The rarA gene as part of an expanded RecFOR recombination pathway: Negative epistasis and synthetic lethality with ruvB, recG, and recQ

The RarA protein, homologous to human WRNIP1 and yeast MgsA, is a AAA+ ATPase and one of the most highly conserved DNA repair proteins. With an apparent role in the repair of stalled or collapsed replication forks, the molecular function of this protein family remains obscure. Here, we demonstrate that RarA acts in late stages of recombinational DNA repair of post-replication gaps. A deletion of most of the rarA gene, when paired with a deletion of ruvB or ruvC, produces a growth defect, a strong synergistic increase in sensitivity to DNA damaging agents, cell elongation, and an increase in SOS induction. Except for SOS induction, these effects are all suppressed by inactivating recF, recO, or recJ, indicating that RarA, along with RuvB, acts downstream of RecA. SOS induction increases dramatically in a rarA ruvB recF/O triple mutant, suggesting the generation of large amounts of unrepaired ssDNA. The rarA ruvB defects are not suppressed (and in fact slightly increased) by recB inactivation, suggesting RarA acts primarily downstream of RecA in post-replication gaps rather than in double strand break repair. Inactivating rarA, ruvB and recG together is synthetically lethal, an outcome again suppressed by inactivation of recF, recO, or recJ. A rarA ruvB recQ triple deletion mutant is also inviable. Together, the results suggest the existence of multiple pathways, perhaps overlapping, for the resolution or reversal of recombination intermediates created by RecA protein in post-replication gaps within the broader RecF pathway. One of these paths involves RarA.

Extending the gap and loading RecA: the presynaptic phase plays a pivotal role in modulating lesion tolerance pathways

During replication, the presence of unrepaired lesions results in the formation of single stranded DNA (ssDNA) gaps that need to be repaired to preserve genome integrity and cell survival. All organisms have evolved two major lesion tolerance pathways to continue replication: Translesion Synthesis (TLS), potentially mutagenic, and Homology Directed Gap Repair (HDGR), that relies on homologous recombination. In Escherichia coli, the RecF pathway repairs such ssDNA gaps by processing them to produce a recombinogenic RecA nucleofilament during the presynaptic phase. In this study, we show that the presynaptic phase is crucial for modulating lesion tolerance pathways. Indeed, impairing either the extension of the ssDNA gap (mediated by the nuclease RecJ and the helicase RecQ) or the loading of RecA (mediated by the RecFOR complex) leads to a decrease in HDGR. We suggest a model where defects in the presynaptic phase delay the formation of the D-loop and increase the time window allowed for TLS. We indeed observe an increase in TLS independent of SOS induction. In addition, we revealed an unexpected synergistic interaction between recF and recJ genes, that results in a recA deficient-like phenotype in which HDGR is almost completely abolished.

Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

Elevated Expression of Toxin TisB Protects Persister Cells against Ciprofloxacin but Enhances Susceptibility to Mitomycin C

Bacterial chromosomes harbor toxin-antitoxin (TA) systems, some of which are implicated in the formation of multidrug-tolerant persister cells. In Escherichia coli, toxin TisB from the tisB/istR-1 TA system depolarizes the inner membrane and causes ATP depletion, which presumably favors persister formation. Transcription of tisB is induced upon DNA damage due to activation of the SOS response by LexA degradation. Transcriptional activation of tisB is counteracted on the post-transcriptional level by structural features of tisB mRNA and RNA antitoxin IstR-1. Deletion of the regulatory RNA elements (mutant Δ1-41 ΔistR) uncouples TisB expression from LexA-dependent SOS induction and causes a ‘high persistence’ (hip) phenotype upon treatment with different antibiotics. Here, we demonstrate by the use of fluorescent reporters that TisB overexpression in mutant Δ1-41 ΔistR inhibits cellular processes, including the expression of SOS genes. The failure in SOS gene expression does not affect the hip phenotype upon treatment with the fluoroquinolone ciprofloxacin, likely because ATP depletion avoids strong DNA damage. By contrast, Δ1-41 ΔistR cells are highly susceptible to the DNA cross-linker mitomycin C, likely because the expression of SOS-dependent repair systems is impeded. Hence, the hip phenotype of the mutant is conditional and strongly depends on the DNA-damaging agent.

