Exploring the links between SOS response, mutagenesis, and resistance during the recovery period.

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.

Regulation of ssb Gene Expression in Escherichia coli

Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by an ssb gene. Although the ssb gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether ssb gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the ssb gene expression kinetics in UV- and γ-irradiated E. coli and revealed that ssb gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the sulA gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the ssb gene was overexpressed in the absence of DNA damage. Furthermore, we measured ssb gene expression by droplet digital PCR during unaffected bacterial growth and revealed that ssb gene expression was equal in wild-type and SOS- bacteria, whereas sulA expression was higher in the former. This study thus reveals a complex pattern of ssb gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The E. coli ssb gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria.

Azithromycin can induce SOS response and horizontal gene transfer of SXT element in Vibrio cholerae.

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.

Genetically stable CRISPR-based kill switches for engineered microbes.

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.

Division of labor between SOS and PafBC in mycobacterial DNA repair and mutagenesis.

DNA repair systems allow microbes to survive in diverse environments that compromise chromosomal integrity. Pathogens such as Mycobacterium tuberculosis must contend with the genotoxic host environment, which generates the mutations that underlie antibiotic resistance. Mycobacteria encode the widely distributed SOS pathway, governed by the LexA repressor, but also encode PafBC, a positive regulator of the transcriptional DNA damage response (DDR). Although the transcriptional outputs of these systems have been characterized, their full functional division of labor in survival and mutagenesis is unknown. Here, we specifically ablate the PafBC or SOS pathways, alone and in combination, and test their relative contributions to repair. We find that SOS and PafBC have both distinct and overlapping roles that depend on the type of DNA damage. Most notably, we find that quinolone antibiotics and replication fork perturbation are inducers of the PafBC pathway, and that chromosomal mutagenesis is codependent on PafBC and SOS, through shared regulation of the DnaE2/ImuA/B mutasome. These studies define the complex transcriptional regulatory network of the DDR in mycobacteria and provide new insight into the regulatory mechanisms controlling the genesis of antibiotic resistance in M. tuberculosis.

Coexistence of SOS-Dependent and SOS-Independent Regulation of DNA Repair Genes in Radiation-Resistant Deinococcus Bacteria.

Deinococcus bacteria are extremely resistant to radiation and able to repair a shattered genome in an essentially error-free manner after exposure to high doses of radiation or prolonged desiccation. An efficient, SOS-independent response mechanism to induce various DNA repair genes such as recA is essential for radiation resistance. This pathway, called radiation/desiccation response, is controlled by metallopeptidase IrrE and repressor DdrO that are highly conserved in Deinococcus. Among various Deinococcus species, Deinococcus radiodurans has been studied most extensively. Its genome encodes classical DNA repair proteins for error-free repair but no error-prone translesion DNA polymerases, which may suggest that absence of mutagenic lesion bypass is crucial for error-free repair of massive DNA damage. However, many other radiation-resistant Deinococcus species do possess translesion polymerases, and radiation-induced mutagenesis has been demonstrated. At least dozens of Deinococcus species contain a mutagenesis cassette, and some even two cassettes, encoding error-prone translesion polymerase DnaE2 and two other proteins, ImuY and ImuB-C, that are probable accessory factors required for DnaE2 activity. Expression of this mutagenesis cassette is under control of the SOS regulators RecA and LexA. In this paper, we review both the RecA/LexA-controlled mutagenesis and the IrrE/DdrO-controlled radiation/desiccation response in Deinococcus.

Learning Yeast Genetics from Miro Radman.

Miroslav Radman's far-sighted ideas have penetrated many aspects of our study of the repair of broken eukaryotic chromosomes. For over 35 years my lab has studied different aspects of the repair of chromosomal breaks in the budding yeast, Saccharomyces cerevisiae. From the start, we have made what we thought were novel observations that turned out to have been predicted by Miro's extraordinary work in the bacterium Escherichia coli and then later in the radiation-resistant Dienococcus radiodurans. In some cases, we have been able to extend some of his ideas a bit further.

The central role of the SOS DNA repair system in antibiotics resistance: A new target for a new infectious treatment strategy.

Bacteria have a considerable ability and potential to acquire resistance against antimicrobial agents by acting diverse mechanisms such as target modification or overexpression, multidrug transporter systems, and acquisition of drug hydrolyzing enzymes. Studying the mechanisms of bacterial cell physiology is mandatory for the development of novel strategies to control the antimicrobial resistance phenomenon, as well as for the control of infections in clinics. The SOS response is a cellular DNA repair mechanism that has an essential role in the bacterial biologic process involved in resistance to antibiotics. The activation of the SOS network increases the resistance and tolerance of bacteria to stress and, as a consequence, to antimicrobial agents. Therefore, SOS can be an applicable target for the discovery of new antimicrobial drugs. In the present review, we focus on the central role of SOS response in bacterial resistance mechanisms and its potential as a new target for control of resistant pathogens.

