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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., (© 2024. The Author(s).)

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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

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 Fe 2+ , 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 Fe 2+ 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

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

In Escherichia coli, the SulA protein is synthesized during the SOS response to arrest cell division. Two possible models of SulA action were proposed: the sequestration and the capping. In current paper, to clarify which model better reflects the SulA effect on cell division upon the SOS response, the FtsZ/SulA ratio was estimated inside cells based on fusion of both FtsZ and SulA to fluorescent protein mNeonGreen. This allowed to quantify this ratio by fluorescence microscopy as well as western blotting; moreover, the effect of SulA on FtsZ distribution patterns in cells was analyzed based on fluorescence microscopy images. The SulA concentration in cells under the SOS response was shown to be several times (about 10) lower than that of FtsZ. The effect of SulA was unequal to corresponding decrease in FtsZ concentration. These results are supported by uneven FtsZ distribution in cells under the SOS response. Together the results of current work indicate that the division arrest by SulA protein in E. coli cells could not be explained by the sequestration model., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The SulA protein of Escherichia coli and related bacteria interacts directly with FtsZ, blocking cell division by disrupting Z-ring formation, yet the precise mechanism remains not fully understood. Previous demonstrations of Pseudomonas aeruginosa SulA's dimerization capability were confined to X-ray crystallography and lacked confirmation under in vivo conditions. Additionally, uncertainty persisted regarding the dimerization potential of E. coli's SulA protein. This paper employs a bacterial two-hybrid system to establish that both P. aeruginosa and E. coli SulA proteins indeed possess the capacity for dimerization., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

RecA inhibition could be an important strategy to combat antimicrobial resistance because of its key role in the SOS response, DNA repair and homologous recombination contributing to bacterial survival. This study evaluated the impact of RecA inactivation on heteroresistance in clinical isolates of Escherichia coli and their corresponding recA-deficient isogenic strains to multiple classes of antimicrobial agents. A high frequency (>30%) of heteroresistance was observed in this collection of clinical isolates. Deletion of the recA gene led to a marked reduction in heteroresistant subpopulations, especially against quinolones or β-lactams. The molecular basis of heteroresistance was associated with an increase in copy number of plasmid-borne resistance genes (bla TEM-1B ) or tandem gene amplifications (qnrA1). Of note, in the absence of the recA gene, the increase in copy number of resistance genes was suppressed. This makes the recA gene a promising target for combating heteroresistance., Competing Interests: Competing interests None declared., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Aims: Naringin is a flavonoid with a polyphenolic structure which induces formation of reactive oxygen species (ROS). Although the antibacterial effect of naringin has been demonstrated, the mechanism underlying this effect has not yet been elucidated. We focused on investigating the antibacterial mode of action of naringin in Escherichia coli following ROS generation. The contributions of ROS, hydroxy radicals (OH - ), super oxide (O 2 - ), and hydrogen peroxide (H 2 O 2 ) were investigated., Main Methods: ROS accumulation was detected using fluorescence dyes, and all experiments were conducted using the scavenger including tiron, sodium pyruvate, and thiourea to assess the contribution of each ROS. Western blotting assays were used to observe the activation of the SOS response for DNA repair. DNA fragmentation, membrane depolarization, and phosphatidylserine exposure were estimated using TUNEL, DiBAC 4 (3), and Annexin V/PI., Key Findings: Accumulation of ROS was observed in Escherichia coli after treatment with naringin. Oxidative stress induced cellular dysfunction including DNA damage, which results in SOS response activation. Eventually, apoptosis-like death occurred in cells treated with naringin. The cells had different contributions of each ROS and accompanying apoptotic factors. The ROS most destructive to E. coli was OH - , followed by H 2 O 2 and O 2 - ., Significance: Due to its efficacy, naringin is a useful antimicrobial agent. An initial investigation into the antibacterial mode of action of naringin is presented in this paper. The contribution of each ROS to apoptosis-like cell death (ALD) was investigated, and the results enhanced our understanding of the correlation between the SOS response and oxidative stress in bacteria., (Copyright © 2022. Published by Elsevier Inc.)

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., Competing Interests: AA, GP, NM, AV, GC, AY, DB, MK No competing interests declared, (© 2022, Alekseev, Pobegalov et al.)

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., (© 2022. The Author(s).)

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., (© 2022 The Societies and John Wiley & Sons Australia, Ltd.)

