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Escherichia coli K-12 substr. MG1655 Transporter: multidrug efflux transporter MdfA (multifunctional)



Gene: mdfA Accession Numbers: G6440 (EcoCyc), b0842, ECK0832

Synonyms: cmlA, cmr

Regulation Summary Diagram: ?

Summary:
The MdfA protein, also known as Cmr, is a multidrug efflux protein belonging to the major facilitator superfamily (MFS) [Paulsen96a]. Overexpression of MdfA has demonstrated that it confers resistance to tetracycline, chloramphenicol, erythromycin, some aminoglycosides and fluoroquinolones, and organic cations such as ethidium bromide [Edgar97]. Deletion of mdfA resulted in increased susceptibility to ethidium bromide and benzalkonium chloride [Sulavik01]. Transport experiments suggest that MdfA confers resistance via an efflux mechanism dependent on the proton motive force [Edgar97]. Consistent with this, chloramphenicol/proton antiport was observed in membrane vesicles prepared from cells overexpressing mdfA [Mine98]. Overexpression of mdfA also results in spectinomycin sensitivity and isopropyl-Β-D-thiogalactopyranoside (IPTG) exclusion due to unknown mechanisms [Bohn98]. 12 transmembrane domains (TMs) have been predicted from the hydropathy plot of the protein and confirmed by gene fusion analysis [Edgar99, Adler02].

MdfA catalyzes both electrogenic and electroneutral transport. MdfA-catalyzed transport of neutral substrates is electrogenic, resulting in a net movement of electric charges, whereas transport of cationic substrates is electroneutral with no net movement of charges, driven primarily by proton chemical gradient [Lewinson03]. The negatively charged, membrane embedded, amino acid residues glutamic acid 26 (E26) and aspartic acid 34 (D34) and the positively charged arginine residue at position 112 (R112) have been implicated in transport function [Edgar99, Adler04, Adler05, Sigal05]. R112 is located within a predicted drug binding pocket and a positive charge at this position is essential for transport function [Sigal05]. No single replacement of any acidic residue with a neutral residue abolishes activity of the transporter, however an E26C/D34C double mutation disrupts MdfA-mediated resistance to a variety of test drugs [Sigal06, Sigal09]. Aspartic acid at position 34 (D34) is the proton binding carboxylic residue [Fluman12]. Substrate and protons compete for binding to MdfA but they bind at different sites within the protein [Fluman12].

Insertion of an additional acidic residue into the predicted substrate binding pocket of MdfA broadens the substrate specificity of the transporter to include divalent cationic compounds [Tirosh12].

In addition to its role as a multidrug resistance transporter, MdfA also mediates alkaline resistance. In deletion mutation experiments mdfA mutants were sensitive even to mild alkaline conditions, and the wild-type phenotype is restored fully by mdfA expressed from a plasmid. mdfA expressed from a multicopy plasmid was found to confer extreme alkaline pH resistance, allowing the growth of cells under conditions that are close to those used normally by alkaliphiles (up to pH 10). Inverted vesicle fluorescence studies demonstrated that MdfA catalyzes Na+ or K+-dependent proton transport [Lewinson04]. mdfA has been implicated in arabinose efflux [Koita12].

Reviews: [Bibi01, Sigal06a, Krulwich05]

Citations: [Sigal07]

Gene Citations: [Nilsen96]

Locations: inner membrane

Map Position: [882,896 -> 884,128] (19.03 centisomes)
Length: 1233 bp / 410 aa

Molecular Weight of Polypeptide: 44.321 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0002871 , DIP:DIP-48116N , EchoBASE:EB3460 , EcoGene:EG13696 , EcoliWiki:b0842 , Mint:MINT-1227586 , ModBase:P0AEY8 , OU-Microarray:b0842 , PortEco:cmr , PR:PRO_000022304 , Protein Model Portal:P0AEY8 , RefSeq:NP_415363 , RegulonDB:G6440 , SMR:P0AEY8 , String:511145.b0842 , UniProt:P0AEY8

Relationship Links: InterPro:IN-FAMILY:IPR005829 , InterPro:IN-FAMILY:IPR011701 , InterPro:IN-FAMILY:IPR016196 , InterPro:IN-FAMILY:IPR020846 , Pfam:IN-FAMILY:PF07690 , Prosite:IN-FAMILY:PS50850

In Paralogous Gene Group: 17 (37 members)

