A staphylococcal multidrug resistance gene product is a member of a new protein family

PLASMID

27, 119-129

(1992)

A Staphylococcal Multidrug Resistance Gene Product Is a Member of a New Protein Family L.GRINKJs,*+'

G. DREGUMENE,* E. B. GOLDBERG,? C.-H. LIAO,$ AND% J. PROJAN+

*Department of Biochemistry and Biophysics, Vilnius University, Vilnius 232009, Lithuania; fDepartment of Molecular Biology and Microbiology, Tujis University School of Medicine, Boston, Massachusetts 0211I; and tipplied Microbiology, Inc.. Public Health Research Institute, 455 First Avenue, New York, New York 10016 Received June 3, 1991; revised September 30, I99 1 The complete nucleotide sequence (32 1 bp) of smr (staphylococcal multidrug resistance), a gene coding for efflux-mediated multidrug resistance of Staphylococcus aureus, was determined by using two different plasmids as DNA templates. The smr gene product (identical to products of ebr and qacC/D genes) was shown to be homologous to a new family of small membrane proteins found in Escherichia coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Proteus vulgaris. The smr gene was subcloned and expressed in S. aureus and E. coli and its ability to confer the multidrug resistant phenotype was demonstrated for two different lipophilic cation classes:phosphonium derivatives and quartemary amines. Expression of smr gene leads to the efflux of tetraphenylphosphonium and to a net decrease in the uptake of lipophilic cations. The deduced polypcptide sequence (107 amino acid residues, 11,665 kDa) has 46% hydrophobic residues (Phe, Ile, Leu, and Val) and 20% hydroxylic residues (Ser and Thr). Four transmembrane segments are predicted for smr gene product. Of the charged amino acid residues, only Glu 13 is located in a transmembrane segment. This Glu 13 is conserved in all members of the family of small membrane proteins. We propose a mechanism whereby exchange of protons at the Glu 13 is a key in the efflux of the lipophilic cation. This mechanism includes the idea that protons are transported to the Glu 13 via an appropriate chain of hydroxylit residues in the transmembrane segments of Smr. 0 1992 Academic Press, Inc.

Eukaryotic and prokaryotic cell membranes possessefflux-mediated transport systems for the export of a number of lipophilic cations. In multidrug-resistant mammalian cells, the P-glycoprotein responsible for extrusion of lipophilic toxic compounds has been identified (Chen et al., 1986; Gros et al., 1986). The yeast STE6 gene has been shown to encode a homologue of the mammalian multidrug resistance P-glycoprotein (McGrath and Varshavsky, 1989). In bacteria, there are a number of effluxmediated systems which are encoded by transmissible plasmids (Tisa and Rosen, 1990). Genetic determinants for resistance to ethidium bromide were identified on a staph’ To whom correspondence should be addressedat Department of Molecular Biology and Microbiology, Tufts University Medical School, 136 Harrison Avenue, Boston, MA 02111.

ylococcal plasmid (Johnson and Dyke, 1969) and the sequence of the ethidium bromide resistance (ebr) determinant has been published (Sasatsuet al., 1989) but no data about the range of the specificity of its product were presented. A plasmid-mediated resistance to quaternary amine compounds (qac) has been found in methicillin-resistant Staphylococcus aureus (Emslie et al., 1986; Gillespie and Skurray, 1986; Tennent et al., 1985). Different transport systems for the elllux of lipophilic cations encoded by the qac genes have been determined in plasmids of 5’. aureux (see Lyon and Skurray, 1987; Tennent et al., 1989). The qacA determinant shares restriction site identity and DNA sequence similarity with the qacB determinant, as based on DNA hybridization experiments. The other qacdeterminants (Cand D) seemto be genetically distant from qacA and qacB and are re-

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0147-619X/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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GRINIUS ET AL.