The bacterial DNA damage response pathway (SOS response) is composed of a network of genes regulated by a single transcriptional repressor, LexA. The lexA promoter, itself, contains two LexA operators, enabling negative feedback. In Escherichia coli, the downstream operator contains a conserved DNA cytosine methyltransferase (Dcm) site that is predicted to be methylated to 5-methylcytosine (5mC) specifically during stationary phase growth, suggesting a regulatory role for DNA methylation in the SOS response. To test this, we quantified 5mC at the lexA locus, and then examined the effect of LexA on Dcm activity, as well as the impact of this 5mC mark on LexA binding, lexA transcription, and SOS response induction. We found that 5mC at the lexA promoter is specific to stationary phase growth, but that it does not affect lexA expression. Our data support a model where LexA binding at the promoter inhibits Dcm activity without an effect on the SOS regulon., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Hydrogen peroxide (H 2 O 2 ) is a debriding agent that damages the microbial structure and function by generating various reactive oxygen species (ROS). H 2 O 2 -produced hydroxyl radical (OH∙) also exerts oxidative stress on microorganisms. The spread of antibiotic-resistance in bacteria is a serious issue worldwide, and greater efforts are needed to identify and characterize novel antibacterial mechanisms to develop new treatment strategies. Therefore, this study aimed to clarify the relationship between H 2 O 2 and Escherichia coli and to elucidate a novel antibacterial mechanism(s) of H 2 O 2 . Following H 2 O 2 exposure, increased levels of 8-hydroxydeoxyguanosine and malondialdehyde indicated that H 2 O 2 accelerates oxidation of bacterial DNA and lipids in E. coli. As oxidative damage worsened, the SOS response was triggered. Cell division arrest and resulting filamentous cells were identified in cells, indicating that LexA was involved in DNA replication. It was also verified that RecA, a representative SOS gene, helps self-cleavage of LexA and acts as a bacterial caspase-like protein. Our findings also showed that dinF is essential to preserve E. coli from H 2 O 2 -induced ROS, and furthermore, demonstrated that H 2 O 2 -induced SOS response and SOS genes participate differently in guarding E. coli from oxidative stress. As an extreme SOS response is considered apoptosis-like death (ALD) in bacteria, additional experiments were performed to examine the characteristics of ALD. DNA fragmentation and membrane depolarization appeared in H 2 O 2 -treated cells, suggesting that H 2 O 2 causes ALD in E. coli. In conclusion, our investigations revealed that ALD is a novel antibacterial mode of action(s) of H 2 O 2 with important contributions from SOS genes., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The SOS response-associated peptidase (SRAP) is an ancient protein superfamily in all domains of life. The mammalian SRAP was recently reported to covalently bind to the abasic sites (AP) in single stranded (ss) DNA to shield the chromosome integrity. YedK, the Escherichia coli SRAP, is not functionally characterized. Here we report the fortuitous pull-down of YedK from bacterial cell lysates by short (<20 bp) double stranded (ds) DNAs, further enrichment of YedK was observed when single stranded (ss) DNA was added. YedK can bind multiple DNA substrates, particularly with a high affinity to DNA duplex with single strand segment. As a SRAP protein, the involvement of YedK in SOS response was extensively examined, however yedK mutant of Escherichia coli showed no difference from the wild type strain upon the treatments with UV and various DNA damaging reagents, indicating its non-essentiality or redundancy in E. coli. Surprisingly, yedK mutants derived from Escherichia coli and Samonella enterica both showed an increased plasmid DNA transformation efficiency compared to the wild types. In accordance with this, induction of YedK effectively decreased the copy number of plasmid DNA. Site-directed mutagenesis of YedK demonstrated that residues involved in single strand DNA binding and cysteine residue at position 2 from N-terminus can discharge the repression of the plasmid transformation efficiency., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

When subcloned into low-copy-number expression vectors, rumAB, encoding polV R391 (RumA' 2 B), is best characterized as a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in dramatic contrast to the poorly mutable phenotype when polV R391 is expressed from the native 88.5 kb R391, suggesting that R391 expresses cis-acting factors that suppress the expression and/or the activity of polV R391 . Indeed, we recently discovered that SetR R391 , an ortholog of λ cI repressor, is a transcriptional repressor of rumAB. Here, we report that CroS R391 , an ortholog of λ Cro, also serves as a potent transcriptional repressor of rumAB. Levels of RumA are dependent upon an interplay between SetR R391 and CroS R391 , with the greatest reduction of RumA protein levels observed in the absence of SetR R391 and the presence of CroS R391 . Under these conditions, CroS R391 completely abolishes the high levels of mutagenesis promoted by polV R391 expressed from low-copy-number plasmids. Furthermore, deletion of croS R391 on the native R391 results in a dramatic increase in mutagenesis, indicating that CroS R391 plays a major role in suppressing polV R391 mutagenesis in vivo. Inactivating mutations in CroS R391 therefore have the distinct possibility of increasing cellular mutagenesis that could lead to the evolution of antibiotic resistance of pathogenic bacteria harboring R391., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Bacterial type IV secretion systems (T4SSs) mediate the conjugative transfer of mobile genetic elements (MGEs) and their cargoes of antibiotic resistance and virulence genes. Here, we report that the pED208-encoded T4SS (Tra pED208 ) translocates not only this F plasmid but several plasmid-encoded proteins, including ParA, ParB1, single-stranded DNA-binding protein SSB, ParB2, PsiB, and PsiA, to recipient cells. Conjugative protein translocation through the Tra pED208 T4SS required engagement of the pED208 relaxosome with the TraD substrate receptor or coupling protein. T4SSs translocate MGEs as single-stranded DNA intermediates (T-strands), which triggers the SOS response in recipient cells. Transfer of pED208 deleted of psiB or ssb , which, respectively, encode the SOS inhibitor protein PsiB and single-stranded DNA-binding protein SSB, elicited a significantly stronger SOS response than pED208 or mutant plasmids deleted of psiA , parA , parB1 , or parB2 . Conversely, translocation of PsiB or SSB, but not PsiA, through the Tra pED208 T4SS suppressed the mating-induced SOS response. Our findings expand the repertoire of known substrates of conjugation systems to include proteins with functions associated with plasmid maintenance. Furthermore, for this and other F-encoded Tra systems, docking of the DNA substrate with the TraD receptor appears to serve as a critical activating signal for protein translocation. Finally, the observed effects of PsiB and SSB on suppression of the mating-induced SOS response establishes a novel biological function for conjugative protein translocation and suggests the potential for interbacterial protein translocation to manifest in diverse outcomes influencing bacterial communication, physiology, and evolution. IMPORTANCE Many bacteria carry plasmids and other mobile genetic elements (MGEs) whose conjugative transfer through encoded type IV secretion systems (T4SSs), or "mating" channels, can lead to a rapid intra- and interspecies proliferation of genes encoding resistance to antibiotics or heavy metals or virulence traits. Here, we show that a model IncF plasmid-encoded T4SS translocates not only DNA but also several proteins intercellularly. The repertoire of translocated proteins includes the plasmidic SOS inhibitor protein PsiB, single-stranded DNA-binding protein SSB, and several partitioning proteins. We demonstrate that intercellular transmission of PsiB and SSB suppresses the SOS response, which is triggered in recipient cells upon acquisition of the single-stranded DNA transfer intermediate during mating. Our findings identify a new biological function for conjugative protein translocation in mitigating potentially deleterious consequences to plasmid and genome integrity resulting from SOS-induced recombination and mutation events.

