Molecular cloning and characterization of acrA and acrE genes of Escherichia coli

Ma, Dzwokai; Cook, David N.; Alberti, Marie; Pon, Ning G.; Nikaido, Hiroshi; Hearst, John E.

doi:10.1128/jb.175.19.6299-6313.1993

Cited by 424 publications

(359 citation statements)

References 40 publications

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“…All of these are so-called membrane fusion proteins (MFPs) of RNDtype multidrug efflux transporters. The analysis also showed VmeB to share 63, 63, 62, 62 and 61 % identities, respectively, with AcrB, AcrD and AcrF of E. coli (Klein et al, 1991;Ma et al, 1993Ma et al, , 1994, SdeY of S. marcescens (Chen et al, 2003) and MexB of P. aeruginosa (Poole et al, 1993). These are inner-membrane proteins of RND-type multidrug efflux transporters.…”

Section: Sequencingmentioning

confidence: 99%

“…All these differences were translationally silent, except two: changing amino acid residue Gly33 in VP1091 to Ala33 in VmeA, and changing Ala1046 in VP1092 to Asp1046 in VmeB. A BLAST search for protein sequence similarities (NCBI) showed VmeA to share, respectively, 49, 49, 47 and 46 % identities with AcrA and AcrE of E. coli (Klein et al, 1991;Ma et al, 1993), SdeX of Serratia marcescens (Chen et al, 2003) and MexA of Pseudomonas aeruginosa (Poole et al, 1993). All of these are so-called membrane fusion proteins (MFPs) of RNDtype multidrug efflux transporters.…”

Section: Sequencingmentioning

confidence: 99%

“…All these differences were translationally silent, except two: changing amino acid residue Gly33 in VP1091 to Ala33 in VmeA, and changing Ala1046 in VP1092 to Asp1046 in VmeB. A BLAST search Ma et al, 1993Ma et al, , 1994, SdeY of S. marcescens (Chen et al, 2003) and MexB of P. aeruginosa (Poole et al, 1993). These are inner-membrane proteins of RND-type multidrug efflux transporters.…”

Section: Sequencingmentioning

confidence: 99%

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VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus

et al. 2007

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Genes vmeA and vmeB, encoding a multidrug efflux transporter in the halophilic bacterium Vibrio parahaemolyticus, have been cloned using a drug-hypersusceptible Escherichia coli strain as the host. Cells of E. coli KAM33 (DacrAB DydhE) carrying the vmeAB region from V. parahaemolyticus conferred much higher MICs for a variety of antimicrobial agents than did control cells. Cells possessing VmeAB under energized conditions maintained very low intracellular concentrations of ethidium. This was as expected for an energy-dependent efflux system, and supports the notion -based on sequence homology -that VmeAB belongs to the resistance nodulation cell division (RND) family of multidrug efflux transporters. It is likely that VmeAB forms functional complexes with the outer-membrane protein TolC in E. coli, because introduction of vmeAB into cells of E. coli KAM43, which lacks the tolC gene, failed to elevate the MICs for any of the antimicrobial agents tested. Therefore, a V. parahaemolyticus homologue of tolC was also cloned, designated vpoC, and was introduced together with vmeAB into cells of E. coli KAM43. The MICs of all agents tested were raised and were comparable to the values observed in E. coli KAM33 harbouring a plasmid carrying vmeAB. Finally, a vmeAB-deficient mutant of V. parahaemolyticus was constructed (designated TM3). TM3 showed slightly higher susceptibility than the parental V. parahaemolyticus to some antimicrobial agents. Survival rate of the TM3 when exposed to deoxycholate decreased compared with that of the parent. INTRODUCTIONVibrio parahaemolyticus, a slightly halophilic marine bacterium, is a major cause of food poisoning in Japan (Obata et al., 2001; WHO, 1999;Yamazaki et al., 2003) and many other countries (Cabanillas-Beltran et al., 2006; CDC, 2006;Lozano-Leon et al., 2003;McLaughlin et al., 2005; Sen et al., 2007;Su et al., 2005a). The organism has some peculiar physiological characteristics, such as a very fast growth rate, a Na + requirement for growth (Baumann & Schubert, 1984) and an ability to live in diverse environments including brackish water (Kumazawa & Kato, 1985), on estuarine algae (Kumazawa et al., 1991), and in mammalian hosts, including humans (Yamamoto & Yokota, 1989).In achieving human infection, V. parahaemolyticus survives in the intestine and especially in the duodenum, where bile acids and their conjugates are abundant. Bile acids are anionic detergents and support the digestion of fats in the intestine. In addition, bile has bactericidal effects due to its membrane-solvent property (Provenzano et al., 2000). Thus, bile resistance of enteropathogenic bacteria is thought to be important for their survival in the intestine. Some enteropathogenic bacteria have been shown to possess systems to protect from the actions of bile. In the case of Vibrio cholerae, the outer-membrane component TolC Vc , which had already been identified as a component of some multidrug efflux transporters (sometimes called multidrug efflux pumps), was found to contribute to resistance to bile,...

