Genes acrA and acrB encode a stress‐induced efflux system of Escherichia coli

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

doi:10.1111/j.1365-2958.1995.tb02390.x

Cited by 569 publications

(533 citation statements)

References 38 publications

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“…Alternatively, as permeability barriers and active efflux are both essential for preventing drug access to bacterial targets (30,37), the sanA gene could code for an efflux pump (of broad specificity), normally unexpressed, activated only in extreme conditions (or when artificially expressed from a medium-copynumber plasmid), like the stress-induced efflux system encoded by genes acrA and acrB in E. coli (29). The behavior of our null mutant sanA::Tc strain (Fig.…”

Section: Discussionmentioning

confidence: 99%

Amplification of a novel gene, sanA, abolishes a vancomycin-sensitive defect in Escherichia coli

Rida

Caillet

1996

J Bacteriol

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We have isolated an Escherichia coli gene which, when overexpressed, is able to complement the permeability defects of a vancomycin-susceptible mutant. This gene, designated sanA, is located at min 47 of the E. coli chromosome and codes for a 20-kDa protein with a highly hydrophobic amino-terminal segment. A strain carrying a null mutation of the sanA gene, transferred to the E. coli chromosome by homologous recombination, is perfectly viable, but after two generations at high temperature (43؇C), the barrier function of its envelope towards vancomycin is defective.An essential function of the outer membrane (39), an additional bilayer located outside the peptidoglycan in gram-negative bacteria such as Escherichia coli, is to act as a selective permeability barrier (38) towards hydrophobic (52) and hydrophilic compounds if the latter are larger than 600 Da (3). The outer membrane also prevents leakage of periplasmic components and controls direct passage of secreted proteins which lack classical amino-terminal signal sequences (e.g., hemolysin) from the cytoplasm to the external medium (sec-independent secretory pathway) (47). Also the outer membrane is probably responsible for the initiation of signals ultimately resulting in changes in gene expression (32).The inner leaflet of the outer membrane is composed, like the cytoplasmic membrane, of phospholipid molecules (42), whereas its outer leaflet is a unique structure composed of lipopolysaccharide (LPS) (50), porins (36), lipoproteins (14), and small amounts of enzymes (5, 26) and other minor proteins (65). The structures and biosynthetic pathways of these components are well-known, but many features of their insertion into the outer membrane and of their function are still obscure. The barrier function of the outer membrane in relation to its structure has been studied mainly by using membrane-damaging drugs (cation chelators, dyes, detergents, and local anesthetics, etc.) which modify its permeability (24, 57) and by the isolation and characterization of mutants with altered permeability towards various antibiotics (both hydrophobic and hydrophilic), dyes, and detergents (54).Gram-positive bacteria are sensitive to vancomycin, a glycopeptide antibiotic which inhibits late steps in the extracytoplasmic synthesis of cell wall peptidoglycan (44). Although hydrophilic (35), vancomycin cannot enter gram-negative bacteria because of its large size (1.4 kDa); thus, wild-type E. coli is naturally vancomycin resistant. We have undertaken the isolation, from a library of wild-type E. coli genes inserted in a bacteriophage vector, of one or several genetic determinants which can complement the defect of a recently isolated vancomycin-sensitive mutant of E. coli. Here we report the cloning, the mapping at min 47 of the E. coli chromosome, and the nucleotide sequence of a novel gene, named sanA, which when expressed from a medium-copy-number plasmid suppresses the vancomycin susceptibility as well as some other phenotypic characteristics of this mutant. The hydrophobic na...

