those of the antibiotic-free group. The copy numbers of the tigecycline resistance gene of tet(X4) in the OMVs of most antimicrobial-treated groups were higher than that of the control group. Moreover, transcriptome analysis on ciprofloxacin-treated E. coli 47EC indicated that the SOS response and prophage activation might participate in the ciprofloxacin-induced OMV formation. In conclusion, the clinical application of antibiotics in treating bacterial infections, especially multi-drug resistant bacteria, might lead to the increased secretion of bacterial OMVs and the enrichment of antimicrobial-resistant genes in the OMVs.

Our laboratory recently reported that induction of the SOS response, triggered by SOS-inducing drugs, was accompanied by a large release of DNA from enteric bacteria. The SOS response release had not previously been reported to include release of extracellular DNA from bacterial cells. We followed up on those observations in this current study and found that not just double-stranded DNA was being released, but also single-stranded DNA, RNA, and protein. SOS-inducing drugs also triggered formation of biofilm at the air–fluid interface on glass, and the biofilms contained DNA. We extended our study to test whether inhibitors of the SOS response would block DNA release and found that SOS inhibitors, including zinc salts, nitric oxide donors, and dequalinium, inhibited SOS-induced DNA release. The understanding that SOS-induced DNA release is associated with formation of biofilms increases our appreciation of the role of the SOS response in pathogenesis, as well as in emergence of new antibiotic resistance. Our findings with SOS inhibitors also suggest that regimens might be devised that could block the deleterious effects of the SOS response, at least temporarily, when this is desired.

The molecular and ecological factors shaping horizontal gene transfer (HGT) via natural transformation in microbial communities are largely unknown, which is critical for understanding the emergence of antibiotic‐resistant pathogens. We investigate key factors shaping HGT in a microbial co‐culture by quantifying extracellular DNA release, species growth, and HGT efficiency over time. In the co‐culture, plasmid release and HGT efficiency are significantly enhanced than in the respective monocultures. The donor is a key determinant of HGT efficiency as plasmids induce the SOS response, enter a multimerized state, and are released in high concentrations, enabling efficient HGT. However, HGT is reduced in response to high donor lysis rates. HGT is independent of the donor viability state as both live and dead cells transfer the plasmid with high efficiency. In sum, plasmid HGT via natural transformation depends on the interplay of plasmid properties, donor stress responses and lysis rates, and interspecies interactions. Synopsis: Plasmid multimerization, donor stress response and donor lysis rate are major determinants of horizontal gene transfer via natural competence in a microbial co‐culture.Horizontal gene transfer (HGT) via natural competence is highly efficient in a co‐culture of the recipient B. subtilis and donor E. coli and is modulated by initial species abundance.The presence of RecA in the donor species substantially enhances HGT efficiency in the co‐culture by enhancing plasmid multimerization and eDNA release via the SOS response.Efficient HGT can occur for both intact dead and live donor cells.High donor cell lysis can reduce HGT efficiency in the co‐culture. [ABSTRACT FROM AUTHOR]

In response to DNA damage, bacterial RecA protein forms filaments with the assistance of DinI protein. The RecA filaments stimulate the autocleavage of LexA, the repressor of more than 50 SOS genes, and activate the SOS response. During the late phase of SOS response, the RecA filaments stimulate the autocleavage of UmuD and λ repressor CI, leading to mutagenic repair and lytic cycle, respectively. Here, we determined the cryo-electron microscopy structures of Escherichia coli RecA filaments in complex with DinI, LexA, UmuD, and λCI by helical reconstruction. The structures reveal that LexA and UmuD dimers bind in the filament groove and cleave in an intramolecular and an intermolecular manner, respectively, while λCI binds deeply in the filament groove as a monomer. Despite their distinct folds and oligomeric states, all RecA filament binders recognize the same conserved protein features in the filament groove. The SOS response in bacteria can lead to mutagenesis and antimicrobial resistance, and our study paves the way for rational drug design targeting the bacterial SOS response. [ABSTRACT FROM AUTHOR]

