Abstract
The QacC polypeptide is a member of a family of small membrane proteins which confer resistance to toxic compounds. The staphylococcal qacC gene confers resistance to toxic organic cations via proton-dependent export. The membrane topology of the QacC polypeptide was investigated by constructing and analyzing a series of qacC-phoA and qacC-lacZ fusions. From these analyses, most of the predicted features of the QacC protein were verified, although data regarding the possible orientation of the COOH region were not conclusive. The role of the sole cysteine residue, Cys-42, in QacC was studied by using the sulfhydryl reagent N-ethylmaleimide and site-directed mutagenesis. N-Ethylmaleimide was shown to inhibit qacC-mediated ethidium export. Multiple amino acid substitutions were made for Cys-42, and mutations at this location had various effects on resistance specificity. This suggests that the Cys-42 residue may be located near a region of QacC that is involved in substrate recognition. Mutagenesis of conserved residues in QacC indicated that Tyr-59 and Trp-62 also play an essential structural or functional role in QacC.
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- Brickman E., Beckwith J. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol. 1975 Aug 5;96(2):307–316. doi: 10.1016/0022-2836(75)90350-2. [DOI] [PubMed] [Google Scholar]
- Evans J., Dyke K. G. Characterization of the conjugation system associated with the Staphylococcus aureus plasmid pJE1. J Gen Microbiol. 1988 Jan;134(1):1–8. doi: 10.1099/00221287-134-1-1. [DOI] [PubMed] [Google Scholar]
- Firth N., Ridgway K. P., Byrne M. E., Fink P. D., Johnson L., Paulsen I. T., Skurray R. A. Analysis of a transfer region from the staphylococcal conjugative plasmid pSK41. Gene. 1993 Dec 22;136(1-2):13–25. doi: 10.1016/0378-1119(93)90442-6. [DOI] [PubMed] [Google Scholar]
- Fox C. F., Kennedy E. P. Specific labeling and partial purification of the M protein, a component of the beta-galactoside transport system of Escherichia coli. Proc Natl Acad Sci U S A. 1965 Sep;54(3):891–899. doi: 10.1073/pnas.54.3.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gottesman M. M., Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem. 1993;62:385–427. doi: 10.1146/annurev.bi.62.070193.002125. [DOI] [PubMed] [Google Scholar]
- Griffith J. K., Baker M. E., Rouch D. A., Page M. G., Skurray R. A., Paulsen I. T., Chater K. F., Baldwin S. A., Henderson P. J. Membrane transport proteins: implications of sequence comparisons. Curr Opin Cell Biol. 1992 Aug;4(4):684–695. doi: 10.1016/0955-0674(92)90090-y. [DOI] [PubMed] [Google Scholar]
- Grinius L., Dreguniene G., Goldberg E. B., Liao C. H., Projan S. J. A staphylococcal multidrug resistance gene product is a member of a new protein family. Plasmid. 1992 Mar;27(2):119–129. doi: 10.1016/0147-619x(92)90012-y. [DOI] [PubMed] [Google Scholar]
- Gutierrez C., Devedjian J. C. A plasmid facilitating in vitro construction of phoA gene fusions in Escherichia coli. Nucleic Acids Res. 1989 May 25;17(10):3999–3999. doi: 10.1093/nar/17.10.3999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Leelaporn A., Paulsen I. T., Tennent J. M., Littlejohn T. G., Skurray R. A. Multidrug resistance to antiseptics and disinfectants in coagulase-negative staphylococci. J Med Microbiol. 1994 Mar;40(3):214–220. doi: 10.1099/00222615-40-3-214. [DOI] [PubMed] [Google Scholar]