lated to each other on the basis of both DNA hybridization studies (Lyon and Skurray, 1987; Tennent et al., 1989) and qacC/D gene nucleotide sequence (Littlejohn et al., 1991). The qacA gene has been sequenced recently and its common ancestry with tetracycline and sugar transport systems has been demonstrated (Rouch et al., 1990). In Bacillus subtilis, the geneencoding multidrug resistance (bmr) has been identified and sequenced (Neyfakh et al., 1991). The product of this gene is responsible for the efflux of ethidium and also displays a remarkable sequence similarity to tetracycline-efflux pumps and, to a lesser extent, to arabinose and xylose-Hf symporters (Neyfakh et al., 1991). Thus, these proteins are members of a family of prokaryotic proteins, most of which have 12 or more transmembrane segments. The body of experimental evidence indicates that efflux system(s) for lipophilic cations could be induced in Escherichia coli liquid cultures (see Midgley, 1987) and a gene encoding an ethidium efflux system in E. coli has been cloned recently (Purewall et al., 1990). These findings raise two fundamental questions: why did bacteria develop transport systems with a broad range of specificity and what is the molecular mechanism of their function? To address these issues, our two groups independently identified and sequenced the same gene for staphyloccocal multidrug resistance (smr) from two different plasmids of S. aureus. It was shown to be identical to both the ebr (Sasatsuet al., 1989) and the qacC/D genes (Littlejohn et al., 1991). These authors reported resistance only to quarternary amine compounds (i.e., ethidium bromide, antiseptics, and disinfectants). In this article we tested the resistance phenotype of smr for tetraphenylphosphonium and its derivatives aswell. To this end we subcloned this gene in both S. aureus and E. coli and demonstrated its ability to confer multidrug resistance. The putative smr gene product is homologous to a new family of small membrane proteins also found in E.

coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Proteus vulgaris. The effect of smr gene expression on the accumulation of lipophilic cations has been assayed in vivo. MATERIALS

AND METHODS

Bacterial strains, plasmids, and culture media. The methicillin-resistant S. aureus strain WG365 containing plasmid pWBG32 (Emslie et al., 1986) was isolated at the Royal Perth Hospital, Perth, Australia, and was obtained from Dr. W. B. Grubb (School ofMedical Technology, Curtin University of Technology, Perth, Australia). E. coli strain KO 1489 used in this study was a sodium dodecyl sulfate-sensitive derivative of MC4 100 (araD139, A(argF-lac), U169, rpsL150, relA7, deoCl, ptsF25, rbsR, thi, supF, Zla:tnlOsdsl6). The strain was kindly supplied by Dr. A. Wright (Department of Molecular Biology and Microbiology, Tufts University, Boston, MA). Plasmid pBgc2 containing the 2-kb insert from the plasmid present in a clinical isolate of S. aureus Al 18 was constructed by Jones and Midgley (1985) and was kindly provided by Dr. M. Midgley (Department of Biochemistry, University of Hull, Hull, UK). The CY broth (Novick and Brodsky, 1972) was used for liquid cultures of S. aureus grown at 37°C on a shaker. Cultures of E. coli K01489 were grown at 37°C with aeration in the minimal medium (Vogel and Bonner, 1956), containing ampicillin (100 &ml) where appropriate. Ethidium bromide and gramicidin S were from Sigma (St. Louis, MO); methyltriphenylphosphonium (MTP+) bromide and tetraphenylphosphonium (TPP+) bromide were purchased from Aldrich (Milwaukee, WI). Assay of multidrug resistant phenotype. The multidrug-resistant phenotype of S. aureus and E. coli K01489 was determined as resistance both to ethidium and to TPP+ and MTP+. These lipophilic cations have almost nothing in common except their electric charge and moderately hydrophobic nature.

