Molecular Microbiology (1995) 16(2), 333-343

A second ABC transporter is involved in oleandomycinresistance and its secretion by Streptomycesantibioticus

Carfos Olano, Ana Maria Rodriguez, Carmen Mendezand Jose A. Salas*Departamento de Biotogia Funcional e tnstitutoUniversitario de Biotecnologia (I.U.B.A.). Universidad deOviedo. 33006 Oviedo, Spain.

Summary

A 3.2 kb Sst\-Sph\ DNA fragment of Streptomycesantibioticus, an oleandomycin producer, conferringresistance to oleandomycin was sequenced and foundto contain an open reading frame of 1710 bp {oieB).Its deduced gene product (OteB) showed a highdegree of similarity with other proteins belonging tothe ABC-transporter superfamily including the geneproduct of another oleandomycin-resistance gene(OleC). The OleB protein contains two ATP-bindingdomains, each of approximately 200 amino acids inlength, and no hydrophobic transmembrane regions.Functional analysis of the oleB gene was carried outby deleting specific regions of the gene and assayingfor oleandomycin resistance. These experimentsshowed that either the first or the second half of thegene containing only one ATP-binding domain wassufficient to confer resistance to oleandomycin. Thegene oleB was expressed in Escherichia coli fusedto a maltose-binding protein (MBP) using the pMal-c2vector. The MBP-OleB hybrid protein was purifiedby affinity chromatography on an amylose resin andpolyclonal antibodies were raised against the fusionprotein. These were used to monitor the biosynthesisand physical location of OleB during growth. ByWestern anaiysis, the OleB protein was detectedboth in the soluble and in the membrane fraction andits synthesis paralleled oleandomycin biosynthesis.It was also shown that a Streptomyces albus strain,containing both a glycosyltransferase (OleD) able toinactivate oleandomycin and the OleB protein, wascapable of glycosylating oleandomycin and secretingthe inactive glycosylated molecule. It is proposed

Received 28 August. 1994; revised 11 December, 1994; accepted4 January, 1995. 'For correspondence. E-mail JASF@DWARF1.QUIMICA.UNIOVI.ES; Tel. (85) 103652; Fax (85) 103148.

that OleB constitutes the secretion system by whicholeandomycin or its inactive glycosylated form couldbe secreted by S. antibioticus.

Introduction

Three different mechanisms of self-resistance in macrolide-producing organisms have so far been described. Modifi-cation of the antibiotic target site by mono- or dimethyla-tion of a single adenine residue in the 23S rRNA hasbeen reported in several macrolide producers. Thismechanism has been found to confer self-resistance tothe producers of erythromycin (Skinner and Cundliffe,1982), tylosin (Zaiacain and Cundliffe. 1989; 1991) andcarbomycin (Zaiacain and Cundliffe, 1990). In contrast, inthe oleandomycin (OM) producer Streptomyces antibioti-cus. a ribosomal-mediated resistance mechanism hasnot been found (Fierro et at., 1987). However, this organ-ism possesses an intraceliular glycosyltransferase thatinactivates OM by glycosylation of a hydroxyi group inone of the sugars (desosamine) attached to the macro-lactone ring (Vilches et al.. 1992). The gene encodingthis glycosyltransferase has been cioned and sequenced(Hernandez ef at.. 1993) and the enzyme purified (L M.Quiros, C. Hernandez and J. A. Salas, submitted). Possi-bly this glycosyltransferase is part of the antibiotic biosyn-thetic pathway acting on an OM biosynthesis intermediate.Interestingly, a second enzyme that reactivates the inac-tive glycosylated OM (GS-Of\/l) (Vilches ef at.. 1992) hasalso been detected and purified from the culture super-natants of this organism (Quiros ef at.. 1994). The cellmembrane also plays a role in resistance. In severalmacroiide producers, impermeability of the cell membraneto the produced antibiotic has been found to participate inthe outside-inside impermeability (Fierro et at., 1987;1988). Furthermore, in recent years, several macrolide-resistance genes have been cloned and characterized atthe DNA-sequencing level. These have been found toencode proteins that have been suggested to participatein the export of macrolides through ATP-dependent pro-cesses thus conferring resistance to tytosin (ttrC\ Rostecketal., 1991), carbomycin {carA, Schoner etal., 1992) andspiramycin {srmB; Geistlich ef al., 1992; Schoner ef al.,1992),

1995 Blackwell Science Ltd

334 C. Otano. A. M. Rodriguez, C. Mendez and J. A. Salas

pOR501

0.5 kb

2 3 5 6 7 8 910 11 1213

ill

14 15 181718

Resistant

pOR502

pOR503

pOR504(pQBi 2)

pOH505

pOR506(pOLEB)

Sensitive

Sensitive

Resistant

Sensitive

Resistant

Fig. 1. Restriction map of pOR501 and localization of the resistance gene. pOR501 is equivalent to plJ702 containing a 4.4kb Sst\ fragmentthat confers resistance to oleandomycin (Rodn'guez etat. 1993). pOR502 was constructed by subcloning the Sst\~Sph) fragment (sites 1-8)into the Sst\-Sph\ sites of plJ2921 and rescued as a Bgt\\ fragmeni for subcloning into the BgH\ site of plJ702. pOR503 was constructed byfirst subcloning the 4.4kb Sst\ fragment (sites 1-18) into the same site of plJ2921 resulting in pOR5001 and then digesting Ihis constructionwith Bgtw and subcloning the Bgl\\-Sst\ fragment (siles 11-18) as a Bgt\\ fragment into the BglW site of pi702. pOR504 (also designated aspQB12) contains the BamH\-Bgl\\ fragment (sites 5-11) in the BgtW site of plJ702. pOR505 was constnjcted by subcloning the Sph\ fragment(siles 8-14) into the unique $ph\ site of plJ702. For construction of pOR506 (also designated pOLEB) the 8amH\-Stu\ fragment (sites 5-17)was initially subcioned into the BamH\~Sma\ sites of plJ2921 and rescued tor subcloning, as a Psfl-Ssfl fragment, into the Psfi-Ssf I sites ofplJ702. Only relevant sites are shown. The dashed boxes in pOR501 correspond to plJ702.

