LINDE HH, ARNOLD M, IRTENKAUF C, DECKER J, NOTKA F, LEHN N; Interscience Conference on Antimicrobial Agents and Chemotherapy (43rd: 2003: Chicago, Ill.).
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2003 Sep 14-17; 43: abstract no. C1-2067.
Inst. Med. Microbiol., Univ. Regensburg, Regensburg, Germany.
BACKGROUND: Active efflux is increasingly recognized as a resistance mechanism in Enterobacteriaceae. In E. coli quinolones are effluxed by the AcrAB-TolC system (among others), which is regulated by MarA and other transcription factors (TF) like SoxS or Rob. Here, we investigated regulation of the mar operon in E. cloacae using a mar promotor-gfp reporter construct (pmar-gfp). METHODS: Two clinical isolates of E. cloacae (1) were transposed with pmar-gfp. Second-step random knockout clones were screened for altered expression of GFP and mapped for putative TFs using BLAST search. RT-PCR was used to confirm transcription in vivo. Candidate genes were expressed in pCC1, and MICs to ciprofloxacin (CIP) and chloramphenicol (CHA) were determined using Etest. E. cloacae DSMZ 3264 and a susceptible clinical isolate were used as controls. GenBank accession number of roh: AY291074. RESULTS: Screening of the knockout library of pmar-gfp transformed E. cloacae detected an isolate with diminished fluorescence. Mapping of the transposon insertion site revealed a 405 bp open reading frame, which demonstrated 47% aa homology to the C-terminal part of Rob of E. coli, now named roh (rob-homologue). Overexpression of Roh in E. coli resulted in >/= 4-fold/2-fold increase of MIC to CIP/CHA, compared to a control strain containing pCC1 without Roh. CONCLUSION: The N-terminal part of Rob of E. coli was shown to contain the DNA-binding region (2). Here, using a pmar-gfp reporter construct and a subsequent knockout strategy in E. cloacae, we identified the putative TF Roh with 47% aa homology to the N-terminus of Rob of E. coli. Overexpression of Roh in E. coli resulted in increased MICs to CIP and CHA, agreeable with an efflux phenotype. Possible binding sites of Rho are the promoter regions of mar and or acrAB, or other efflux-related loci. References: Linde, H.J., et al. 2002; J. Antimicrob. Chemother. 49:625. Rosner, J.L., et al. 2002; J. Bacteriol. 184:1407.
Publication Types:
Keywords:
- Ciprofloxacin
- Enterobacter cloacae
- Escherichia coli
- Escherichia coli Proteins
- Genes, Regulator
- Microbial Sensitivity Tests
- Promoter Regions (Genetics)
- Transcription Factors
- Transcription, Genetic
- genetics
Other ID:
UI: 102264672
From Meeting Abstracts