Identification of a DNA-Damage-Inducible Regulon in Acinetobacter baumannii

Abstract

dThe transcriptional response of Acinetobacter baumannii, a major cause of nosocomial infections, to the DNA-damaging agent mitomycin C (MMC) was studied using DNA microarray technology. Most of the 39 genes induced by MMC were related to either prophages or encoded proteins involved in DNA repair. Electrophoretic mobility shift assays demonstrated that the product of the A. baumannii MMC-inducible umuD gene (umuDAb) specifically binds to the palindromic sequence TTGAAAATGTAAC TTTTTCAA present in its promoter region. Mutations in this palindromic region abolished UmuDAb protein binding. A comparison of the promoter regions of all MMC-induced genes identified four additional transcriptional units with similar palindromic sequences recognized and specifically bound by UmuDAb. Therefore, the UmuDAb regulon consists of at least eight genes encoding seven predicted error-prone DNA polymerase V components and DddR, a protein of unknown function. Expression of these genes was not induced in the MMC-treated recA mutant. Furthermore, inactivation of the umuDAb gene resulted in the deregulation of all DNA-damage-induced genes containing the described palindromic DNA motif. Together, these findings suggest that UmuDAb is a direct regulator of the DNA damage response in A. baumannii. In Escherichia coli and many other bacterial species, extensive DNA damage that cannot be repaired by other cellular mechanisms induces a mutagenic repair pathway known as the SOS response (1). As part of the SOS response, the RecA protein binds to the single-stranded region of the damaged DNA, where it is activated and forms a nucleoprotein filament (2). Activated RecA induces the autoprotease activity of LexA, which in the absence of activated RecA represses the expression of SOS genes by binding to specific sequences in their promoters. As cleaved LexA is unable to bind to these regulator sequences, the SOS genes are derepressed (1). The proteins encoded by SOS genes mediate DNA repair and replication in addition to pausing the cell cycle. The activities in the SOS response include those of highly error-prone polymerases (1). Once the damaged DNA is repaired, RecA no longer induces the autocleavage of LexA, which then accumulates and shuts down the SOS response (3).The SOS genetic network is widely present in Eubacteria (4), but in Acinetobacter spp. the DNA damage response is characterized by several atypical features: (i) there is no damage-induced mutagenesis response to DNA damage, with the remarkable exception of the opportunistic pathogens Acinetobacter baumannii, Acinetobacter ursingii, and Acinetobacter beijerinckii (5-7); (ii) after DNA damage, further induction of recA does not require the RecA protein (8); (iii) none of the promoters of the DNAdamage-inducible genes of Acinetobacter spp. contain a known SOS box (7, 9, 10); (iv) a canonical LexA homologue has not been identified (8, 10, 11); and (v) a UmuD homologue in A. baumannii (UmuDAb) has been proposed as a putative indirect regulator of the DNA damage response (6). Given the...

Differential Roles of Antimicrobials in the Acquisition of Drug Resistance through Activation of the SOS Response in Acinetobacter baumannii

“…baumannii contains multiple umuDC operons and unlinked umuC and umuD genes, all of which encode components of errorprone DNA polymerase V (6). In A. baumannii strain ATCC 17978, the induction of these components after DNA damage leads to the introduction of point mutations in the bacterial genome, including those able to generate antibiotic resistance (7,8). Previous studies showed that the umuD and umuC genes of A. baumannii are induced by ciprofloxacin, which is also a strong inducer of the SOS response (6).…”

“…Previous studies showed that the UmuDAb regulon, encoding several error-prone DNA polymerase V subunits, is an important component of the SOS response and mutagenesis in A. baumannii (6)(7)(8). Point mutations in the bacterial genome caused by these error-prone DNA polymerases can result in the acquisition of drug resistance.…”

“…Gene expression was determined by reverse transcription-quantitative real-time PCR (RT-qPCR) using the LightCycler RNA master SYBR green I (Roche) on a LightCycler 480 instrument (LC480) (Roche), according to the manufacturer's instructions. Specific oligonucleotides (Roche) were used to quantify the genes encoding error-prone DNA polymerase components belonging to the UmuDAb regulon of A. baumannii: A1S_0636, A1S_1174, A1S_1389, A1S_2008, and A1S_2015 (8). The relative mRNA concentration obtained from the genes studied was determined according to a standard curve generated by amplifying an internal fragment of the gyrB gene, which does not belong to the SOS regulon (8).…”

“…The relative mRNA concentration obtained from the genes studied was determined according to a standard curve generated by amplifying an internal fragment of the gyrB gene, which does not belong to the SOS regulon (8). The expression factor was calculated as the ratio between the mRNA concentration of the genes expressed in the treated cultures versus that from the untreated culture (8). The results showed that genes encoding components of the error-prone DNA polymerase V of A. baumannii were induced by either ciprofloxacin or tetracycline but not by meropenem or colistin (Fig.…”

“…Specific oligonucleotides (Roche) were used to quantify the genes encoding error-prone DNA polymerase components belonging to the UmuDAb regulon of A. baumannii: A1S_0636, A1S_1174, A1S_1389, A1S_2008, and A1S_2015 (8). The relative mRNA concentration obtained from the genes studied was determined according to a standard curve generated by amplifying an internal fragment of the gyrB gene, which does not belong to the SOS regulon (8). The expression factor was calculated as the ratio between the mRNA concentration of the genes expressed in the treated cultures versus that from the untreated culture (8).…”