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Section: Sequencingmentioning

confidence: 99%

Section: Sequencingmentioning

confidence: 99%

“…All these differences were translationally silent, except two: changing amino acid residue Gly33 in VP1091 to Ala33 in VmeA, and changing Ala1046 in VP1092 to Asp1046 in VmeB. A BLAST search Ma et al, 1993Ma et al, , 1994, SdeY of S. marcescens (Chen et al, 2003) and MexB of P. aeruginosa (Poole et al, 1993). These are inner-membrane proteins of RND-type multidrug efflux transporters.…”

Section: Sequencingmentioning

confidence: 99%

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VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus

et al. 2007

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“…Likewise, other tetracycline derivatives were similarly affected by AcrAB and/or AcrEF, with two-to fourfold increases in the MICs (Table 3). AcrB is the highly potent MDR transporter in E. coli, and AcrF is its close homologue; the amino acid identity between the two is 77% (13). Bcr expression did not result in any change in the tigecycline MIC.…”

Section: Induction Of Tet(b) By Tigecycline In E Coli W3104/plgt2 [Tmentioning

confidence: 99%

Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)

Hirata

Saito

Nishino

et al. 2004

Antimicrob Agents Chemother

108

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The activity of tigecycline, 9-(t-butylglycylamido)-minocycline, against Escherichia coli KAM3 (acrB) strains harboring plasmids encoding various tetracycline-specific efflux transporter genes, tet(B), tet(C), and tet(K), and multidrug transporter genes, acrAB, acrEF, and bcr, was examined. Tigecycline showed potent activity against all three Tet-expressing, tetracycline-resistant strains, with the MICs for the strains being equal to that for the host strain. In the Tet(B)-containing vesicle study, tigecycline did not significantly inhibit tetracycline efflux-coupled proton translocation and at 10 M did not cause proton translocation. This suggests that tigecycline is not recognized by the Tet efflux transporter at a low concentration; therefore, it exhibits significant antibacterial activity. These properties can explain its potent activity against bacteria with a Tet efflux resistance determinant. Tigecycline induced the Tet(B) protein approximately four times more efficiently than tetracycline, as determined by Western blotting, indicating that it is at least recognized by a TetR repressor. The MICs for multidrug efflux proteins AcrAB and AcrEF were increased fourfold. Tigecycline inhibited active ethidium bromide efflux from intact E. coli cells overproducing AcrAB. Therefore, tigecycline is a possible substrate of AcrAB and its close homolog, AcrEF, which are resistance-modulation-division-type multicomponent efflux transporters.One approach to overcoming the clinical drug resistance problem in bacterial infections is to modify existing antibiotics to avoid the presence of a resistance determinant. This approach is being used for tetracyclines. Tigecycline is a broadspectrum glycylcycline derivative (2) and is efficacious against highly resistant bacteria (1), including methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae. However, it is less active against clinically problematic opportunistic pathogens, such as Pseudomonas aeruginosa (6) and Proteus mirabilis (3, 28).The most common mechanism for tetracycline resistance is an efflux pump-mediated one in both gram-positive and gramnegative organisms (5), i.e., a metal-tetracycline/proton antiporter encoded on plasmids (4, 14, 32). Besides tetracyclinespecific transporters, multidrug transporters are also becoming a problem in clinical situations (27).We recently constructed a library of all 37 possible genes for efflux pumps in Escherichia coli and found that 20 of them conferred resistance to one or more antibiotics, detergents, or dyes (17). Although many of them are not expressed under the usual culture conditions, they may cause clinical resistance in the future, and it is thus meaningful to explore their characters and prepare for the possibility of the development of resistance before it becomes a real problem (17).We were interested in determining whether in the future tigecycline will pose a resistance problem due to these efflux mechanisms. In this study, we studied the effects of efflux pumps on susceptibil...

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“…Many PEPs, such as the hemolysin translocator accessory protein HlyD (Felmlee et al, 1985), have a stretch of hydrophobic residues near the N terminus believed to span the cytoplasmic membrane (Schu È lein et al, 1992). Others, like AcrA and AcrE of E. coli (Ma et al, 1993) and MexA of P. aeruginosa (Poole et al, 1993) have a lipoprotein modi®cation signal and are likely to insert in the cytoplasmic membrane via an attached lipid moiety (Dinh et al, 1994;Seiffer et al, 1993). PEPs appear to form trimers (Thanabalu et al, 1998), and have been shown to interact speci®cally with OEPs (Akatsuka et al, 1997;Binet & Wandersman, 1995;Hwang et al, 1997;Le Âtoffe Â et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

Johnson

Church

1999

Journal of Molecular Biology

155

147

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Broad-speci®city ef¯ux pumps have been implicated in multidrug-resistant strains of Pseudomonas aeruginosa and other Gram-negative bacteria. Most Gram-negative pumps of clinical relevance have three components, an inner membrane transporter, an outer membrane channel protein, and a periplasmic protein, which together coordinate ef¯ux from the cytoplasmic membrane across the outer membrane through an unknown mechanism. The periplasmic ef¯ux proteins (PEPs) and outer membrane ef¯ux proteins (OEPs) are not obviously related to proteins of known structure, and understanding the structure and function of these proteins has been hindered by the dif®culty of obtaining reasonable multiple alignments. We present a general strategy for the alignment and structure prediction of protein families with low mutual sequence similarity using the PEP and OEP families as detailed examples. Gibbs sampling, hidden Markov models, and other analysis techniques were used to locate motifs, generate multiple alignments, and assign PEP or OEP function to hypothetical proteins in several species. We also developed an automated procedure which combines multiple alignments with structure prediction algorithms in order to identify conserved structural features in protein families. This process was used to identify a probable a-helical hairpin in the PEP family and was applied to the detection of transmembrane b-strands in OEPs. We also show that all OEPs contain a large tandem duplication, and demonstrate that the OEP family is unlikely to adopt a porin fold, in contrast to previous predictions.

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Molecular cloning and characterization of acrA and acrE genes of Escherichia coli

Abstract: The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test (4,24

Cited by 424 publications

References 40 publications

VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus

VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus

Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)

Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

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