Colicins are bacterial toxins targeting Gram-negative bacteria, including E. coli and related Enterobacteriaceae strains. Some colicins form ion-gated pores in the inner membrane of attacked bacteria that are lethal to their target. Colicin Ia was the first pore-forming E. coli toxin, for which a high-resolution structure of the monomeric full-length protein was determined. It is so far also the only colicin, for which a low-resolution structure of its membrane-inserted pore was reported by negative-stain electron microscopy. Resolving this structure at the atomic level would allow an understanding of the mechanism of toxin pore formation. Here, we report an observation that we made during an attempt to determine the Colicin Ia pore structure at atomic resolution. Colicin Ia was natively expressed by mitomycin-C induction under a native SOS promotor and purified following published protocols. The visual appearance in the electron microscope of negatively stained preparations and the lattice parameters of 2D crystals obtained from the material were highly similar to those reported earlier resulting from the same purification protocol. However, a higher-resolution structural analysis revealed that the protein is Dps (DNA-binding protein from starved cells), a dodecameric E. coli protein. This finding suggests that the previously reported low-resolution structure of a "Colicin Ia oligomeric pore" actually shows Dps., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The bacterial SOS response to DNA damage induces an error-prone repair program that is mutagenic. In Escherichia coli, SOS-induced mutations are caused by the translesion synthesis (TLS) activity of two error-prone polymerases (EPPs), Pol IV and Pol V. The mutational footprint of the EPPs is confounded by both DNA damage and repair, as mutations are targeted to DNA lesions via TLS and corrected by the mismatch repair (MMR) system. To remove these factors and assess untargeted EPP mutations genome-wide, we constructed spontaneous SOS mutator strains deficient in MMR, then analyzed their mutational footprints by mutation accumulation and whole genome sequencing. Our analysis reveals new features of untargeted SOS-mutagenesis, showing how MMR alters its spectrum, sequence specificity, and strand-bias. Our data support a model where the EPPs prefer to act on the lagging strand of the replication fork, producing base pair mismatches that are differentially repaired by MMR depending on the type of mismatch., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ribonucleotides are frequently incorporated into DNA and can be used as a marker of DNA replication enzymology. To investigate on a genome-wide scale, how E. coli pol V accesses undamaged chromosomal DNA during the SOS response, we mapped the location of ribonucleotides incorporated by steric gate variants of pol V across the entire E. coli genome. To do so, we used strains that are deficient in ribonucleotide excision repair (ΔrnhB), deficient in pol IV DNA polymerase, constitutively express all SOS-regulated genes [lexA(Def)] and constitutively "activated" RecA* (recA730). The strains also harbor two steric gate variants of E. coli pol V (Y11A, or F10L), or a homolog of pol V, (pol V R391 -Y13A). Ribonucleotides are frequently incorporated by the pol V-Y11A and pol V R391 -Y13A variants, with a preference to the lagging strand. In contrast, the pol V-F10L variant incorporates less ribonucleotides and no strand preference is observed. Sharp transitions in strand specificity are observed at the replication origin (oriC), while a gradient is observed at the termination region. To activate RecA* in a recA + strain, we treated the strains with ciprofloxacin and genome-wide mapped the location of the incorporated ribonucleotides. Again, the pol V-Y11A steric gate variant exhibited a lagging strand preference. Our data are consistent with a specific role for pol V in lagging strand DNA synthesis across the entire E. coli genome during the SOS response., (Copyright © 2021 Elsevier B.V. All rights reserved.)

