Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones

Yoshida, Hiroaki; Bogaki, M; Nakamura, Shinichi; Ubukata, Kimiko; Konno, Masatoshi

doi:10.1128/jb.172.12.6942-6949.1990

Cited by 430 publications

(324 citation statements)

References 39 publications

(27 reference statements)

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“…These mechanisms include export of the metabolite out of the producing cell. The phlE gene, located at the 39 end of the phlACBD operon in P. fluorescens strains (Bangera & Thomashow, 1996Delany, 1999) encodes a putative transmembrane permease with 12 predicted transmembrane segments (TMS) (Bangera & Thomashow, 1999 transporter mediating resistance to a range of structurally dissimilar drugs (Paulsen & Sukurray, 1993;Yoshida et al, 1990). PhlE also has structural similarity with integral membrane proteins associated with resistance which are encoded within clusters responsible for polyketide biosynthesis (Brautaset et al, 2000; Fernandez-Moreno et al, 1991;Guillfoile & Hutchinson, 1992;Marger & Saier, 1993;Molnar et al, 2000).…”

Section: Introductionmentioning

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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Section: Introductionmentioning

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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“…In Gram-negative bacteria, tetracycline resistance is mostly assigned to the drug etitux systems, which are classified into A-H [11]. They have a conserved sequence motif, G(K/R)XSD(R/K)XGR(R/K), in the first cytoplasmic loop (named loop2-3), which is common not only in bacterial drug export proteins [12,13] but also in the secondary transporters and belongs to a major facilitator family [14,15]. Tet(K) and Tet(L) also contain a derivative of this motif, GKXSDXX(X/G)XK(K/R), in the corresponding loop, indicating that they belong to a major facilitator family.…”

Section: Introductionmentioning

confidence: 99%

Transmembrane glutamic acid residues play essential roles in the metal‐tetracycline/H⁺ antiporter of Staphylococcus aureus

Fujihira¹,

Kimura²,

Shiina³

et al. 1996

FEBS Letters

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; however, it has no transmembrane aspartyl residue. On the other hand, Tet(K) has three glutamyl residues, Glu-30, Glu-152 and Glu-397, in the putative transmembrane regions. In the present work, tet(K) gene was expressed in Escherichia coli and the transport activity was measured in everted membrane vesicles. When these glutamyl residues were replaced with Gin, the tetracycline transport activity was almost completely lost, indicating the important roles of these residues in Tet(K). In the case of Glu-397, even the charge-conserved mutation to Asp caused complete loss of the activity. On the other hand, the mutation of Glu-30 and Glu-152 to Asp resulted in significant retention of transport activity. These results are similar to those on the mutation of the three transmemhrane aspartyl residues in Tet(B), indicating that the transmembrane glutamyl residues in Tet(K) play roles similar to those of the transmemhrane aspartyl residues in Tet(B).

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“…For example, human P-glycoprotein, the product of the MDR1 gene, is highly homologous to the protein MDR2, which does not efflux drugs but is a specific phosphatidylcholine flippase (6). Similarly, the bacterial multidrug transporters Bmr and Blt of Bacillus subtilis and NorA of Staphylococcus aureus are highly homologous to the efflux transporters whose only known substrate is tetracycline (7)(8)(9)(10).…”

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confidence: 99%

Efflux of the Natural Polyamine Spermidine Facilitated by the Bacillus subtilis Multidrug Transporter Blt

Woolridge

Vázquez‐Laslop

Markham

et al. 1997

Journal of Biological Chemistry

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Multidrug transporters pump structurally dissimilar toxic molecules out of cells. It is not known, however, if detoxification is the primary physiological function of these transporters. The chromosomal organization of the gene encoding the Bacillus subtilis multidrug transporter Blt suggests a specific function for this protein; it forms a single operon with another gene, bltD, whose protein product is identified here as a spermine/spermidine acetyltransferase, an enzyme catalyzing a key step in spermidine degradation. Overexpression of the Blt transporter in B. subtilis leads not only to the multidrug-resistance phenotype but also to the efflux of large amounts of spermidine into the medium; this efflux is supressed by an inhibitor of Blt, reserpine. Taken together, these results strongly suggest that the natural function of the Blt transporter is the efflux of spermidine, whereas multiple drugs may be recognized by Blt merely opportunistically.Multidrug transporters, first discovered in mammalian cells (P-glycoprotein) (1) and later found in yeast (2) and many species of bacteria (3-5), pump structurally dissimilar toxic molecules, including many anticancer, antifungal, and antibacterial agents, out of cells. The ability of each multidrug transporter to recognize dozens of compounds that have no apparent structural consensus seemingly contradicts basic dogmas of biochemistry and remains enigmatic.Another unresolved question associated with multidrug transporters concerns their normal physiological functions. Because overexpression of these proteins causes an increase in cellular resistance to toxins, it is commonly believed that they have evolved to protect cells from diverse environmental toxic molecules. It is possible, however, that each multidrug transporter has a specific, presently unidentified natural substrate, e.g. some cellular metabolite, whereas multiple drugs are effluxed by them merely opportunistically. This latter hypothesis is indirectly supported by the analysis of the primary structures of multidrug transporters. More than two dozen known transporters capable of effluxing multiple drugs share no common sequence motif and belong to at least four distinct superfamilies of membrane proteins (1-5). Furthermore, many multidrug transporters display strong homology to substrate-specific transporters. For example, human P-glycoprotein, the product of the MDR1 gene, is highly homologous to the protein MDR2, which does not efflux drugs but is a specific phosphatidylcholine flippase (6). Similarly, the bacterial multidrug transporters Bmr and Blt of Bacillus subtilis and NorA of Staphylococcus aureus are highly homologous to the efflux transporters whose only known substrate is tetracycline (7-10).It has recently been demonstrated that P-glycoprotein transports not only drugs but also structurally diverse natural lipids across the membrane (11). Here we demonstrate that the activity of the B. subtilis multidrug transporter Blt leads to the efflux of a natural cellular constituent, the polyamine spermi...

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Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones

Cited by 430 publications

References 39 publications

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

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

Transmembrane glutamic acid residues play essential roles in the metal‐tetracycline/H⁺ antiporter of Staphylococcus aureus

Efflux of the Natural Polyamine Spermidine Facilitated by the Bacillus subtilis Multidrug Transporter Blt

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Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones

Cited by 430 publications

References 39 publications

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

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

Transmembrane glutamic acid residues play essential roles in the metal‐tetracycline/H+ antiporter of Staphylococcus aureus

Efflux of the Natural Polyamine Spermidine Facilitated by the Bacillus subtilis Multidrug Transporter Blt

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Transmembrane glutamic acid residues play essential roles in the metal‐tetracycline/H⁺ antiporter of Staphylococcus aureus