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The ability of the cell to grow was assayedon strands were sequencedin their entirety using GL agar (Novick and Brodsky, 1972) or on circular plasmid DNA as a template, and all agarized minimal medium (Vogel and Bon- junctions were overlapped. The oligodeoxyner, 1956), respectively, supplemented with nucleotide primers were synthesized either at either ethidium bromide, MTP’, or TPP+. the PHRI Microchemistry laboratory operThe agar dilution method (Emslie et al., ated by Dr. Y. K. Yip or at Fermentas. RecombinantDNA techniques.Restriction 1986) was used to determine a value of the minimal inhibitory concentration (MIC) of endonucleasesand DNA modifying enzymes the lipophilic cations. The multidrug-resis- either were purchased from New England tant phenotype was assayed as a IO-fold in- Biolabs (Beverly, MA) and BoehringerMannheim Biochemicals (Indianapolis, IN) creasein the MIC. Assay of lipophilic cation uptake and mem- or were obtained from Fermentas and were brane potential. E. coli K01489 cells were used according to manufacturer’s instrncwashed once with 10 mM Hepes buffer (pH tions. All manipulations with DNA and E. 7.0), resuspended in the same buffer, and coli were performed as described (Maniatis et kept on ice during the experiment. The cells al., 1982). Restriction mapping of pWBG32 were resuspended in buffer containing 10 was done with the ethidium bromide-CsClmu sodium phosphate (pH 7.5) 5 mM so- purified plasmid DNA samples (Clewel and dium succinate, and 2 mM TPP+ at 37°C Helinski, 1969). The method of the protoand TPP+ uptake was assayed with a plast transformation (Chang and Cohen, TPP+-specific electrode (Grinius, 1987). The 1979) as modified by Murphy (1983) was membrane potential was calculated from the used to transform S. aureus cells. Computeranalysis of sequencedata. DNA, distribution of TPP+ across the cell memprotein sequenceanalysis, and sequencedata brane as described (Grinius, 1987). DNA sequencedetermination. The strategy base searching were conducted with profor the sequencing of the pWBG32 plasmid grams contained within the SequenceAnalyin New York (Fig. IA) was to clone the TaqI sis Software Package (version 6.0) licensed fragments into the appropriate sites of phage from the Genetics Computer Group (UniverMl3mpl8 and Ml3mpl9 DNA (Messing sity of Wisconsin, Madison) (Devereux et al., and Vieira, 1982; Yanisch-Perron et al., 1984). This package is mounted on a DEC 1985) for sequencing by the dideoxy chain VAX computer in the Image Analysis Labotermination method (Sanger et al., 1977) us- ratory at Tufts University (Boston, MA). The ing the Sequenase 2.0 DNA sequencing kit deduced amino acid sequenceof the putative (U.S. Biochemicals, Cleveland, OH) and [a- Smr polypeptide was compared by the algo35S]dATP(New England Nuclear). The DNA rithm of Pearson and Lipman (1988) fragments were recovered from gels as de- (TFASTA implementation of GenBank) scribed by Maxam and Gilbert ( 1980). The with polypeptide sequencesin the GenBank 1113-bp HpaI-SalI fragment of the pBgc2 (version 68.0) database. (Fig. 1B) was cloned in Vilnius into an analog Nucleotide sequence accession number. of pUC 19 vector for sequencing by the enzy- The DNA sequence reported here is held in matic dideoxy chain termination method the GenBank under Accession No. M33479. (Sanger et al., 1977) using the Sequencing Kit (Pharmacia, Upsala, Sweden) and [aRESULTS AND DISCUSSION 32P]dATP from Isotop (Leningrad, USSR). A set of overlapping unidirectional deletions Identljication of the smr gene.The plasmid was generated by serial Ba131treatment us- pWBG32 was sequenced in its entirety and ing the nested deletion kit (Fermentas, Vil- the presence of two open reading frames nius, Lithuania), the overlapping DNA (ORFs) larger than 200 bp and capable of en-

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GRINIUS ET AL.

A smr

ORF

rep ORF

OrI

4 I

I

I

e I

I

I

1.6

I

I

I

0.8 4

0.0

4

Jaq I

4

Rsa I

Hfnd

Taq I

I

4 III

kbp

4 Taq I

I 91 Obp Taq I fragment

B Sal

I

Hpa

+

I

I

xORF I

4 Taq I

M DRS

1.0 4 Dra

I

’

’

’

0.5

ORF ’

Taq I fragment

I

i l

4 Taq I

DRS

l

’ 0.0 a

kbp

4

Jaql

I I

I 440bp

smr

6a/31 I

600bp

Taq I fragment

FIG. I. Restriction endonuclease cleavage maps of the 1600-bp TuqI-TuqI fragment of pWBG32 plasmid (A) and the 1113-bp HpuI-Sufl fragment of pBgc2 plasmid (B). Sites of putative open reading frames (ORF) and a replication origin (ori) and landmark restriction sites are shown.

coding proteins was found (Fig. IA). Each ORF was associated with a typical initiation codon (ATG), a potential ribosome binding site (RBS), and an RNA polymerase binding site. The nucleotide sequence of the larger ORF resembled the replication gene of the pC194, a plasmid which encodes the resistance to chloramphenicol (Horinouchi and Weisblum, 1982). We found an 83% identity of amino acids between the putative products of these ORFs in the pWBG32 and in the pC 194 plasmids. The smr determinant in the pWBG32 was identified in two ways. A chloramphenicol resistance (cmr) gene was isolated on a 1121-bp TaqI fragment from the pC221 plasmid (Projan et al., 1985) and ligated to the single RsaI site in the putative smr gene of the pWBG32 (Fig. 1A). The resulting plasmids carrying the cmr determinant no longer conferred the smr phenotype upon the transformed hosts. In a parallel experiment, the 910-kbp Tag1 fragment was isolated from the pWBG32 plasmid. This fragment contains both the putative smr determinant and the putative origin of replica-

