Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli.
To understand better the mechanisms of resistance-nodulation-division (RND)-type multidrug efflux pumps, we examined the Escherichia coli AcrD pump, whose typical substrates, aminoglycosides, are not expected to diffuse spontaneously across the lipid bilayer. The hexahistidine-tagged AcrD protein was purified and reconstituted into unilamellar proteoliposomes. Its activity was measured by the proton flux accompanying substrate transport. When the interior of the proteoliposomes was acidified, the addition of aminoglycosides to the external medium stimulated proton efflux and the intravesicular accumulation of radiolabeled gentamicin, suggesting that aminoglycosides can be captured and transported from the external medium in this system (corresponding to cytosol). This activity required the presence of AcrA within the proteoliposomes. Interestingly, the increase in proton efflux also occurred when aminoglycosides were present only in the intravesicular space. This result suggested that AcrD can also capture aminoglycosides from the periplasm to extrude them into the medium in intact cells, acting as a "periplasmic vacuum cleaner."
Aires
Julio Ramos
JR
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
Nikaido
Hiroshi
H
D000143:Acids / Q000378:metabolism; D000617:Aminoglycosides / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000493:pharmacokinetics; D003593:Cytoplasm / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial / Q000502:physiology*; D048168:Escherichia coli K12 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005839:Gentamicins / Q000493:pharmacokinetics; D008074:Lipoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D013307:Streptomycin / Q000493:pharmacokinetics; D014316:Tritium
D000143:Acids; C083497:AcrA protein, E coli; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D005839:Gentamicins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; D014316:Tritium; D013307:Streptomycin
J Bacteriol. 2000 Mar;182(6):1754-6
10692383
J Biol Chem. 1999 Sep 10;274(37):26065-70
10473554
J Bacteriol. 2000 Aug;182(15):4264-7
10894736
Mol Microbiol. 2000 Jul;37(2):219-25
10931319
J Mol Microbiol Biotechnol. 1999 Aug;1(1):107-25
10941792
Antimicrob Agents Chemother. 2001 Apr;45(4):1126-36
11257026
J Bacteriol. 2001 Oct;183(20):5803-12
11566977
J Struct Biol. 2001 Oct;136(1):81-8
11858709
Nature. 2002 Oct 10;419(6907):587-93
12374972
J Bacteriol. 2002 Dec;184(23):6490-8
12426336
Science. 2003 May 9;300(5621):976-80
12738864
J Bacteriol. 2003 Sep;185(18):5349-56
12949086
J Bacteriol. 2003 Oct;185(19):5657-64
13129936
J Biol Chem. 2003 Dec 12;278(50):50474-82
14523004
J Biol Chem. 2004 Jun 18;279(25):25939-42
15117957
Proc Natl Acad Sci U S A. 2004 Jul 6;101(27):9994-9
15226509
Biochemistry. 1981 Mar 17;20(6):1534-8
6261798
J Biol Chem. 1988 Jan 25;263(3):1182-7
2447086
J Biol Chem. 1991 May 15;266(14):8946-51
2026607
Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11367-70
1454821
J Bacteriol. 1994 Jul;176(13):3825-31
8021163
J Biol Chem. 1994 Jul 8;269(27):17981-7
7517935
Antimicrob Agents Chemother. 1994 Aug;38(8):1742-52
7986004
Mol Microbiol. 1995 Apr;16(1):45-55
7651136
J Bacteriol. 1996 Jan;178(1):306-8
8550435
J Bacteriol. 1996 Oct;178(19):5803-5
8824631
J Bacteriol. 1996 Oct;178(20):5853-9
8830678
Clin Infect Dis. 1998 Aug;27 Suppl 1:S32-41
9710669
Methods Enzymol. 1998;292:3-20
9711542
J Bacteriol. 1998 Sep;180(17):4686-92
9721312
J Mol Biol. 1999 Jan 8;285(1):409-20
9878415
Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7190-5
10377390