- Levy S. B. Active efflux mechanisms for antimicrobial resistance. Antimicrob Agents Chemother. 1992 Apr;36(4):695–703. doi: 10.1128/aac.36.4.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis K. Multidrug resistance pumps in bacteria: variations on a theme. Trends Biochem Sci. 1994 Mar;19(3):119–123. doi: 10.1016/0968-0004(94)90204-6. [DOI] [PubMed] [Google Scholar]
- Littlejohn T. G., DiBerardino D., Messerotti L. J., Spiers S. J., Skurray R. A. Structure and evolution of a family of genes encoding antiseptic and disinfectant resistance in Staphylococcus aureus. Gene. 1991 May 15;101(1):59–66. doi: 10.1016/0378-1119(91)90224-y. [DOI] [PubMed] [Google Scholar]
- Littlejohn T. G., Paulsen I. T., Gillespie M. T., Tennent J. M., Midgley M., Jones I. G., Purewal A. S., Skurray R. A. Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus. FEMS Microbiol Lett. 1992 Aug 15;74(2-3):259–265. doi: 10.1016/0378-1097(92)90439-u. [DOI] [PubMed] [Google Scholar]
- Lomovskaya O., Lewis K. Emr, an Escherichia coli locus for multidrug resistance. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8938–8942. doi: 10.1073/pnas.89.19.8938. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loo T. W., Clarke D. M. Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein. J Biol Chem. 1993 Sep 25;268(27):19965–19972. [PubMed] [Google Scholar]
- Lyon B. R., May J. W., Skurray R. A. Analysis of plasmids in nosocomial strains of multiple-antibiotic-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1983 Jun;23(6):817–826. doi: 10.1128/aac.23.6.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lyon B. R., May J. W., Skurray R. A. Tn4001: a gentamicin and kanamycin resistance transposon in Staphylococcus aureus. Mol Gen Genet. 1984;193(3):554–556. doi: 10.1007/BF00382099. [DOI] [PubMed] [Google Scholar]
- Lyon B. R., Skurray R. Antimicrobial resistance of Staphylococcus aureus: genetic basis. Microbiol Rev. 1987 Mar;51(1):88–134. doi: 10.1128/mr.51.1.88-134.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marger M. D., Saier M. H., Jr A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci. 1993 Jan;18(1):13–20. doi: 10.1016/0968-0004(93)90081-w. [DOI] [PubMed] [Google Scholar]
- Martinez E., Bartolomé B., de la Cruz F. pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. Gene. 1988 Aug 15;68(1):159–162. doi: 10.1016/0378-1119(88)90608-7. [DOI] [PubMed] [Google Scholar]
- Midgley M. The phosphonium ion efflux system of Escherichia coli: relationship to the ethidium efflux system and energetic studies. J Gen Microbiol. 1986 Nov;132(11):3187–3193. doi: 10.1099/00221287-132-11-3187. [DOI] [PubMed] [Google Scholar]
- Neyfakh A. A., Bidnenko V. E., Chen L. B. Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4781–4785. doi: 10.1073/pnas.88.11.4781. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nikaido H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science. 1994 Apr 15;264(5157):382–388. doi: 10.1126/science.8153625. [DOI] [PubMed] [Google Scholar]
- Paulsen I. T., Littlejohn T. G., Rådström P., Sundström L., Sköld O., Swedberg G., Skurray R. A. The 3' conserved segment of integrons contains a gene associated with multidrug resistance to antiseptics and disinfectants. Antimicrob Agents Chemother. 1993 Apr;37(4):761–768. doi: 10.1128/aac.37.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Paulsen I. T., Skurray R. A. Topology, structure and evolution of two families of proteins involved in antibiotic and antiseptic resistance in eukaryotes and prokaryotes--an analysis. Gene. 1993 Feb 14;124(1):1–11. doi: 10.1016/0378-1119(93)90755-r. [DOI] [PubMed] [Google Scholar]