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Infection with Mycobacterium tuberculosis continues to cause substantial human mortality, in part because of the emergence of antimicrobial resistance. Antimicrobial resistance in tuberculosis is solely the result of chromosomal mutations that modify drug activators or targets, yet the mechanisms controlling the mycobacterial DNA-damage response (DDR) remain incompletely defined. Here, we identify RecA serine 207 as a multifunctional signaling hub that controls the DDR in mycobacteria. RecA S207 is phosphorylated after DNA damage, which suppresses the emergence of antibiotic resistance by selectively inhibiting the LexA coprotease function of RecA without affecting its ATPase or strand exchange functions. Additionally, RecA associates with the cytoplasmic membrane during the mycobacterial DDR, where cardiolipin can specifically inhibit the LexA coprotease function of unmodified, but not S207 phosphorylated, RecA. These findings reveal that RecA S207 controls mutagenesis and antibiotic resistance in mycobacteria through phosphorylation and cardiolipin-mediated inhibition of RecA coprotease function.

Abstract The molecular and ecological factors shaping horizontal gene transfer (HGT) via natural transformation in microbial communities are largely unknown, which is critical for understanding the emergence of antibiotic‐resistant pathogens. We investigate key factors shaping HGT in a microbial co‐culture by quantifying extracellular DNA release, species growth, and HGT efficiency over time. In the co‐culture, plasmid release and HGT efficiency are significantly enhanced than in the respective monocultures. The donor is a key determinant of HGT efficiency as plasmids induce the SOS response, enter a multimerized state, and are released in high concentrations, enabling efficient HGT. However, HGT is reduced in response to high donor lysis rates. HGT is independent of the donor viability state as both live and dead cells transfer the plasmid with high efficiency. In sum, plasmid HGT via natural transformation depends on the interplay of plasmid properties, donor stress responses and lysis rates, and interspecies interactions.

The increase in antibiotic resistance represents a major global challenge for our health systems and calls for alternative treatment options, such as antimicrobial light-based therapies. Blue light has shown promising results regarding the inactivation of a variety of microorganisms; however, most often, antimicrobial blue light (aBL) therapy is performed using wavelengths close to the UV range. Here we investigated whether inactivation was possible using blue light with a wavelength of 475 nm. Both Gram-positive and -negative bacterial strains were treated with blue light with fluences of 7.5–45 J/cm2. Interestingly, only some bacterial strains were susceptible to 475 nm blue light, which was associated with the lack of RecA, i.e., a fully functional DNA repair mechanism. We demonstrated that the insertion of the gene recA reduced the susceptibility of otherwise responsive bacterial strains, indicating a protective mechanism conveyed by the bacterial SOS response. However, mitigating this pathway via three known RecA inhibiting molecules (ZnAc, curcumin, and Fe(III)-PcTs) did not result in an increase in bactericidal action. Nonetheless, creating synergistic effects by combining a multitarget therapy, such as aBL, with an RecA targeting treatment could be a promising strategy to overcome the dilemma of antibiotic resistance in the future. [ABSTRACT FROM AUTHOR]

Abstract Bacteria can evolve rapidly by acquiring new traits such as virulence, metabolic properties, and most importantly, antimicrobial resistance, through horizontal gene transfer (HGT). Multidrug resistance in bacteria, especially in Gram-negative organisms, has become a global public health threat often through the spread of mobile genetic elements. Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact. Conjugative plasmids, a major vehicle for the dissemination of antimicrobial resistance, are selfish elements capable of mediating their own transmission through conjugation. To spread to and survive in a new bacterial host, conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids. Such mechanisms have mostly been studied in model plasmids such as the F plasmid, rather than in conjugative plasmids that confer antimicrobial resistance (AMR) in important human pathogens. A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance. Here, we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria, by following the life cycle of conjugative plasmids.