We have cloned three OM-resistance genes from S.antibioticus. one of which, oleC, was characterized andfound to encode an ATP-binding protein (Rodriguez etat., 1993) belonging to the superfamily of the ABC (ATP-binding cassette) transporters (Hyde ef al., 1990; Hig-gins. 1992). Here we report the characterization of a sec-ond OM-resistance gene (oleB) that also encodes aprotein simiiar to the ABC transporters. The oleB genehas been expressed as a fusion protein in Escherichiacoti and its functionai properties analysed by gene dele-tion. We also present experimental evidence that thisABC transporter can recognize and participate in thesecretion of inactive GS-OM.

Results

Sequencing of the oieB gene

In a previous paper, we reported the cioning and expres-sion in both Streptomyces tividans and Streptomyces atbusof three OM-resistance genes {oleA. oleB and oleC) fromS. antibioticus (Rodriguez et at., 1993). The originai oleBclone (pOR500) contained an insert of about 14 kb withtwo internai Ssri sites. By subcloning the Sst\ fragmentsof pOR500 in piJ702. the resistance determinant wasreduced to a 4.4 kb Sst\ fragment (clone designatedpOR501). The restriction map of this insert is shown inFig. 1. Subcloning of different fragments of this cione

showed that the smallest DNA fragment conferring resis-tance was the 1.4 kb BamH\-Bgt\\ fragment (sites 5-11in Fig. 1), However, ciones containing the 3.1 kb BamH\-Stu\ fragment (sites 5-17 in Fig. 1), which include theBamH\-Bgl\\ fragment, grew more rapidly and wereslightly more resistant to OM, The BamH\-Bgl\\ fragmentconferred resistance when subcioned in both orienta-tions, thus indicating that the resistance gene was beingexpressed from its own promoter. A 3.2 kb Sst\-Sph\ frag-ment from pOR501 (sites 1-15 in Fig. 1) was sequenced,and the sequence was deposited in the GenBank data-base under the accession number L36601. Anaiysis of thenucieotide sequence for coding regions using the CODON-pREFERENce program (Devereux ef ai, 1984) reveaied theexistence of an open reading frame (oleB) and a non-coding region (1420bp in length) upstream of tiie oleBgene. The oleB gene (1710bp) starts with an ATG codon(nt 1421-1423) which is preceded by a weak potentialribosomai binding site and ends in a TGA stop codon (nt3128-3130). It could code for a polypeptide of 570amino acids and an estimated mass of 61 747 Da. Noapparent coding region was detected in the 1420bp pre-ceding OleB. A search in databases (GenBank, release84.0; EMBLasof 1 October1994;andSwissprotein,reiease29,0) using the deduced amino acid sequence of oleBreveaied significant similarity to proteins belonging to theABC-transporter superfamily. The percentages of similar/identical amino acids with several ABC-transporters

1995 Blackwall Sdence Ltd, Molecular Microbiology, 16, 333-343

Walker A Motif

G X X G X G K

HB B

ABC transporters in Streptomyces 335

Walker B Motif

hhhh DE PT

HLL L L D E f r N H LL L L L DE PT N —L L L L C ' S P T M H L .L L LL DE PT M^jL

L L L I. D E F' T '•( H I Ml

G A I|A Lii A

IA L/' A

IV IR A

IA L111 I

Fig. 2. Alignment of the regions encoding potential ATP-binding domains of oteB with those of homologous ABC transporters invotved inmacroiide resistance. The amino acid region corresponding to the Walker A and B motifs (Walker et at.. 1982) and to loop 3 (Hyde efa/., 1990)are aligned and the amino acids are indicated. carA. carbomycin-resistance gene from S, (rter/noto/erans (Schoner et at. 1992); srmB,spiramycin-resistance gene from S. ambofaderis (Geistlich et al.. 1992); tIrC. tylosin-resistanoe gene from S. fradiae (Rosteok et at., 1991);oteC, oleandomycin-resistance gene from S. antibioticus (Rodriguez et at., 1993),

involved in resistance to macrolides in producer organismswere 68.5%/50.5% with carA from Streptomyces thermo-tolerans (Schoner et at., 1992), 68.5%/50.9% with srmBfrom Streptomyces ambofaciens {Geisflich et at.. 1992),69.5%/53.6% with tIrC from Streptomyces fradiae (Ros-teck ef at.. 1991) and 51.2%/29.3% with oteC from S.antibioticus (Rodriguez et at., 1993). The OleB alsoshowed similarity to other resistance genes from non-producer bacteria: 51.1 %/26.9% with msrA from Staphylo-coccus epidermidis (Ross etai, 1990), 44.2%/23.3% withtmrB from Bacillus subtiiis (Noda et ai, 1992) and 54.0%/29.0% with vga from Staphylococcus aureus (Allignet etat.. 1992). The oleB product contains a duplicated ATP-binding domain with two well-conserved amino acid regionsaround the so-called Walker A and B motifs (Walker etaL.

1982) characteristic of the ABC transporters (Fig. 2), Inaddition, the colinearity with the ABC transporters fromother macroiide producers containing two ATP-bindingdomains extended along the entire length of the polypep-tide (Fig. 3).

Construction of ptasmids containing different domainsof OleB

In order to characterize the importance of the differentWali<er A and B motifs of the oleB product in OM resis-tance, various gene constructions were generated andassayed for OM resistance (henceforth, we will designatethe two Walker A motifs as Al and A3 and the two WalkerB motifs as B2 and B4). A 3.1 kb BamH\-Stu\ fragment

1400

200

400

200

Fig. 3. C0MPAB6/DDTPL0T Comparison of theOteB product with other ABC transporters frommacroiide producers. carA. carbomycin-resislance gene trom S. thermottioterans(Schbner et al.. 1992); srmB, spiramycin-resistance gene from S. amtxrfaciens(Geistlich etat.. 1992); ttrC. tylosin-resistancegene Irom S. fradiae (Rosteck et al.. 1991);oleC. oleandomycin-resistance gene (rom S.antibioticus (Rodriguez el at.. 1993). Thewindow used was 40 with a stringency of 20.Numbers on the axis ot the graph indicateamino acids from the N-terminus of theprotein.

