Escherichia coli K-12 substr. MG1655 Polypeptide: AcrAD-TolC multidrug efflux system - permease subunit

Gene: acrD Accession Numbers: EG10014 (EcoCyc), b2470, ECK2465

Synonyms: yffA

Regulation Summary Diagram: ?

Regulation summary diagram for acrD

Component of: AcrAD-TolC multidrug efflux system (extended summary available)

AcrD is a member of the resistance-nodulation-division (RND) superfamily [Saier14] and a component of the AcrAD-TolC multidrug efflux transport system in E. coli K-12. By analogy with the AcrB RND permease, AcrD is shown as a homotrimer in the complex.

Locations: inner membrane

Map Position: [2,585,617 -> 2,588,730] (55.73 centisomes, 201°)
Length: 3114 bp / 1037 aa

Molecular Weight of Polypeptide: 113.05 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0008136 , CGSC:35697 , DIP:DIP-9050N , EchoBASE:EB0014 , EcoGene:EG10014 , EcoliWiki:b2470 , Mint:MINT-1256458 , ModBase:P24177 , OU-Microarray:b2470 , PortEco:acrD , Pride:P24177 , Protein Model Portal:P24177 , RefSeq:NP_416965 , RegulonDB:EG10014 , SMR:P24177 , String:511145.b2470 , Swiss-Model:P24177 , UniProt:P24177

Relationship Links: InterPro:IN-FAMILY:IPR001036 , InterPro:IN-FAMILY:IPR004764 , InterPro:IN-FAMILY:IPR027463 , Pfam:IN-FAMILY:PF00873 , Prints:IN-FAMILY:PR00702

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for acrD

GO Terms:

Biological Process: GO:0006855 - drug transmembrane transport Inferred from experiment [Rosenberg00]
GO:0042493 - response to drug Inferred from experiment [Aires05]
GO:0046618 - drug export Inferred from experiment [Aires05]
GO:0006810 - transport Inferred by computational analysis [UniProtGOA11, GOA01]
Molecular Function: GO:0015238 - drug transmembrane transporter activity Inferred from experiment [Rosenberg00]
GO:0015307 - drug:proton antiporter activity Inferred from experiment [Aires05]
GO:0005215 - transporter activity Inferred by computational analysis [GOA01]
Cellular Component: GO:0005886 - plasma membrane Inferred from experiment Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, DiazMejia09, Daley05]
GO:0016020 - membrane Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, Aires05]
GO:0016021 - integral component of membrane Inferred by computational analysis [UniProtGOA11, GOA01]

MultiFun Terms: cell processes protection drug resistance/sensitivity
cell structure membrane
transport Electrochemical potential driven transporters Porters (Uni-, Sym- and Antiporters)

Essentiality data for acrD knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]

Subunit of: AcrAD-TolC multidrug efflux system

Subunit composition of AcrAD-TolC multidrug efflux system = [AcrD]3[AcrA][(TolC)3]
         AcrAD-TolC multidrug efflux system - permease subunit = AcrD (summary available)
         AcrAB-TolC multidrug efflux system - membrane fusion protein = AcrA (extended summary available)
         TolC outer membrane channel = (TolC)3 (extended summary available)
                 TolC monomer = TolC

AcrD is a member of the resistance-nodulation-division (RND) family [Saier94]. RND family transporters interact with membrane fusion proteins (MFPs) and outer membrane channels for transport of their substrates into the external medium. AcrD is believed to form a tripartite complex with AcrA (an MFP) and TolC for multidrug transport. AcrD shows amino acid sequence similarity with the E. coli RND multidrug efflux proteins AcrB and AcrF.

AcrD was shown to require AcrA for aminoglycoside transport when reconstituted into proteoliposomes [Aires05]. AcrAD are believed to form a complex with TolC for multidrug export [Aires05]. Experiments using proteoliposomes show the AcrAD-TolC complex is able to transport drugs from both the cytoplasm and the periplasm to the extracellular space [Aires05]. AcrD was shown to export streptomycin, but only from the periplasm [Aires05]. AcrD was also shown to bind cisplatin [Will08]. Linear α-helical cathelicidin LL-37 and β-sheet defensins HNP-1-3, hBD-2/-3 and HD-5 are not substrates for AcrAB [Rieg09].