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process. • RecN increases E. coli recombination rate. • E. coli RecN has intrinsic and ssDNA-stimulated ATPase activity. • E. coli RecN binds preferentially to ssDNA. • RecN does not associate with nucleoid in the absence of SOS response. [ABSTRACT FROM AUTHOR]

Microbial ferroptosis has been proved to combat drug-resistant pathogens, but whether this pattern can be applied to the prevention and control of Escherichia coli remains to be further explored. In this study, ferrous gluconate (FeGlu) showed remarkable efficacy in killing E. coli MG1655 with a mortality rate exceeding 99.9%, as well as enterotoxigenic E. coli H10407 (ETEC H10407) and enterohemorrhagic E. coli O157:H7 (EHEC O157:H7). Bacteria death was instigated by the infiltration of Fe2+, accompanied by a burst of intracellular reactive oxygen species (ROS) and lipid peroxidation. Notably, mitigating lipid peroxidation failed to alleviate death of E. coli. Further findings confirmed that FeGlu induced DNA damage, and ΔrecA mutant showed more sensitive, implicating that DNA damage was involved in the death of E. coli. The direct interaction of Fe2+ with DNA was demonstrated by fluorescent staining, gel electrophoresis, and circular dichroism (CD). Moreover, proteomic analysis unveiled 50 differentially expressed proteins (DEPs), including 18 significantly down-regulated proteins and 32 significantly up-regulated proteins. Among them, the down-regulation of SOS-responsive transcriptional suppressor LexA indicated DNA damage induced severely by FeGlu. Furthermore, FeGlu influenced pathways such as fatty acid metabolism (FadB, FadE), iron-sulfur cluster assembly (IscA, IscU, YadR), iron binding, and DNA-binding transcription, along with α -linolenic acid metabolism, fatty acid degradation, and pyruvate metabolism. These pathways were related to FeGlu stress, including lipid peroxidation and DNA damage. In summary, FeGlu facilitated ferroptosis in E. coli through mechanisms involving lipid peroxidation and DNA damage, which presents a new strategy for the development of innovative antimicrobial strategies targeting E. coli infections. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance‐breaker compounds, which inhibit resistance mechanisms and thus resensitize bacteria to existing antibiotics. In the current study, we identify bacterial DNA double‐strand break repair as a promising target for the development of resistance‐breaking co‐therapies. We examined genetic variants of Escherichia coli that combined antibiotic‐resistance determinants with DNA repair defects. We observed that defects in the double‐strand break repair pathway led to significant resensitization toward five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full resensitization. For ciprofloxacin and nitrofurantoin, sensitization manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the promutagenic SOS response. Disruption of double‐strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break‐repair inhibitors can be developed they could resensitize antibiotic‐resistant bacteria to multiple classes of existing antibiotics and may suppress the development of de novo antibiotic‐resistance mutations.

Abstract The term “SOS response” was first coined by Radman in 1974, in an intellectual effort to put together the data suggestive of a concerted gene expression program in cells undergoing DNA damage. A large amount of information about this cellular response has been collected over the following decades. In this review, we will focus on a few of the relevant aspects about the SOS response: its mechanism of control and the stressors which activate it, the diversity of regulated genes in different species, its role in mutagenesis and evolution including the development of antimicrobial resistance, and its relationship with mobile genetic elements.

Antibiotics have a primary mode of actions, and most of them have a common secondary mode of action via reactive species (ROS and RNS) mediated DNA damage. Bacteria have been able to tolerate this DNA damage by SOS (Save-Our-Soul) response. RecA is the universal essential key protein of the DNA damage mediated SOS repair in various bacteria including ESKAPE pathogens. In addition, antibiotics also triggers activation of various other bacterial mechanisms such as biofilm formation, host dependent responses, persister subpopulation formation. These supporting the survival of bacteria in unfriendly natural conditions i.e. antibiotic presence. This review highlights the detailed mechanism of RecA mediated SOS response as well as role of RecA-LexA interaction in SOS response. The review also focuses on inter-connection between DNA damage repair pathway (like SOS response) with other survival mechanisms of bacteria such as host mediated RecA induction, persister-SOS interplay, and biofilm-SOS interplay. This understanding of inter-connection of SOS response with different other survival mechanisms will prove beneficial in targeting the SOS response for prevention and development of therapeutics against recalcitrant bacterial infections. The review also covers the significance of RecA as a promising potent therapeutic target for hindering bacterial SOS response in prevailing successful treatments of bacterial infections and enhancing the conventional antibiotic efficiency. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3–4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates. [ABSTRACT FROM AUTHOR]

