VOL. 53 NO. 4, APR. 2000 THE JOURNAL OF ANTIBIOTICS pp.373 - 384

ABCTransporter Genes, kasKLM,Responsible for Self-resistance of a Kasugamycin Producer Strain

Souichi Ikeno*, Yasuhiro Yamane, Yoko Ohishi, Naoko Kinoshita1*, Masa Hamada1", Kayoko S. Tsuchiya and Makoto Hori Showa College of Pharmaceutical Sciences, 3-3 1 65 Higashi Tamagawagakuen^ Machida-shi, Tokyo 194-8543, Japan institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021 , Japan (Received for publication November 19, 1999)

Wepreviously reported that a 7.6-kb DNAfragment from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSMacetyltransferase gene (kac33S) and some other genes possibly involved in KSMbiosynthesis. As an extension of that study, a 10-kb Sacl-Kpnl DNAfragment, located 5~ 15-kb upstream of kac?3%, was cloned and a 4.2-kb Sacl- EcoRl fragment there from was sequenced, revealing one incomplete (designated ORFJ) and three complete open reading frames (designated kasK, kasL and kasM). The coding frames of kasK, L and Moverlap one another with terminator/initiator ATGAsequence. RT-PCRanalysis of a DNAregion including kasKLMindicated the presence of one transcript that is long enough to span the three genes. The kasK gene potentially encodes an ATP-binding protein of the ATP- binding cassette (ABC) transporter super family. Homology search for the deduced KasK protein shows similarity to other ABCtransporters involved in self-resistance of a mithramycin and possibly doxorubicin producer strain. The kasL and kasMgenes encode different integral membraneproteins, both having six putative transmembrane helices. Anexpression plasmid for kasKLM(pTV^KLM)was constructed and these genes were expressed in E. coli JM109, which had been sensitive to KSM.The transformant acquired resistance to KSM,suggesting that KasK, L and Mproteins as a set in S. kasugaensis M338-M1pumpout KSMto protect the producer from its toxic metabolite.

Organisms producing potentially autotoxic antibiotics producers, utilizing the energy from ATPhydrolysis to possess basically three types of self-resistance mechanisms pumpout the toxic metabolites across the membraneeven to avoid suicide: (1) modification of the target site that the against a concentration gradient. Some genes encoding antibiotic acts on, (2) intracellular inactivation of the ABC transporters have been cloned and characterized: antibiotic and (3) exclusion of the antibiotic from the cell1^ drrAB3^ from S. peucetius (daunorubicin/doxorubicin The last resistance mechanism is designated "membrane- resistance), mtrABA) from S. argillaceus (mithramycin associated system" consisting of two classes. In one class, resistance) and OleC,C55) from S. antibioticus resistance is mediated by membrane proteins, which (oleandomycin resistance). The mechanism of drrAB is are believed to energize export of antibiotic molecules similar to P-glycoprotein6) for multidrug resistance of by proton-dependent transmembrane electrochemical human cancers, suggesting that understanding the gradients. The other class belongs to the ABCtransporter mechanisms of bacterial ABCtransporters mayshed light superfamily2) comprising many membrane-associated on how to solve the drug-resistance problems associated export and import systems, which are present both in with some humandiseases. prokaryotic and in eukaryotic cells. ABC transporters We previously cloned and characterized some KSM possess a highly conserved ATP-binding cassette, which is biosynthetic genes7) from S. kasugaensis M338-M18). In the the most characteristic feature of this super family. They present paper wereport the cloning, characterization and participate in the secretion of antibiotics from the functional analysis of ABC transporter type genes 374 THE JOURNAL OF ANTIBIOTICS APR. 2000 (kasKLM) from S. kasugaensis M338-M1. DNASIS-Mac version 3.6 (Hitachi Software Engineering Co. Ltd.). Amino acid sequences of potential gene products were compared with those in the databases (SWISS-PROT Materials and Methods and PIR) by means ofFASTA16) and BLAST17). Bacterial Strains and Plasmids Nucleotide Sequence Accession Number Bacterial strains and plasmids used in this work are The nucleotide sequence data reported in this paper will described in Table 1. S. kasugaensis M338-M1has been appear in the DDBJ, EMBL and GenBank nucleotide maintained at the Institute of Microbial Chemistry. The sequence databases with the accession number AB033992. following strains and plasmids were of commercial origins: Escherichia coli TH29) (TaKaRa, Code No. 9056), E. coli Preparation of RNA DH5a10) (TOYOBO, Code No. DNA-903), E. coli S. kasugaensis M338-M1was cultured for 24 hours as JM10910'n) (TaKaRa, Code No. 9052), cloning vector PKF described above. Approximately 1 g of wet mycelia was 39'12) (TaKaRa, Code No. 3100), pUC1 1813) (TaKaRa, Code resuspended in 10ml of the extraction buffer (see below) No. 3318) and expression vector pTV118N14) (TaKaRa, containing 4 m guanidinium thiocyanate. The mycelia were Code No. 3328). Other plasmids were produced in the broken in a sonicator (Branson Sonifier 350) by 5 cycles of present study. 30 seconds beating at 6 watts at 0°C and 30 seconds of chilling at 0°C. The cell lysate was treated as described in Growth Conditions the instructions of RNA extraction kit (Amersham S. kasugaensis M338-M1was grown in TSB medium Pharmacia Biotech, 27-9270-0 1) to prepare total RNA. (DIFCO, 0370-17-3) and/or MRmedium (KSM producing medium: 2% glycerol, 2% dextrin, 1% Bacto soyton, 0.3% RT-PCR yeast extract, 0.2% ammonium sulfate, 0.2% calcium Reverse transcription (RT) and PCRamplification were carbonate pH 7.0) under shaking at 27°C for 24-96 hours. performed with RNALA PCRá"Kit (AMV) Ver. 1.1 E. coli TH2transformants were grown at 37°C in L-broth (TaKaRa, RR012A) according to the instructions of the containing chloramphenicol (12 ^g/ml) and streptomycin supplier. One fig of total RNAfrom S. kasugaensis M338- (50jag/ml). E. coli DH5a and JM109 transformants were Ml, which was used as template for CDNAsynthesis, was grown at 37°C in YT medium containing ampicillin treated with 7.5 units ofRNase-free DNase I for 2 hours at (lOO ^g/ml). 37°C to eliminate residual genomic DNA. 3'-pKS15 (5'- GCGACACAGACCTCCAGCCCCAGTT-3', Fig. 4A) was Cloning and Sequencing used as a primer for first-strand CDNAsynthesis. The Isolation of genomic DNAfrom S. kasugaensis M338- RT reaction was performed with avian myeloblastosis Ml was conducted as described previously15). The E. coli virus reverse transcriprase for 30 minutes at 60°C. For TH2/PKF3 cloning system was used to clone PSKE4 and the following PCR amplification, 5'-pKS23b (5'- 5. TCCGGGATTTCCGAAGGAACGGCGT-3') and the 3'- The cloned DNAregion (4.2-kb SacI-EcoW fragment) pKS15were used as primers. A 100/il PCRmixture was digested with appropriate restriction endonucleases, contained 20ji\ of the RT mix, 20pmol each of the two and the fragments were subcloned into pUC118 (pKS15, primers, 2.5 units of LA Taq DNApolymerase (TaKaRa) 22-25, Table 1) and sequenced with an automated laser and IXLA PCR buffer. The PCR involved 30 cycles of fluorescence sequencer (ALFredá" DNA Sequencer, denaturation (30 seconds at 97°C), annealing (1 minute AmershamPharmacia Biotech), using the ALFexpressá" at 65°C) and extension (1 minute at 72°C). Control AutoCycleá" Sequencing Kit (Amersham Pharmacia amplification using equivalent amounts of RNA that Biotech, Code No. 27-2693-02) according to the supplier's had not been incubated with reverse transcriptase was instructions. Sequencing primers used were Ml3universal conducted to confirm that genomic DNA was not and reverse primers and somesynthesized oligonucleotide contributing to the PCR amplification. The RT-PCR primers (labeled with Cy5, purchased from Amersham products were electrophoresed on a 0.6% agarose gel and Pharmacia Biotech). visualized using ethidium bromide staining (Fig. 4B).

Computer Analysis of DNAand Protein Sequences DNA and protein sequences were analyzed with the VOL. 53 NO.4 THE JOURNAL OF ANTIBIOTICS 375

Table 1. Strains andplasmids.

Strains and plasmids Genotype and genetic construct j Reference Strains

S. kasugaensis 8 M338-M1 Kasugamycinproducing strain E. coli DH 5ot <|)80/acZAM15 A(lacZYA-argF) U169 deoR recAl endAl hidR17(rK~, mK+)phoA supE44 X'thi-1 gyrA96 relAl I 10 TH2 supEU hsdS2Q(rB~, mB+) recA13 ara-U proAl lacYl galK2 rpsLlQ xyl-5 mtl-1 thi-1 trpR624 9 JM109 recAl endAl gyrA96 thi hsdRll supEAA relAl A(lac-proAB) / F{[traD36proAB+ lacF lacZAMIS] \ 10, ll Plasmids

Cloning vector. Ampr. 3.1-kb. pUC118 13 pTV118N 1 Expression vector derived from pUCl18, containing a unique Nco I site. Ampr. 3.1-kb. 14 pKF3 | Cloningvector. Sms, Cmr. 2.2-kb. | 9, 12 PKF 3 derivative incorporating 8.4-kb Kpnl fragment from S. kasugaensis M338-M1. 10.6-kb. SKE 5 | j)KF 3 derivative incorporating 5.5-kb Sad fragment from S. kasugaensis M338-M1. 7.7-kb. pUC118 derivative containing 718 bp Kpnl-EcoRl fragment from PSKE 5. 3.9-kb. pUCl18 derivative containing 577 bp Sacl-Smal fragment from PSKE 5. 3.7-kb. pUC118 derivative containing 1135 bp Smal-EcoKl fragment from PSKE5. 4.3-kb. KS24 pUC118derivative containing 846 bp EcoRl-Sphl fragment from PSKE5. 4.0-kb. This work KS25pUCl18 derivative containing 965 bp Sphl-Kpnl fragment fromPSKEThis5. 4.1-kb.work

PTV-NE 1 95 pTV1 18N derivative containing a PCR fragment (195 bp Ncol-EcoRl a head region of kasK). This work Negative control for PTV-Kgal; lacZ' being fused out of frame. PTV- KLM pTV118Nderivative containing kasKLM( 2.7-kb Ncol-EcoRl fragment ) genes. This work OTV- Ke al pTV118Nderivative carrying a chimera gene consisting of a head region of kasK fused by lacZ' in frame. I This work PTV-KLgal PTV-KLMderivative in which LMregion is replaced by a chimera gene consisting of a head region of kasL fused bv lacZ' in frame. This work PTV-KLgal(-) PTV-KLMderivative lacking 1.7-kb Sphl fragment. Negative control for PTV-KLgal; lacZ' being fused out of frame. This work PTV-KLMgal PTV-KLMderivative carrying complete kasKL and a head region of kasM fused by lacZ' in frame. This work PTV- KLMgal(-) PTV-KLMNegativederivativecontrol lackingfor PTV-KLMgal;0.7-kb KpnllacZ'fragment.being fused out of frame. This work

Subcloning of kasK, L and Minto E. coli Expression DNAand its boundaries ofpTV-NE195 were confirmed by Vector the sequence analysis. To introduce a Ncol site including the start codon The 2.5-kb EcoRl fragment from PSKE 5 (Fig. 1) was of the kasK gene, The 5' region of the kasK gene (nt subcloned into the EcoRl site of PTV-NE195, yielding 1513-1708, Fig. 2) was amplified by PCR. 5'-ORF K PTV-KLM(Table 1, Fig. 5). The orientation of the inserted (Sense: 5'-GGCCATGGTCGAGGTCAC-3 ') and 3 '-ORF K fragment was confirmed by sequence analysis. (Antisense: 5 '-GGGAATTCTGACCAGGGT-3') were used as PCRprimers. PCRwas performed using a MiniCyclerá" Confirmation of Separate Translation of KasK, L and M (MJ Research). The reaction mixture contained 1 ng pKS23 Proteins (Table 1), 20pmol of each primer, 50 jiu each dNTP, 20 mM To confirm separate translational initiation of KasK, L Tris-HCl pH 8.0, 25mM KC1, 1.5mM MgCl2 and 0.05% and Mproteins, PTV-KLMwas modified to encode a fusion Tween 20 in a final volume of 100ji\. Amplification protein consisting of a short TV-terminal peptide of either involved 30 cycles of denaturation (30 seconds at 97°C), KasK, L or M and /3-galactosidase a peptide (Fig. 5). To annealing (1 minutes at 65°C) and extension (1 minute at produce these derivatives, for example, a DNAregion of nt 72°C). The resulting PCR product was digested with 1513-1591 (Fig. 2), encoding the TV terminal region of NcoI/EcoRI and inserted into pTV118N, yielding pTV- KasK, was PCR-amplified using as primers 5'-ORF K NE195(Table 1). The nucleotide sequences of the inserted (Sense: 5'-GGCCATGGTCGAGGTCAC-3') and 3'-ORF 376 THE JOURNAL OF ANTIBIOTICS APR. 2000

K2 (Antisense: 5'-GGGAATTCGGTCCGCTCT-3'). The underlined sequences in the primers are Ncol site and Results EcoRl sites, respectively. The PCRproduct was digested with Ncol and EcoRl and inserted into pTV118N to obtain Cloning and Sequence Analysis a recombinant plasmid (PTV-Kgal) that would encode a An 8.4-kb Kpnl fragment from S. kasugaensis M338-M1 fusion protein consisting of Met1-Pro26 of KasK and chromosomewas cloned using as a probe the 850bpPstl- Asn4~His106 of j8-galactosidase a peptide. As a negative Kpnl fragment from PSKE2. The resultant recombinant control, PTV-NE195 was used, which would encode a plasmid was designated PSKE 4. A 5.5-kb Sad fragment fusion protein consisting of Met1-lie65 of KasKand a from S. kasugaensis M338-M1 chromosome was also frameshift (inactive) protein. Similarly, plasmids designed cloned using as a probe the 594bp Kpnl-Sphl fragment to encode a fusion protein consisting of Me^-Ser17of from PSKE4. The plasmid was named PSKE 5 (Fig. 1). We KasL and His19~His106 of /J-galactosidase a peptide (pTV- sequenced the 4236bp SacI-EcoRI DNAregion from PSKE KLgal) and another fusion protein consisting of 5 (Fig. 1). The GCcontent of the region was calculated to Me^-Gly14 of KasM and Leu13~His106 of jS-galactosidase be 67.7% from the sequence. Open reading frames (ORFs) a peptide (PTV-KLMgal), and their negative controls (pTV- were searched for based on the codon usage and third KLgal(-) and PTV-KLMgal(-), respectively), were also codon position bias which are characteristic of constructed (Table 1, Fig. 5). Streptomyces genes18). Within this region we recognized four ORFs, i.e. ORF J, kasK, kasL and kasM, though ORF J /3-Galactosidase Assay was incomplete. All these ORFsrun in the same direction E. coli JM109 transformants were cultured as described (Fig. 1). There were inverted repeat sequences downstream above. The expression of fused j8-galactosidase genes was of both ORF J (nt 722-752, -32.30kcal/mol) and kasM induced with 1 mMisopropylthiogalactopyranoside (IPTG) (nt 4062-41 1 1, -63.80 kcal/mol), possibly functioning as when the cell density reached 0.8-1.0 (OD660). The cells transcriptional terminators (Fig. 2). In the intergenic space were collected by centrifugation for 5 minutes at 5,000Xg, between ORF J and kasK (nt 646-1512), there is a resuspended in 5ml of PBS and disrupted by sonication. relatively low GCcontent region for Streptomyces DNA(nt The lysates were centrifuged for 5 minutes at 5,000Xg to 1033-1303, GC%=57.2%), suggesting that DNA unwinds remove the cell debris. To measure the /3-galactosidase there to initiate transcription of the downstreamgenes. activity of the fused proteins, 70^1 of 4mg/ml o- There are three possible translational start points for nitrophenyl-/3-D-galactopyranoside and 200 jA of PBS were kasK i.e., nt 1297 ATG, nt 1468 ATG and nt 1513 ATG added to the cell lysate (30^1). The reaction was carried (Fig. 2). Wefavor the nt 1513 ATGas the translational start out for 30 minutes at 37°C and stopped by the addition of site for kasK, because the ATGis closely preceded by a 0.5 ml of 1 msodium carbonate. The absorbance at 420nm potential ribosome binding site (RBS) (AAGGAG) and by released o-nitrophenol was read. because the GCbias of third codon positions near that point is typical of Streptomyces codons. The coding frames of Induction ofKSMresistance in E. coli JM109 kasK, L and M overlap at their boundaries (ATGA), E. coll JM109 carrying pTV118N.br PTV-KLM was possibly suggesting translational coupling, as in many other grown at 37°C in YT medium containing ampicillin bacterial genes19'20). The stop codon for the kasK is nt 2500 (100jUg/ml) under shaking. Growth was monitored by TGA,while the start for kasL is nt 2499 ATG,preceded by reading OD660 of the cultures. When the OD660 reached an RBS (GGGGG).The stop codon for kasL is nt 3270 0.5-0.7, 0.2mM IPTG was added to the cultures and TGA,while the start codon for kasM is nt 3269 ATG, cultivation was continued at 37°C for 3 hours. Twenty ji\ preceded by an RBS (GGTGA). The stop codon for kasMis portions of the cultures were inoculated into culture tubes nt 3989 TGA. containing 2 ml of YT medium with ampicillin (lOO ^ug/ml) and KSM(200 jUg/ml) and the growth was monitored by reading OD660 of the cultures at 37°C for 42 hours (Fig. 6). Characterization of Putative KasK, L and MProteins The kasK gene is deduced to encode a protein (KasK) containing 329 amino acids with a molecular mass of 35,862Da and a pi of 6.58. Kyte-Doolittle analysis21) of KasK suggests it is hydrophilic (data not shown). The possible function(s) of KasK was deduced from the result VOL. 53 NO. 4 THE JOURNAL OF ANTIBIOTICS 377

Fig. 1. Restriction map of the cloned DNAregion from S. kasugaensis M338-M1, including the has gene cluster.

On the restriction map, the striped region (left, also shown in an enlarged scale) is dealt with in the present paper. The open region (middle) remains to be studied. The solid region (right) has been reported7). The open arrows indicate the deduced ORFsand direction of transcription. There are two ATGA'sat the boundaries between kasK and kasL and between kasL and kasM. Both ATGAare bifunctional; as the stop codon (TGA) for each preceding gene and as the initiation codon (ATG)for each following gene. The inserts ofplasmids PSKE1, PSKE2, PSKE4 and PSKE5 are indicated above the map. Abbreviations: B, BamHI; E, EcoRl; K, Kpnl; P, Pstl; S, Sad; Sm, Smal; Sp, Sphl.

of a homology search using the FASTA and BLAST masses of 27,497 and 26,431 Da and with pi's of 8.2 and program. The deduced amino acid sequence of KasK 9.56, respectively. Kyte-Doolittle analysis of these putative showed 39% similarity with DrrA3) protein which is proteins indicate that the two proteins would be extremely responsible for self-resistance of S. peucetius to its own hydrophobic and both contain six transmembrane helices, toxic metabolites, daunorubicin and doxorubicin. KasK was therefore, would cross the cell membranesix times (Fig. 3). found to be similar to some other ATP-binding proteins; The deduced amino acid sequence of KasL showed 33% OleC5) from S. antibioticus (39%) responsible for similarity with NodJ25) from Rhizobium leguminosarum oleandomycin resistance, MtrA4) from S. argillaceus (37%) and 31% similarity with MtrB4) from S. argillaceus. for mithramycin resistance, Nodi from Rhizobium galegae Comparison of KasMwith other proteins also revealed (37%) for oligosaccharide export (unpublished data, L. 30% similarity with NodJ25) from Rhizobium Paulin et al., Accession No. P50332), NatA22) from leguminosarum. These known proteins are membrane- Bacillus subtilis (36%) for extrusion of Na ion and spanning subunits in support of the above described ATP- TnrB223) from S. longisporoflavus (35%) for tetronasin binding subunits to form each transporter system. resistance. The deduced amino acid sequence of KasK contains an ATP-binding motif24) which is commonto the large family of ABCtransporters (Fig. 2). Detection ofmRNAof kasK, L and Mby RT-PCR The kasL and kasM encode proteins (KasL and KasM) We expect that kasK, L and Mare transcribed into a containing 257 and 240 amino acids, with molecular polycistronic mRNAbecause these coding regions overlap 378 THE JOURNAL OF ANTIBIOTICS APR. 2000

Fig. 2. Nucleotide sequence of a 4.2-kb SacI-EcoRI region from PSKE5 and deduced amino acid sequences of ORF J, kasK, kasL, and kasM. VOL. 53 NO. 4 THE JOURNAL OF ANTIBIOTICS 379

Fig. 2. (Continued)

Shadowed amino acids represent the conserved motifs (Walker A, loop 3, and Walker B) for an ATP-binding cassette21). Double underlines and dots indicate putative ribosome binding site (RBS) and putative terminator of transcription, respectively.

one after another. To test the possibility, we analyzed the RNA template used for PCR amplification was not transcriptional product of kasK, L and M by RT-PCR. The significantly contaminated with genomic DNA. hybridization positions and the orientations of the primers are shown in Fig. 4A, and the result ofRT-PGRis shown in Fig. 4B. When the CDNA, synthesized with 3'-pKS15 Confirmation of Codon-framing of kasK, L and M primer, was used as the template for PCR, a product with Wenext tested if the codon-framings of kasK, L and M the expected length was amplified (Fig. 4B, 2643bp were correct. For this purpose, we constructed plasmids fragment, Lane 2). This result suggests that kasK, L and M that included TV-terminal 14-26 codons of either kasK, L or are transcribed as a polycistronic mRNA.Thecontrol run Mfused in frame with /3-galactosidase a peptide gene (Fig. of PCRusing equivalent amounts of RNAthat had not been 5). Translational initiation usually needs RBS that is incubated with reverse transcriptase did not give any located closely upstream of ATG. RBSfrom the expression amplified product (Fig. 4B, Lane 3), indicating that the vector, pTVl 18N, was expected to work for kasK, while nt 380 THE JOURNAL OF ANTIBIOTICS APR. 2000

Fig. 3. Hydrophobicity analysis of the deduced KasL and KasMproteins.

Numerals on the absissas indicate the numbering of amino acids. Window size 10. Range of relative hydrophobicity 5 to -5.

2486 GGGGGand nt 3257 GGTGA(Fig. 2) might function from antibiotic-producing actinomycetes. Salas et al. as RBSfor kasL and M, respectively. E. coli transformants classified these genes into three groups, type I, II and III, harboring these plasmids showed /J-galactosidase activity on the basis of gene structures and arrangement26). A type I when induced with IPTG (Table 2). Introduction of a gene consists of two contiguous genes; the upstreamone frameshift at the fusing point of any of the chimera genes encodes a hydrophilic protein including an ATPbinding resulted in loss of /3-galactosidase activity of the site and the downstream one encodes a hydrophobic transformant s. membrane-binding protein (e.g., drrAB3\ mtrAB4\ tnrB2B323) etc.). A type II gene encodes a hydrophilic protein including two ATP binding sites (e.g. , tlrC, tylosin Resistance to KSMInduced by kasK, L and M resistance27); oleB, oleandomycin resistance28); imrC, inE. coli JM109 lincomycin resistance29) etc.). A type III gene encodes a E. coli JM109 transformed with PTV-KLMacquired membrane-crossing (6 times) protein including an ATP resistance to KSM(200 fig/ml) (Fig. 6) suggesting that the binding site on the carboxy terminal region (e.g., ABCtransporter, consisting of the products of kasK, L and strVW, streptomycin resistance30); ble-orfl, bleomycin Mgenes, is functional in exporting KSMout of the cell. resistance31)). Our KSMtransporter genes, kasK, L and M, should belong to type I because kasL and M, resembling each other and both encoding hydrophobic membrane- Discussion binding proteins, seem to have originated from a downstreamgene of type I. Similar gene structures were Morethan ten ABCtransporter genes have been cloned reported for lantibiotic ABCtransporters, such as those in THEVOL.53JOURNALOFANTIBIOTICS 381 NO.4

Fig. 4. Detection of the mRNAofkasKLMby RT-PCR.

(A) Diagram showing the positions and orientation of the oligonucleotide primers for RT-PCR.(B) Lane 1 and 4: Molecular size markers. Lane 2: The amplified 2643bp fragment. Lane 3: Negative control (minus reverse transcriptase).

Fig. 5. Schematic representation of the fusion ofkasK, hash or kasMwith lacZ'.

pTV118N is an expression vector. DNAsegments including proposed translational start codons of kasK, kasL and teM were fused in frame with lacZ', to produce PTV-Kgal, PTV-KLgal and PTV-KLMgal,respectively. PTV- KLMis the parent clone. Q indicates the putative terminator of transcription behind kasM (see the dots from 4062 to 4111inFig.2). 382 THE JOURNAL OF ANTIBIOTICS APR. 2000

Fig. 6. KSMresistance ofE. coli JM109 expressing kasKLMgenes.

Proliferation profiles ofE. coli JM109 harboring the pTVl 1 8N and PTV-KLM, grown in YT medium containing KSM (200 ^g/ml).

Staphylococcus epidermidis (epidermin32)), Lactococcus translation of the upstream gene through the overlapping lactis (lacticin 48133)), etc. ATGA.As shown in Fig. 4B, we proved that the kasK, L The kasL start codon overlaps with the kasK stop codon and Mgenes were transcribed in a polycistronic mRNA. in nt 2499 ATGA,and kasM start codon overlaps the kasL Studies are in progress to elucidate how the initiation of the stop codon in nt 3269 ATGA (Fig. 2). An overlapping transcription is controlled. ATGAwas also found at the boundary of the drrAB3) As shown in Fig. 6, KSM transporter conferred genes. Such a structure should induce a translational resistance to KSMon E. coli JM109 possibly by pumping coupling19'20) of kasK, L and M. Translational coupling out KSM that somehow permeated into the cells. We refers to situations where translation of a gene in a previously reported that kacm, the gene encoding KSM polycistronic mRNAis more or less dependent on the acetyltransferase, is a gene responsible for self-resistance of VOL.53 NO.4 THE JOURNAL OF ANTIBIOTICS 383 KSMproducers7). All the KSMproducer strains so far 25-33, 1992 13) Vieira, J. & J. Messing: Production of single-stranded tested were positive with fee338 in Southern analysis. The plasmid DNA. Methods in Enzymology 153: 3-ll, same wastrue with kasK, L and M. It is tempting to 1987 presume that KSMproducers developed the double-safety 14) Maki, M.; E. Takano, H. Mori, A. Sato, T. Murachi & system, one is inactivation and the other secretion, against M. Hatanaka: All four internally repetitive domains of their own toxic metabolite, KSM. pig calpastatin possess inhibitory activities against calpains I and II. FEBS 223: 174-180, 1987 15) Ikeno, S.; K. Higashide, N. Kinoshita, M. Hamada & Acknowle dgments M. Hori: Correlation between the presence of kac, kasugamycin acetyltransferase gene, and the productivity of kasugamycin in Strepromyces. Actinomycetologica We thank Miss Shizue Myokai and Miss Yukiko Aoki for 10(2): 73-79, 1996 excellent technical assistance. 16) Pearson, W. R. & D. J. Lipman: Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. References USA 85(8): 2444-2448, 1988 17) Altschul, S. R; T. L. Madden, A. A. Schaffer, J. 1) Cundliffe, E.: How antibiotic-producing organisms Zhang, Z. Zhang, W. Miller & D. J. Lipman: Gapped avoid suicide. Annu. Rev. Microbiol. 43: 207-233, 1989 BLASTand PSI-BLAST: a new generation of protein 2) Higgins, F. C: ABCtransporters: from microorganisms database search programs. Nucleic Acids Res. 25(17): to man. Annu. Rev. Cell Biol. 8: 67-113, 1992 3389-3402, 1997 3) Guilfoile, G. P. & R. C. Hutchinson: A bacterial 18) Bibb, M. X; P. R. Findlay & M. W. 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