Induction of the SOS response of Escherichia coli in repair-defective strains by several genotoxic agents.

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.

Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori.

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 (H2O2) 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. H2O2-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 H2O2 is strain-specific, and the mode of action of H2O2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

The cis-encoded antisense RNA IsrA from Salmonella Typhimurium represses the expression of STM0294.1n (iasE), an SOS-induced gene coding for an endoribonuclease activity.

Toxin-antitoxin systems are known to be involved in many bacterial functions that can lead to growth arrest and cell death in response to stress. Typically, toxin and antitoxin genes of type I systems are located in opposite strands, where the antitoxin is a small antisense RNA (sRNA). In the present work we show that the sRNA IsrA from Salmonella Typhimurium down-regulates the expression of its overlapping gene STM0294.1n. Multiple sequence alignment and comparative structure analysis indicated that STM0294.1n belongs to the SymE toxin superfamily, and the gene was renamed iasE (IsrA-overlapping gene with similarity to SymE). The iasE expression was induced in response to mitomycin C, an SOS-inducing agent; conversely, IsrA overexpression repressed the iasE expression even in the presence of mitomycin C. Accordingly, the inactivation of IsrA with an anti-IsrA RNA expressed in trans abrogated the repressive effect of IsrA on the iasE expression. On the other hand, iasE overexpression, as well as the blockage of the antisense IsrA function, negatively affected bacterial growth, arguing for a toxic effect of the iasE gene product. Besides, a bacterial lysate obtained from the iasE-overexpressing strain exhibited endoribonuclease activity, as determined by a fluorometric assay based on fluorescent reporter RNAs. Together, these results indicate that the IasE/IsrA pair of S. Typhimurium constitutes a functional type I toxin-antitoxin system.

Global mistranslation increases cell survival under stress in Escherichia coli.

Mistranslation is typically deleterious for cells, although specific mistranslated proteins can confer a short-term benefit in a particular environment. However, given its large overall cost, the prevalence of high global mistranslation rates remains puzzling. Altering basal mistranslation levels of Escherichia coli in several ways, we show that generalized mistranslation enhances early survival under DNA damage, by rapidly activating the SOS response. Mistranslating cells maintain larger populations after exposure to DNA damage, and thus have a higher probability of sampling critical beneficial mutations. Both basal and artificially increased mistranslation increase the number of cells that are phenotypically tolerant and genetically resistant under DNA damage; they also enhance survival at high temperature. In contrast, decreasing the normal basal mistranslation rate reduces cell survival. This wide-ranging stress resistance relies on Lon protease, which is revealed as a key effector that induces the SOS response in addition to alleviating proteotoxic stress. The new links between error-prone protein synthesis, DNA damage, and generalised stress resistance indicate surprising coordination between intracellular stress responses and suggest a novel hypothesis to explain high global mistranslation rates.

Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status.

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.

SosA inhibits cell division in Staphylococcus aureus in response to DNA damage.

Inhibition of cell division is critical for viability under DNA-damaging conditions. DNA damage induces the SOS response that in bacteria inhibits cell division while repairs are being made. In coccoids, such as the human pathogen, Staphylococcus aureus, this process remains poorly studied. Here, we identify SosA as the staphylococcal SOS-induced cell division inhibitor. Overproduction of SosA inhibits cell division, while sosA inactivation sensitizes cells to genotoxic stress. SosA is a small, predicted membrane protein with an extracellular C-terminal domain in which point mutation of residues that are conserved in staphylococci and major truncations abolished the inhibitory activity. In contrast, a minor truncation led to SosA accumulation and a strong cell division inhibitory activity, phenotypically similar to expression of wild-type SosA in a CtpA membrane protease mutant. This suggests that the extracellular C-terminus of SosA is required both for cell division inhibition and for turnover of the protein. Microscopy analysis revealed that SosA halts cell division and synchronizes the cell population at a point where division proteins such as FtsZ and EzrA are localized at midcell, and the septum formation is initiated but unable to progress to closure. Thus, our findings show that SosA is central in cell division regulation in staphylococci.

Betulinic Acid Prevents the Acquisition of Ciprofloxacin-Mediated Mutagenesis in Staphylococcus aureus.

