Relevance of DNA Alkylation Damage Repair Systems in Salmonella enterica Virulence

Abstract

Systematic inactivation of pathways involved in DNA alkylation damage repair demonstrated that inactivation of the ada, ogt, tag, uvrA, and mfd genes is required to detect a Salmonella enterica virulence decrease. Furthermore, the fitness of S. enterica, defective in these genes, is lowered only when the bacterium is orally, but not intraperitoneally, inoculated.Bacteria are exposed to a wide variety of DNA-injuring agents, including alkylating agents, and their effects have been well studied (12,17,18). The repair of alkylated DNA in bacterial cells has been described mainly in Escherichia coli (12,18), which presents the following two different mechanisms to eliminate alkyl radicals from its DNA: (i) the alkylinduced expression of genes encoding the necessary repair enzymes (12, 17) and (ii) the constitutive synthesis of such proteins (9).Many bacterial species have a genetic network, known as the adaptive response, which functions to repair alkyl lesions in the bacterial DNA (18,19) and is regulated by the Ada protein, which also possesses methyltransferase activity (18). Transfer of a methyl from DNA-methylated phosphates to the Cys-37 residue of Ada (17) triggers a conformational change in this protein that converts it into a positive transcriptional regulator. Once activated, Ada stimulates the expression of its own transcriptional unit, including the alkB gene, which encodes an N 1 -meA-DNA dioxygenase, and the alkA and aidB genes, which encode a N 3 -meA-DNA-glycosylase and a flavin-containing DNA binding protein, respectively (9).Bacteria also possess the following two additional enzymes involved in the repair of alkylated DNA: Ogt (O 6 -meG-DNA methyltransferase) and Tag (N 3 -meA-DNA glycosylase) (9). Expression of the ogt and tag genes is constitutive and does not depend on the presence of DNA alkylation (9). Despite the importance of alkylated DNA repair, there is very little information concerning the relevance of this process in pathogenic bacteria.To determine the implications of DNA alkylation damage repair in Salmonella enterica virulence, mutants defective in the ada gene, or genes under its control (alkA, alkB and aidB), or mutants defective in the ogt and tag genes were constructed using the one-step PCR-based gene replacement method (6).As expected, survival assays showed that each of these single mutants was more sensitive than the wild-type strain to several alkylating agents, including N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), and diethyl sulfate (DES) (data not shown). Furthermore, competition assays between each of these mutants and the wild-type strain, carried out as reported previously (3, 4), demonstrated that the inactivation of neither ada, ogt, nor tag had any effect on the virulence of S. enterica cells, regardless of whether the bacteria were inoculated orally or intraperitoneally (i.p.) in BALB/c mice (data not shown).In light of these results, S. enterica strains carrying all possible double combinations of the ada, tag, and ogt mutations we...

Biofilm formation and transcriptome analysis of Streptococcus gallolyticus subsp. gallolyticus in response to lysozyme

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Added insult to injury? The response of meat-associated pathogens to proposed antimicrobial interventions

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“…However, questions regarding the effectiveness of transcriptomic studies with UV damage response can be raised given the disruptive effect that DNA damage related to UV treatment has on cell transcription, with lower abundances of cDNA transcripts isolated from UV-treated cells than other treatments employed in this study (McKay et al 2004 ). Some DNA damage responses such as the alkylation response tag and ogt genes thus tend to be constitutively expressed in the cytoplasm and might be better assessed in proteomic-based studies (Àlvarez et al 2010 ).…”

Occurrence and repair of alkylating stress in the intracellular pathogen Brucella abortus

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