DNA polymerase IV (pol IV) is expressed at increased levels in Escherichia coli cells that suffer DNA damage. In a recent live-cell single-molecule fluorescence microscopy study, we demonstrated that the formation of pol IV foci is strongly recB-dependent in cells treated with the DNA break-inducing antibiotic ciprofloxacin. The results of that study support a model in which pol IV acts to extend D-loop structures during recombinational repair of DNA double-strand breaks. In the present study, we extend upon this work, investigating the UmuD and UmuD' proteins as potential modulators of pol IV activity in ciprofloxacin-treated cells. We found that the non-cleavable mutant UmuD(K97A) promotes long-lived association of pol IV with the nucleoid, whereas its cleaved form, UmuD', which accumulates in DNA-damaged cells, reduces binding. The results provide additional support for a model in which UmuD and UmuD' directly modulate pol IV-binding to the nucleoid.

Feedback mechanisms are fundamental to the control of physiological responses. One important example in gene regulation, termed negative autoregulation (NAR), occurs when a transcription factor (TF) inhibits its own production through transcriptional repression. This enables more-rapid homeostatic control of gene expression. NAR circuits presumably evolve to limit the fitness costs of gratuitous gene expression. The key biochemical reactions of NAR can be parameterized using a mathematical model of promoter activity; however, this model of NAR has been studied mostly in the context of synthetic NAR circuits that are disconnected from the target genes of the TFs. Thus, it remains unclear how constrained NAR parameters are in a native circuit context, where the TF target genes can have fitness effects on the cell. To quantify these constraints, we created a panel of Escherichia coli strains with different lexA- NAR circuit parameters and analyzed the effect on SOS response function and bacterial fitness. Using a mathematical model for NAR, these experimental data were used to calculate NAR parameter values and derive a parameter-fitness landscape. Without feedback, survival of DNA damage was decreased due to high LexA concentrations and slower SOS "turn-on" kinetics. However, we show that, even in the absence of DNA damage, the lexA promoter is strong enough that, without feedback, high levels of lexA expression result in a fitness cost to the cell. Conversely, hyperfeedback can mimic lexA deletion, which is also costly. This work elucidates the lexA -NAR parameter values capable of balancing the cell's requirement for rapid SOS response activation with limiting its toxicity. IMPORTANCE Feedback mechanisms are critical to control physiological responses. In gene regulation, one important example, termed negative autoregulation (NAR), occurs when a transcription factor (TF) inhibits its own production. NAR is common across the tree of life, enabling rapid homeostatic control of gene expression. NAR behavior can be described in accordance with its core biochemical parameters, but how constrained these parameters are by evolution is unclear. Here, we describe a model genetic network controlled by an NAR circuit within the bacterium Escherichia coli and elucidate these constraints by experimentally changing a key parameter and measuring its effect on circuit response and fitness. This analysis yielded a parameter-fitness landscape representing the genetic network, providing a window into what gene-environment conditions favor evolution of this regulatory strategy., (Copyright © 2020 Kozuch et al.)

Induction of the SOS response, a cellular system triggered by DNA damage in bacteria, depends on DNA replication for the generation of the SOS signal, ssDNA. RecA binds to ssDNA, forming filaments that stimulate proteolytic cleavage of the LexA transcriptional repressor, allowing expression of > 40 gene products involved in DNA repair and cell cycle regulation. Here, using a DNA replication system reconstituted in vitro in tandem with a LexA cleavage assay, we studied LexA cleavage during DNA replication of both undamaged and base-damaged templates. Only a ssDNA-RecA filament supported LexA cleavage. Surprisingly, replication of an undamaged template supported levels of LexA cleavage like that induced by a template carrying two site-specific cyclobutane pyrimidine dimers. We found that two processes generate ssDNA that could support LexA cleavage. 1) During unperturbed replication, single-stranded regions formed because of stochastic uncoupling of the leading-strand DNA polymerase from the replication fork DNA helicase, and 2) on the damaged template, nascent leading-strand gaps were generated by replisome lesion skipping. The two pathways differed in that RecF stimulated LexA cleavage during replication of the damaged template, but not normal replication. RecF appears to facilitate RecA filament formation on the leading-strand ssDNA gaps generated by replisome lesion skipping., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Myka and Marians.)

