ImuA facilitates SOS mutagenesis by inhibiting RecA-mediated activity in Myxococcus xanthus

Bacteria have two pathways to restart stalled replication forks caused by environmental stresses, error-prone translesion DNA synthesis (TLS) catalyzed by TLS polymerase and error-free template switching catalyzed by RecA, and their competition on the arrested fork affects bacterial SOS mutagenesis. DnaE2 is an error-prone TLS polymerase, and its functions require ImuA and ImuB. Herein, we investigated the transcriptions of imuA , imuB and dnaE2 in UV-C irradiated Myxococcus xanthus and found that the induction of imuA occurred significantly earlier than that of the other two genes. Mutant analysis showed that unlike that of imuB or dnaE2 , the deletion of imuA significantly delayed bacterial regrowth and slightly reduced the bacterial mutation frequency and UV-resistance. Transcriptomic analysis revealed that the absence of ImuA released the expression of some known SOS genes, including recA1 , recA2 , imuB , and dnaE2 . Yeast two-hybrid and pull down analysis proved ImuA interact physically with RecA1 besides ImuB. Protein activity analysis indicated that ImuA had no DNA binding activity, but inhibited the DNA binding and recombinase activity of RecA1. These findings indicate the new role of ImuA in SOS mutagenesis, that is, ImuA inhibits the recombinase activity of RecA1, thereby facilitating SOS mutagenesis in M. xanthus . Importance : DnaE2 is responsible for bacterial SOS mutagenesis in nearly one-third of sequenced bacterial strains. However, its mechanism, especially the function of its accessory protein ImuA, is still unclear. Here we reported that M. xanthus ImuA could affect SOS mutagenesis by inhibiting the recombinase activity of RecA1, which helps to explain the mechanism of DnaE2-dependent TLS and the selection of the two restart pathways to repair the stalled replication fork.

Inactivation of Umuc Protein Significantly Reduces Resistance to Ciprofloxacin and SOS Mutagenesis in Escherichia coli Mutants Harboring Intact Umud Gene

Background: Ciprofloxacin induces SOS response and mutagenesis by activation of UmuD’2C (DNA polymerase V) and DinB (DNA polymerase IV) in Escherichia coli, leading to antibiotic resistance during therapy. Inactivation of DNA polymerase V can result in the inhibition of mutagenesis in E. coli. Objectives: The aim of this research was to investigate the effect of UmuC inactivation on resistance to ciprofloxacin and SOS mutagenesis in E. coli mutants. Methods: Ciprofloxacin-resistant mutants were produced in a umuC- genetic background in the presence of increasing concentrations of ciprofloxacin. The minimum inhibitory concentration of umuC-mutants was measured by broth dilution method. Alterations in the rifampin resistance-determing region of rpoB gene were assessed by PCR amplification and DNA sequencing. The expression of SOS genes was measured by quantitative real-time PCR assay. Results: Results showed that despite the induction of SOS response (overexpression of recA, dinB, and umuD genes) following exposure to ciprofloxacin in E. coliumuC mutants, resistance to ciprofloxacin and SOS mutagenesis significantly decreased. However, rifampicin-resistant clones emerged in this genetic background. One of these clones showed mutations in the rifampicin resistance-determining region of rpoB (cluster II). The low mutation frequency of E. coli might be associated with the presence and overexpression of umuD gene, which could somehow limit the activity of DinB, the location and type of mutations in the β subunit of RNA polymerase. Conclusions: In conclusion, for increasing the efficiency of ciprofloxacin against Gram-negative bacteria, use of an inhibitor of umuC, along with ciprofloxacin, would be helpful.

ImuA participates in SOS mutagenesis by interacting with RecA1 and ImuB in Myxococcus xanthus

Bacteria have two pathways to restart stalled replication forks caused by environmental stresses, error-prone translesion DNA synthesis (TLS) catalyzed by TLS polymerase and error-free template switching catalyzed by RecA, and their competition on the arrested fork affects bacterial SOS mutagenesis. DnaE2 is an error-prone TLS polymerase, and its functions require ImuA and ImuB. Here we investigated the function of imuA, imuB and dnaE2 in Myxococcus xanthus and found that imuA showed differences from imuB and dnaE2 in bacterial growth, resistance and mutation frequency. Transcriptomics analysis found that ImuA were associated with bacterial SOS response. Yeast-two-hybrid scanning revealed that ImuA interacted with RecA1 besides ImuB. Protein activity analysis proved that ImuA had no DNA binding activity, but inhibited the DNA binding and recombinase activity of RecA1. These findings highlight that ImuA not only participates in TLS by binding ImuB, but also inhibits the recombinase activity of RecA1 in M. xanthus, suggesting a role of ImuA in the two replication restart pathways.ImportanceDnaE2 is responsible for bacterial SOS mutagenesis in nearly one third of sequenced bacterial strains. However, its mechanism, especially the function of its accessory protein ImuA, is still unclear. Here we reported that M. xanthus ImuA might facilitate DnaE2 TLS by inhibiting the recombinase activity of RecA1, which helps to explain the mechanism of DnaE2-dependent TLS and the scientific problem of choosing one of the two restart pathways to repair the stalled replication fork.

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.

Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis

ABSTRACT Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found to increase mutations to antibiotic resistance in Streptococcus uberis cultures. The mutational spectra revealed mainly G:C→A:T transitions, and Northern analyses demonstrated increased expression of a Y-family DNA polymerase resembling UmuC under DNA-damaging conditions. In the absence of the Y-family polymerase, S. uberis cells were sensitive to UV light and to mitomycin C. Furthermore, the UV-induced mutagenesis was almost completely abolished in cells deficient in the Y-family polymerase. The gene encoding the Y-family polymerase was localized in a four-gene operon including two hypothetical genes and a gene encoding a HdiR homolog. Electrophoretic mobility shift assays demonstrated that S. uberis HdiR binds specifically to an inverted repeat sequence in the promoter region of the four-gene operon. Database searches revealed conservation of the gene cassette in several Streptococcus species, including at least one genome each of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Streptococcus sanguinis, and Streptococcus thermophilus strains. In addition, the umuC operon was localized in several mobile DNA elements of Streptococcus and Lactococcus species. We conclude that the hdiR-umuC-ORF3-ORF4 operon represents a novel gene cassette capable of mediating SOS mutagenesis among members of the Streptococcaceae.