Characterization of regulatory element Rp3 of regulation of β-lactamases fromRalstonia pickettii

Abstract

The two chromosomally encoded beta-lactamases, OXA-22 and OXA-60, from Ralstonia pickettii are inducible by beta-lactam molecules. Disruption of RP3 abolished induction of both beta-lactamases and the resistance to pH, osmolarity and survival in the stationary phase, suggesting that RP3 might be a global regulator. Interactions between RP3, OXA-22 and OXA-60 were investigated at a transcript and protein level using 5'-rapid amplification of cDNA ends experiments, real-time reverse transcription (RT)-PCR and footprinting assays. The rp3 gene was actively transcribed and the promoter sequences corresponded to a nontypical sigma(70)-type promoter. RT-PCR analysis showed that rp3 expression as well as that of the bla(OXA) genes was positively regulated: the level of transcripts of rp3, bla(OXA-22) and bla(OXA-60) genes were, respectively, increased 20-, 100- and 2000-fold upon imipenem induction. DNAse I footprinting showed that RP3 specifically bound to tandem repeats centered at positions -55.5 and -73.5 upstream from the bla(OXA-22) and bla(OXA-60) transcriptional start sites. Interestingly, the binding site at bla(OXA-60) overlapped the -35 region of the rp3 promoter, although the region essential for induction lies at the beginning of the orf-rp3. This result indicates that RP3 is most probably only one component of a novel regulatory system involved in the expression of beta-lactamases in R. pickettii.

Role of IS Kpn7 and Deletions in bla KPC Gene Expression

“…a Number of bla KPC RNA molecules. The results were normalized using the 16S rRNA gene as a standard (8).…”

“…In order to quantify expression of the bla KPC gene in K. pneumoniae KN2303, K. pneumoniae YC, K. pneumoniae 475, and E. coli (carrying pKPCprom1,2,3, pKPCprom1,2, or pKPCprom2), one-step qRT-PCR was used with the respective forward (qRT PCR 1) and reverse (qRT PCR 2) primers listed in Table 1. Expression levels were standardized relative to the transcription level of the constitutively expressed 16S rRNA gene (8). DNA traces were removed by treating RNA extracts with DNase I during the extraction protocol, as recommended by the manufacturer (Qiagen).…”

A Case of Ventriculoperitoneal Shunt Infection Due to Ralstonia pickettii

“…Birbirinden bağımsız, kromozomal olarak kodlanan OXA-22 ve OXA-60 olmak üzere iki oksasilinaz enzimi üretir. Oksasilinaz enzimi (oksasilini hidrolize eden β-laktamaz) genellikle oksasilin, metisilin ve kloksasilini benzil penisilinden daha iyi hidrolize eder ve aktivitesi sodyum klorürle inhibe olur (17). Bu enzimlerden OXA-60 sadece β-laktam indüksiyonu sonrasında açığa çıkar.…”

Carbapenem Resistance in Elizabethkingia meningoseptica Is Mediated by Metallo-β-Lactamase BlaB

“…These results suggest that E. meningoseptica increases BlaB production after sensing stress conditions of growth, probably resembling those encountered when causing human meningitis (the cerebrospinal fluid is a nutrientlimiting medium) or in its natural habitats. Cases of complex growth-dependent regulation were observed for SBLs such as E. coli AmpC and Ralstonia pickettii OXA-22 and OXA-60 (19,38). The gene bolA, a regulator of cell wall biosynthetic enzymes and ampC expression, is induced in stationary phase or under sudden carbon starvation.…”

“…The gene bolA, a regulator of cell wall biosynthetic enzymes and ampC expression, is induced in stationary phase or under sudden carbon starvation. In R. pickettii, the bla OXA-22 and bla OXA-60 SBL genes are inducible by ␤-lactam molecules (19). Disruption of the regulatory protein RP3 abolishes induction of both ␤-lactamases, together with resistance to pH and osmolarity, and survival in the stationary phase.…”