Disruption of the acrD gene did not result in hypersusceptibility to lipophilic and amphiphilic drugs, but did result in hypersusceptibility to a variety of aminoglycosides including amikacin, gentamicin, tobramycin, kanamycin, neomycin, erythromycin, and polymyxin B [Rosenberg00]. The mutants also accumulated more dihydrostreptomycin and gentamicin than the parental strain [Rosenberg00]. Treatment with CCCP increased aminoglycoside accumulation suggesting that efflux of aminoglycosides is energized by the proton-motive force [Rosenberg00]. Expression of acrD on a multicopy plasmid from native or IPTG inducible promoters in an acrAB mutant resulted in increased resistance to deoxycholate, SDS, novobiocin, kanamycin, tetracycline, nalidixic acid, norfloxacin, fosfomycin [Nishino01], bile acids, fusidic acid [Elkins02], and progesterone [Elkins06]. AcrADTolC confers stronger resistance to anionic β-lactams (aztreonam, carbenicillin, sulbenicillin) than AcrABTolC [Nishino03]. The two large periplasmic loops of AcrD are implicated in substrate specificity [Elkins02, Kobayashi14].

In one study, disruption of acrA or tolC did not result in hypersensitivity to aminoglycosides [Rosenberg00]. Since aminoglycosides do not cross the inner membrane spontaneously, it was suggested that AcrD simply transports the hydrophilic molecules to the periplasm and does not require other proteins for aminoglycoside transport across the outer membrane [Rosenberg00]. Later studies showed AcrA was required for transport of cholic acid, taurocholic acid, and novobiocin by AcrD [Elkins02], and that acrA mutants were as susceptible to aminoglycosides as acrD mutants [Aires05]. AcrD and AcrA have also been shown to interact through chemical crosslinking studies [Elkins02]. These results suggest that AcrD interacts with AcrA and TolC for transport of at least some substrates [Elkins02].

BaeR is responsible for activation of acrD transcription [Hirakawa03a], and this activation is enhanced by CpxR [Hirakawa05]. Indole was shown to activate BaeR and CpxR leading to acrD transcription [Hirakawa05]. Iron depletion led to reduced expression of acrD [Bleuel05].

Citations: [Nakamura79, Ma94, Fralick96, Nikaido96, Yamada06]

Last-Curated ? 12-May-2008 by Johnson A , JCVI

Enzymatic reaction of: multidrug transport (AcrAD-TolC multidrug efflux system)

Transport reaction diagram for multidrug transport

AcrADTolC exports substrates from the periplasm and from the cytoplasm according to the following reactions:
drug(periplasmic space) + H+(periplasmic space) ===> drug(extracellular) + H+(cytoplasm)

drug(cytoplasm) + H+(periplasmic space) ===> drug(extracellular) + H+(cytoplasm)

Sequence Features

Protein sequence of AcrAD-TolC multidrug efflux system - permease subunit with features indicated