tion; it was subcloned onto the pRN5 101, the temperature-sensitive plasmid carrying the cmr determinant. The resulting clones expressed both the smr and the cmr determinants. In the control experiment, neither smr nor cmr was expressedat nonpermissive temperature. The nucleotide sequence of 1113-bp within the pBgc2 plasmid was determined between the HpaI/Bal31 site and the MI site. Two ORFs larger than 200 bp were found in this region (Fig. 1B). Each of them was preceded by typical signals for the initiation of both transcription and translation. The larger ORE (321 bp), a putative smr determinant, was flanked with direct repeat sequences (DRS). The other ORF (207 bp) was located downstream of the first one (Fig. 1B). To identify the smr gene in this plasmid, the ORFs with their potential RBS and promoters were separated by TaqI digestion from the 1113-bp fragment (Fig. 1B) and the two fragments (440 and 600 bp) were subcloned into pUC 19. Twelve subclones transformed with the pUC 19 plasmid which con-

BACTERIAL E.co/i /put

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123

19 E.co/i/puc

19smr

Ir

Grarnlcldln

:

Gramicldln

FIG. 2. TPP+ uptake by E. co/i cells and determination of membrane potential. The TPP’ uptake was assayedand the membrane potential was determined as described under Materials and Methods. Curve 1, cells transformed with the control plasmid. Curve 2, cells transformed with the srnr genetic determinant. Cells were added to concentrations of 3-4 x lo9 cells/ml and gramicidin S to 4 &ml.

tained the x ORF were isolated and not one of them was found to be resistant to ethidium bromide. On the other hand, all plasmids containing the SMTdeterminant were able to render E. coli resistant to ethidium bromide, MTP+, and TPP+ (data not shown). An alignment of the corresponding nucleotide sequences in the plasmids pWBG32 and pBgc2 has revealed the 100% identity of their smr ORFs (data not shown). To elucidate an effect of the expression of the smr determinant on the uptake of lipophilic cations, we used a sodium dodecyl sulfate-sensitive mutant of E. culi (KO1489), whose outer membrane is permeable to TPP+. This mutation permitted us to detect TPP+ uptake in intact cells that contained the control plasmid (curve 1, Fig. 2). With these cells we determined the initial rate of TPP+ uptake, which is driven by the membrane potential, and the steady-state value of the membrane potential, which was calculated in terms of TPP+ distribution across cell mem-

brane. The membrane potential value in the control cells reached 166 mV. Depolarization of the membrane with gramicidin (which forms ion channels in the cell membrane) caused TPP+ efflux. When E. coli (KO1489) cells were transformed to multidrug resistance using pUC19smr, the TPP+ uptake was followed by efflux (curve 2, Fig. 2). The apparent. membrane potential only reached 128 mV. This is due to the rapid efflux of TPP+ caused by product of the smr gene. If these transformed cells were depolarized with gramicidin, the final level of TPP+ concentration in the cell suspension was higher than that in the control cell suspension (curve 2, Fig. 2). smr-transformed cells also accumulated less MTP+ (data not shown). These experiments indicate that expression of the smr gene in bacteria interferes with the measurement of the membrane potential because of the TPP+ efflux. Further studies on the bioenergetics of ion-drug exchange are required to determine actual

124

GRINIUS ET AL. 1 61

AlU I 7T%kh GGT;GGCTTAi~ACGCTATG&GACATTCiTCTCCAAGT&AGTTAAG GGTTCTTCTCAAACAATCAATAAATTTTCTCGGCATRAAT -44

121

60

ATACTGATA&=AT-

fi TAiAAGGATAGTTGCAAATGAWAAiACTTAGAAT-T

. Rss TAAATAAAATACGAAAATT~TTd,AATGCCT+ATATTTATTd.TAATAGC& mPlYiYliia smr +

120 .-10 180 240

241

420

421

ACAACCGTA~TCTCAATAA~TATTTTCAAiGAACAMTAiATCTAATAA~TATAGTATC~ ttvvoiiiflkoqinllitivs