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

When its DNA is damaged, Escherichia coli induces the SOS response, which consists of about 40 genes that encode activities to repair or tolerate the damage. Certain alleles of the major SOS-control genes, recA and lexA, cause constitutive expression of the response, resulting in an increase in spontaneous mutations. These mutations, historically called "untargeted", have been the subject of many previous studies. Here we re-examine SOS-induced mutagenesis using mutation accumulation followed by whole-genome sequencing (MA/WGS), which allows a detailed picture of the types of mutations induced as well as their sequence-specificity. Our results confirm previous findings that SOS expression specifically induces transversion base-pair substitutions, with rates averaging about 60-fold above wild-type levels. Surprisingly, the rates of G:C to C:G transversions, normally an extremely rare mutation, were induced an average of 160-fold above wild-type levels. The SOS-induced transversion showed strong sequence specificity, the most extreme of which was the G:C to C:G transversions, 60% of which occurred at the middle base of 5'GGC3'+5'GCC3' sites, although these sites represent only 8% of the G:C base pairs in the genome. SOS-induced transversions were also DNA strand-biased, occurring, on average, 2- to 4- times more often when the purine was on the leading-strand template and the pyrimidine on the lagging-strand template than in the opposite orientation. However, the strand bias was also sequence specific, and even of reverse orientation at some sites. By eliminating constraints on the mutations that can be recovered, the MA/WGS protocol revealed new complexities of SOS "untargeted" mutations., Competing Interests: Declaration of Competing Interest Brittany A. Niccum is currently employed at a life-sciences company but was not so employed when the research was done and analyzed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The SOS response is considered to be an extremely important feature of bacterial cells. It helps them to survive bad times, including helping to develop resistance to antibiotics. The SOS response blocks the cell division. For Escherichia coli it is well known that the SulA protein directly interacts with FtsZ - a key division protein. Now it is believed that fission blocking is based on FtsZ sequestration by the SulA protein, which leads to decrease in effective concentration of FtsZ in the cell below a critical value, which in vitro leads to dismantling of FtsZ polymers. In this work, we demonstrate that in order to block the division of E. coli, it is sufficient to have a relatively small amount of SulA in the cell. Moreover, the analysis of structures formed by FtsZ in E. coli cells under the conditions of SulA protein expression or the SOS response showed that there is no complete disassembly of FtsZ polymers, although Z-rings indeed are not formed. The results of the work indicate that the well-known sequestration mechanism is not comprehensive to explain blocking of the division process by SulA in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Restriction-modification systems (R-M) are one of the antiviral defense tools used by bacteria, and those of the Type II family are composed of a restriction endonuclease (REase) and a DNA methyltransferase (MTase). Most entering DNA molecules are usually cleaved by the REase before they can be methylated by MTase, although the observed level of fragmented DNA may vary significantly. Using a model EcoRI R-M system, we report that the balance between DNA methylation and cleavage may be severely affected by transcriptional signals coming from outside the R-M operon. By modulating the activity of the promoter, we obtained a broad range of restriction phenotypes for the EcoRI R-M system that differed by up to 4 orders of magnitude in our biological assays. Surprisingly, we found that high expression levels of the R-M proteins were associated with reduced restriction of invading bacteriophage DNA. Our results suggested that the regulatory balance of cleavage and methylation was highly sensitive to fluctuations in transcriptional signals both up- and downstream of the R-M operon. Our data provided further insights into Type II R-M system maintenance and the potential conflict within the host bacterium., (© The Author(s) 2020. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

In the study presented here, we tested, how large a fraction of lysogenic culture was undergoing filamentation, which could indicate triggering of the SOS response or SOS-independent prophage induction that is also known to cause cell filamentation. Here, antibiotic stress was triggered by adding mitomycin C and oxidative stress was induced by hydrogen peroxide. Observation of bacterial cells under an optical microscope revealed more filamenting cells for lysogenic Escherichia coli than for strains not carrying a prophage. Moreover, the amount of filamenting cells depended not only on the stress agents used and the type of the prophage, but also on the host. During induction of the 933W prophage, the resulting phage titer and the amount of elongating cells were different when using E. coli O157:H7 EDL933 clinical isolate and the E. coli MG1655 laboratory strain. The amount of filamenting cells correlates well with the observed phage titers.

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

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform., Competing Interests: HG, BP, JL, SJ, KG, ZR, EW, RW, MC, Av No competing interests declared

Arenicin-1, a 21-mer antimicrobial peptide exerts significant broad-spectrum antimicrobial activity with membrane-active mechanisms. However, owing to multiple mechanisms of cell death, the antibacterial mechanism of arenicin-1 requires detailed analysis. In the present study, arenicin-1-treated bacteria underwent an apoptosis-like response, which was mechanistically and morphologically similar to eukaryotic apoptosis. Changes in the physiological status of arenicin-1-treated bacterial cells involved accumulation of reactive oxygen species, imbalance of intracellular calcium gradients, disruption of membrane potential, bacterial caspase-like protein activation, and DNA damage. In arenicin-1-induced apoptosis-like death, autocleavage of LexA was observed because of the activation of the caspase-like activity of RecA. Additionally, typical reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals, were scavenged in arenicin-1-treated cells to assess the role of specific reactive oxygen species. Scavenging of hydrogen peroxide interfered with the activity of arenicin-1 in Escherichia coli, whereas the superoxide and hydroxyl radicals level did not affect arenicin-1-induced apoptosis-like death activity. Furthermore, inhibition of Fenton reaction attenuated apoptosis-like response. In conclusion, arenicin-1-induced apoptosis like death requires SOS response proteins and is mediated by hydrogen peroxide and Fenton reaction in E. coli. Arenicin-1 may be a representative antimicrobial peptide with potent apoptotic response against E. coli.