- Purewal A. S. Nucleotide sequence of the ethidium efflux gene from Escherichia coli. FEMS Microbiol Lett. 1991 Aug 1;66(2):229–231. doi: 10.1016/0378-1097(91)90338-b. [DOI] [PubMed] [Google Scholar]
- Rampal A. L., Jung C. Y. Substrate-induced conformational change of human erythrocyte glucose transporter: inactivation by alkylating reagents. Biochim Biophys Acta. 1987 Jan 26;896(2):287–294. doi: 10.1016/0005-2736(87)90189-1. [DOI] [PubMed] [Google Scholar]
- Rouch D. A., Cram D. S., DiBerardino D., Littlejohn T. G., Skurray R. A. Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins. Mol Microbiol. 1990 Dec;4(12):2051–2062. doi: 10.1111/j.1365-2958.1990.tb00565.x. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sasatsu M., Shibata Y., Noguchi N., Kono M. High-level resistance to ethidium bromide and antiseptics in Staphylococcus aureus. FEMS Microbiol Lett. 1992 Jun 1;72(2):109–113. doi: 10.1016/0378-1097(92)90514-o. [DOI] [PubMed] [Google Scholar]
- Sasatsu M., Shima K., Shibata Y., Kono M. Nucleotide sequence of a gene that encodes resistance to ethidium bromide from a transferable plasmid in Staphylococcus aureus. Nucleic Acids Res. 1989 Dec 11;17(23):10103–10103. doi: 10.1093/nar/17.23.10103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith R. L., Banks J. L., Snavely M. D., Maguire M. E. Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coli. Identification of a new class of transport protein. J Biol Chem. 1993 Jul 5;268(19):14071–14080. [PubMed] [Google Scholar]
- Smith R. L., O'Toole J. F., Maguire M. E., Sanders C. R., 2nd Membrane topology of Escherichia coli diacylglycerol kinase. J Bacteriol. 1994 Sep;176(17):5459–5465. doi: 10.1128/jb.176.17.5459-5465.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tennent J. M., Lyon B. R., Gillespie M. T., May J. W., Skurray R. A. Cloning and expression of Staphylococcus aureus plasmid-mediated quaternary ammonium resistance in Escherichia coli. Antimicrob Agents Chemother. 1985 Jan;27(1):79–83. doi: 10.1128/aac.27.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tennent J. M., Lyon B. R., Midgley M., Jones I. G., Purewal A. S., Skurray R. A. Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus. J Gen Microbiol. 1989 Jan;135(1):1–10. doi: 10.1099/00221287-135-1-1. [DOI] [PubMed] [Google Scholar]
- Traxler B., Boyd D., Beckwith J. The topological analysis of integral cytoplasmic membrane proteins. J Membr Biol. 1993 Feb;132(1):1–11. doi: 10.1007/BF00233047. [DOI] [PubMed] [Google Scholar]
- Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
- Yan R. T., Maloney P. C. Identification of a residue in the translocation pathway of a membrane carrier. Cell. 1993 Oct 8;75(1):37–44. [PubMed] [Google Scholar]
- Yun C. H., Van Doren S. R., Crofts A. R., Gennis R. B. The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides. J Biol Chem. 1991 Jun 15;266(17):10967–10973. [PubMed] [Google Scholar]
- van Beilen J. B., Penninga D., Witholt B. Topology of the membrane-bound alkane hydroxylase of Pseudomonas oleovorans. J Biol Chem. 1992 May 5;267(13):9194–9201. [PubMed] [Google Scholar]
- van Iwaarden P. R., Driessen A. J., Konings W. N. What we can learn from the effects of thiol reagents on transport proteins. Biochim Biophys Acta. 1992 Aug 14;1113(2):161–170. doi: 10.1016/0304-4157(92)90037-b. [DOI] [PubMed] [Google Scholar]
- von Wachenfeldt C., Hederstedt L. Bacillus subtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli. FEBS Lett. 1990 Sep 17;270(1-2):147–151. doi: 10.1016/0014-5793(90)81255-m. [DOI] [PubMed] [Google Scholar]