IDEw

400

200

o® 200O

200 400

OleB

1995 Blackwell Science Ltd. Motecular Microbiology, 16, 333-343

200 400

OleB

336 C. Otano, A. M. Rodriguez, C. Mendez and J. A. Satas

ASgI 1

N Bg Ss1

StI RESISTANCE

OleB I Al Bg A3 B4>

QB1234 (olaB) r~ -——--•• , - ; ;i ^. + +

Q B 2 3 a I —I t •••, sL,, , , : ;^ ' • •• „ " " ^ 1 ±

QB134 I: . 1 T::':. :: Z\ + +QB34 r~' ~[ : •" I ^n + +

QB14 L̂ I 1 -.. . — JQB12 1 H + +

Fig. 4. Oleandomycin resistance ot the clones in S. atbus containing different deletions of oleB. The different constructions were generated asdescribed in the text and introduced into S. atbus. Assays of oleandomycin resistance were carried out as described in the Experimentalprocedures. The dotted lines indicate the regions that were deleted from the gene in each constnjction. +++, growth after 72 h in platescontaining 100^lgml ' oleandomycin; ++, growth after 72 h in plates containing between 50 and 75 tig ml ' ; +, growth after 72 h in platescontaining between 30 and 40 ng mi ^ - , no growth after 72 h. S. atbus containing only the vector plJ702 was used as a control and did notgrow on plates containing 20ngml ' oleandomycin. A, Age\, B. SamHI; Bg, BglW: Bs. SssHII; N, Notl. Sg, SgrAl; St, Stu\.

(sites 5-17 in Fig. 1) that contains the entire oleB genewas subcioned into the BamH\-Sma\ sites of plJ2921resulting in piasmid pOLEB. This construction was linear-ized with SssHII, blunt ended wifh fhe Klenow fragmenfof DNA poiymerase i and digested with SamHl to releasethe 1.8 kb SamHt-SssHII fragment. This fragment wasfhen subcioned into the SamHI-Smal sites of pUCi9resulting in pOLEB'. This plasmid, which lacks the B4motif, was the starting construction for fhe deletion experi-ments. The different Walker A and B motifs were thendeleted by double digestion of pOLEB" using the followingresfricfion sites thaf flanked fhe differenf motifs which wereto be deleted: (i) Age\ plus SgrAl, religated to produce Aldeletion plasmid pQB23; (Ii) SprAI plus Not\, both bluntended with Klenow polymerase and religated to produceB2 deletion plasmid pQB13; (iii) Agel plus Not\, bofhblunt ended with Klenow and religated to produce pQB3,deleted in Al and B2. Each of these constructions wasthen digested with BgtW plus Sst\ (A3 is deleted with thisdouble-digestion) then ligated with a Bgl\\-Sst\ fragmentcontaining the A3 and B4 motifs from pOLEB. The result-ing plasmids were designated pQB234. pQB134 andpQB34, respectively. To create the fourth construction,which was designed to delete the B2 and A3 motifs,pOLEB" was digested with SgrAl, blunt-ended with Kle-now and again digested with Sst\. Finally, a SssHII(blunt ended)-Ssfl fragment from pOLEB (containing theB4 motif) was ligated with this vector fragment, generatingpQB14. The fifth construction, deleted in the A3 and B4motifs, was already achieved in plasmid pOR504 (alsonamed pBQ12, for convenience) (Fig. 1).

In al! constructions, the final step was the subcloningof the different deleted oteB genes into the Pst\-Sst\sites of the Streptomyces plasmid vector plJ702 usingthe Pst\ and Sst\ sites of the polylinker of pUC19. Thesefinal constructions were then introduced into S. atbus G(R M ) protoplasts and the transformants selected forthiostrepton resistance. These constructions were then

analysed for OM resistance (Fig. 4). Some deletionsmore drastically affected the function of the ATP-bindingprotein. Thus, the simultaneous deletion of Walker motifsB2 and A3 (QB14) completely abolished resistancewhereas constructions lacking Walker Al motif (QB234)only gave a low level of resistance. In the firat case(QB14), it is worthy of mention that this deletion involvesa number of amino acids from the interdomain regionfhat could be affecting the functionality of the protein. Incontrast, clones containing fhe constructions QB134,QB34 and QB12 grew in the presence of OM but theirresistance to the antibiotic was lower fhan the originalclone (pOR506).

Heteroiogous expression of oleB in E. coli andgeneration of polyclonal antibodies

The OleB product was expressed in E coti as a fusion pro-tein to a MBP encoded by the ma/Egene using the expres-sion vector pMal-c2 (Guan etai. 1987). This was achievedby creating a gene construction (pMALOR32) where theOleB gene was fused in frame to the 3' end of the malEgene. The starting construction was plasmid pALOR26E(a pALTER derivative), which contains the first half of theOteB gene with an EcoRV site at the initiation codon ofthe gene introduced by site-directed mutagenesis. A 3 kbSg/ll-SamHI fragment Isolated from cosAB35 (Swan e^ai. 1994), containing the second half of the oleB gene,was subcioned into the Sg/ll-SamHI sites of pALOR26Egenerating plasmid pAL0R31. The 4kb £coRV-6amHIfragment from fhis construction was subcioned into theAsp700-BamH\ sites of pMal-c2 where the entire oleBgene was fused in frame to the 3' end of the matE gene(pMALOR32). After induction, by IPTG, of clones contain-ing this construction, the expression of a 103 kDa OleB(61 kDa)-MBP (42 kDa) fusion protein was observedin Coomassie-blue-stained gels (Fig. 5C). The fusion pro-tein was purified by affinity chromatography on an amylose

1995 Blackwell Science Ltd, Motecutar Microbiology, 16, 333-343

ABC transporters in Streptomyces 337

A B C D

20.1

Fig. 5. Expression of the MBP-OleB fusion protein in £. cott.PMALOR32 was used as a source of MBP-OleB fusion protein.The gel was stained with Coomassie blue- Lane A, molecularweight markers; lane B, uninduced celi-free extract; lane C, inducedcell-free extract; lane D, elution from the amylose resin column,

resin (Fig. 5D) and injected inte New Zealand rabbits toraise polycional antibodies.

