Understanding LexA as a central regulator of the bacterial SOS response.

Two Modes of Binding of DinI to RecA Filament Provide a New Insight into the Regulation of SOS Response by DinI Protein

Functional consequences of inducible genetic elements from the p53 SOS response in a mammalian organ system

The bacterial SOS response is an elaborate program for DNA repair, cell cycle regulation and adaptive mutagenesis under stress conditions. Using sensitive sequence and structure analysis, combined with contextual information derived from comparative genomics and domain architectures, we identify two novel domain superfamilies in the SOS response system. We present evidence that one of these, the SOS response associated peptidase (SRAP; Pfam: DUF159) is a novel thiol autopeptidase. Given the involvement of other autopeptidases, such as LexA and UmuD, in the SOS response, this finding suggests that multiple structurally unrelated peptidases have been recruited to this process. The second of these, the ImuB-C superfamily, is linked to the Y-family DNA polymerase-related domain in ImuB, and also occurs as a standalone protein. We present evidence using gene neighborhood analysis that both these domains function with different mutagenic polymerases in bacteria, such as Pol IV (DinB), Pol V (UmuCD) and ImuA-ImuB-DnaE2 and also other repair systems, which either deploy Ku and an ATP-dependent ligase or a SplB-like radical SAM photolyase. We suggest that the SRAP superfamily domain functions as a DNA-associated autoproteolytic switch that recruits diverse repair enzymes upon DNA damage, whereas the ImuB-C domain performs a similar function albeit in a non-catalytic fashion. We propose that C3Orf37, the eukaryotic member of the SRAP superfamily, which has been recently shown to specifically bind DNA with 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, is a sensor for these oxidized bases generated by the TET enzymes from methylcytosine. Hence, its autoproteolytic activity might help it act as a switch that recruits DNA repair enzymes to remove these oxidized methylcytosine species as part of the DNA demethylation pathway downstream of the TET enzymes.ReviewersThis article was reviewed by RDS, RF and GJ.

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

Bacterial SOS response: a food safety perspective

The bacterial SOS response is not only a vital reply to DNA damage but also constitutes an essential mechanism for the generation of genetic variability that in turn fuels adaptation and resistance development in bacterial populations. Despite the extensive depiction of the SOS regulon itself, its activation by stresses different from typical DNA damaging treatments remains poorly characterized. Recently, we reported the RecA- and LexA-dependent induction of the SOS response in Escherichia coli MG1655 after exposure to high hydrostatic pressure (HP, approximately 100 MPa), a physical stress of which the cellular effects are not well known. We now found this HP mediated SOS response to depend on RecB and not on RecF, which is a strong indication for the involvement of double strand breaks. As the pressures used in this work are thermodynamically unable to break covalent bonds in DNA, we hypothesized the involvement of a cellular function or pathway in the formation of this lesion. A specialized screening allowed us to identify the cryptic type IV restriction endonuclease Mrr as the final effector of this pathway. The HP SOS response and its corresponding phenotypes could be entirely attributed to the HP triggered activation of Mrr restriction activity. Several spontaneously occurring alleles of mrr, incapable of triggering the HP-induced SOS response, were isolated and characterized. These results provide evidence for a specific pathway that transmits the perception of HP stress to induction of the SOS response and support a role for Mrr in bacterial stress physiology.

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

The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.

The SOS response of bacteria is a global regulatory network targeted at addressing DNA damage. Governed by the products of the lexA and recA genes, it co-ordinates a comprehensive response against DNA lesions and its description in Escherichia coli has stood for years as a textbook paradigm of stress-response systems in bacteria. In this paper we review the current state of research on the SOS response outside E. coli. By retracing research on the identification of multiple diverging LexA-binding motifs across the Bacteria Domain, we show how this work has led to the description of a minimum regulon core, but also of a heterogeneous collection of SOS regulatory networks that challenges many tenets of the E. coli model. We also review recent attempts at reconstructing the evolutionary history of the SOS network that have cast new light on the SOS response. Exploiting the newly gained knowledge on LexA-binding motifs and the tight association of LexA with a recently described mutagenesis cassette, these works put forward likely evolutionary scenarios for the SOS response, and we discuss their relevance on the ultimate nature of this stress-response system and the evolutionary pressures driving its evolution.

Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.

