Lysine g-aminotransferase Activity in Escherichia coli

Transcription

1 JOURNAL OF BACTERIOLOGY, Oct. 1991, p /91/ $02.00/0 Copyright X) 1991, American Society for Microbiology Vol. 173, No. 19 Localization of the Lysine -Aminotransferase (lat) and 8-(L-oL-Aminoadipyl)-L-Cysteinyl-D-Valine Synthetase (pcbab) Genes from Streptomyces clavuligerus and Production of Lysine g-aminotransferase Activity in Escherichia coli MATTHEW B. TOBIN,1 STEVEN KOVACEVIC,1 KRISHNA MADDURI,2 JO ANN HOSKINS,' PAUL L. SKATRUD,1 L. C. VINING,3 COLIN STUTTARD,2 AND JAMES R. MILLER'* Department of Molecular Genetics Research, Lilly Research Laboratories, Indianapolis, Indiana 46285,1 and Departments of Microbiology2 and Biology,3 Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 Received 22 April 1991/Accepted 16 July 1991 Lysine e-aminotransferase (LAT) in the,i-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a I(-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an -M, 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of 8-(L-a-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbab suggests that the coding region occupies 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the I8-lactam biosynthetic cluster between pcbc and cefe (-25 kbp) is nearly complete. Many naturally occurring P-lactam antibiotics (isopenicillin N, cephalosporin C, and cephamycins) contain a side chain derived from a-aminoadipic acid (a-aaa). When the producer is a filamentous fungus (Penicillium chrysogenum, Aspergillus nidulans, Cephalosporium acremonium), this amino acid is formed as an obligate intermediate in the synthesis of lysine. However, in procaroytes, lysine is synthesized via the diaminopimelic acid pathway without the production of a-aaa (for reviews, see references 23 and 36). Whitney et al. (37) established that in the P-lactamproducing Streptomyces spp., ot-aaa is derived from the breakdown of lysine. Although the catabolism of lysine in bacteria is diverse, conversion of lysine to a-aaa by removal of the E-amino group was tentatively linked in Streptomyces lipmanii to an aminotransferase (16) which was later conclusively shown to be present in Nocardia (previously Streptomyces) lactamdurans (15). Recently, Madduri et al. (21) reported that in these actinomycetes, L-lysine-e-aminotransferase (LAT) is specific to P-lactam (cephamycin C) producers and provides the precursor for antibiotic synthesis; the pathway via cadaverine is obligatory for lysine catabolism (Fig. 1). Moreover, a gene governing LAT production was found exclusively in Streptomyces spp. producing P-lactams (22) and mapped within the P-lactam biosynthetic gene cluster of Streptomyces clavuligerus (18, 24, 33). Romero et al. (28) had also described mutants of S. clavuligerus that have no (nccl) or reduced (ncal) LAT activity and suggested that reduction of cephamycin biosynthesis was due to a deficiency of LAT. In this study, the gene that complements LAT activity (lat) was sequenced, and the similarity of the derived amino * Corresponding author acid sequence to other aminotransferases is discussed in this report. The open reading frame (ORF) was cloned in an Escherichia coli expression vector to investigate the properties of the putative protein. Sequencing of the region immediately downstream from the lat gene identified another ORF that has similarities to 8-(L-(x-aminoadipyl)-L-cysteinyl-Dvaline (ACV) synthetases (ACVSs) encoded by pcbab from P. chrysogenum and C. acremonium. The regulation of,b-lactam biosynthesis in the actinomycetes by lat is considered in light of the close physical linkage between lat and pcbab. MATERIALS AND