Time course for the biosynthesis of Of\/! and OleBand the intracellular location of the OleB protein

During growth of S. antibioticus in Trypticase-Soy broth

0 4 • I I IB 20 34 Zl 33 IS 40 44 4B S2 M SO

TME (hauti)

(TSB) medium, OM was detected in the culture superna-tant after 24 h of growth (Fig. 6A). To monitor fhe biosynth-esis of the OieB protein and its location in the mycellium,ceil-free extracts of S. antibioticus were obtained at differ-ent times during the growth cycle and fractionated intomembrane and soiubie fractions. Profeins in these frac-tions were solubilized and eiectrophoresed by SDS-PAGE. The gel was fhen transferred fo a nylon membraneby Western blotting and assayed using the anti-OleBserum. A single band giving a positive reaction with theantiserum was detected both in the soiubie (Fig. 6B) andin the membrane fraction (Fig. 6C), and its mobility wasequivalent to thaf corresponding to the estimated moiecu-iar weight of OleB. Its defection during fhe growth cyclecoincided with the time at which OM was detected in fheculture supematants.

Glycosylated oteandomycin can be secreted by theOleB protein

An experiment was designed to prove whether or not GS-OM can be secreted by an ABC transporter comprising fheOleB protein. A S. albus strain (poieB/D) was generatedthat contains the oleD gene, which encodes a S. antibioti-cus glycosyIfransferase fhat inactivates OM by glyco-sylation (Viiches etai, 1992; Hernandez etai, 1993), andthe OteB gene. These genes were harboured in two differ-ent and compatibie plasmids {oteD in cosmid pKC505, andOleB in plasmid plJ702). The rationale behind this experi-ment was as foliows: incubation of fhis cione in fhe pre-sence of OM would allow the incorporation of fhe

Fig. 6. Biosynthesis of oteandomycin andlocation of the OleB protein during growth nfS. arMibioticus.A. S. antibioticus was grown in TSB mediumand the presence of oleandomycin in theCulture supernatant determined at differenttime intervals by bioassay against M. luteus.B. C. Location ol the OleB protein. At differenttimes of growth in TSB medium (Indicated inthe figure), mycelia of S. antibioticus weredisrupted by ultrasonication and fractionated

I into the soluble fraction (B) and the membranefraction (C). Profeins were solubilized andseparated by SDS-PAGE and analysed byWestern blotting against rabbit anti-OleBsenjm.

B12 16 20 24 32 40 48

94 kD«67 kDa

43 kDa30 kDa

12 16 20 24 32 40 48

C 1995 Btackweil Science Ltd, Molecular Microbiology, 16, 333-343

338 C. Olano, A. M. Rodriguez, C. Mendez and J. A. Salas

-0.110

Tlma (mln)

Fig. 7. iHPLC analysis of chloroform-extracted culture supematants ot S. albus ciones- After incubating the clones with 70 tig ml ^oleandomycin, (he cutture supematants were extracted with chtorofom and analysed by HPLC. Peaks corresponding to the mobilities of OMand GS-OM are indicated by arrows. The peak corresponding to GS-OM was recovered, lyophilized and incubated with purified glycosidase{Quiros ef al.. 1994) as described in the Experimental procedures.A. HPLC analysis of S. albus culture supernatant containing piasmid vectors (piJ702 and pKC505).B. HPLC anaiysis ot S. albus poleB/D cione culture supernatant.C. HPLC anaiysis of the GS-OM peak before incubation with glycosidase.D. HPLC analysis ot the GS-OM after incubation with the giycosidase.E. Bioassay against /W. tuteus of the QS-OM (ieft) and OM (right) peaks coliected from the HPLC analysis shown In (D).

anfibiotic iiito fhe S. atbus mycellium and its infracellularglycosylafion by the OleD profein; if fhe OleB profein wasable fo recogiiize the GS-OM and facilitate its secretion,the gtycosylated form of the antibiotic would be detectedIn the culture supernatant. To test this hypothesis, theclone was grown on TSB medium in the presence ofapramycin (25).igml ') and thiostrepton (5|.igml ') tomaintain the derivatives of pKC505 and plJ702, respec-tively. After 48h incubation at 30 C, the mycelium wascollected by cenfrifugation, washed twice with 10.3%sucrose and resuspended in minimal medium (Vilches etai, 1990) containing 0.1% (NH4)2S04 and 1% glucose.After addition of 70ngmr ^ OM. fhe mycellium was incu-bated for seven additional hours. Then, the culture super-natant was recovered after centrifugafion and both OM andGS-OM were extracted with chloroform and anaiysedby high-performance liquid chromatography (HPLC).

Several control experiments were run in parallel, withclones containing either both vectors or the oteB or theOteD genes alone. Under the HPLC conditions employed,OM and GS-OM can easily be separated wifh retentiontimes of approximately 8.7 and 3.7 min, respectively. Afterincubation of the S. albus po!eB/D clone with OM, most ofthe OM initially added was recovered as GS-OM(Fig. 7B). In clones containing the oteD gene but not theOteB gene, the level of GS-OM extracellularly detectedwas only 10% of that found in the presence of both oleBand oteD genes. Similarly, in clones containing the oleBgene, but not the oteD gene, no extracellular GS-OMwas observed (data not shown). Clear evidence showingthe identity between the material eluted from the HPLC(3.7 min retention time) and GS-OM arose from reactiva-tion assays. The material obtained from the 3.7 min peai<(Fig. 7B) was analysed for its mobility in HPLC before

1995 Blackwell Science Ltd. Motecular Microbiology. 16. 333-343

ABC transporters in Streptomyces 339

(Fig. 7C) and after (Fig. 7D) incubation with the purifiedglycosidase that has been found to convert inactive GS-OM into active OM. The two peaks observed after the gly-cosidase reaction (Fig. 7D) (corresponding to GS-OMand OM) were collected and their antibiotic activity wasassayed against Microeoecus tuteusby bioassay (Fig. 7E).The conclusion drawn was that the inactive GS-OMwas converted by the purified glycosidase into activeOM. All these experiments clearly demonstrate that GS-OM can be secreted by a transporter that contains theOleB protein.