When combined with recombinase defects, chromosome breakage and double-strand break repair deficiencies render cells inviable. However, cells are viable when an SOS response occurs in recAts polA cells in Escherichia coli. Here, we aimed to elucidate the underlying mechanisms of this process. Transposon mutagenesis revealed that the hslO gene, a redox chaperone Hsp33 involved in reactive oxidative species (ROS) metabolism, was required for the suppression of recAts polA lethality at a restricted temperature. Recently, it has been reported that lethal treatments trigger ROS accumulation. We also found that recAts polA cells accumulated ROS at the restricted temperature. A catalase addition to the medium alleviates the temperature sensitivity of recAts polA cells and decreases ROS accumulation. These results suggest that the SOS response and hslO manage oxidative insult to an acceptable level in cells with oxidative damage and rescue cell growth. Overall, ROS might regulate several cellular processes. [ABSTRACT FROM AUTHOR]

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 that 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 conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The effects of ionizing radiation (IR) on the protein dynamics of cold-stressed cells of a radioresistant actinobacterium, Kocuria rhizophila PT10, isolated from the rhizosphere of the desert plant Panicum turgidum were investigated using a shotgun methodology based on nanoflow liquid chromatography coupled to tandem mass spectrometry. Overall, 1487 proteins were certified, and their abundances were compared between the irradiated condition and control. IR of cold-acclimated PT10 triggered the over-abundance of proteins involved in (1) a strong transcriptional regulation, (2) amidation of peptidoglycan and preservation of cell envelope integrity, (3) detoxification of reactive electrophiles and regulation of the redox status of proteins, (4) base excision repair and prevention of mutagenesis and (5) the tricarboxylic acid (TCA) cycle and production of fatty acids. Also, one of the more significant findings to emerge from this study is the SOS response of stressed PT10. Moreover, a comparison of top hits radio-modulated proteins of cold-acclimated PT10 with proteomics data from gamma-irradiated Deinococcus deserti showed that stressed PT10 has a specific response characterised by a high over-abundance of NemA, GatD, and UdgB., Competing Interests: Declaration of competing interest None declared., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

As the availability of data sets increases, meta-analysis leveraging aggregated and interoperable data types is proving valuable. This study leveraged a meta-analysis workflow to identify mutations that could improve robustness to reactive oxygen species (ROS) stresses using an industrially important melatonin production strain as an example. ROS stresses often occur during cultivation and negatively affect strain performance. Cellular response to ROS is also linked to the SOS response and resistance to pH fluctuations, which is important to strain robustness in large-scale biomanufacturing. This work integrated more than 7000 E. coli adaptive laboratory evolution (ALE) mutations across 59 experiments to statistically associate mutated genes to 2 ROS tolerance ALE conditions from 72 unique conditions. Mutant oxyR , fur , iscR , and ygfZ were significantly associated and hypothesized to contribute fitness in ROS stress. Across these genes, 259 total mutations were inspected in conjunction with transcriptomics from 46 iModulon experiments. Ten mutations were chosen for reintroduction based on mutation clustering and coinciding transcriptional changes as evidence of fitness impact. Strains with mutations reintroduced into oxyR , fur , iscR , and ygfZ exhibited increased tolerance to H 2 O 2 and acid stress and reduced SOS response, all of which are related to ROS. Additionally, new evidence was generated toward understanding the function of ygfZ , an uncharacterized gene. This meta-analysis approach utilized aggregated and interoperable multiomics data sets to identify mutations conferring industrially relevant phenotypes with the least drawbacks, describing an approach for data-driven strain engineering to optimize microbial cell factories.