481

ATAGTTTTAATCATAGTCGGCGTAGTTTCGTTAAACATTT irliirgPr slniflgtsh

601

TC

661

GTTCTTCTCAACTTCARTRTTTTCTCGGCATAAATGCATGTTTACTGT-TTGAT~

720

721

CTGATAC&T

780

-F

AlU I AATGGTChGCTTAAT-CGCTATGkGACATTCG&TGCAAGTTiAGTTAAGG

ma 781

AGAAAC-

AAAAAATAAAAGGATAGTTGCAAATGAAAAATACATATAT

660

I 7 93

FIG. 3. Nucleotide sequence of the 793-bp TugI-DruI fragment of pBgc2 and the predicted amino acid sequenceof the smr gene product. The beginning and the end of this sequence correspond to bases9 1 and 884, respectively, on the map (Fig. 1B). The putative promoter (-44, -35, -lo), rib-osomebinding (RBS) sites, and landmark restriction sites are indicated. Predicted transmembrane segments of the Smr protein are boxed. Direct repeats are indicated by thick underlines.

membrane potential as well as to explain the incomplete sensitivity of the TPP+ efflux to gramicidin. DNA sequence of the smr gene. The nucleotide sequence of the smr gene in the pBgc2 plasmid is shown in Fig. 3. The smr ORF is predicted to start with an ATG codon at base 2 14. This initiation codon is associated with a potential RBS (AAAGGAG) at bp 20 1. Translation starting at the first methionine (6 bp from the putative RBS) would yield a protein of 107 amino acids (11,665 molecular weight). The srnr sequence of pBgc2 was flanked both upstream and downstream by an ideal DRS of 92 bp (Fig. 3). A potential promoter for smr is seen upstream at bp 155 to 172 similar to the cannonical bacterial promoter consensus, with a 17-bp

hexamer spacing and 9 of 12 basesidentical to the consensus TTgAca-17-bp TAtAAT for the -35 and - 10 boxes, respectively. The region -41 to -45 of a potential promoter occurs 73 bp upstream from the smr ORF. The nucleotide sequence of the 1113-bp fragment in the pBgc2 plasmid was aligned with the pWBG32 nucleotide sequence and the computer analysis revealed identical sequences at the smr ORF, potential RBS, and the - 10 region of the putative promoter (data not shown). In the pWBG32 plasmid, only fragments of the DRS have been revealed both upstream and downstream the smr gene (data not shown). The smr sequence of the pBgc2 plasmid (Fig. 3) shared regions - 35 and - 10 of potential promoter aswell asthe ORF and the DRS

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MULTIDRUG

with both the ebr (Sasatsu et al., 1989) and the qacD determinants (Littlejohn et al., 1991). The sequence of the ebr determinant lacked 2 bp in the position at bp 73 to 74 of the sequence of the smr and contained cytosine instead of adenine (bp 202 of the smr) in the potential RBS. On the other hand, the smr sequence of the pWBG32 plasmid was identical to the corresponding sequence of the qacC genetic determinant (Littlejohn et al., 1991). Thus ebr, qacC, qacD, and smr are exactly the same genes in different context and they encode for the same 107-aminoacid protein. Because this gene encodes the system for extrusion of lipophilic cations and not only quaternary amine compounds and is responsible for the multiresistant phenotype, we suggestthe designation smr for it. Predicted Smr protein structure. The putative product of the smr gene (Smr) is a hydrophobic polypeptide of 107 amino acid residues with a molecular weight of 11,665. The hydrophobic nature of Smr suggestsits localization in the cytoplasmic membrane. Analysis of the structure of this putative protein reveals (Fig. 3) four hydrophobic segments that are connected together by flexible hydrophilic segments. Compared to other protondependent transporters, the putative Smr protein is small. It has only four transmembrane segments, whereas the others seem to have 12 or more transmembrane segments (Henderson and Maiden, 1990). Comparison of the Smr with products of other genes. Amino acid identity in sequences of Smr and the putative products of the qacA gene of S. aureus (Rouch et al., 1990), the bmr gene of B. subtilis (Neyfakh et al., 199I), and the tetA gene of E. coli (Waters et al., 1983) reached only 22, 31, and 13%, respectively, when a number of gaps were introduced in the Smr sequence (data not shown). This weak homology suggestsan independent origin of the smr gene or a primitive branching in the evolutionary chain. On the other hand, the Smr protein is related to a family of small hydrophobic proteins (Table 1) with highly conserved sequencesof amino