Discussion

Several macrolide-resistance genes from macrolide-producer organisms have been cloned and characterizedin recent years. Some of these have been shown bysequence analysis to encode ATP-binding proteins simi-lar to those of the widely extended ABC-transportersuperfamily (Higgins etai, 1986). In S. antibioticus, atleast two of the three OM-resistance genes cloned, oteC(Rodriguez ef at.. 1993) and oteB (this paper), couldencode ATP-binding proteins, although ATP has not yetbeen shown binding to these proteins. However, there isa major difference between these gene products, i.e. thenumber of putative ATP-binding domains: one and twofor OleC and OleB, respectively. The existent homologybetween the two halves of OleB suggests that it couldhave evolved by tandem duplication of an ancestral ATP-binding gene. Both halves are funcfionally acfive. as canbe deduced from the in-frame deletion experiments ofthe OleB gene: the presence of either the first (0B12) orthe second (OB34) half of the gene is sufficient to conferresistance to oleandomycin.

In most of the ABC transporters studied so far, the ATP-binding domain is either fused to a hydrophobic domain ina singie polypeptide or it interacts with hydrophobic mem-brane proteins for transport to occur (Higgins, 1992). In thecase of OleC, o/eC-ORF5, a gene encoding a hydropho-bic protein that contains six putative membrane-spanningsegments, is present immediately downstream of theOteC gene (Rodriguez et ai, 1993). This gene organiza-tion therefore resembles that represented by the oligopep-tide permease (Hiles et at., 1987) and the maltose- andhistidine-transport systems (Higgins etai, 1986) in Satmo-nella typhimurium. The situation, however, is not as clearfor the OleB protein, because no apparent membrane-protein encoding gene has been found in either the 1.4 kbfragment upstream of the oleB gene or downstream ofOteB. So far, this kind of organization (lacking a membrane-protein gene) has only been found in antibiotic-resistancegenes encoding ABC transporters and could represent anew subgroup of ABC transporters. Possibly, in order tocomplete the transporter system in S. antibioticus, the

OleB protein could use the gene product of o/eC-ORF5,thus resembling the structure of the ribose import systemof Escherichia coli (Bell et ai, 1986). Alternatively,another membrane protein capable of interacting withOleB could form part of the export system.

The oteB gene is probably part of the OM biosyntheticpathway as it is located approximately 8 kb downstreamof the C-terminus of a large open reading frame (ORF)encoding the third subunit of the OM polyketide synthase(Swan et ai. 1994). In addition, the fact that OleB synth-esis parallels that of OM indicates that expression of theOleB gene and OM biosynthesis are co-ordinately regu-lated. The OleB protein has been found both in the solubleand in the membrane fraction of S. antibioticus mycelium.This ambiguity can be explained by assuming thaf ifs nor-mal physiological location would be the cytoplasmic faceof the membrane interacting with the membrane com-ponent of the export system. Its presence in the solublefraction could be due in part to newly synthesized OleBprotein which had not yet been incorporated into the mem-brane and also to protein whose interaction with the mem-brane protein has been lost during cell disruption andfractionation.

ABC transporters have been reported for a wide (andrapidly increasing) number of compounds that are com-posed of many different molecular structures (for a reviewsee Higgins, 1992). However, the mechanism determiningsubstrate recognition and the component(s) of the trans-port system responsible for this specificity are not wellknown. For Gram-negative bacterial uptake systems,there is some evidence that both the periplasmic-bindingproteins and the membrane-associated components insubstrate specificity are involved (Higgins, 1992). How-ever, in most of the systems studied there is no evidencein favour of a role of the ATP-binding proteins as a deter-minant for substrate specificity. In fact, experiments withchimeric mdr genes argue against this role for the ATP-binding domains (Buschman and Gross. 1991). In the caseof the OieB and OleC transporters, the following experi-mental evidence suggests that these two ATP-binding pro-teins could be responsible for substrate recognition: (i) theintroduction into S, atbus or S. lividans of a DNA fragmentcontaining only the oleB gene or a truncated gene compris-ing the first ATP-binding domain, is sufficient to conferresistance to OM; and (ii) a similar situation was foundwhen the oteC gene was subcioned in the absence ofthe o/eC-ORF5 membrane-protein gene (Rodriguez etai, 1993). In addition, there have been reports of othermacrolide-resistance genes conferring resistance to macro-iide antibiotics in the absence of additional membrane-protein genes, This is the case with msrA (Ross et ai,1990), ttrC (Rosteck et al.. 1991) and srmB (Schoner etai, 1992); a particular case could be represented bycar,4 which seems to require additional upstream sequences

C; 1995 Blackwell Science Ltd, Molecular Microbiology. 16, 333-343

340 C. Otano. A. M. Rodriguez, C. Mendez and J. A. Salas

(Schoner et at.. 1992). Among the different macrolides,OteB and oleC confer only high levels of resistance toOM and no resistance to other macrolides. To explainthese results, one should assume either that substratespecificity resides in the ATP-binding protein or thatthere might be an integral membrane protein responsiblefor substrate specificity in the two different hosts used forsubcloning (S. albus and S lividans). Obviously, the latteris difficult to assume on the basis of the high specificityof the transport system for OM. Perhaps the macroiideresistance systems could constitute a new class of ABCtransporters, at least with respect to the substrate recogni-tion aspect.

What then could be the natural substrate of the OMtransporters? OM is an inhibitor of protein synthesis whichinteracts with the 50S ribosomal subunit. In contrast toSaccharopolyspora erythraea (an erythromycin producer)which has ribosomes that are constitutively resistant toerythromycin (Skinner and Cundliffe. 1982), S. antibioticuslacks this resistance mechanism which is mediated bytarget-site modification (Fierro etai. 1987). Therefore, theorganism must avoid the presence of active intracellularOM, which can be achieved by glycosylating a precursor inOM biosynthesis using a glycosyltransferase enzyme(Vitches et ai. 1992; Hernandez er ai, 1993). Conse-quently, the final intracellular product of the pathwaycould be the inactive GS-OM. The oleB and oteC trans-porters could be responsible for secrefion of this inactivemolecule which would then be reactivated by an extracel-lular enzyme capable of removing the glucose moiety ofthe inactive GS-OM, resulting in active OM (Vilches etaL, 1992; Ouiros et ai, 1994). Evidence in favour of thishypothesis is provided in this paper: the OleB protein isable to recognize and to secrete GS-OM.