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

The widespread use of nanoparticles has triggered increasing concern and interest due to the adverse effects on global public health and environmental safety. Whether the presence of nano-metal oxides (NMOs) could facilitate the formation of new antimicrobial resistance genes (ARGs) via de novo mutation is largely unknown. Here, we proved that two widely used NMOs could significantly improve the mutation frequencies of CIP- and CHL-resistant E. coli isolates; however, the corresponding metal ions have weaker effects. Distinct concentration-dependent increases of 1.0-14.2 and 1.1-456.3 folds were observed in the resistance mutations after treatment with 0.16-100 mg/L nano-Al 2 O 3 and 0.16-500 mg/L nano-ZnO, respectively, compared with those in the control. The resistant mutants showed resistance to multiple antibiotics and hereditary stability after sub-culturing for 5 days. We also explored the mechanism underlying the induction of antimicrobial resistance by NMOs. Whole-genome sequencing analysis showed that the mutated genes correlated with mono- and multidrug resistance, as well as undetected resistance to antibiotics. Furthermore, NMOs significantly promoted intracellular reactive oxygen species (ROS), which would lead to oxidative DNA damage and an error-prone SOS response, and consequently, mutation rates were enhanced. Our findings indicate that NMOs could accelerate the mutagenesis of multiple-antibiotic resistance and expanded the understanding of the mechanisms in nanoparticle-induced resistance, which may be significant for guiding the production and application of nanoparticles., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bacterial persisters are subpopulations of phenotypic variants in isogenic cultures that can survive lethal doses of antibiotics. Their tolerances are often attributed to reduced activities of antibiotic targets, which limit corruption and damage in persisters compared with bacteria that die from treatment. However, that model does not hold for nongrowing populations treated with ofloxacin, a fluoroquinolone, where antibiotic-induced damage is comparable between cells that live and those that die. To understand how those persisters achieve this feat, we employed a genetic system that uses orthogonal control of MazF and MazE, a toxin and its cognate antitoxin, to generate model persisters that are uniformly tolerant to ofloxacin. Despite this complete tolerance, MazF model persisters required the same DNA repair machinery (RecA, RecB, and SOS induction) to survive ofloxacin treatment as their nongrowing, WT counterparts and exhibited similar indicators of DNA damage from treatment. Further investigation revealed that, following treatment, the timing of DNA repair was critical to MazF persister survival because, when repair was delayed until after growth and DNA synthesis resumed, survival was compromised. In addition, we found that, with nongrowing, WT planktonic and biofilm populations, stalling the resumption of growth and DNA synthesis after the conclusion of fluoroquinolone treatment with a prevalent type of stress at infection sites (nutrient limitation) led to near complete survival. These findings illustrate that the timing of events, such as DNA repair, following fluoroquinolone treatment is important to persister survival and provide further evidence that knowledge of the postantibiotic recovery period is critical to understanding persistence phenotypes., Competing Interests: The authors declare no conflict of interest.

The use of platinum-based anticancer drugs is limited by both their side effects and their effect on normal microflora's metagenome. Drugs that possess mutagenic and genotoxic properties may cause mutations in microbial genomes that contribute to the emergence of resistance to antimicrobial preparations and the development of complications after chemotherapy. The effects of cisplatin and oxaliplatin on microorganisms were studied using bacterial biosensors - E. coli strains MG1655 pKatG-lux, which reacts to the generation of hydrogen peroxide; MG1655 pSoxS-lux, which reacts to the superoxide anion radical; and the MG1655 pColD-lux strain, which detects DNA damage. The biosensor tests demonstrated high levels of genotoxicity for both drugs and some differences in the spectrum of reactive oxygen species generated. Ascor-bate reduced genotoxicity of cisplatin by 41%. Nonlethal doses of cisplatin induced a three- to sevenfold increase in the frequency of the mutations that confer the resistance of E. coli to rifampicin and ciprof loxacin. Ascorbate also reduced frequency of the mutations by 65%. Thus, the effect of these drugs was probably associated with the generation of reactive oxygen species and induction of SOS response. The risk of secondary antibiotic-resistant infections may be decreased by applying antioxidants and antimutagens. At the same time, these increases may also decrease the anti-tumoral action of these compounds.

