Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli

Molecular Microbiolcgy (1995) 16(1), 45-55

system of Escherichia coli Dzwokai Wla,^'^ David N. Cook,^ Wlarie Albert!,^ NingG. Pon,^ Hiroshi Nikaido'^'* and John E. 'Department of Chemistry, University of Gatlfornia, Berkeley, California 94720. USA. ^Division of Structural Biology. Lawrence Berkeley Laboratory, Berkeley. California 94720. USA. '^Sferitech inc., 2525 Stanwell Drive, Suite 300, Concord, California 94520, USA. '^Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA. Summary Defined mutations of acrA or acrB (formerly acrE) genes increased the susceptibility of Escherichia coli to a range of small inhibitor molecules. Deletion of acrAB increased susceptibility to cephalothin and cephaloridine, but the permeability of these |i-lactams across the outer membrane was not increased. This finding is inconsistent with the eariier hypothesis that acrAB mutations increase drug susceptibility by increasing the permeability of the outer membrane, and supports our model that acrAB codes for a multi-drug efflux pump. The natural environment of an enteric bacterium such as E. coli is enriched in bile salts and fatty acids. An acrAB deletion mutant was found to be hypersusceptible to bile salts and to decanoate. In addition, acrAB expression was elevated by growth in 5mM decanoate. These resuits suggest that one major physiological function of AcrAB is to protect E. coli against these and other hydrophobic inhibitors. Transcription of acrAB is increased by other stress conditions including 4% ethanol, 0.5 Wl NaCl, and stationary phase in LuriaBertani medium. Finally, acrAB expression was shown to be increased in mar (multiple-antibioticresistant) mutants.

Introduction Mutations in the acrA locus are known to render Escherichia coli hypersusceptible to basic dyes, detergents and antibiotics (Nakamura, 1965; 1968), The observation that Received 23 September t994; revised 30 November 1994; accepted 13 December, 1994. ^'For correspondence, Tel. (510) 642 2027; Fax (510)643 9290, © 1995 Biackweil Science Ltd

acrA mutants are hypersusceptibte to a broad spectrum of agents, which have different structures and different intracellular targets, suggested a general role for the acrA locus tn determining the intrinsic drug resistance of E. coll. One explanation is that acrA mutations increase the permeability of the outer membrane of E. coli. In several studies to date that have addressed this question, however, an unambiguous change has not been detected in the chemical composition of the outer membrane of acrA strains (see Sukupolvi and Vaara, 1989), We have previously cloned the acrA locus by complementing the drug-hypersusceptible phenotype of an acrA mutant, N43 (Ma efai, 1993a; also see Xu etai, 1993), Sequence analysis has revealed at least two genes, acrA and acrB (formerly acrE), which are located within a single operon. Gene acrA appeared to encode a periptasmic fipoprotein whose amino-terminus was anchored to the inner membrane, and gene acrB appeared to encode an integral inner membrane protein with 12 transmembrane segments. The predicted topologies of AcrA and AcrB were supported by the construction of phoA fusions to acrA and acrB (Ma ef ai. 1993a). These predictions gained further credence by the comparison with several AcrAB homologues found in E. co//or Pseudomonas aeruginosa {Se\iier etai. 1993; Poole efai. 1993), Strain N43 contained an \S2 element inserted near tine 5' end of acrA, and a plasmid containing only the acrA* allele was able to complement the drug hypersusceptibility ot N43, Therefore, AcrA is required for the intrinsic drug resistance of E, coli. Based on operon organization and sequence comparison with other transporter proteins, we have further suggested that AcrB contributes to the intrinsic drug resistance as a component of an energy-dependent drug efflux pump. Direct experimental evidence for the involvement of AcrB has been lacking, however. Initial experiments strongly suggested that AcrAB pumped out drugs such as acriflavine (Ma et ai. 1993a). However, there was much residual efflux activity left even in the acrA mutant N43, presumably because of the existence of other multi-drug efflux pumps with overlapping specificities. Consistent with this hypothesis, three AcrAB homologues — AcrEF (formerly EnvCD). OrfAB, and AcrD — have now been identified in E. coli (for a review, see Ma et ai. 1994). Mutations in orfAB result in drug hypersusceptibiiity (G- Storz, personal covnniunication). Moreover, drug-hypersusceptibility of an acrAB