Experimental procedures

Bacteriat strains, plasmids and phages

S. antibioticus ATCC 11891, an OM producer, was usedthroughout this study. S. tividans TK21 (Hopwood et ai,1985) and S. altjus J1074 {ilv-1. sat-2, R M ) (Chater andWilde, 1980) were used as strepfomycete cloning hosts. E.CO//TGI recO1504::Tn5 (Kolodner et ai, 1985) was used ashost for subcloning. High-copy-number plJ702 {Katz et ai.1984) was kindly provided by Professor D. A. Hopwood.plJ2921, a pUCia derivative (Janssen and Bibb, 1993), wasprovided by G. R. Janssen. pMal-c2 vector was obtainedfrom Bioiabs. Culture conditions for Streptomyces strainswere as described (Hopwood etal., 1985) and for E. coti wereas described by Sambrook ef ai (1989), When plasmid-containing clones were grown, the medium was supplementedwith the appropiate antibiotics: 5 or 50 f,ig ml" ̂ thiostrepton forliquid or solid cultures, respectively; and 25 (,tg ml" ̂ ampicillin.The M13-derivative phages M13mp18 and M13mp19(Yanisch-Perron et at., 1985) were used as vectors for DNAsequencing.

DNA maniputations

Plasmid DNA preparations, restriction endonuclease diges-tions, alkaline phosphatase treatments, Iigations and otherDNA manipulations were performed according to standard pro-cedures for E. coli (Sambrook et al.. 1989) and Streptomyces(Hopwood er at., 1985). Preparation of Streptomyces profo-plasts, transformation and selection of transformants werecarried out as described by Hopwood er ai (1985), wifh theexception of S. atbus profoplasts where the organism wasgrown in TSB medium supplemented with 1% glycine.

DNA sequencing

DNA sequencing was performed on single-stranded templatesderived from differenf clones in either M13mp18 or M13mp19,using the dideoxynudeotide chain-termination method (San-ger et ai. 1977) with [ct-̂ ^Sl-dATP (1200Cimmol \ Amer-sham) and modified T7 DNA polymerase (Sequenase version2,0; US Biochemicals). In some clones, and in order to over-come band-compression artefacts, 7-deaza-dGTP was usedinstead of dGTP (Mizusawa er a/,, 1986). Single-strandedDNA was prepared by polyethylene-glycol precipitation asdescribed (Sambrook ef ai 1989). Both DNA strands weresequenced with primers supplied in the Sequenase kit orwith internal oligonucleotide primers (17-mer), In somecases, in order to check the constructions (gene fusions andin-frame deletions) or fo confirm the introduction of a muta-tion, sequencing was performed on double-stranded DNA(Sambrook er ai. 1989) using primers derived from themalE or the T7 promoters where appropiate. Computer-aided database searching and sequence analyses werecarried out using the University of Wisconsin Genetics Com-puter Group programs package (UWGCG; Devereux et ai,1984).

Site-directed mutagenesis

An EcoRV restriction site was introduced, by site-directedmutagenesis, into the initiation codon of the oteB gene toallow subcloning of the gene in the fusion vector. For muta-genesis we used the 'Altered sites in vitro mutagenesis sys-tem' kit (Promega), A previously cloned 1.5 kb Sma\ fragment(sites 6-12 in Fig. 1) in M13mp18 containing the first half ofOteB gene was subcioned in the vector pALTER (Promega)as an EcoRI-H/ndlll fragment using these restriction sitesfrom the polylinker (pALOR26). A 25-mer mutagenic oligo-nucleofide (5-TGGCGGATCAGATATCCAGAACGCA-3) con-taining an EcoRV site (underlined) was synthesized and themutation introduced into the fragment using the Promegamutagenesis kit. The presence of the new EcoRV site in theresulting construction (pALOR26E) was confirmed by restric-tion digestions and DNA sequencing using the T7 promoterprimer.

Expression and purification of the fusion protein

An overnight culture of clone pMALOR32 was used to inocu-late fresh 2'< TY medium consisfing of (gl ') tryptone (16),yeast extract (10), and sodium chloride (S), and containing

1995 Blackwell Science Ltd, Motecular Microbiology. 16, 333-343

ABC transporters in Streptomyces 341

100 \ig ml ^ ampiciilin, and the culture was incubated at 37'Cuntil the ODeoo was approximateiy 0.4. iPTG was then addedto a final concentration of 0.3 mM to induce the expression ofthe tac promoter present in the vector. After an additional 2 h ofincubation, the cells were harvested by centrifugation at4000 X g for 15 min and resuspended in lysis buffer (20 mMTris-HCI pH7.4, 0.2 M NaCI, 10mM 2-mercaptoethano!.1 mM EDTA and 1 mM sodium azide). The cells were dis-rupted by ultrasonication (10 pulses of 30s each, with inter-mittent cooling) in a 150-W Ultrasonic disintegrator, andcentrifuged at 14000x g for 20 min. The supernatant wasloaded onto an amylose-resin column at a low flow rate(7mlh"'). The column was then washed with 10 volumes ofthe same buffer and with a linear gradient of NaCi from 0.2-1 M. The protein fusion was then eluted with the same buffercontaining 10mM maltose.