Lesions to DNA compromise chromosome integrity, posing a direct threat to cell survival. The bacterial SOS response is a widespread transcriptional regulatory mechanism to address DNA damage. This response is coordinated by the LexA transcriptional repressor, which controls genes involved in DNA repair, mutagenesis and cell-cycle control. To date, the SOS response has been characterized in most major bacterial groups, with the notable exception of the Bacteroidetes. No LexA homologs had been identified in this large, diverse and ecologically important phylum, suggesting that it lacked an inducible mechanism to address DNA damage. Here, we report the identification of a novel family of transcriptional repressors in the Bacteroidetes that orchestrate a canonical response to DNA damage in this phylum. These proteins belong to the S24 peptidase family, but are structurally different from LexA. Their N-terminal domain is most closely related to CI-type bacteriophage repressors, suggesting that they may have originated from phage lytic phase repressors. Given their role as SOS regulators, however, we propose to designate them as non-canonical LexA proteins. The identification of a new class of repressors orchestrating the SOS response illuminates long-standing questions regarding the origin and plasticity of this transcriptional network.

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.

Exploration of inhibitors of the bacterial LexA repressor-protease

Antimicrobial agentshave revolutionized the treatment of infectiousdiseases; however, their widespread and extensive use has acceleratedthe emergence of antimicrobial resistance (AMR). While resistancedriven by mutation and gene transfer poses a major threat, the transientlydormant subpopulation, called persisters, poses an equally significantchallenge. The bacterial SOS response, triggered by extensive DNAdamage, promotes resistance by inducing mutations in the target protein,activating drug efflux systems and horizontally transferring geneticmaterial. Furthermore, SOS has been implicated in controlling toxin-antitoxinmodules, as observed in the tisB/istR system in Escherichia coli, whichfurther facilitates the emergence of persister populations. Althoughpersistence and resistance are often studied separately, their sharedregulation by the SOS system remains unexplored. Targeting SOS regulators,such as RecA and LexA, and DNA damage responders, including error-pronepolymerases, can significantly help control the emergence and spreadof AMR, although such approaches are in their early translationalstages. This review explores the role of the SOS response as a unifyingmechanism linking resistance and persistence, while evaluating emergingtherapeutic strategies targeting SOS regulators and associated stressresponse pathways as antievolution approaches to combat AMR effectively.

Bacterial SOS response is an inducible system of DNA repair and mutagenesis. Streptococci lack a canonical SOS response, but an SOS-like response was reported in some species. The mef(A)-msr(D)-carrying prophage Ф1207.3 of Streptococcus pyogenes contains a region, spanning orf6 to orf11, showing homology to characterized streptococcal SOS-like cassettes. Genome-wide homology search showed the presence of the whole Φ1207.3 SOS-like cassette in three S. pyogenes prophages, while parts of it were found in other bacterial species. To investigate whether this cassette confers an SOS-mutagenesis phenotype, we constructed Streptococcus pneumoniae R6 isogenic derivative strains: (i) FR172, streptomycin resistant, (ii) FR173, carrying Φ1207.3, and (iii) FR174, carrying a recombinant Φ1207.3, where the SOS-like cassette was deleted. These strains were used in survival and mutation rate assays using a UV-C LED instrument, for which we designed and 3D-printed a customized equipment, constituted of an instrument support and swappable-autoclavable mini-plates and lids. Upon exposure to UV fluences ranging from 0 to 6,400 J/m2 at four different wavelengths, 255, 265, 275, and 285 nm, we found that the presence of Φ1207.3 SOS-like cassette increases bacterial survival up to 34-fold. Mutation rate was determined by measuring rifampicin resistance acquisition upon exposure to UV fluence of 50 J/m2 at the four wavelengths by fluctuation test. The presence of Φ1207.3 SOS-like cassette resulted in a significant increase in the mutation rate (up to 18-fold) at every wavelength. In conclusion, we demonstrated that Φ1207.3 carries a functional SOS-like cassette responsible for an increased survival and increased mutation rate in S. pneumoniae. IMPORTANCE Bacterial mutation rate is generally low, but stress conditions and DNA damage can induce stress response systems, which allow for improved survival and continuous replication. The SOS response is a DNA repair mechanism activated by some bacteria in response to stressful conditions, which leads to a temporary hypermutable phenotype and is usually absent in streptococcal genomes. Here, using a reproducible and controlled UV irradiation system, we demonstrated that the SOS-like gene cassette of prophage Φ1207.3 is functional, responsible for a temporary hypermutable phenotype, and enhances bacterial survival to UV irradiation. Prophage Φ1207.3 also carries erythromycin resistance genes and can lysogenize different pathogenic bacteria, constituting an example of a mobile genetic element which can confer multiple phenotypes to its host.