Antibiotics and disinfectants resistance is acquired by activating RecA‐mediated DNA repair, which maintains ROS‐dependent DNA damage caused by the antimicrobial molecules. To increase the efficacy of different antimicrobials, an inhibitor can be developed against RecA protein. The present study aims to design a denovo inhibitor against RecA protein of Acinetobacter baumannii. Pharmacophore‐based screening, molecular mechanics, molecular dynamics simulation (MDS), retrosynthetic analysis, and combinatorial synthesis were used to design lead VTRA1.1 against RecA of A. baumannii. Pharmacophore models (structure‐based and ligand‐based) were created, and a phase library of FDA‐approved drugs was prepared. Screening of the phase library against these pharmacophore models selected thirteen lead molecules. These filtered leads were used for the denovo fragment‐based design, which produced 253 combinations. These designed molecules were further analyzed for its interaction with active site of RecA that selected a hybrid VTRA1. Further, retrosynthetic analysis and combinatorial synthesis produced 1000 analogs of VTRA1 by more than 100 modifications. These analogs were used for XP docking, binding free energy calculation, and MDS analysis which finally select lead VTRA1.1 against RecA protein. Further, mutations at the interacting residues of RecA with VTRA1.1, alter the unfolding rate of RecA, which suggests the binding of VTRA1.1 to these residues may alter the stability of RecA. It is also found that VTRA1.1 had reduced interaction of RecA with LexA and ssDNA polydT, showing the lead's efficacy in controlling the SOS response. Further, it was also observed that VTRA1.1 does not contain any predicted human off‐targets and no cytotoxicity to cell lines. As functional RecA is involved in antimicrobial resistance, denovo designed lead VTRA1.1 against RecA may be further developed as a significant combination for therapeutic uses against A. baumannii. [ABSTRACT FROM AUTHOR]

Background: The emergence and spread of drug resistance in Vibrio cholerae are mainly attributed to horizontal gene transfer of mobile genetic elements, especially the SXT (sulfamethoxazole and trimethoprim) element, an integrative conjugative element carrying multiple drug resistance genes. SOS (save our souls) bacterial stress response in Vibrio cholerae plays a pivotal role in inducing the SXT element transfer and induction of the CTX prophage, carrying the important virulence factor cholera toxin encoded by the ctxAB gene. Methods: This study investigated whether the subinhibitory concentration of antibiotics like ciprofloxacin, tetracycline, and azithromycin induce SOS response by detecting the expression of recA and lexA, the key genes of SOS response by reverse transcriptase real time PCR (RT-qPCR). We also studied the transfer of SXT element in response to these three antibiotics by bacterial conjugation. Transfer of SXT elements was confirmed by detecting the SXT element-specific conserved genes. Results: The results of the real-time PCR showed that all three antibiotics induced SOS response with more robust induction by tetracycline and azithromycin relative to ciprofloxacin. We observed a higher frequency of transfer of SXT elements in cultures exposed to these antibiotics and the control mitomycin C compared to unexposed cultures. Conclusion: Our study indicates that antibiotics including azithromycin, ciprofloxacin, and tetracycline activate SOS response in Vibrio cholerae and demonstrates a robust mechanism for wide dissemination of drug resistance. [ABSTRACT FROM AUTHOR]