Feature Class Location Citations Comment
Transmembrane-Region 10 -> 28
UniProt: Helical; Name=1;; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 303
[Nilles94, UniProt15]
UniProt: (in Ref. 1; AAA20584).
Transmembrane-Region 340 -> 359
UniProt: Helical; Name=2;; Non-Experimental Qualifier: by similarity;
Transmembrane-Region 366 -> 385
UniProt: Helical; Name=3;; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 372
[Nilles94, UniProt15]
UniProt: (in Ref. 1; AAA20584).
Sequence-Conflict 385
[Ma95a, UniProt15]
UniProt: (in Ref. 2; AAA74741).
Transmembrane-Region 392 -> 413
UniProt: Helical; Name=4;; Non-Experimental Qualifier: by similarity;
Transmembrane-Region 442 -> 460
UniProt: Helical; Name=5;; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 461
[Nilles94, UniProt15]
UniProt: (in Ref. 1; AAA20584).
Transmembrane-Region 474 -> 496
UniProt: Helical; Name=6;; Non-Experimental Qualifier: by similarity;
Transmembrane-Region 538 -> 556
UniProt: Helical; Name=7;; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 665
[Nilles94, UniProt15]
UniProt: (in Ref. 1; AAA20584).
Sequence-Conflict 763
[Nilles94, UniProt15]
UniProt: (in Ref. 1; AAA20584).
Sequence-Conflict 775
[Nilles94, UniProt15]
UniProt: (in Ref. 1; AAA20584).
Sequence-Conflict 778
[Nilles94, UniProt15]
UniProt: (in Ref. 1; AAA20584).
Transmembrane-Region 871 -> 890
UniProt: Helical; Name=8;; Non-Experimental Qualifier: by similarity;
Transmembrane-Region 897 -> 916
UniProt: Helical; Name=9;; Non-Experimental Qualifier: by similarity;
Transmembrane-Region 923 -> 944
UniProt: Helical; Name=10;; Non-Experimental Qualifier: by similarity;
Transmembrane-Region 972 -> 990
UniProt: Helical; Name=11;; Non-Experimental Qualifier: by similarity;
Transmembrane-Region 1004 -> 1026
UniProt: Helical; Name=12;; Non-Experimental Qualifier: by similarity;

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Units:

Transcription-unit diagram

Transcription-unit diagram


10/20/97 Gene b2470 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10014; confirmed by SwissProt match.


Aires05: Aires JR, Nikaido H (2005). "Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli." J Bacteriol 187(6);1923-9. PMID: 15743938

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Bleuel05: Bleuel C, Grosse C, Taudte N, Scherer J, Wesenberg D, Krauss GJ, Nies DH, Grass G (2005). "TolC is involved in enterobactin efflux across the outer membrane of Escherichia coli." J Bacteriol 187(19);6701-7. PMID: 16166532

Daley05: Daley DO, Rapp M, Granseth E, Melen K, Drew D, von Heijne G (2005). "Global topology analysis of the Escherichia coli inner membrane proteome." Science 308(5726);1321-3. PMID: 15919996

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Elkins02: Elkins CA, Nikaido H (2002). "Substrate specificity of the RND-type multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops." J Bacteriol 184(23);6490-8. PMID: 12426336

Elkins06: Elkins CA, Mullis LB (2006). "Mammalian steroid hormones are substrates for the major RND- and MFS-type tripartite multidrug efflux pumps of Escherichia coli." J Bacteriol 188(3);1191-5. PMID: 16428427

Fralick96: Fralick JA (1996). "Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli." J Bacteriol 1996;178(19);5803-5. PMID: 8824631

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

Hirakawa03a: Hirakawa H, Nishino K, Yamada J, Hirata T, Yamaguchi A (2003). "Beta-lactam resistance modulated by the overexpression of response regulators of two-component signal transduction systems in Escherichia coli." J Antimicrob Chemother 52(4);576-82. PMID: 12951338

Hirakawa05: Hirakawa H, Inazumi Y, Masaki T, Hirata T, Yamaguchi A (2005). "Indole induces the expression of multidrug exporter genes in Escherichia coli." Mol Microbiol 55(4);1113-26. PMID: 15686558

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kobayashi14: Kobayashi N, Tamura N, van Veen HW, Yamaguchi A, Murakami S (2014). "β-Lactam Selectivity of Multidrug Transporters AcrB and AcrD Resides in the Proximal Binding Pocket." J Biol Chem. PMID: 24558035

Ma94: Ma D, Cook DN, Hearst JE, Nikaido H (1994). "Efflux pumps and drug resistance in gram-negative bacteria." Trends Microbiol 2(12);489-93. PMID: 7889326

Ma95a: Ma D., Cook D.N., Alberti M., Nikaido H., Hearst J.E. (1995). Data submission to EMBL/GenBank/DDBJ databases on 1995-08.