Genome-Wide Identification and Expression Analysis of SOS Response Genes in Salmonella enterica Serovar Typhimurium

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

RexAB Is Essential for the Mutagenic Repair of Staphylococcus aureus DNA Damage Caused by Co-trimoxazole

ABSTRACT Co-trimoxazole (SXT) is a combination therapeutic that consists of sulfamethoxazole and trimethoprim that is increasingly used to treat skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the use of SXT is limited to the treatment of low-burden, superficial S. aureus infections and its therapeutic value is compromised by the frequent emergence of resistance. As a first step toward the identification of approaches to enhance the efficacy of SXT, we examined the role of bacterial DNA repair in antibiotic susceptibility and mutagenesis. We found that mutants lacking the DNA repair complex RexAB had a modest SXT MIC that was 2-fold lower than that seen with wild-type strains but were killed 50-fold to 5,000-fold more efficiently by the combination antibiotic at the breakpoint concentration. SXT-mediated DNA damage occurred via both thymidine limitation and the generation of reactive oxygen species and triggered induction of the SOS response in a RexAB-dependent manner. SOS induction was associated with a 50% increase in the mutation rate, which may contribute to emergence of resistant strains during SXT therapy. In summary, this work determined that SXT caused DNA damage in S. aureus via both thymidine limitation and oxidative stress and that the damage was repaired by the RexAB complex, leading to induction of the mutagenic SOS response. Small-molecule inhibitors of RexAB could therefore have therapeutic value by increasing the efficacy of SXT and decreasing the emergence of drug resistance during treatment of infections caused by S. aureus.

THE EFFECT ON THE GENETIC APPARATUS OF THE MICROBIAL CELL OF COMPOUNDS BASED ON SUBSTITUTED 1H-INDOL-4-, 5-, 6-, 7-YLAMINES

The study of new antimicrobial compounds includes determining the mechanism of their effect on the microbial cell. As a rule, the effect of most modern synthetic antimicrobials is associated either with the suppression of DNA synthesis, or with the suppression of bacterial protein synthesis at the level of translation or transcription.There are sensitive and simple methods for screening and monitoring the potential genotoxic activity of a wide range of natural and synthetic compounds. To date, the Ames test has been widely used, based on the sensitivity of Salmonella strains to carcinogenic chemicals, although some compounds that cause Ames negative reactions could actually be carcinogenic to animals.Similarly, the SOS chromotest is an SOS transcriptional analysis that can evaluate DNA damage caused by chemical and physical mutagens. It measures the expression of a reporter gene (β-galactosidase). The β-galactosidase enzyme processes ortho-nitrophenyl galactopyranoside to form a yellow compound detected at 420 nm. Then, the induction of β-galactosidase normalizes the activity of alkaline phosphatase, an enzyme expressed constitutively by Escherichia coli. SOS chromotest is also widely used for genotoxicological studies. The answer is quick (several hours) and does not require the survival of the test strain. Dose response curves for various chemicals include a linear region. The slope of this area is taken as a measure of SOS induction.Therefore, an SOS chromotest was selected for the study, which allows one to identify the DNA-mediated effect of the studied compounds.The aim of the work was to evaluate the SOS-inducing activity of antimicrobial compounds based on substituted 1H-indol-4-, 5-, 6-, 7-ylamines.The strain Escherichia coli PQ 37 with the genotype F-thr leu his-4 pyrD thi galE lacΔU169 srl300 :: Th10 rpoB rpsL uvrA rfa trp :: Mis + sfi A :: Mud (Ar, lac) cts, Due to the presence of sfi A genes :: lac Z, lacZ β-galactosidase gene expression in strain PQ 37 is controlled by the promoter of the sfiA gene, one of the components of the E. coli SOS regulon. The indicator of the SOS-inducing activity of the studied compounds in the SOS chromotest is the activity of β-galactosidase, which evaluates the activity of active microorganisms - alkaline phosphatase, which also allows you to control the toxic effect of the studied compounds on bacterial cells.The results showed that 4,4,4-trifluoro-N-(6-methoxy-1,2,3-trimethyl-1H-indol-5-yl)-3-oxobutanamide (1), 4,4,4-trifluoro-N-(6-methyl-2-phenyl-1H-indol-5-yl)-3-oxobutanamide (2) and N-(1,5-dimethyl-2-phenyl-1H-indol-6-yl)-4,4,4-trifluoro-3-oxobutanamide (3) does not possess SOS-inducing activity in the studied concentrations. 4-Hydroxy-8-phenyl-4-(trifluoromethyl)-1,3,4,7-tetrahydro-2H-pyrrolo [2,3-h] -quinolin-2-one (4), 9-hydroxy-5-methyl-2-phenyl-9-(trifluoromethyl)-1,6,8,9-tetrahydro-7Н-pyrrolo-[2,3-f]quinolin-7-one (5), 6-hydroxy-2,3-dimethyl-6-(trifluoromethyl)-1,6,7,9-tetrahydro-8H-pyrrolo[3,2-h]quinolin-8-one (6) and 1,2,3,9-tetramethyl-6-(trifluoromethyl)-1,9-dihydro-8H-pyrrolo [3,2-h]quinolin-8-one (7) showed dose-dependent SOS-inducing activity in bactericidal concentrations. The obtained research results allowed us to identify compounds 4, 5, 6, 7, the mechanism of action of which includes exposure to DNA of a microbial cell.