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006855 - drug transmembrane transport Inferred from experiment [Edgar97, Mine98]
GO:0030641 - regulation of cellular pH Inferred from experiment [Lewinson04]
GO:0035725 - sodium ion transmembrane transport Inferred from experiment [Lewinson04]
GO:0046618 - drug export Inferred from experiment [Edgar97]
GO:0046677 - response to antibiotic Inferred from experiment Inferred by computational analysis [UniProtGOA11, Edgar97]
GO:0071805 - potassium ion transmembrane transport Inferred from experiment [Lewinson04]
GO:0006810 - transport Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0055085 - transmembrane transport Inferred by computational analysis [GOA01]
Molecular Function: GO:0015238 - drug transmembrane transporter activity Inferred from experiment [Edgar97, Mine98]
GO:0015307 - drug:proton antiporter activity Inferred from experiment [Edgar97]
GO:0015385 - sodium:proton antiporter activity Inferred from experiment [Lewinson04]
GO:0015386 - potassium:proton antiporter activity Inferred from experiment [Lewinson04]
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, Adler02]
GO:0005887 - integral component of plasma membrane Inferred from experiment [Adler02]
GO:0016020 - membrane Inferred by computational analysis [UniProtGOA11, GOA01]
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 mdfA 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]
Yes [Feist07, Comment 4]

Credits:
Last-Curated ? 07-Mar-2013 by Mackie A , Macquarie University


Enzymatic reaction of: K+:H+ antiporter (multidrug efflux transporter MdfA (multifunctional))

Summary:
Km for K+ is in the range 50-100mM [Lewinson04]


Enzymatic reaction of: Na+:H+ antiporter (multidrug efflux transporter MdfA (multifunctional))

Summary:
Km for Na+ is in the range 50-100mM [Lewinson04]


Enzymatic reaction of: multidrug efflux transporter MdfA


Sequence Features

Feature Class Location Common Name Citations Comment
Transmembrane-Region 15 -> 32  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Mutagenesis-Variant 26  
[Adler04, UniProt11]
Alternate sequence: E → T; UniProt: Strong decrease in chloramphenicol efflux activity. Loss of ethidium bromide efflux activity.
Alternate sequence: E → Q; UniProt: Slight decrease in chloramphenicol efflux activity. Loss of ethidium bromide efflux activity.
Alternate sequence: E → N; UniProt: Strong decrease in chloramphenicol efflux activity. Loss of ethidium bromide and TPP efflux activities.
Alternate sequence: E → L; UniProt: Strong decrease in chloramphenicol efflux activity. Loss of ethidium bromide and TPP efflux activities.
Alternate sequence: E → K; UniProt: Decrease in TPP efflux activity. Exhibits low ethidium bromide efflux activity.
Alternate sequence: E → V; UniProt: Slight decrease in chloramphenicol efflux activity. Exhibits low ethidium bromide efflux activity.
Alternate sequence: E → I; UniProt: Slight decrease in chloramphenicol efflux activity. Exhibits low ethidium bromide efflux activity.
Alternate sequence: E → H; UniProt: Almost no chloramphenicol efflux activity. Loss of ethidium bromide efflux activity.
Alternate sequence: E → D; UniProt: No change in ethidium bromide efflux activity.
Alternate sequence: E → A; UniProt: Loss of ethidium bromide efflux activity.
Amino-Acid-Sites-That-Bind 34 proton binding site
[Fluman12]
 
Transmembrane-Region 54 -> 72  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Sequence-Conflict 59  
[Edgar97, UniProt10]
Alternate sequence: T → N; UniProt: (in Ref. 2; CAA69997);
Transmembrane-Region 83 -> 103  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Transmembrane-Region 110 -> 130  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Transmembrane-Region 145 -> 165  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Transmembrane-Region 167 -> 187  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Sequence-Conflict 225 -> 240  
[Nilsen96, UniProt10]
Alternate sequence: ALALGFVSLPLLAWIA → GAGAADSLVCRCWRGSP; UniProt: (in Ref. 1; AAC44147);
Transmembrane-Region 227 -> 247  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Transmembrane-Region 256 -> 276  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Transmembrane-Region 288 -> 308  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Transmembrane-Region 315 -> 335  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Transmembrane-Region 347 -> 367  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;
Transmembrane-Region 379 -> 399  
[UniProt10a]
UniProt: Helical;; Non-Experimental Qualifier: probable;


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.


References

Adler02: Adler J, Bibi E (2002). "Membrane topology of the multidrug transporter MdfA: complementary gene fusion studies reveal a nonessential C-terminal domain." J Bacteriol 184(12);3313-20. PMID: 12029048

Adler04: Adler J, Lewinson O, Bibi E (2004). "Role of a conserved membrane-embedded acidic residue in the multidrug transporter MdfA." Biochemistry 43(2);518-25. PMID: 14717607

Adler05: Adler J, Bibi E (2005). "Promiscuity in the geometry of electrostatic interactions between the Escherichia coli multidrug resistance transporter MdfA and cationic substrates." J Biol Chem 280(4);2721-9. PMID: 15557318