METHODS Bacterial strains, growth conditions, DNA manipulations, and DNA sequence determination. E. coli strains were grown in TY broth (18) supplemented with either 5,ug of tetracycline per ml or 80 jig of ampicillin per ml under standard conditions, except as noted. Restriction endonuclease digestion, ligation, and determination of DNA restriction fragments by agarose or acrylamide gel electrophoresis followed established procedures (29). DNA fragments were subcloned into puc or M13 vectors for further analysis. DNA sequencing was performed by the dideoxy method with TAQ-TRACK (Promega Biotec) and followed the supplier's recommendations. Fragments were sequenced either directly from the plasmid or after subcloning into M13 bacteriophage vectors, using forward and reverse sequencing primers for puc and M13 templates, as well as several custom DNA oligonucleotide primers. Custom DNA oligonucleotide primers were synthesized by using an Applied Biosystems DNA Synthesizer (model 380A) according to the manufacturer's instructions. Computing. DNA sequences were analyzed with the GCG

2 6224 TOBIN ET AL. J. BACTERIOL. 1-Piperideine- -6-carboxylate R.CO. R.CHNH2. 2H20 a-amino adipate J 2H 4 I (CH2)3 HCNH2 (CH2)3 HCNH2 I P-lactam antibiotics " Lysine Co2 (CH2)3 2 R.CO. Cadaverine 4 RCHNH2. + H20 I. " 2H20 2H CO2 + H20 + NH3 1-Piperideine 8-Aminovalerate ScaI and -3.3-kbp ScaI-BamHI fragments and was cloned in the -5.8-kbp NcoI-BamHI fragment from pow241, resulting in plasmid pow407. Gene expression in E. coli. For LAT production, E. coli JM109 cells containing pow407 were grown overnight at 25 C, diluted 20-fold, induced at 42 C for 6 h, and examined by phase-contrast microscopy for the appearance of granules. Cell extracts were prepared as described previously (19) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LAT enzyme assay. Cells were harvested from a culture by centrifugation 1, 2, and 6 h after induction at 42 C. They were resuspended in 0.2 M potassium phosphate buffer (ph 7.5) and disrupted with ultrasound (six 10-s pulses with 1-min cooling intervals). The supernatant fluid from centrifugation (20 min, 10,000 x g, 4 C) was assayed for LAT activity by measuring the formation of 1-piperideine-6-carboxylate with an o-aminobenzaldehyde reagent as described previously (20). Nucleotide sequence accession number. These data have been submitted to GenBank and have been assigned accession number M RESULTS Sequence and molecular characterization of lat. Cloning in S. lividans, a host that does not produce 1-lactam antibiotics, of DNA fragments linked to pcbc and cefe of S. clavuligerus led to the production of LAT activity (22). Moreover, the gene governing LAT production was shown to reside on a single 4.7-kbp SstI-EcoRI fragment from S. FIG. 1. Catabolic pathways for lysine in Strelptomyces spp clavuligerus. An indication that this gene encoded the amino Enzyme activities are as follows: 1, lysine decarbox ylase; 2, cada- acid sequence of the enzyme rather than serving a regulatory verine aminotransferase; 3, 1-piperideine-6-carboxylate dehydroge- function might come from DNA sequence analysis. Althe growth of though the exact location of the gene for LAT was unknown, nase. The pathway on the right is essential for Streptomyces spp. on lysine (7); the pathway on the left is present spontaneous deletions of pdq301d (22) in a pool of primary only in,-lactam-producing Streptomyces spp. Anoth4erpathway, for transformants and concurrent loss of LAT activity in S. the catabolism of a-aaa (---), exists in S. lividans clavuligerus. Bracketed intermediate is hypothetical Lbut not in S. lividans