Evidence for Chloramphenicol/H+ Antiport in Cmr (MdfA) System of Escherichia coli and Properties of the Antiporter

Abstract

We detected chloramphenicol/H+ antiport activity in membrane vesicles of Escherichia coli and cloned a gene for the antiporter from chromosomal DNA of E. coli. Introduction of the gene into E. coli cells conferred resistance to chloramphenicol and ethidium. A slight increase in resistance to acridine orange was also observed. Elevated chloramphenicol efflux and ethidium efflux were observed in cells harboring a plasmid carrying the gene. Addition of chloramphenicol to the assay mixture reduced the efflux of ethidium. Elevated chloramphenicol/H+ antiport activity was observed in membrane vesicles prepared from cells harboring the plasmid. The pH optimum for the activity was 6.5. We sequenced the gene and deduced the amino acid sequence of its product. A sequence homology search revealed that it was same as that of Cmr (or MdfA). Thus, it became clear that Cmr (MdfA) is the chloramphenicol(and ethidium)/H+ antiporter.

Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7

“…Although the DmexXY mutant (PA7 DXY) showed the same antimicrobial susceptibility pattern as the DmexXY-oprA mutant (PA7 DXYA), the DoprA mutant (PA7 DA) did not show any significant difference to the wild-type strain PA7, suggesting that another outer-membrane protein(s) complements the oprA deletion. In P. aeruginosa PAO1, the MexXY system utilizes OprM of the multidrug efflux operon mexAB-oprM as its outer membrane component (Mine et al, 1999;Aires et al, 1999). In fact, the DoprA DoprM double mutant (PA7 DA DM) showed the same susceptibility to the aminoglycosides, although the DoprM mutant (PA7 DM) did not; therefore, MexXY utilizes OprM in addition to OprA as one of its outer membrane components in PA7.…”

“…This strain is very useful for the analysis of various multidrug efflux pumps from micro-organisms (Morita et al, 1998;Mine et al, 1999;Masaoka et al, 2000) and plants (Li et al, 2002). E. coli KAM3 containing the mexXY or mexXY-oprA genes of PA7 showed increased resistance to a number of antibiotics, including erythromycin, ciprofloxacin, cefpirome, tetracycline and gentamicin (Table 2) to the same extent as E. coli KAM3 containing the mexXY genes of P. aeruginosa PAO1 (data not shown).…”

“…The mexXY genes from PAO1 (Mine et al, 1999) were released via the digestion of pSportI : : mexXY (Morita et al, 2006) with EcoRI and cloned into pSTV28 and pYM101, digested with EcoRI, and dephosphorylated to yield pYM115 and pYM120, respectively.…”

“…MexX (397 aa) and MexY (1045 aa) of PA7 showed strong sequence similarities to MexXY (www.pseudomonas. com) of PAO1 [95 (98)% and 97 (99)% identity (similarity), respectively] and other P. aeruginosa strains, while OprA (467 aa) of PA7 showed higher sequence similarity to OprA of Burkholderia species [56-58 (70-71)% identity (positives)], such as Burkholderia thailandensis, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia multivorans and Burkholderia cenocepacia, than to OprM encoded by the mexAB-oprM multidrug efflux operon of P. aeruginosa PAO1 [44 (59)% identity (similarity)], which MexXY of P. aeruginosa PAO1 requires as an outer membrane component to function (Mine et al, 1999;Aires et al, 1999). OprA of B. pseudomallei is an outer membrane component of AmrABOprA, which is a major multidrug efflux pump that is implicated in the pan-aminoglycoside resistance of the organism and is closely related to MexXY of P. aeruginosa (Moore et al, 1999;Mima & Schweizer, 2010).…”

Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa

“…In several clinically isolated mutants of P. aeruginosa, higher expression of RND-type efflux pumps was observed (Hirai et al, 1987;Rella & Haas, 1982;Wolter et al, 2004). Of the 11 RND efflux pumps identified in this organism, 10 pumps, MexAB-OprM (Poole et al, 1993), MexCD-OprJ (Poole et al, 1996), MexEF-OprN (Kohler et al, 1997), MexGHI-OpmD (Aendekerk et al, 2002;Sekiya et al, 2003), MexJK (Chuanchuen et al, 2002), MexMN (Mima et al, 2005), MexPQ-OpmE (Mima et al, 2005), MexVW (Li et al, 2003), MexXY (Mine et al, 1999;Westbrock-Wadman et al, 1999) and TriABCOpmH (Mima et al, 2007), have been experimentally confirmed, and their properties have been reported. The RND-type efflux pump usually consists of three components to fulfil the function properly and includes an innermembrane component (RND component), a periplasmic component (MFP component), and an outer-membrane component (OMP component) (Touze et al, 2004), and the three-dimensional structures of MexA, MexB and OprM have been reported (Akama et al, 2004a, b;Higgins et al, 2004;Sennhauser et al, 2009).…”