Suppression of the SOS response has been postulated as a therapeutic strategy for potentiating antimicrobial agents. We aimed to evaluate the impact of its suppression on reversing resistance using a model of isogenic strains of Escherichia coli representing multiple levels of quinolone resistance. E. coli mutants exhibiting a spectrum of SOS activity were constructed from isogenic strains carrying quinolone resistance mechanisms with susceptible and resistant phenotypes. Changes in susceptibility were evaluated by static (MICs) and dynamic (killing curves or flow cytometry) methodologies. A peritoneal sepsis murine model was used to evaluate in vivo impact. Suppression of the SOS response was capable of resensitizing mutant strains with genes encoding three or four different resistance mechanisms (up to 15-fold reductions in MICs). Killing curve assays showed a clear disadvantage for survival (Δlog 10 CFU per milliliter [CFU/ml] of 8 log units after 24 h), and the in vivo efficacy of ciprofloxacin was significantly enhanced (Δlog 10 CFU/g of 1.76 log units) in resistant strains with a suppressed SOS response. This effect was evident even after short periods (60 min) of exposure. Suppression of the SOS response reverses antimicrobial resistance across a range of E. coli phenotypes from reduced susceptibility to highly resistant, playing a significant role in increasing the in vivo efficacy. IMPORTANCE The rapid rise of antibiotic resistance in bacterial pathogens is now considered a major global health crisis. New strategies are needed to block the development of resistance and to extend the life of antibiotics. The SOS response is a promising target for developing therapeutics to reduce the acquisition of antibiotic resistance and enhance the bactericidal activity of antimicrobial agents such as quinolones. Significant questions remain regarding its impact as a strategy for the reversion or resensitization of antibiotic-resistant bacteria. To address this question, we have generated E. coli mutants that exhibited a spectrum of SOS activity, ranging from a natural SOS response to a hypoinducible or constitutively suppressed response. We tested the effects of these mutations on quinolone resistance reversion under therapeutic concentrations in a set of isogenic strains carrying different combinations of chromosome- and plasmid-mediated quinolone resistance mechanisms with susceptible, low-level quinolone resistant, resistant, and highly resistant phenotypes. Our comprehensive analysis opens up a new strategy for reversing drug resistance by targeting the SOS response., (Copyright © 2017 Recacha et al.)

The rnhAB mutant Escherichia coli, deficient in two RNase H enzymes that remove both R-loops and incorporated ribonucleotides (rNs) from DNA, grow slowly, suggesting accumulation of rN-containing DNA lesions (R-lesions). We report that the rnhAB mutants have reduced viability, form filaments with abnormal nucleoids, induce SOS, and fragment their chromosome, revealing replication and/or segregation stress. R-loops are known to interfere with replication forks, and sensitivity of the double rnhAB mutants to translation inhibition points to R-loops as precursors for R-lesions. However, the strict specificity of bacterial RNase HII for RNA-DNA junctions indicates that R-lesions have rNs integrated into DNA. Indeed, instead of relieving problems of rnhAB mutants, transient inhibition of replication from oriC kills them, suggesting that oriC-initiated replication removes R-loops instead of compounding them to R-lesions. Yet, replication from an R-loop-initiating plasmid origin kills the double rnhAB mutant, revealing generation of R-lesions by R-loop-primed DNA synthesis. These R-lesions could be R-tracts, contiguous runs of ≥4 RNA nucleotides within DNA strand and the only common substrate between the two bacterial RNase H enzymes. However, a plasmid relaxation test failed to detect R-tracts in DNA of the rnhAB mutants, although it readily detected R-patches (runs of 1-3 rNs). Instead, we detected R-gaps, single-strand gaps containing rNs, in the chromosomal DNA of the rnhAB mutant. Therefore, we propose that RNase H-deficient mutants convert some R-loops into R-tracts, which progress into R-gaps and then to double-strand breaks-explaining why R-tracts do not accumulate in RNase H-deficient cells, while double-strand breaks do., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Aims: The aim of this study was to investigate the influence of 20-hydroxyecdysone (20E) on the susceptibility of growing Escherichia coli to antibiotics., Methods and Results: Susceptibility of E. coli to antibiotics in the presence of 20E was estimated by determination of the colony-forming ability and the specific growth rate. Pretreatment with 20E decreased the bactericidal effect of ciprofloxacin (0·3 and 3·0 μg ml -1 ), streptomycin (10 and 40 μg ml -1 ) and kanamycin (10 μg ml -1 ) and increased the bactericidal action of 0·03 μg ml -1 ciprofloxacin. To study the influence of 20E on gene expression, we used strains of E. coli carrying fusions of promoters of relevant genes with the structural gene of β-galactosidase. 20E had no marked effect on the expression of antioxidant genes katG, katE, sodA and the rpoS gene (general stress response), while it (alone or combined with ciprofloxacin) markedly stimulated expression of the sulA gene belonging to the SOS regulon (DNA damage response)., Conclusion: In growing E. coli, 20E modulated the bactericidal action of antibiotics and stimulated the SOS response., Significance and Impact of the Study: The results of this study may be used to enhance the efficiency of antibacterial therapies., (© 2016 The Society for Applied Microbiology.)

Introduction: Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation., Objective: The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin., Methods: Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing., Results: Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated., Conclusion: It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The dnaQ gene of Escherichia coli encodes the ε subunit of DNA polymerase III, which provides the 3' → 5' exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of ε leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our laboratory identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response after quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby, in addition to proofreading, ε plays a distinct role in replisome disassembly and/or processing of stalled replication forks. To explore this model, we generated a pentapeptide insertion mutant library of the dnaQ gene, along with site-directed mutants, and screened for separation of function mutants. We report the identification of separation of function mutants from this screen, showing that proofreading function can be uncoupled from SOS phenotypes (partially constitutive SOS and the nalidixic acid SOS defect). Surprisingly, the two SOS phenotypes also appear to be separable from each other. These findings support the hypothesis that ε has additional roles aside from proofreading. Identification of these mutants, especially those with normal proofreading but SOS phenotype(s), also facilitates the study of the role of ε in SOS processes without the confounding results of high mutator activity associated with dnaQ knockout mutants., (Copyright © 2015 Whatley and Kreuzer.)