indicated that the gene governing LAT production began near the EcoRI site and was transcribed toward pcbc. EcoRI-KpnI, KpnI, and KpnI-SstI fragments from the 4.7- kbp SstI-EcoRI fragment were introduced into M13 vectors Sequence Analysis Software Package (4). The Frames pro- and sequenced. The DNA sequence from the EcoRI site and gram was used to identify ORFs in the DNA ssequence and the derived translation products suggested by the analysis indicate rare codon choices for actinomycetes, using codon described below are shown in Fig. 2. preferences based on those described by Serno and Baltz The analysis of the DNA sequence (Frames [4]) indicated (30). that of six possible reading frames, two were candidates for S. clavuligerus lat expression vector cloning. LAT activity protein-coding regions. These ORFs had a strong Streptohad previously been recovered by cloning a 4.7-kbp SstI- myces codon bias (2, 30) with a G+C content greater than EcoRI fragment from S. clavuligerus in a StrE eptomyces-e. 90% in the third position. TestCode, a program that identifies coli shuttle vector (pdq302) and transforming Streptomyces protein-coding sequences independent of codon bias or lividans, a LAT- host (22). The shuttle vector c,ontaining the reading frame, gave identical results. The initiation codon 4.7-kbp fragment was used as the source o)f all S. cla- was selected on the basis of this analysis and identification of vuligerus DNA. A thermoinducible lambda ddl promoter- a putative ribosome binding site immediately upstream of the based E. coli expression plasmid, pow241, wa: s constructed start site. An inverted repeat that may act as a transcription to clone NcoI-BamHI fragments. Plasmid I pow382, an termination signal was also located immediately following expression vector previously described (19), w{as linearized the translation stop codon. by digestion with NcoI, and the ends were filled in by using Comparison of sequences from a protein data bank with Klenow polymerase and were ligated to f4orm plasmid the putative protein revealed a strong similarity (50%, data pow240. An -5.3-kbp BamHI-EcoRI fra.gment from not shown) to rat as well as human ornithine aminotransligated to an ferase (OAT; (25, 26), an enzyme of like function (3). One pow240 containing the vector elements was -1.5-kbp EcoRI-BamHI fragment isolated fromipit353 (18), region of human OAT (Fig. 3) with no computer-assisted creating pow241, which combines the A pl regulatory gaps that shows the highest similarity to the ORF (38% elements with a unique NcoI site at a positic n for proper identical amino acid residues; 63% related) contains the translation initiation and a unique BamHI site. OAT active-site lysine that binds the pyridoxal cofactor The DNA region encoding the putative LAIr polypeptide (Lys-292 [25, 26, 31]). This alignment places the ORF was isolated from pdq302 on contiguous -1.1-kbp NcoI- Lys-304 as the candidate for cofactor binding. These data

3 VOL. 173, 1991 LOCALIZATION OF lat AND pcbab FROM S. CLAVULIGERUS GAATTCCCCTGAACACGAAGCTGAGCAACAGCTCGTCACGCGCTCCCGAGCTGGCCATTC 61 AGGCGCAGTTCACAAAGAGCCATCGAGAGGCGTCCGAGAGAGCTGGAAGAGGGGTCCAAGA 121 GCATGGTGGGTCATTATTGTGATCCTAAAATGTCCAGTTCACCGCCATGACAGCAGAGGC >lat 181 TGGAAAG;TCCCCCATAATTC'AGCCTGATCCCCCAGGATTCTCACCCATGGGCGAAGCAG M G E A A 241 CACGCCACCCCGACGGCGATTTCTCGGACGTGGGAAACCTCCACGCTCAGGACGGCACC R HP DGD F SDV G NL SAQ0D V HQ AGGCACTTGAGCAGCATATGCTCGTCGACGGGTACGACCTCGTTCTCGACCTCGACGCCA A L E Q H M. L V D G Y D L V L D L D A S GCCGC4TGTG~AGCTACCGACGACCACTTC S G V L V DA V TQ RY L DL F SF TCTGC F A SA PLG I NPP SI V E DPAF M TCCGGGAGCTGCCGGCGGTACACGCACcGTTTTCG R