Polyacrylamide get electrophoresis andimmunobtotting of proteins

Protein analysis was carried out by polyacrylamide gel elec-trophoresis in the presence of SDS (Laemmli, 1970). Westernblotting was carried out as described (Burnette, 1981) but thefitters were developed with alkaline phosphatase (Sambrooketai, 1989).

determined by bioassay against M. tuteus as described byVilches etai (1990). in other cases, OM and GS-OM wereextracted from the culture supematants with two volumes ofchloroform after adjusting the pH to 9. 8 with KOH. After rotaryvapour dessication, the residue was suspended in a smallvolume of methanol and the products analysed by HPLCusing a nBondapak Ci8 column with an isocratic gradient com-posed of 30% acetonitrile and 70% 50 mM phosphate buffer{pH6.8) at a flow rate of 2mimin" \ Detection was carriedout at 260 nm.

Reactivation of OM

Inactive GS-OM was converted to active OM by incubationwith purified glycosidase as previously described (Ouiros etai, 1994). The products were analysed finally by HPLC andbioassay.

Minimal inhibitory concentration

Susceptibility to OM by the different clones was tested bydetermining the minimal inhibitory concentration by replicaplating on agar plates containing different concentrations ofOM.

Generation of potyctonat antitiodies

The purified fusion protein (MBP-OleB) was used to immun-ize New Zealand rabbits (Biocentre, Spain) by subcutaneousinjection of the fusion protein in Freund's complete adjuvant.These were boosted intramuscularly with Ihe same dose ofantigen in Freund's incomplete adjuvant two weeks later,and bled 15days after the last immunization.

The animal work was carried out according to the rulesapproved by the Spanish Ministry of Health.

Cellular fractionation

To determine the location of the OleB protein in the myceliumand to follow its biosynthesis during the growth cycle, S. anti-bioticus was grown on TSB medium at 30 C with orbital shak-ing. At different times during growth, samples were cen-trifuged and washed once with 2 M NaCI and then twice with50 mM Tris-HCI (pH7,5) containing 2mM EDTA and 2mMdithiothreitol. The mycelium was disrupted by ultrasonication(10 pulses of 30 s each with intermittent cooling) in a 150-WUltrasonic disintegrator. The extract was then centrifuged at25000 y. g for 30 min and the supernatant again centrifugedat 100000 X p for 1 h. The resulting supernatant was desig-nated the 'soluble fraction'. The pellet containing the cellmembranes was resuspended in a small volume of theabove-mentioned buffer, washed again by ultracentrifugationand after resuspension in the same buffer was designatedthe membrane fraction'.

Extraction of OM and GS-OM and their anatysis

In some experiments, OM in the culture supematants was

© 1995 Blackwell Science Ltd, Molecular Microbiology. 16, 333-343

Ac k nowledgements

Work in this project was supported by grants from the CICYT(PB91-0336) and from the European Union (BIOTECH Pro-grame. Project PL930145). CO, and A.M.R. were each recipi-ents of a pre-doctoral grant from the FICYT, Asturias. We wishto thank Juan R. Toyos and Alfredo F. Brana for their kind helpin the preparation of the antibodies and in the HPLC analysis,respectively. We are also very grateful to Luis M. Ouiros forproviding help with the protein purification procedures andfor providing a sample of purified glycosidase. We are alsograteful to Mr S. J. Lucania (Squibb Co.) for the kind gift ofthiostrepton.

References

Allignet, J . . Londe, V. , and Sohl, N.EI. (1992) Sequence of astaphylococcal plasmid gene vga. encoding a putativeATP-binding protein involved in resistance to virginiamycinA-like antibiotics. Gene 117: 45-51.

Bell, A.W., Buckel, S.D., Groarke, J.M-, Hope, J.N., Kingsley,D.H., and Hermodson, M.A. (1986) The nucleotide sequenceof the rbsD, rbsA and rbsC genes of Escherichia coli. JBiol Chem 261: 7652-7658.

Burnette, W.M. (1981) Western-blotting: electrophoretictransfer of proteins from sodium dodecyl sulphate-poiy-acrylamide gels to unmodified nitrocelulose and radio-graphic detection with radioiodinated protein A. AnalBiochem^^2•. 195-203.

Buschman, E., and Gross, P. (1991) Functional analysis ofchimeric genes obtained by exchanging homologous domainsof the mouse mdr1 and mdr2 genes. Mol Celt Biol 11: 595-603.

Chater, K.F., and Wilde, L.C. (1980) Streptomyces albus G

342 C. Olano, A. M. Rodriguez, C. Mendez and J. A. Salas

mutants defective in the Sa/GI restriction-modificationsystem. J Gen Microbiot 116: 323-334.

Devereux, J., Haeberli, P., and Smithies, O. (1984) Acomprehensive set of sequence anatysis programs forthe VAX. Nuct Acids Res 12: 387-395.

Fierro, J.F., Hardisson, C., and Salas, J.A. (1987) Resistanceto oleandomycin in Streptomyces antibioticus, the producerorganism. J Gen Microbiot 133: 1931-1939.

Fierro, J.F., Hardisson, C. , and Salas, J.A. (1988) Involve-ment of cell impermeability in resistance to macrolidesin some producer streptomycetes. J Antibiot 41: 142-144.

Geistlich, M., Losick, R., Turner. J.R., and Rao, NR. (1992)Characterization of a novel regulatorv gene governing theexpression of a polyketide synthase gene in Streptomycesambofaciens. Mot Microbiot 6: 2019-2029.

Guan. C, Li, P., Riggs, P.D., and Inouye, H. (1987) Vectorsthat facilitate the expression and purification of foreignpeptides in Escherichia ooli by fusion to maltose-bindingprotein. Gene 67: 21-30.

Hernandez, C, Olano, C, Mendez, C, and Salas, J.A. (1993)Characterization of a Streptomyces antibioticus genecluster encoding a glycosyltransferase involved in olean-domycin inactivation. Gene 134: 139-140.

Higgins, C.F. (1992) ABC transporters; from microorganismsto man. Annu Rev Cett Bioehem 8: 67-113.

Higgins, C.F., Hiles, I.D.. Salmond, G.P.C.. Gill, D.R.,Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel,S.D., Bell, A.W., and Hermodson, M.A. (1986) A family ofrelated ATP-binding subunits coupled to many distinctbiological processes in bacteria. Nature 323: 448-450.

Htles, I.D., Gallagher, M.P,, Jamieson, D., and Higgins, C.F.(1987) Moiecular characterization of the oligopeptidepermease of Satmonelta typhimurium. J Mot Biol 195:125-142.

Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Bnjton,C.J,, Kieser, H.M., Lydiate, D.J., Smith, C.P.,,Ward, J.M.,and Schrempf, H. (1985) Genetic Manipulation of Strepto-myces: A Laboratory Manuat. Norwich: John InnesInstitute.

Hyde, S.C, Elmsley, P., Hartshorn, M., Mimmack, MM.,Gileadi, U., Pearce, S.R., Gallagher, M.P., Hubbard, R.,and Higgins, C.F. (1990) Structural model of ATP-bindingproteins associated with cystic fibrosis, multi-drug resis-tance and bacterial transport. Nature 346: 362-365.

Janssen, G.R., and Bibb, M.J. (1993) Derivatives of pUC18that have BgtW sites flanking a modified multiple cloningsite and that retain the ability to identify recombinant clonesby visual screening of Escherichia coli colonies. Gene 124:133-134.

Katz, E., Thompson, CJ,, and Hopwood, D.A. (1984) Cioningand expression of the tyrosinase gene from Streptomycesantibioticus in Streptomyces lividans. J Gen Microbiof\29:2703-2714.

Kolodner, R., Fishel, R.A., and Howard, M. (1985) Geneticrecombination of bacterial plasmid DNA: effect of RecFpathway mutations on plasmid recombination in Escher-ichia coti J Bacteriot 163: 1060-1066,

Laemmli, U.K. (1970) Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature227:680-685.

Mizusawa, S., Nishimura. S., and Seela, F. (1986) Improve-ment of the dideoxy chain-termination method of DNAsequencing by use of deoxy-7-deazaguanosine triphos-phate in place of dGTP. Nuct Acids Res 14: 1319-1324.

Noda, Y., Yoda, K., Takatsuki, A., and Yamasaki, M. (1992)TmrB protein, responsible for tunicamycin resistance ofBacillus subtiiis, is a novel ATP-binding membrane protein.J Bacteriol 174: 4302-4307.

Quiros, L.M., Hernandez, C, and Salas, J.A. (1994)Purification and characterization of an extracellularenzyme from Streptomyces antibioticus that convertsinactive glycosylated oleandomycin into the active anti-biotic. Eur J Bioehem 222: 129-135.

Rodriguez, A.M., OSano, C , Vilches, C, t\flendez, C , andSalas, J.A, (1993) Streptomyces antibioticus contains atleast three oleandomycin resistance determinants, one ofwhich shows similarity with proteins of the ABC transportersuperfamily. Mol Microbiol 8: 571-582.

Ross, J.I., Eady, E.A., Cove, J,H., Cundtiffe, W.J,, Baumberg,S., and Wootton, J.C. (1990) Inducible erythromycinresistance in staphylococci is encoded by a member ofthe ATP-binding transport super-gene family. Mot Microbiot4: 1207-1214.

Rosteck, P.R., Reynolds, P.A., and Hersberger, C.L (1991)Homology between proteins controlling Streptomycesfradiae resistance and ATP-binding transport. Gene 102:27-32.

Sambrook, J,, Fritsch, E.F., and Maniatis, T. (1989) MotecutarCtoning. A Laboratory Manuat. Cold Spring Harbor, NewYork: Cold Spring Harbor Laboratory Press.

Sanger, F., NicKlen, S., and Coulson, A.R. (1977) DNAsequencing with chain terminating inhibitors. Proc NattAcad Sci USA 74: 5463-5467.

Schoner, B.. Geistlich, M., Rosteck. Jr, P., Rao, R.N., Seno,E., Reynolds, P., Cox, K., Burgett, S., and Hershberger, C.(1992) Sequence similarity between macrolide-resistancedeterminants and ATP-binding transport proteins. Gene115:93-96.

Skinner, R.H.. and Cundliffe, E. (1982) Dimethylation ofadenine and the resistance of Streptomyces erythreus toerythromycin. J Gen Microbiot 123: 2411 -2416.

Swan, D.G., Rodriguez, A.M., Vilches, C, Mendez, C, andSalas, J.A. (1994) Characterisation of a Streptomycesantibioticus gene encoding a type I polyketide synthasewhich has an unusual coding sequence. Mot Gen Genet242: 358-362,

Viiches, C, Mendez, C, Hardisson, C, and Salas, J.A.(1990) Biosynthesis of oieandomycin by Streptomyces anti-bioticus. influence of nutritional conditions and develop-ment of resistance. J Gen Microbiot 136: 1447-1454.

Viiches, C, Hernandez, C, Mendez, C, and Salas, J.A.(1992) Role of glycosylation and deglycosylation in thebiosynthesis of and resistance to oleandomycin in theproducer organism, Streptomyces antibioticus. J Bacteriol174: 161-165.

Walker. J.E., Saraste. M., Runswick, M.J., and Gray, N.J.(1982) Distantly related sequences in the ct- and p-subunitsof ATP synthase, myosin kinases and other ATP-requiringenzymes and a common nucieotide binding fold. EMBO J8: 945-951.

Yanisch-Perron, C, Vieira, J., and Messing, J. (1985)

1995 Blackwell Science Ltd. Mot&cutar Microbiology. 16, 333-343

ABC transporters in Streptomyces 343

Improved M13 phage cloning vectors and host strains: Zaiacain, M., and Cundliffe. E. (1990) Methylation of 23Snucleotide sequences ofthe M13mp18and pUCI 9 vectors. ribosomal RNA due to carB. an antibiotic-resistanceGene 33: 103-119. determinant from the carbomycin producer. Streptomyces

Zaiacain. M.. and Cundiiffe, E. (1989) Methyiation of 23S ttiermotolerans. Eur J Biochem 189: 67-72.rRNA caused by tirA (ermSF). a tylosin resistance Zaiacain, M., and Cundliffe, E. (1991) Cloning of WrD, a fourthdeterminant from Streptomyces fradiae. J Bacterioi 171: resistance gene, from the tyiosin producer. Streptomyces4254-4260. fradiae. Gene 97: 137-142.

1995 Blackwell Science Ltd, Molecular Microbiology. 16. 333-343