Characterization of the RND family of multidrug efflux pumps: in silico to in vivo confirmation of four functionally distinct subgroups.
We have developed a generalized profile that identifies members of the root-nodulation-cell-division (RND) family of efflux pumps and classifies them into four functional subfamilies. According to Z-score values, efflux pumps can be grouped by their metabolic function, thus making it possible to distinguish pumps involved in antibiotic resistance (group 1) from those involved in metal resistance (group 3). In silico data regarding efflux pumps in group 1 were validated after identification of RND efflux pumps in a number of environmental microbes that were isolated as resistant to ethidium bromide. Analysis of the Pseudomonas putida KT2440 genome identified efflux pumps in all groups. A collection of mutants in efflux pumps and a screening platform consisting of 50 drugs were created to assign a function to the efflux pumps. We validated in silico data regarding efflux pumps in groups 1 and 3 using 9 different mutants. Four mutants belonging to group 2 were found to be more sensitive than the wild-type to oxidative stress-inducing agents such as bipyridyl and methyl viologen. The two remaining mutants belonging to group 4 were found to be more sensitive than the parental to tetracycline and one of them was particularly sensitive to rubidium and chromate. By effectively combining in vivo data with generalized profiles and gene annotation data, this approach allowed the assignment, according to metabolic function, of both known and uncharacterized RND efflux pumps into subgroups, thereby providing important new insight into the functions of proteins within this family.
Consejo Superior de Investigaciones Científicas, Department of Environmental Protection, E-18008 Granada, Spain.
Patricia
P
Godoy
Antonio J
AJ
Molina-Henares
Jesús
J
de la Torre
Estrella
E
Duque
Juan L
JL
Ramos
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D016000:Cluster Analysis; D019295:Computational Biology; D024881:Drug Resistance, Bacterial; D010802:Phylogeny; D016958:Pseudomonas putida / Q000187:drug effects* / Q000235:genetics*
D018528:ATP-Binding Cassette Transporters; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins
Nucleic Acids Res. 2004 Jan 1;32(Database issue):D138-41
14681378
J Bacteriol. 2005 Oct;187(19):6804-15
16166543
Proc Int Conf Intell Syst Mol Biol. 1994;2:53-61
7584418
J Bacteriol. 1993 Oct;175(19):6299-313
8407802
J Bacteriol. 1996 Oct;178(20):5853-9
8830678
Biochim Biophys Acta. 2009 May;1794(5):769-81
19026770
Mol Microbiol. 1997 Jan;23(2):345-54
9044268
J Bacteriol. 2008 Jul;190(13):4447-52
18469108
Environ Microbiol. 2010 Jun;12(6):1427-38
20050871
Environ Microbiol. 2002 Dec;4(12):799-808
12534463
Mol Microbiol. 1996 Aug;21(4):713-24
8878035
Nature. 2000 Jun 22;405(6789):914-9
10879525
Antimicrob Agents Chemother. 2001 Dec;45(12):3375-80
11709311
J Bacteriol. 2001 Apr;183(8):2646-53
11274125
Mol Microbiol. 2001 Feb;39(4):1100-6
11251828
Appl Microbiol Biotechnol. 1999 Jun;51(6):730-50
10422221
Antimicrob Agents Chemother. 2003 Oct;47(10):3067-72
14506010
Lancet. 1975 Feb 1;1(7901):235-40
46385
J Bacteriol. 1998 Jul;180(13):3323-9
9642183
Appl Environ Microbiol. 2004 Aug;70(8):4424-31
15294769
Microbiol Mol Biol Rev. 2005 Jun;69(2):326-56
15944459
Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8
3162770
J Mol Biol. 2008 May 16;378(5):1155-73
18433769
Comput Chem. 1996 Mar;20(1):3-23
8867839
Environ Microbiol Rep. 2009 Dec;1(6):469-76
23765924
J Bacteriol. 2010 Apr;192(8):2169-81
20139187
Microb Biotechnol. 2009 Jan;2(1):62-74
21261882
Microb Biotechnol. 2008 Mar;1(2):158-69
21261833
Nature. 2002 Oct 10;419(6907):587-93
12374972
Environ Microbiol Rep. 2009 Dec;1(6):518-23
23765930
Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7190-5
10377390
J Bacteriol. 1994 Nov;176(22):6957-64
7961458
Environ Microbiol. 2009 Dec;11(12):2970-88
19601960
Microb Biotechnol. 2009 May;2(3):304-7
21261925
Antimicrob Agents Chemother. 2008 Jul;52(7):2455-62
18474583
J Bacteriol. 1999 Feb;181(4):1203-10
9973347
Environ Microbiol. 2007 May;9(5):1135-45
17472630
Antimicrob Agents Chemother. 1995 Sep;39(9):1948-53
8540696
Structure. 2007 Dec;15(12):1663-73
18073115
Nucleic Acids Res. 2001 Jan 1;29(1):22-8
11125040
Science. 1996 Aug 2;273(5275):595-603
8662544
Nat Med. 1999 Feb;5(2):183-8
9930866
Structure. 2006 Mar;14(3):577-87
16531241
Mol Microbiol. 2007 Mar;63(5):1307-18
17244194
J Bacteriol. 2009 Mar;191(6):1729-37
19060146
Nucleic Acids Res. 1999 Jan 1;27(1):263-7
9847197
FEMS Microbiol Rev. 2003 Jun;27(2-3):313-39
12829273
J Bacteriol. 1998 Jun;180(11):2987-91
9603892
J Bacteriol. 2001 Jan;183(1):367-74
11114937
Environ Microbiol. 2005 Feb;7(2):225-8
15658989
Microb Biotechnol. 2009 Mar;2(2):287-94
21261922
FEMS Microbiol Rev. 2003 Jun;27(2-3):341-53
12829274
Curr Opin Microbiol. 1998 Oct;1(5):516-23
10066525
Protein Sci. 1994 Jan;3(1):139-46
7511453
Trends Microbiol. 2000 Nov;8(11):481-3
11121749
Nucleic Acids Res. 2006 Jan 1;34(Database issue):D227-30
16381852
J Bacteriol. 2001 Jul;183(13):3967-73
11395460
J Bacteriol. 1989 Dec;171(12):6782-90
2687253
J Mol Biol. 1990 Dec 20;216(4):813-8
2176240
Microb Biotechnol. 2008 May;1(3):202-7
21261839
Microb Biotechnol. 2009 Jan;2(1):40-61
21261881
Antimicrob Agents Chemother. 2004 Oct;48(10):4054-5
15388479
J Biol Chem. 2002 Apr 12;277(15):12507-15
11756427
Microbiol Mol Biol Rev. 1997 Dec;61(4):393-410
9409145
N Engl J Med. 1998 May 7;338(19):1376-8
9571260
Infect Immun. 2006 Jul;74(7):3845-52
16790756
Appl Environ Microbiol. 2005 Jan;71(1):580-6
15640241