EL A VAA VNK P S NP DL Y SVP CGTACGCCCGTTCGCAAGACCTTCGCCCGGGTCCTCGGCGACcCCCCGGCTGCGGCGGC Y AR FV KT FA RV LGD P RL R RL TGTTCTTCGTGGACGGCGGGGCGCTGGCCGTGGAGAACGCGCTCAAGGCGGCCCTCGACT F FV D GGA L A VE NAL KAA LD W GGAAGGCCCAGAAGCTGGGCCTCGCCGAGCCGGACACCatACCGGCTCCAGGTGCTGCATC K A0K L GL AEP DT D RL QV L HL TGGAGCGCTCGTTCCACGGCCGCAGCGGCTACACCATGTCGCTGACGAACACCGAGCCGT ER SF H GR SG YT M SLT N TEP S CCAAGACCGCCCGCTTCCCCAAGTTCGGCTGGCCACGGATCTCGTCCCCCGCCCTCCAGC K TA R FP KF GWP RI S SP AL Q ACCCGCCGGCCGAGCACACCGGCGCCAACCAGGAGGCCGAGCGACGGGCGCTGGAGGCCG P PAEHBT GA NQ E A E RRAL EA A CCCGGGAGGCGTTCGCAGCGGCGGACGGCATGATCGCCTGCTTCATCGCGGAGCCCATCC R EA FA AA DG MI AC F IA E P I AGGGCGAGGC,rGGCGACAACCACCTCAGCGCGGAG;TTcCTCCAGGCCAT'GCAGCGGCTCT G EG GD N HL SA E FL QA MQRL C GCCACGAGAACGACGCCCTGTTCGTCCTGGACGAGGTGCAGAGCGGCTGCGGCATCACCG H END A LPV L DEV Q SG CG I TG GTACCGCCTGGGCCTACCA'GCAGCTCGGC~TCCAGCCCGACCTGGTGGCCTTCGGCAAG T A WA YQ QL GLQ P DL VAFPG KK AGACCCAGGTCTGCGGGGTGATGGGCGGCGGCCGGATCGACGAGGTCCCCGAGACGTC TQ0V CG V MG GG R IDE V PEHNVV TCGCCGTCTCCTCCCGGAT'CAGCTCCACCTGGGGCGGCAACCTCGCCGACATGGTCCGCG A V S SRI S ST WGGHNL A D MVRA CCACCCGGCTGCTGGAGACGATCGAGCGCACCCAGGTCTTCGACACCGTCGTCCAGCGCG T R LL E T I E R T Q V F D T V V Q R G GCAAGTACTTCCGGGACGGCCTGGAGGACCTGGCCGCCCGCCACCCCTCCGTCGTGACCA K YF RD G LE DL AA R HP SV V TN ACGCCCGCGGCCGGGGCCTGATGTGCGCGGTCGACCTGCCGGACACCCGGACCCGCAATG AR G RG L MCA V DLP DT R TR NE AGGTGCTGCGGCTCATGTACACGGAGCACCAGGTCATCGCCCTGCCCTGCGGCGGGCGCA V L R LMY TE H QV I A LP CG GR S GCCTCCGGTTCCGCCCCGCGCTGACGATCGCGGAGCACGAGATCGACCAGGCCCTTCAGG L.LRF RP A LT IA E HE I DQ0ALQ0A CGCTGGCGAGCAGTGTCACGCCGTCGCC'GAGAGCGTCTGACGCCCGGCCGGCCGG-GTGC L AS S V TP VA E SV 450 CCCAGCGGGGCCCGGCCGGCCGCACCGCGGAACGGAACACCCCTGGCGCGTCGCCCGGTG ACACGCTCTCCGGGCGGCTCAACCGCGCCACCCCCACGGCACGTTCGCCTTCACCCGCAC >acv synthetase. GGAGAGCCCACGAATGATGTCAGCACGGTACCCGAGGACCGCAGCGGAGTGGACCACTCG M M S AR YP RT A AE WT TR CATTCAAGGAGTGTCGAGCGAGCGTTGCG'ATCTTGAAAT'GCTGCTGAAG'GACGAGTGGCG IQ0G VS SE R C DLE MLL K DE WR CAACAGGATCGCGGTACGGGACGACGACCCCGGTGTCCGTGCGACGAGGCAGCGGGACAT 720 N R IAV RD D DP G VRA TRQ0RDDI CGTCGTCGACGGGCGGGAGTACACCGCTCTGAAGGACGCGCTGCGCGCCGCCGACGGGGT V V DGR E Y TA L KD A L RAADA G 1981 CTCGGCGGGCGCGCTCGCGCTGGCCTCCCTGCACAGCGTGATGCGCGCGTACGGCCATGG S A GA LA LAS L HSV MR A YG H G 2041 CGAGCAGACGGTGGCGGCG'TTCGTCGACGC.GACGGCGAC'GGCGGAGCTGAAGACGGCCGC EQ0T VA A FV DA TA TA EL KTA A CGTGCTGCCGGTGATCGTCGACCATATCGAGCACACCCGGCTGACCTGCGCCGAGGCGAT V LP V IV D HI E H T RL T CA EA I CCGGGAGCTGGACGAGACGCTGCGCCGCAAGGACTCCTACACCCGTGCGGACGAGGTGCT R E L D ET L RRKD S Y T RA DE VL CCGGG6TTCA'CCGTGGTGCACCAGGCCGCG QR GL FDA LL VL AE R E VAL S E GCTGCCGTCGGCCCCGCTGG6TCATGGTCG'TCCGGGCGACGCGGCCCGGGGCCGGCT~GT LP0 S AP L VM V V RD DAA R GRL C CTGGACGATGGCGTACGCGGGCGAGCTGTTCGGACACGACGGTCGCCGGTGTGCTGGA W T MA YAG EL FE DT T VAG VL E GG;TCGTCCGGG-AGGTGCTC'GGGCAGTACGCCGGGCGGCCGGGACCGGGCGCCGAGAT V V RE VL GQYA G RP GD R VA E I CGAGCTGGCCTCGCGGGAGCAGCGCGAGCGGCTTCACGTGGACGCGACCGATGGCG E LA SR EQ RE R LQR W NA TD GD CTCCG6ACGG'TACACGTGGCGCTCGGTGCG F P ADQ0R L ND L VE AA VR RS P D 2581 CCGTGAGGCGGTCGTCTTCGGGACACAGCGGCTGACCTATCGGGAGGTCGAC 1260 R E A V V F G T Q R L T Y R E V D FIG. 2. Nucleotide and derived amino acid sequence of S. clavuligerus lat and partial sequence of pcbab. The putative translation start sites for LAT and ACVS are +228 and , respectively. The asterisk following LAT amino acid 457 denotes a stop codon. Overlined sequences -10 bp upstream of both ORFs are potential ribosome binding sites, and the overlined sequences following lat represent an inverted repeat. suggested that the ORF was related to other known aminotransferases and may be a structural gene (lat) for LAT activity. Expression of lat in E. coli. Most aminotransferases (transaminases), e.g., OAT and aspartate transaminase, are homopolymeric proteins with a subunit Mr of -50,000 (3). To determine whether the S. clavuligerus sequence might code for a single subunit leading to enzyme activity, we cloned the entire coding region and downstream sequences in E. coli looked for the production of LAT activity. The putative aod initiation start codon was part of an NcoI site, and the entire OV$F was inserted as a 4.4-kbp NcoI-BamHI fragment in a p '-driven expression vector (pow407; Fig. 4A). After temporature induction of the PL promoter, crude cell extracts wf,re analyzed for foreign protein; supernatants from clarified sonicates were assayed for LAT activity. As judged by SDS-PAGE, a band with an Mr of --48,000 was present (pow407 [42'C]; Fig. 4B). Also, cells containing a mock plasmid (pow241) produced the correct protein (isopenicillin N synthetase [IPNS]; 38 kda) without the induction of a 48-kDa band, indicating that the 48-kDa protein was not expressed from a vector gene. LAT activity, absent in E. coli, was present 1 and 2 h after induction of cells containing pow407. At 6 h, the activity decreased, perhaps as a result of granule formation and loss of material by centrifugation. Because the protein produced was no larger than 48 kda, we suggest that the translation stop codon in Fig. 2 is correctly assigned and that the single ORF was sufficient for LAT activity. However, because pow407 contained nearly 3 kbp of S. clavuligerus DNA downstream of the putative lat gene, we could not exclude the presence of other ORFs that be expressed in E. coli. mnight

4 6226 TOBIN ET AL. J. BACTERIOL. 242 LAT E P I Q G E G D N H L S A E F Q A M Q R L CTH E N D ALF V OAT E P I Q G E A J V V V P D P G Y M G V R E T R H Q V I LAT L D V S C G I T T A W j Y Q Q L G L Q f L A F[T OAT A I T L A R T R W L V D Y E N V RLH I L L 292 FIG. 3. Amino acid identities between LAT and OAT, located with assistance from the GCG Wordsearch, Segments, and Bestfit programs. The sequences were aligned by the pyridoxal phosphate binding site (lysine 292) in OAT. Identical amino acids are enclosed in boxes. Location of ACVS (pcbab) N terminus. The Frames analysis and other parameters (G+C content in the third position; streptomycete codon bias; TestCode program) indicated another ORF immediately downstream from lat. Smith et al. (33) reported that S. clavuligerus pcbab coding for ACVS mapped between cefe and pcbc. Comparison of the amino acid sequence encoded by DNA immediately downstream from lat with the amino acid sequences encoded by pcbab of P. chrysogenum (5, 34) and C. acremonium (9-11) revealed a region of identity between the streptomycete ORF and the corresponding fungal ORFs (Fig. 5A). That this identity continued further downstream was shown by sequencing a contiguous restriction fragment (Fig. 5B). Also, a previously reported ORF (6) located just upstream of pcbc in S. clavuligerus is similar to the C terminus of the fungal ACVS (13). Since P. chrysogenum pcbab is approximately 12 kbp (5, 34) and lat maps approximately 12 kbp from pcbc (22), we believe that pcbab has been precisely located in S. clavuligerus and that ACVS is closely related in size to the fungal protein. Furthermore, the direction of transcription for pcbab is toward pcbc in S. clavuligerus. DISCUSSION The gene for LAT (lat), previously shown to be closely linked to other genes of the P-lactam biosynthetic pathway in S. clavuligerus (22), was characterized at the molecular level A. al from the DNA sequence. Our data establish that lat is the LAT structural gene and confirm that it maps approximately midway between pcbc and cefe (19, 22, 24) (Fig. 6). Moreover, cloning of DNA leading to the production of LAT activity in E. coli is associated with the presence of a single, overproduced protein with an Mr of 48,000. LAT from S. clavuligerus is apparently derived from a single ORF and is active as either a monomer or a homopolymer, as are most of the aminotransferases (3). This contrasts with the bestcharacterized LAT protein (from Flavobacterium lutescens), which is composed of four nonidentical subunits and has a molecular weight of about 110,000 (38). Other LAT activities have been identified in Pseudomonas spp. that metabolize lysine (7), but these have yet to be characterized biochemically. In actinomycetes, LAT is found only in the P-lactamproducing species and is not needed for sustaining growth on L-lysine (21). The gram-negative bacterium Flavobacterium sp. strain SC 12,154 can presumably express LAT activity because it produces the,-lactam deacetoxycephalosporin C (32). However, although many P-lactam biosynthetic genes are known to be physically linked in this organism (33), LAT activity has not been investigated and it is not known whether lat is included in this cluster. It is noteworthy that only 1 kbp of DNA remains uncharacterized in the P-lactam cluster of Flavobacterium sp. strain SC 12,154. This would B..Kpn 9 CL 42 25' LAT Ncol - IPNS 31 - BamHI FIG. 4. Expression vector pow407 and production of LAT polypeptide in E. coli. (A) Restriction map of plasmid pow407. ci857, the constitutively expressed gene for the temperature-sensitive lambda repressor, tet, the tetracycline resistance gene, are shown as cross-hatched boxes. PLI the leftward promoter of bacteriophage lambda, is shown as an open box. The solid box indicates the ORF for lat (228 to 1601; Fig. 2). (B) SDS-PAGE. Total protein was electrophoresed and stained with Coomassie blue. Lanes: 1 and 2, JM109(pOW241) (mock plasmid); 4 and 5, JM109(pOW407); 6 and 7, JM109 (no plasmid). Protein was obtained from cultures grown at 25 C or induced at 42 C for 6 h. The proteins induced in the expression system and their positions in the gel are indicated at the right. Standard proteins are in lane 3 (sizes on the left in kilodaltons)

5 VOL. 173, 1991 LOCALIZATION OF lat AND pcbab FROM S. CLAVULIGERUS 6227 A. Strep R E A TTY Q L R E R P R H W H G P G V R R Pen K I A V V CE RE L T G E L N AIQ G NL AY L R I G I.. L P E Q L V Ceph Q H V D K T Y E E L N AM A N R L AHIH L V S G.. Q TE Q L V Strep G A A P Pen LF L D K S L IVTILGWKSGAAYVP I D P T Y PDERVRFVL Ceph [EL F L D K Tn M S1TILGIWKSGAAF P I D P G YPDER1 B. Strep L L S F R V N V N G KA Pen L L S F L E K K L P M P T R L V Q L S I P V N V N G K A D L R A L P A Ceph L L S F L E K K L P R Y M V P T R L V Q L A A Strep LER P DWG G GA[1GI T T T[G V R G E R[E I S A LG L[V S RIGD G Pen I S.... NSTERR JDLR DT EI A L GE I W A D L la R Q R S V S R N Ceph V[DV A P H K Q D G E R G N Q L E S D L A Ai G N V[D A Q D G S E Strep R L G LI V F V G VE D V F T L R L Pen NFFR L G G H S TC I RQRL VS jsisv E D V F A R T L Ceph F R L G G H S I AC I Q L I ARG TL t Q T FIG. 5. Partial amino acid comparisons of ACVSs from S. clavuligerus (Strep), P. chrysogenum (Pen), and C. acremonium (Ceph). (A) Aligned on the N-terminal portion of the P. chrysogenum A-domain core (residues 307 to 385) described by Smith et al. (34). (B) Aligned on the C-terminal portion of the A-domain core (residues 768 to 884). be insufficient to encode LAT if the enzyme is a heteropolymer similar to that of F. lutescens. Therefore, lat in the Flavobacterium spp. may not be within the,-lactam gene cluster; it may be unrelated to the actinomycete lat gene, the origin of which is uncertain. The nucleotide sequence in contiguous S. clavuligerus restriction fragments containing DNA immediately downstream from lat encodes amino acid sequences similar to those of the N and C termini of the "core" polypeptide found as three repeated domains in both C. acremonium and P. chrysogenum ACVS (5, 10, 34). The C. acremonium pebab sequence is near completion (11; see Addendum below); in the region encoding the N terminus, it contains a sequence for one of the three repeated domains that is similar to the streptomycete ORF reported here, as well as to tyrocidine A and gramicidin S synthetases. The report by Doran et al. (6) that an ORF immediately upstream of pcbc is similar to a sequence at the C terminus of the fungal ACVSs (13) also supports the close relationship between S. clavuligerus ACVS and this class of peptide synthetases. Intergenic hybridization indicates a close evolutionary relationship between the fungal ACVSs of P. chrysogenum and A. nidulans, even though the Mr of P. chrysogenum ACVS derived from the DNA sequence is >420,000 and the size by gel chromatography of purified A. nidulans ACVS is only 230,000 Da (35). Smith et al. (34) have pointed out that the Mr of ACVSs may be wrongly estimated by twofold. The C. acremonium ACVS may also be closely related to these in size and functional organization (1, 9-11; see Addendum below). Results reported by two different laboratories indicate that the ACVS from S. clavuligerus has a subunit with an Mr of approximately 300,000 that shows aggregation (1, 14), and Zhang and Demain (40) have reported a subunit size of 360,000 Da. Both Zhang and Demain (40) and Baldwin et al. (1) report identical migration patterns on SDS-PAGE for Cephalosporium and Streptomyces ACVSs. Jensen et al. (14) have also shown that ACVS may be multimeric with two large (identical?) subunits but may also contain one small subunit per complex. Because our DNA sequence is incomplete, we cannot rule out that ACVS from S. clavuligerus is composed of subunits with individual enzyme activities. However, we anticipate that it will prove to be closely related in size to the fungal enzyme, and therefore, we propose that the region between lat and pcbc in S. clavuligerus represents a single ORF coding for a multifunctional ACVS. This does not preclude the possibility that the ACVS is multimeric with a small thioesterase subunit (Mr -32,000) as proposed by Jensen et al. (14). EJ -.,-. -_.,_ IPNS ACVS LAT DACS EPI DAOCS FIG. 6. Linear map of the P-lactam biosynthetic gene cluster in S. clavuligerus. The open boxes are the approximatesizes of each coding region. The gene products (and genes) are: IPNS, pcbc; ACVS, pcbab; LAT, lat; deacetylcephalosporin C synthetase (DACS), ceff (17); isopenicillin N epimerase (EPI), cefd (18); and deacetoxycephalosporin C synthetase (DAOCS), cefe (18). Arrows represent direction of transcription and derived amino acid sequence. The transcript for ACVS is represented as a dashed line in the region where the DNA sequence is not available but is proposed to code for a single polypeptide. The dashed region downstream from deacetoxycephalosporin C synthetase (ceje) has been reported to be part of a large transcript and contains an ORF of unknown function (18). The region between deacetylcephalosporin C synthetase and isopenicillin N epimerase has been reported to contain the gene for O-carbamoyl deacetylcephalosporin C hydroxylase (33), but the DNA sequence was not reported.

6 6228 TOBIN ET AL. In S. clavuligerus, pcbc and pcbab are transcribed in the same direction, yet in the fungi they are transcribed in an opposite orientation (9, 10). Also, lat and pcbab map between pcbc and ceje in the gram-positive procaryote, but pcbab lies outside this region in the gram-negative Flavobacterium sp. strain SC 12,154 (33), and the location of lat is unknown. If the pathway genes from actinomycetes (specifically S. clavuligerus), Flavobacterium sp. strain SC 12,154, and the fungi are related evolutionarily by horizontal transfer of the DNA as suggested (12, 24, 33), then they have undergone rearrangement yet remained tightly linked. The position of genes and the placement of promoter sequences may be a way to regulate the synthesis of the blocks of enzymes during antibiotic formation. We have already shown that the expression of cefd and cefe as a single message may coordinately control the enzymes necessary for cephalosporin (cephamycin) biosynthesis after growth has ceased (18). Two reports have described mutants of S. clavuligerus that may involve the regulation of lat. Romero et al. (28) described mutants that have no (nccl) or reduced (ncal) LAT activity. They suggested that reduction of cephamycin biosynthesis was due to a deficiency of LAT, yet the mutations may not be within lat since these mutants were also pleiotropic with respect to the production of other enzymes in the cephamycin C pathway or the clavulanic acid pathway. For example, arginase, a proposed enzyme of the clavulanic acid pathway (an oxycephem) was also reduced in nccl, the mutant completely blocked in cephamycin C production. Because the levels of epimerase, expandase, and IPNS were also reduced in this mutant, they also suggested that these enzymes might be regulated in a concerted manner. Yet pcbab, which maps between lat and pcbc, was seemingly not regulated because the level of ACV (and therefore the level of ACVS?) did not differ from that of the cephamycin-producing parent strain. Although an inverted repeat sequence indicative of a transcription terminator is present between lat and pcbab (Fig. 1), transcription analysis with specific gene probes is needed to determine whether pcbab and lat can be coordinately expressed. Moreover, complementation of the blocked mutants may help determine the regions of biosynthetic control. Piret et al. (27) reported that S. clavuligerus NP1, a mutant severely impaired in cephalosporin synthesis and with lower (but not absent) ACVS and IPNS activity, was complemented by DNA that mapped close to pcbc. Interestingly, although pcbc from Streptomyces griseus (8) and S. clavuligerus (13) is transcribed as a monocistronic message, IPNS is down-regulated in the mutant and activity is only partially restored in the transformants. We judge from the published restriction fragment analysis that the complementing DNA fragment contained lat as well as one-third of the putative pcbab region; therefore, the lesion in S. clavuligerus NP1 may have been present in the lat coding region or within the lat promoter. While further sequence, transcriptional, and biochemical analyses are needed to establish whether lat has a role in the regulation of antibiotic synthesis, cloning lat on a multicopy plasmid without supplying exogenous lysine to the host may lead to lysine auxotrophy (22) resulting from excessive lysine catabolism. This may explain the anomalous plasmid copy number effect seen with pnbr1 reported by Piret et al. (27). LAT activity, and its gene, are specific to P-lactamproducing actinomycetes (20-22), and LAT now represents the first enzyme directed toward cephamycin C synthesis. Like ACVS and IPNS, two other early enzymes in the J. BACTERIOL. biosynthetic pathway, LAT in N. lactamdurans (15) and S. clavuligerus (20, 21) peaks in activity before exponential growth ends (prior to cephamycin production) and then declines. Other activities, epimerase and expandase, are subsequently produced and sustained during antibiotic production. As pointed out by Piret et al. (27), ACVS is a major target for nutritional control of cephalosporin biosynthesis, but it now seems that regulation of LAT and the possible coordinated regulation of LAT and ACVS should be considered in the overall control system. ACKNOWLEDGMENTS We thank Susan E. Jensen for valuable discussions and disclosing results before publication. 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