46 a Ma et al. mutant can be suppressed by mutations mapping close to acrEP, which presumably increase the expression of acrEFgenes (Xu etai, 1993). In addition, evidence has recently been presented suggesting the existence of multi-drug efflux pumps of another class, I.e. EmrAB (Lomovskaya and Lewis, 1992). The proposal that AcrAB encodes a drug-efflux pump has been supported by several recent discoveries. First, by functional complementation, Poole ef ai (1993) have cloned a putative effiux system (the MexAB-OprK system) for the secretion of siderophores during iron starvation from P. aeruginosa. MexAB is homologous to AcrAB, and mutations in the genes encoding MexAB-OprK also make P. aeruginosa hypersusceptible to hydrophobic antibiotics and inhibitors. Second, Dinh ef ai (1994) have proposed that AcrA belongs to the family of 'membrane fusion proteins' (MFP), Many members of this family are known to function in conjunction with inner membrane transporters to pump out peptides, proteins, carbohydrates or drugs across the two membranes of Gram-negative baoteria. EmrA is also a member of the MFP family. Although AcrAB may be hypothesized to contribute to the intrinsic drug resistance of E. coll, most of its known substrates, such as antibiotics and cationic dyes, are not found in the natural environment of E, coll. It seems likely that the AcrAB efflux system may have evolved for other purposes. In this regard, it is noteworthy that the natural environment of enterfc bacteria such as E. co//is enriched in bile salts and fatty acids (Lenlner, 1981). Both bile salts and fatty acids are hydrophobic compounds that inhibit the growth of Gram-positive bacteria (Freese etai, 1973). However, Gram-negative bacteria such as E. coll are known to be intrinsically resistant to bile salts and fatty acids. Traditionally, this resistance has primarily been attributed to the oufer membrane, which is the major barrier for the entry of hydrophobic inhibitors into the cell (Sheu ef al., 1973). However, recent studies have shown that the outer membrane of Gram-negative bacteria can only slow down the influx of hydrophobic inhibitors, and the equilibration time for many hydrophobic inhibitors across the outer membrane is much shorter than the doubting time of the bacteria (Plesiat and Nikaido, 1992). As a result, Gram-negative bacteria need additional mechanism(s), such as active efflux, for protection against these hydrophobic inhibitors. We show here that AcrB, as well as AcrA, is necessary for the intrinsic resistance of E coli to hydrophobic antibiotics and detergents. Our measurernent of the permeability of |ii-lactams across the outer membrane of an acrAB null mutant was inconsistent with the earlier hypothesis that acrAB mutations increase drug susceptibility by increasing the permeability of the outer membrane and supports the more recent modei that acrAB encodes a drug-eftlux pump. Moreover, the acrAB null mutant is

hypersusceptible to both bile salts and decanoate (a CIO fatty acid), suggesting that one physiological function of AcrAB is to protect E. coli against these naturally occurring hydrophobic inhibitors. Finally, we report that trarv scription of acrAB is increased not only by decanoate but also by conditions of general stress, and in marR (multiple-antibiotic-resistant) mutants. Results Both AcrA and AcrB are required for the intrinsic drug resistance of E. coii In order to delineate the role of AcrB, we have constructed three mutants (Fig. 1) and determined their drug susceptibilities (Table 1). Mutants J2M100 or J2M130 contairied the insertion of a 1.3 kb kanamycin-resistance gene cartridge near the 5' end of acrB or acrA, respectively. For mutant JZM120, a large portion of acrAt3 was deleted and replaced by the same 1.3 kb gene cartridge. As shown in Table 1, all three mutants are hypersusceptibie to novobiocin, erythromycin and SDS, when compared with their isogenic parent sirain (JC7623). These results

acrA

iurB

JC7&23

acrB

JZM130

ggliaiH^SSi::;:;:!:::

ilcrA

JZM too

Mscl JZM 120

Fig, •!. The construction of acrAB mutants, twtutations of acrAB were tirst introduced into ptasmid pUCK151A, wtiich contained the witd-tyiDe acrAB genes. Mutations were Ihen moved to E. coli JC7623 by tiomotocjous recombination (tor detaits, ^ee the Experimentai procedures). Strain JZM100 or J2M130 contains the insertion ot •1,3 kb Tn903kan' gene cartridge at the Nae\ site of acrB or the Msc\ site of acrA, respectivety. t=or JZtV!i20, the Msc\-Hpa] fragmenl of acrAB was deleted and replaced by the 1,3kb Tr\903kan' gene cartridge. C) 1995 Biackweil Science Ltd, Molecular fiiiicroblology. 16, 45-55

stress-induced efflux system ot Esctterichia coli Tabie 1. Comparison of lhe drug susceptibility in LB medium between the acrAB mutants and Iheir isogenic wild lypes.

MtC (ll gml ') Foi': Compound

JC7623

JZtVitOO

JZM120

JZM130

K4401

KZM120

Novobiocirt Er/thromycin SDS Sodium cholale Sodium taurodeoxychoiate

64 128 >10000