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acid residues (Fig. 4). These proteins are encoded by short (ca 330 bp) ORFs that start with a typical initiation codon (ATG) and end with a stop codon (data not shown). Each ORF is preceded by a typical RBS. Most ORFs identified so far do not overlap with other genes except for the ORF encoding a Smr-like protein which precedesthe suU(sulfonamide resistance) gene (Sundstroem et al., 1988; Guerineau and Mullineaux, 1989). In this case, there is 4-bp overlap between the 3’-terminus of the smr-like ORF and the 5’terminus of the sufl gene (data not shown). Some of these ORFs are under a promoter which is common to a whole operon, but some of them, for example, an ORF in thefrd operon of P. vulgaris (identified by Cole, 1987) seem to be under their own promoters. Smr is most closely related to the product of E. coli mvrC (methyl viologen resistance; Morimyo, 1991) gene and to an ORF which precedesthe SUNgene. There is 42% identity in a 105-amino-acid overlap and 4 1% identity in a 95-amino-acid overlap (lines M62732 and Xl 5024, respectively, Fig. 4). An ORF from the frd operon of P. vulgaris encodesa protein (line X06 144, Fig. 4) which shows 33% identity in a lOZamino-acid overlap. It seems remarkable that each of these four proteins (including Smr) has four putative transmembrane segments. Two other ORFs (an ORF in T-DNA of A. tumefaciens (Gheysen et al., 1990) and an ORF in plasmid DG 100 of E. coli (Cameron et al., 1986)) encode polypeptides of 83 amino acid residues which are almost identical in the Nand C-termini. These two putative proteins show 39 and 37% identity with Smr in 61and 45-amino-acid overlaps, respectively (lines M35007 and X04555, Fig. 4). No resistance to lipophilic cations has been related with these polypeptides so far. They may form just two transmembrane segments and the position of the second transmembrane segment in A. tumefaciens polypeptide is shifted toward the C-terminus in comparison to other polypeptides (Fig. 4). An ORF,

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GRINIUS ET AL. TABLE 1 PROTEINS OF SMR-LIKE FAMILY

Source of DNA, GenBank accession number

Number of amino acid residues in the polypeptide chain

Identity with Smr (W)

Amino acid overlap with Smr

104 110

33 42

l-102 2-107

115

41

5-100

83

39

4-65

83

50

5-47

30”

58

5-30

frd operon of P. vulgaris.

X06144 mvrC gene of E. coli, M62132 Plasmids R46, R388 of E. coli, X12868, Xl5024 T-DNA of A. tumefacients, M3.5007 Plasmid DGlOO of E. coli, x04555 sac (&)-II gene of P. aeruginosa, M29695

a The complete sequence of this ORF is not available.

which is located downstream of the sac (6’)-II gene, encodes a short sequenceof amino acid residues (line M29695, Fig. 4). This sequence is identical to the first transmembrane segment of three polypeptides listed above while the rest of this polypeptide is unknown because its DNA sequence is not yet available (Shaw et al., 1989). Experimental data presented in Fig. 4 demonstrate a number of conserved motifs. The unique sequencesEVI in the first transmembrane segment and KSSEGF(S/T)K(L/F) and in the first loop are conserved in six poly10

20

30

40

50

peptides. The second transmembrane segment possessesa conserved PS sequence, while a Pro residue is conserved in the second loop. The third transmembrane segment contains the motif YAXWXGXG which is generally conserved, whereas in the fourth transmembrane segment, only an LI sequence is conserved (Fig. 4). Summarizing data presented in Fig. 4, it seemsremarkable that the conserved amino acid residues include a Glu in the first transmembrane segment, a Pro in the second, and a Gly XC sequence in the third. 60