Nakamura79: Nakamura H (1979). "Novel acriflavin resistance genes, acrC and acrD, in Escherichia coli K-12." J Bacteriol 139(1);8-12. PMID: 378962

Nikaido96: Nikaido H (1996). "Multidrug efflux pumps of gram-negative bacteria." J Bacteriol 178(20);5853-9. PMID: 8830678

Nilles94: Nilles M.L., Bertrand K.P. (1994). Data submission to EMBL/GenBank/DDBJ databases on 1994-07.

Nishino01: Nishino K, Yamaguchi A (2001). "Analysis of a complete library of putative drug transporter genes in Escherichia coli." J Bacteriol 2001;183(20);5803-12. PMID: 11566977

Nishino03: Nishino K, Yamada J, Hirakawa H, Hirata T, Yamaguchi A (2003). "Roles of TolC-dependent multidrug transporters of Escherichia coli in resistance to beta-lactams." Antimicrob Agents Chemother 47(9);3030-3. PMID: 12937021

Rieg09: Rieg S, Huth A, Kalbacher H, Kern WV (2009). "Resistance against antimicrobial peptides is independent of Escherichia coli AcrAB, Pseudomonas aeruginosa MexAB and Staphylococcus aureus NorA efflux pumps." Int J Antimicrob Agents 33(2);174-6. PMID: 18945595

Rosenberg00: Rosenberg EY, Ma D, Nikaido H (2000). "AcrD of Escherichia coli is an aminoglycoside efflux pump." J Bacteriol 2000;182(6);1754-6. PMID: 10692383

Saier14: Saier MH, Reddy VS, Tamang DG, Vastermark A (2014). "The transporter classification database." Nucleic Acids Res 42(1);D251-8. PMID: 24225317

Saier94: Saier MH, Tam R, Reizer A, Reizer J (1994). "Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport." Mol Microbiol 1994;11(5);841-7. PMID: 8022262

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

Will08: Will J, Sheldrick WS, Wolters D (2008). "Characterisation of cisplatin coordination sites in cellular Escherichia coli DNA-binding proteins by combined biphasic liquid chromatography and ESI tandem mass spectrometry." J Biol Inorg Chem 13(3):421-34. PMID: 18157731

Yamada06: Yamada S, Awano N, Inubushi K, Maeda E, Nakamori S, Nishino K, Yamaguchi A, Takagi H (2006). "Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli." Appl Environ Microbiol 72(7);4735-42. PMID: 16820466

Other References Related to Gene Regulation

Barrios99: Barrios H, Valderrama B, Morett E (1999). "Compilation and analysis of sigma(54)-dependent promoter sequences." Nucleic Acids Res 27(22);4305-13. PMID: 10536136

Huerta03: Huerta AM, Collado-Vides J (2003). "Sigma70 promoters in Escherichia coli: specific transcription in dense regions of overlapping promoter-like signals." J Mol Biol 333(2);261-78. PMID: 14529615

MedinaRivera11: Medina-Rivera A, Abreu-Goodger C, Thomas-Chollier M, Salgado H, Collado-Vides J, van Helden J (2011). "Theoretical and empirical quality assessment of transcription factor-binding motifs." Nucleic Acids Res 39(3);808-24. PMID: 20923783

Viveiros07: Viveiros M, Dupont M, Rodrigues L, Couto I, Davin-Regli A, Martins M, Pages JM, Amaral L (2007). "Antibiotic stress, genetic response and altered permeability of E. coli." PLoS ONE 2;e365. PMID: 17426813

Zhao10a: Zhao K, Liu M, Burgess RR (2010). "Promoter and regulon analysis of nitrogen assimilation factor, sigma54, reveal alternative strategy for E. coli MG1655 flagellar biosynthesis." Nucleic Acids Res 38(4);1273-83. PMID: 19969540

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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