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.

The response of Sphingopyxis granuli strain TFA to the hostile anoxic condition.

Sphingomonads comprises a group of interesting aerobic bacteria because of their ubiquity and metabolic capability of degrading many recalcitrant contaminants. The tetralin-degrader Sphingopyxis granuli strain TFA has been recently reported as able to anaerobically grow using nitrate as the alternative electron acceptor and so far is the only bacterium with this ability within the sphingomonads group. To understand how strain TFA thrives under anoxic conditions, a differential transcriptomic analysis while growing under aerobic or anoxic conditions was performed. This analysis has been validated and complemented with transcription kinetics of representative genes of different functional categories. Results show an extensive change of the expression pattern of this strain in the different conditions. Consistently, the most induced operon in anoxia codes for proteases, presumably required for extensive changes in the protein profile. Besides genes that respond to lack of oxygen in other bacteria, there are a number of genes that respond to stress or to damage of macromolecules, including genes of the SOS DNA-damage response, which suggest that anoxic conditions represent a hostile environment for this bacterium. Interestingly, growth under anoxic conditions also resulted in repression of all flagellar and type IV pilin genes, which suggested that this strain shaves its appendages off while growing in anaerobiosis.

ImuB and ImuC contribute to UV-induced mutagenesis as part of the SOS regulon in Pseudomonas aeruginosa.

DNA damage-induced mutagenesis is a process governed by the SOS system that requires the activity of specialized DNA polymerases. These polymerases, which are devoid of proof-reading activity, serve to increase the probability of survival under stressful conditions in exchange for an error-prone DNA synthesis. As an opportunistic pathogen of humans, Pseudomonas aeruginosa employs adaptive responses that originally evolved for survival in many diverse and often stressful environmental conditions, where the action of error-prone DNA polymerases may be crucial. In this study, we have investigated the role of the polymerases ImuB and ImuC in P. aeruginosa DNA-damage induced mutagenesis. UV irradiation of imuB- and imuC-deletion mutants showed that both genes contribute to UV-induced mutagenesis in this bacterium. Furthermore, we confirmed that UV treatment significantly increase the expression levels of the imuB and imuC genes and that they are co-transcribed as a single transcriptional unit under the control of LexA as part of the SOS regulon in P. aeruginosa. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Oxidative damage induced by H2O2 reveals SOS adaptive transcriptional response of Dietzia cinnamea strain P4.

The oxidative stress response of the highly resistant actinomycete Dietzia cinnamea P4 after treatment with hydrogen peroxide (H2O2) was assessed in order to depict the possible mechanisms underlying its intrinsic high resistance to DNA damaging agents. We used transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA levels after exposure to different concentrations of H2O2 at 10 min and 1 h following the addition of the stressor. Catalase and superoxide dismutase genes were induced in different ways, according to the condition applied. Moreover, alkyl hydroperoxide reductase ahpCF, thiol peroxidase, thioredoxin and glutathione genes were upregulated in the presence of H2O2. Expression of peroxidase genes was not detected during the experiment. Overall results point to an actinomycete strain endowed with a set of enzymatic defenses against oxidative stress and with the main genes belonging to a functional SOS system (lexA, recA, uvrD), including suppression of lexA repressor, concomitantly to recA and uvrD gene upregulation upon H2O2 challenge.

The response of Escherichia coli to the alkylating agents chloroacetaldehyde and styrene oxide.

DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.

Temporal order and precision of complex stress responses in individual bacteria.

Sudden stress often triggers diverse, temporally structured gene expression responses in microbes, but it is largely unknown how variable in time such responses are and if genes respond in the same temporal order in every single cell. Here, we quantified timing variability of individual promoters responding to sublethal antibiotic stress using fluorescent reporters, microfluidics, and time-lapse microscopy. We identified lower and upper bounds that put definite constraints on timing variability, which varies strongly among promoters and conditions. Timing variability can be interpreted using results from statistical kinetics, which enable us to estimate the number of rate-limiting molecular steps underlying different responses. We found that just a few critical steps control some responses while others rely on dozens of steps. To probe connections between different stress responses, we then tracked the temporal order and response time correlations of promoter pairs in individual cells. Our results support that, when bacteria are exposed to the antibiotic nitrofurantoin, the ensuing oxidative stress and SOS responses are part of the same causal chain of molecular events. In contrast, under trimethoprim, the acid stress response and the SOS response are part of different chains of events running in parallel. Our approach reveals fundamental constraints on gene expression timing and provides new insights into the molecular events that underlie the timing of stress responses.