10

80

x04555

~K~~KGF~~G~F*~GKS*~*~K~~S-~"FG~~G~K~S :,:,:,,,,,:,:I , :::::: :,, / I:,:: :: I, ::,,I,, II: I, : :, I ::: ::,:, HNP~FsEGFTRLWPSVGTIFTG~G*K~~~sRSTPH :,::,::/::::/ :I:,,:,:I:/:,I::::I :, ::::, I, :,,,I::::/ III,::: I,:,,,,:: /IIll MP~ssEGFSKFIsK~MGHLPLNsG~N~GTsK ::::,::::/,:,:,,I:,::,:::::: :: : ::::,:,,I :lIl::::IIIIIII:I: II ::II::lI:IIIII ~~~SSGF~KIGYGIRFYFLSLYLKSIP"GYAYAVWSGLGWIGQKLDAWCIFVGHGLISPSWKS :::: , ,: IIIIII III IIIIII I ,I,,,,,,,,,,III,,,,IlIIIIIlIIIIIlIlI~**** KSS~FTKLAFSAWHNRLWH~GLSWllYAVWFGTRRRHNYSHCLVASGSKR MKY ::: ::I/II,III1I,IIII :,, I/:1 / : ,III,,I,IIIIIIIIIIIIIIIIIII:I/IIIlII GSEIHFCRCCLCSLYGTRRRHNYSHCLVASWAK MKGSSEGFAKSAWI

1.129695

“KPSSEGFTKL

X06144 "62732 smr x15024 M35007

,,,,,,,,,,,IIII,,II,IIIIIII:lI

90

100

:,::::,

I::

:,::::,I ::::I::

:,,: /,,::::

:

I

/

3::: :

l ****

FIG. 4. Alignment of Smr and the deduced sequencesof some small hydrophobic proteins. The polypeptide sequences were deduced from ORFs that were found in GenBank under the following accession numbers: MO6 144 (Cole, 1987), M62732 (Morimyo, 1991), Xl 5024 (Guerineau and Mullineaux, 1989), M35007 (Gheysen et al., 1990), X04555 (Cameron et aZ., 1986), M29695 (Shaw et al., 1989). They were aligned with the sequence of Smr (accessionnumber M33479). A vertical bar shows identical amino acid residues. A colon indicates similar residues. Underlined segments indicate potential membrane spanning segments, determined as described under Materials and Methods.

I

BACTERIAL

MULTIDRUG

Close homology between the Smr and the products of both mvrC and the ORF which precedesthe sulk gene suggeststhat these two proteins may also function as efflux systems. Polypeptides that form two transmembrane segments would seem too short to perform ion/drug antiport. The function of the membrane protein encoded by the ORF in P. vulgaris remains to be elucidated. In Smr, the conserved Glu 13 residue could serve as a binding site where lipophilic cations are exchanged for protons. Protons might enter the binding site of Smr if it were connected with the external medium via a proton-transfer pathway in the membrane. Formation of a such pathway would require a chain of oriented groups that are able to exchange protons. In fact, all such groups (except Glu 13 and Cys 42) in the transmembrane segmentsof Smr are hydroxylic amino acid residues. These residues may form the putative proton-transfer pathway in the Smr protein. On the other hand, Pro and Gly residues would permit bending of the Smr polypeptide chain which may be essential for the Smr interaction with lipophilic cations. Based on the data presented in this study and other work on multidrug resistance, it is possible to conclude that at least three different gene families (one eukaryotic and two prokaryotic) are responsible for the phenomenon of multidrug resistance. The eukaryotic family codes for ATP-driven pumps (e.g., Pglycoprotein). These proteins form 12 putative transmembrane segments (see Gottesman and Pastan, 1988). The two prokaryotic families code for proteins which presumably operate as lipophilic cation/H+ antiporters: (i) The bmr gene family encodes the Bmr and the QacA/QacB proteins in B. subtilis and S. aureus, respectively. These proteins share obvious homology to tetracycline efflux pumps and carbohydrate-H+ symporters and seemto form 12- 14 transmembrane segments (Neyfakh et al., 1991, Rouch et al., 1990). (ii) The smr gene family encodes small multidrug resistance proteins. In this family,

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only four (or fewer) transmembrane segments seem to be required. The small size of the Smr protein should facilitate determination of functionally important amino acid residues and experimental verification of the molecular mechanism of its function. ACKNOWLEDGMENTS We are grateful to Dr. A. A. Neyfakh of the University of Illinois at Chicago for insightful discussions. The authors also thank Dr. A. A. Neyfakh and Dr. R. A. Skurray of the University of Sidney, Australia, for sharing the sequencesof the bmr and the qacC/qacD genes prior to publication, respectively, and Dr. M. Midgley for providing plasmid pBgc and Dr. V. Butkus and Dr. R. Lubys for assistanceand advice with DNA sequencing procedures. This work was supported by NIH Grant GM135 11 and by NSF DCB 900 62353 for U.S.-USSR Cooperation from the National Science Foundation to E.B.G. and by 2007 from USSR Academy of Sciencesto L.G.

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