Single-cell imaging and characterization of Escherichia coli persister cells to ofloxacin in exponential cultures

Bacterial persistence refers to the capacity of small subpopulations within clonal populations to tolerate antibiotics. Persisters are thought to originate from dormant cells in which antibiotic targets are less active and cannot be corrupted. Here, we report that in exponentially growing cultures, ofloxacin persisters originate from metabolically active cells: These cells are dividing before the addition of ofloxacin and do endure DNA damages during the treatment, similar to their nonpersister siblings. We observed that growth rate, DNA content, and SOS induction vary among persisters, as in the bulk of the population and therefore do not constitute predictive markers for persistence. Persister cells typically form long polynucleoid filaments and reach maximum SOS induction after removal of ofloxacin. Eventually, cell division resumes, giving rise to a new population. Our findings highlight the heterogeneity of persister cells and therefore the need to analyze these low-frequency phenotypic variants on a case-by-case basis at the single-cell level.

N-acetylcysteine blocks SOS induction and mutagenesis produced by fluoroquinolones in Escherichia coli

BackgroundFluoroquinolones such as ciprofloxacin induce the mutagenic SOS response and increase the levels of intracellular reactive oxygen species (ROS). Both the SOS response and ROS increase bacterial mutagenesis, fuelling the emergence of resistant mutants during antibiotic treatment. Recently, there has been growing interest in developing new drugs able to diminish the mutagenic effect of antibiotics by modulating ROS production and the SOS response.ObjectivesTo test whether physiological concentrations of N-acetylcysteine, a clinically safe antioxidant drug currently used in human therapy, is able to reduce ROS production, SOS induction and mutagenesis in ciprofloxacin-treated bacteria without affecting antibiotic activity.MethodsThe Escherichia coli strain IBDS1 and its isogenic mutant deprived of SOS mutagenesis (TLS−) were treated with different concentrations of ciprofloxacin, N-acetylcysteine or both drugs in combination. Relevant parameters such as MICs, growth rates, ROS production, SOS induction, filamentation and antibiotic-induced mutation rates were evaluated.ResultsTreatment with N-acetylcysteine reduced intracellular ROS levels (by ∼40%), as well as SOS induction (by up to 75%) and bacterial filamentation caused by subinhibitory concentrations of ciprofloxacin, without affecting ciprofloxacin antibacterial activity. Remarkably, N-acetylcysteine completely abolished SOS-mediated mutagenesis.ConclusionsCollectively, our data strongly support the notion that ROS are a key factor in antibiotic-induced SOS mutagenesis and open the possibility of using N-acetylcysteine in combination with antibiotic therapy to hinder the development of antibiotic resistance.

The Crohn’s disease-associated Escherichia coli strain LF82 rely on SOS and stringent responses to survive, multiply and tolerate antibiotics within macrophages

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