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Section: Discussionmentioning

confidence: 99%

Amplification of a novel gene, sanA, abolishes a vancomycin-sensitive defect in Escherichia coli

Rida

Caillet

1996

J Bacteriol

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“…The native promoter was used for AcrAB expression. The trc promoter, which requires induction by IPTG, was used for AcrEF and Bcr because AcrEF is not normally expressed by the native promoter under laboratory culture conditions (12,21). This also eliminates the effect of the putative regulator (yeiD) contained in our clone of pUCbcr containing the Bcr native promoter (17).…”

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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“…Both eukaryotic and prokaryotic cells increase expression of some multidrug transporter genes in response to environmental shock caused by heat (Miyazaki et al, 1992), osmotic (Ma et al, 1995) Levy, 1983) stresses. However, the role that transporters have in stress tolerance has not been extensively examined.…”

Section: Role Of Phle In General Stress Tolerancementioning

confidence: 99%

The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance

et al. 2004

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2,4-Diacetylphloroglucinol (PHL) is the primary determinant of the biological control activity of Pseudomonas fluorescens F113. The operon phlACBD encodes enzymes responsible for PHL biosynthesis from intermediate metabolites. The phlE gene, which is located downstream of the phlACBD operon, encodes a putative permease suggested to be a member of the major facilitator superfamily with 12 transmembrane segments. PhlE has been suggested to function in PHL export. Here the sequencing of the phlE gene from P. fluorescens F113 and the construction of a phlE null mutant, F113-D3, is reported. It is shown that F113-D3 produced less PHL than F113. The ratio of cell-associated to free PHL was not significantly different between the strains, suggesting the existence of alternative transporters for PHL. The phlE mutant was, however, significantly more sensitive to high concentrations of added PHL, implicating PhlE in PHL resistance. Furthermore, the phlE mutant was more susceptible to osmotic, oxidative and heat-shock stresses. Osmotic stress induced rapid degradation of free PHL by the bacteria. Based on these results, we propose that the role of phlE in general stress tolerance is to export toxic intermediates of PHL degradation from the cells. INTRODUCTIONMany Pseudomonas fluorescens strains produce 2,4-diacetylphloroglucinol (PHL) (Bangera & Thomashow, 1996;Boruah & Kumar, 2002;Nowak-Thompson et al., 1994;Reddi & Borovkov, 1970; Sharifi-Tehrani et al., 1998). This phenolic molecule has broad-spectrum antifungal (Levy et al., 1992;Schoonbeek et al., 2002;Tomas-Lorente et al., 1989;Weller & Cook, 1983), antibacterial (Levy et al., 1992), antihelminthic (Bowden et al., 1965; Harrison et al., 1993) and phytotoxic (Keel et al., 1992;Reddi et al., 1969) activities. PHL is the primary determinant of biological control by P. fluorescens F113 (Shanahan et al., 1992). Synthesis of PHL is directed by the phlACBD genes, which are believed to constitute an operon (Bangera & Thomashow, 1996Delany, 1999). PhlD shows structural similarities with plant chalcone synthase, also called polyketide synthase type III, and is necessary but not sufficient for the synthesis of monoacetylphloroglucinol (MAPG) (Bangera & Thomashow, 1999). PhlA, PhlC and PhlB are necessary and sufficient for the transacetylation of MAPG to produce PHL (Bangera & Thomashow, 1999;Shanahan et al., 1992). The available data strongly suggest that PhlA, PhlC, PhlB and PhlD function in a multienzyme complex (Bangera & Thomashow, 1999;Delany, 1999). Expression of the phlACBD operon is subject to complex regulation (Aarons et al., 2000;Abbas et al., 2002;Bangera & Thomashow, 1999;Chou et al., 1993;Corbell & Loper, 1995;Delany, 1999; Delany et al., 2000;Duffy & Defago, 1999;Schnider-Keel et al., 2000). In addition, PHL is an autoregulator, positively influencing its own biosynthesis (Abbas et al., 2002;Schnider-Keel et al., 2000).Micro-organisms have developed various ways to resist the toxic effects of the secondary metabolites they produce. These mechanisms include expo...

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Genes acrA and acrB encode a stress‐induced efflux system of Escherichia coli

Cited by 569 publications

References 38 publications

Amplification of a novel gene, sanA, abolishes a vancomycin-sensitive defect in Escherichia coli

Amplification of a novel gene, sanA, abolishes a vancomycin-sensitive defect in Escherichia coli

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

The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance

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