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Investigating Intracellular RecA Concentrations in the Emerging Pathogen Acinetobacter baumannii
Author(s) -
Downs Margaret J.,
Ching Carly,
Tashjian Tommy F.,
Godoy Veronica G.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.647.4
Subject(s) - acinetobacter baumannii , biology , dna damage , gene , recombinase , microbiology and biotechnology , genetics , dna repair , escherichia coli , sos response , population , dna , pseudomonas aeruginosa , bacteria , recombination , demography , sociology
Acinetobacter baumannii is an opportunistic pathogen responsible for many hospital‐acquired infections. A. baumannii is known for its ability to rapidly gain antibiotic resistances and survive desiccation. This ability to survive adverse conditions has been linked to A. baumannii 's unique DNA‐damage response (DDR), which is modulated by the evolutionarily conserved recombinase, RecA. We have previously shown that in A. baumannii, RecA is needed to upregulate the expression of error‐prone DNA polymerase genes, which in turn results in higher frequency of mutagenesis. A. baumannii lacks a functional homologue for the highly conserved global Escherichia coli DDR transcriptional repressor, indicating that their DDRs are regulated differently. In contrast to E. coli , the DDR in A. baumannii is bimodal. That is, when DNA damage is induced and expression of DDR genes is tracked, two distinct subpopulations are evident, one with low expression of DDR genes and one with high. The two populations survive differently, suggesting that the bacterium can fall back on the population that does survive. The details of the mechanism underlying this regulation remain largely unknown. Our lab has recently shown that the recA gene is regulated by a 5′untranslated region that is important for both its stability and expression. Moreover, preliminary evidence suggests that differential recA expression is needed to maintain the bimodal nature of the DDR. Because of RecA's role in the DDR, we sought to compare the levels of RecA in A. baumannii and E. coli . We hypothesized that RecA intracellular concentration in A. baumannii would be lower than E. coli 's, consistent with the differences in regulatory roles. To test this hypothesis, we performed a semiquantitative Western blot using anti‐ E. coli RecA polyclonal antibody. We first purified His‐tagged RecA from each species. The purified proteins were then used to prepare a standard curve for the Western blot. As expected, we observed a difference in affinity of the anti‐ recA antibody based on differences in the primary protein sequence of RecA from the two species. ( A. baumannii and E. coli RecA are approximately 74% identical). Remarkably, and in agreement with our hypothesis, we find that the levels of basal intracellular RecA were at least three times lower in A. baumannii than in E. coli . This difference in RecA protein concentration may have profound effects on A. baumannii physiology and is consistent with the different regulatory role for RecA in A. baumannii. These results show that the intracellular RecA concentration in bacteria may provide insights into the regulation of the DDR and the mechanism by which A. baumannii is able to survive adverse conditions and readily acquire antibiotic resistances. Support or Funding Information Funding was provided by the National Institute of General Medical Sciences (RO1GM088230 to Veronica Godoy). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .