Abstract
Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA–ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA–ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA–ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA–ramA genes, strongly suggesting that a secondary regulator may control ramA expression.
Highlights
Klebsiella pneumoniae is a major nosocomial pathogen that causes both community and hospital acquired infections
Bioinformatic analyses shows that the palindromic binding sites identified for RamR in Salmonella spp. is conserved within the intergenic region between the ramR and romA coding sequences of K. pneumoniae, Enterobacter and Citrobacter spp. (Fig. 1A)
We surmised that the regulation of the romA-ramA locus mediated by RamR must occur via the palindromic sequence that lies upstream of the romA gene. 209 3.4 RACE Mapping The transcriptional start site of ramA as determined by RACE mapping is shown in Figure 1B. The transcriptional start site for ramA was found upstream of the romA stop codon which implies that romA and ramA are part of an operon and are likely co213 transcribed in K. pneumoniae
Summary
Klebsiella pneumoniae is a major nosocomial pathogen that causes both community and hospital acquired infections. Several studies demonstrate that tigecycline resistance results from the upregulation of AraC-family transcriptional regulators such as MarA or RamA which in turn are linked to increased expression of the acrAB efflux pump [6,7, 10,11]. Like MarA, increased ramA expression is linked to the upregulation of the acrAB efflux pump which confers a multidrug resistant phenotype (MDR) to a variety of different antibiotic classes [7, 10,11, 16] This phenotype is mediated exclusively through the acrAB gene as ramA overexpression in an acrAB-deleted strain does not produce a similar phenotype [11]. S. typhimurium [17] and K. pneumoniae [16], have reported that ramR mutations are directly linked to ramA overexpression
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