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.

Bacteria exposed to bactericidal fluoroquinolone (FQ) antibiotics can survive without becoming genetically resistant. Survival of these phenotypically resistant cells, commonly called "persisters," depends on the SOS gene network. We have examined mutants in all known SOS-regulated genes to identify functions essential for tolerance in Escherichia coli. The absence of DinG and UvrD helicases and the Holliday junction processing enzymes RuvA and RuvB leads to a decrease in survival. Analysis of the respective mutants indicates that, in addition to repair of double-strand breaks, tolerance depends on the repair of collapsed replication forks and stalled transcription complexes. Mutation in recF results in increased survival, which identifies RecAF recombination as a poisoning mechanism not previously linked to FQ lethality. DinG acts upstream of SOS promoting its induction, whereas RuvAB participates in repair only. UvrD directly promotes all repair processes initiated by FQ-induced damage and prevents RecAF-dependent misrepair, making it one of the crucial SOS functions required for tolerance.

Thymineless death (TLD) is the rapid loss of colony-forming ability in bacterial, yeast and human cells starved for thymine, and is the mechanism of action of common chemotherapeutic drugs. In Escherichia coli, significant loss of viability during TLD requires the SOS replication-stress/DNA-damage response, specifically its role in inducing the inhibitor of cell division, SulA. An independent RecQ- and RecJ-dependent TLD pathway accounts for a similarly large additional component of TLD, and a third SOS- and RecQ/J-independent TLD pathway has also been observed. Although two groups have implicated the SOS-response in TLD, an SOS-deficient mutant strain from an earlier study was found to be sensitive to thymine deprivation. We performed whole-genome resequencing on that SOS-deficient strain and find that, compared with the SOS-proficient control strain, it contains five mutations in addition to the SOS-blocking lexA(Ind(-)) mutation. One of the additional mutations, csrA, confers TLD sensitivity specifically in SOS-defective strains. We find that CsrA, a carbon storage regulator, reduces TLD in SOS- or SulA-defective cells, and that the increased TLD that occurs in csrA(-) SOS-defective cells is dependent on RecQ. We consider a hypothesis in which the modulation of nucleotide pools by CsrA might inhibit TLD specifically in SOS-deficient (SulA-deficient) cells., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Geraniol is an important industrial material and a potential candidate of advanced biofuels. One challenge of microbial geraniol production is the toxicity to hosts. However, the poor understanding on geraniol tolerance mechanism is an obstacle for developing geraniol tolerant host. This study genome-widely screened a shot-gun DNA library of Escherichia coli and found that recA is able to confer geraniol tolerance in E. coli. The recA knockout mutant was found extremely sensitive to geraniol. Based on our data, it was deciphered that recA provided tolerance through SOS response network responding to DNA damage caused by geraniol. RecA-mediated SOS response activates the homologous recombinational repair by RecB and RecN for corrective DNA maintenance. This protection mechanism suggests an effective strategy to combat geraniol toxicity in E. coli., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A theoretical study is performed of the possible role of the methyl-directed mismatch repair system in the ultraviolet-induced mutagenesis of Escherichia coli bacterial cells. For this purpose, mathematical models of the SOS network, translesion synthesis and mismatch repair are developed. Within the proposed models, the key pathways of these repair systems were simulated on the basis of modern experimental data related to their mechanisms. Our model approach shows a possible mechanistic explanation of the hypothesis that the bacterial mismatch repair system is responsible for attenuation of mutation frequency during ultraviolet-induced SOS response via removal of the nucleotides misincorporated by DNA polymerase V (the UmuD'2C complex)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells. The type I TA loci also exist on the Escherichia coli chromosome but their biological functions are less clear. Genetic organization and regulatory elements of hok/sok and ldr/rdl families are very similar and the toxins are predicted to contain a transmembrane domain, but otherwise share no detectable sequence similarity. This review will give an overview of the type I TA modules of E. coli K-12, especially hok/sok, ldr/rdl and SOS-inducible symE/symR systems, which are regulated by divergently overlapping cis-encoded antisense RNAs.

Chromosomal DNA is exposed to continuous damage and repair. Cells contain a number of proteins and specific DNA repair systems that help maintain its correct structure. The SOS response was the first DNA repair system described in Escherichia coli induced upon treatment of bacteria with DNA damaging agents arrest DNA replication and cell division. Induction of the SOS response involves more than forty independent SOS genes, most of which encode proteins engaged in protection, repair, replication, mutagenesis and metabolism of DNA. Under normal growth conditions the SOS genes are expressed at a basal level, which increases distinctly upon induction of the SOS response. The SOS-response has been found in many bacterial species (e.g., Salmonella typhimurium, Caulobacter crescentus, Mycobacterium tuberculosis), but not in eukaryotic cells. However, species from all kingdoms contain some SOS-like proteins taking part in DNA repair that exhibit amino acid homology and enzymatic activities related to those found in E. coli. but are not organized in an SOS system. This paper presents a brief up-to-date review describing the discovery of the SOS system, the physiology of SOS induction, methods for its determination, and the role of some SOS-induced genes.

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]