Persisters are a subpopulation that exhibit growth suppression, antibiotic tolerance, and regrowth after antibiotic removal, without any genetic mutations, which causes the recalcitrance and recurrence of infectious diseases. Persisters are majorly induced through the repression of energy metabolism, but some exceptions have been reported. We have previously shown that ldhA, which encodes lactate dehydrogenase, induces Escherichia coli persisters, resulting in a state of high‐energy metabolism. However, the detailed mechanism of persister formation upon ldhA expression remains elusive. In the present study, we focused on the SOS response pathway via the DNA repair pathway that consumes adenosine triphosphate and revealed that the SOS response pathway is activated upon ldhA expression even before antimicrobial treatment. Metabolome analysis of ldhA‐overexpressing cells revealed that nucleotide metabolic pathways, such as de novo purine biosynthesis, were activated to prepare a nucleotide pool, as substrate for repairing ofloxacin‐induced DNA damage. We provide a novel persister model that contributes to survival as a species by "accidentally" activating the SOS response even before receiving antimicrobial stress. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS), resulting from endogenous metabolism and/or environmental exposure, can induce damage to the 2-deoxyribose moiety in DNA. Specifically, a hydrogen atom from each of the five carbon atoms in 2-deoxyribose can be abstracted by hydroxyl radical, and improper chemical repair of the ensuing radicals formed at the C1, C3, and C4 positions can lead to the stereochemical inversion at these sites to yield epimeric 2-deoxyribose lesions. Although ROS-induced single-nucleobase lesions have been well studied, the biological consequences of the C3-epimeric lesions of 2-deoxynucleosides, i.e., 2-deoxyxylonucleosides (dxN), have not been comprehensively investigated. Herein, we assessed the impact of dxN lesions on the efficiency and fidelity of DNA replication in Escherichia coli cells by conducting a competitive replication and adduct bypass assay with single-stranded M13 phage containing a site-specifically incorporated dxN. Our results revealed that, of the four dxN lesions, only dxG constituted a strong impediment to DNA replication, and intriguingly, dxT and dxC conferred replication bypass efficiencies higher than those of the unmodified counterparts. In addition, the three SOS-induced DNA polymerases (Pol II, Pol IV, and Pol V) did not play any appreciable role in bypassing these lesions. Among the four dxNs, only dxA directed a moderate frequency of dCMP misincorporation. These results provided important insights into the impact of the C3-epimeric lesions on DNA replication in E. coli cells.

We examined the mutagenic specificity of the widely used antibiotic ciprofloxacin (CPR), which displays weak to moderate mutagenic activity in several bacteria and generates short in-frame deletions in rpoB in Staphylococcus aureus To determine the spectrum of mutations in a system where any gene knockout would result in a recovered mutant, including frameshifts and both short and long deletions, we examined CPR-induced mutations in the thymidylate synthase-encoding thyA gene. Here, any mutation resulting in loss of thymidylate synthase activity generates trimethoprim (Trm) resistance. We found that deletions and insertions in all three reading frames predominated in the spectrum. They tend to be short deletions and cluster in two regions, one being a GC-rich region with potential extensive secondary structures. We also exploited the well-characterized rpoB-Rif(r) system in Escherichia coli to determine that cells grown in the presence of sublethal doses of CPR not only induced short in-frame deletions in rpoB, but also generated base substitution mutations resulting from induction of the SOS system. Some of the specific point mutations prominent in the spectrum of a strain that overproduces the dinB-encoded Pol IV were also present after growth in CPR. However, these mutations disappeared in CPR-treated dinB mutants, whereas the deletions remained. Moreover, CPR-induced deletions also occurred in a strain lacking all three SOS-induced polymerases. We discuss the implications of these findings for the consequences of overuse of CPR and other antibiotics.

RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on ssDNA in an ATP-dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (Escherichia coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

Bacterial persistence is a phenomenon that is founded by the existence of a subpopulation of multidrug-tolerant cells. These so-called persister cells endure otherwise lethal stress situations and enable restoration of bacterial populations upon return to favorable conditions. Persisters are especially notorious for their ability to survive antibiotic treatments without conventional resistance genes and to cause infection relapse. The persister state is typically correlated with reduction or inhibition of cellular activity. Early on, chromosomal toxin-antitoxin (TA) systems were suspected to induce the persister state in response to environmental stress. However, this idea has been challenged during the last years. Especially the involvement of toxins from type II TA systems in persister formation is put into question. For toxins from type I TA systems the debate has just started. Here, we would like to summarize recent knowledge gained for the type I TA system tisB/istR-1 from Escherichia coli. TisB is a small, membrane-targeting toxin, which disrupts the proton motive force (PMF), leading to membrane depolarization. Based on experimental data, we hypothesize that TisB primarily stabilizes the persister state through depolarization and further, secondary effects. We will present a simple model that will provide a framework for future directions. [ABSTRACT FROM AUTHOR]