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

Bibi01: Bibi E, Adler J, Lewinson O, Edgar R (2001). "MdfA, an interesting model protein for studying multidrug transport." J Mol Microbiol Biotechnol 3(2);171-7. PMID: 11321570

Bohn98: Bohn C, Bouloc P (1998). "The Escherichia coli cmlA gene encodes the multidrug efflux pump Cmr/MdfA and is responsible for isopropyl-beta-D-thiogalactopyranoside exclusion and spectinomycin sensitivity." J Bacteriol 1998;180(22);6072-5. PMID: 9811673

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

Edgar97: Edgar R, Bibi E (1997). "MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition." J Bacteriol 1997;179(7);2274-80. PMID: 9079913

Edgar99: Edgar R, Bibi E (1999). "A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA." EMBO J 18(4);822-32. PMID: 10022825

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Fluman12: Fluman N, Ryan CM, Whitelegge JP, Bibi E (2012). "Dissection of mechanistic principles of a secondary multidrug efflux protein." Mol Cell 47(5);777-87. PMID: 22841484

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."

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

Koita12: Koita K, Rao CV (2012). "Identification and Analysis of the Putative Pentose Sugar Efflux Transporters in Escherichia coli." PLoS One 7(8);e43700. PMID: 22952739

Krulwich05: Krulwich TA, Lewinson O, Padan E, Bibi E (2005). "Do physiological roles foster persistence of drug/multidrug-efflux transporters? A case study." Nat Rev Microbiol 3(7);566-72. PMID: 15953929

Lewinson03: Lewinson O, Adler J, Poelarends GJ, Mazurkiewicz P, Driessen AJ, Bibi E (2003). "The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions." Proc Natl Acad Sci U S A 100(4);1667-72. PMID: 12578981

Lewinson04: Lewinson O, Padan E, Bibi E (2004). "Alkalitolerance: a biological function for a multidrug transporter in pH homeostasis." Proc Natl Acad Sci U S A 101(39);14073-8. PMID: 15371593

Mine98: Mine T, Morita Y, Kataoka A, Mizushima T, Tsuchiya T (1998). "Evidence for chloramphenicol/H+ antiport in Cmr (MdfA) system of Escherichia coli and properties of the antiporter." J Biochem (Tokyo) 1998;124(1);187-93. PMID: 9644262

Nilsen96: Nilsen IW, Bakke I, Vader A, Olsvik O, El-Gewely MR (1996). "Isolation of cmr, a novel Escherichia coli chloramphenicol resistance gene encoding a putative efflux pump." J Bacteriol 1996;178(11);3188-93. PMID: 8655497

Paulsen96a: Paulsen IT, Brown MH, Skurray RA (1996). "Proton-dependent multidrug efflux systems." Microbiol Rev 1996;60(4);575-608. PMID: 8987357

Sigal05: Sigal N, Vardy E, Molshanski-Mor S, Eitan A, Pilpel Y, Schuldiner S, Bibi E (2005). "3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge." Biochemistry 44(45);14870-80. PMID: 16274234

Sigal06: Sigal N, Molshanski-Mor S, Bibi E (2006). "No single irreplaceable acidic residues in the Escherichia coli secondary multidrug transporter MdfA." J Bacteriol 188(15);5635-9. PMID: 16855255

Sigal06a: Sigal N, Cohen-Karni D, Siemion S, Bibi E (2006). "MdfA from Escherichia coli, a model protein for studying secondary multidrug transport." J Mol Microbiol Biotechnol 11(6);308-17. PMID: 17114895

Sigal07: Sigal N, Lewinson O, Wolf SG, Bibi E (2007). "E. coli multidrug transporter MdfA is a monomer." Biochemistry 46(17);5200-8. PMID: 17407265

Sigal09: Sigal N, Fluman N, Siemion S, Bibi E (2009). "The secondary multidrug/proton antiporter MDFA tolerates displacements of an essential negatively charged side chain." J Biol Chem. PMID: 19129186

Sulavik01: Sulavik MC, Houseweart C, Cramer C, Jiwani N, Murgolo N, Greene J, DiDomenico B, Shaw KJ, Miller GH, Hare R, Shimer G (2001). "Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes." Antimicrob Agents Chemother 45(4);1126-36. PMID: 11257026

Tirosh12: Tirosh O, Sigal N, Gelman A, Sahar N, Fluman N, Siemion S, Bibi E (2012). "Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA." Proc Natl Acad Sci U S A 109(31);12473-8. PMID: 22802625

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 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."

Other References Related to Gene Regulation

Eguchi03: Eguchi Y, Oshima T, Mori H, Aono R, Yamamoto K, Ishihama A, Utsumi R (2003). "Transcriptional regulation of drug efflux genes by EvgAS, a two-component system in Escherichia coli." Microbiology 149(Pt 10);2819-28. PMID: 14523115

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


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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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