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Error-prone DNA repair system in enteroaggregative Escherichia coli identified by subtractive hybridization.

ABSTRACT: Enteroaggregative Escherichia coli (EAEC) are etiologic agents of diarrhea. The EAEC category is heterogeneous, but most in-depth experimentation has focused on prototypical strain, 042. We hypothesized that 60A, another EAEC strain, might posses virulence or fitness genes that 042 does not have. Through subtractive hybridization we identified 60A-specific sequences, including loci present in other E. coli and phage DNA. One locus thus identified was impB, a LexA repressed error-prone DNA repair gene that has been identified in plasmids from other enteric organisms and which we detected in 21 of 34 EAEC strains. An isogenic 60A impB mutant showed decreased survival and mutagenesis after exposure to UV, as well as bile salt exposure, compared to the wild-type strain, and these phenotypes could be complemented in trans. The EAEC strain 60A imp operon differs structurally from previously described homologs. A cryptic gene, impC, present in other imp operons, is absent from 60A. In addition, transcription of impAB in strain 60A occurs from a promoter that is dissimilar to the previously described impC promoter but is still triggered by UV-mediated damage. In strain 60A the impAB and the aggregative adherence fimbriae I (AAF/I)-encoding genes are on the same large plasmid, and the 60A version of the operon is predominantly seen in AAF/I-positive EAEC. Supplementary imp SOS-inducible error-prone repair systems are common among EAEC even though they are absent in prototypical strain 042.

SUBMITTER: Joo LM

PROVIDER: S-EPMC1913340 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Error-prone DNA repair system in enteroaggregative Escherichia coli identified by subtractive hybridization.

Joo Lucy M LM Macfarlane-Smith Louissa R LR Okeke Iruka N IN

Journal of bacteriology 20070309 10

Enteroaggregative Escherichia coli (EAEC) are etiologic agents of diarrhea. The EAEC category is heterogeneous, but most in-depth experimentation has focused on prototypical strain, 042. We hypothesized that 60A, another EAEC strain, might posses virulence or fitness genes that 042 does not have. Through subtractive hybridization we identified 60A-specific sequences, including loci present in other E. coli and phage DNA. One locus thus identified was impB, a LexA repressed error-prone DNA repair ...[more]

PMID: 17351038

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Project description:UnlabelledEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication.ImportanceDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

Project description:BackgroundExtraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathotype within the whole ExPEC group.ResultsTwenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDa adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal E. coli strains, yielding a significant association with strains of avian origin.ConclusionsWe identified a chromosomally located autotransporter gene in a highly virulent APEC strain which confers increased adherence of a non-fimbriated E. coli K-12 strain to a chicken fibroblast cell line. Even though flanked by mobile genetic elements and three different genetic regions upstream of the gene, most probably indicating horizontal gene transfer events, the adhesin gene was significantly linked with strains of avian origin. Due to the nucleotide sequence similarity of 98% to a recently published adhesin-related gene, located on plasmid pAPEC-O1-ColBM, the name aatA (APEC autotransporter adhesin A) was adopted from that study.Our data substantiate that AatA might not only be of relevance in APEC pathogenicity but also in facilitating their reservoir life style in the chicken intestine, which might pave the way for future intestinal preventive strategies.

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Error-prone DNA repair system in enteroaggregative Escherichia coli identified by subtractive hybridization.

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Error-prone DNA repair system in enteroaggregative Escherichia coli identified by subtractive hybridization.

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