Vol. 173, No. 19

Localization of the Lysine £-Aminotransferase (lat) and8-(L-oL-Aminoadipyl)-L-Cysteinyl-D-Valine Synthetase (pcbAB)

Genes from Streptomyces clavuligerus and Production ofLysine 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 andDepartments 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 stepin 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 ofLAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a singleopen reading frame encoding a polypeptide with an -M, 49,000. Expression of this coding sequence inEscherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived froma single polypeptide. A second open reading frame began immediately downstream from lat. Comparison ofthis partial sequence with the sequences of 8-(L-a-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases fromPenicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gram-icidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of theputative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies 12 kbp and codesfor a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the I8-lactambiosynthetic cluster between pcbC and cefE (-25 kbp) is nearly complete.

Many naturally occurring P-lactam antibiotics (isopenicil-lin N, cephalosporin C, and cephamycins) contain a sidechain derived from a-aminoadipic acid (a-AAA). When theproducer is a filamentous fungus (Penicillium chrysogenum,Aspergillus nidulans, Cephalosporium acremonium), thisamino acid is formed as an obligate intermediate in thesynthesis of lysine. However, in procaroytes, lysine issynthesized via the diaminopimelic acid pathway without theproduction of a-AAA (for reviews, see references 23 and36). Whitney et al. (37) established that in the P-lactam-producing Streptomyces spp., ot-AAA is derived from thebreakdown of lysine. Although the catabolism of lysine inbacteria is diverse, conversion of lysine to a-AAA byremoval of the E-amino group was tentatively linked inStreptomyces lipmanii to an aminotransferase (16) whichwas later conclusively shown to be present in Nocardia(previously Streptomyces) lactamdurans (15).

Recently, Madduri et al. (21) reported that in these acti-nomycetes, L-lysine-e-aminotransferase (LAT) is specific toP-lactam (cephamycin C) producers and provides the pre-cursor for antibiotic synthesis; the pathway via cadaverine isobligatory for lysine catabolism (Fig. 1). Moreover, a genegoverning LAT production was found exclusively in Strep-tomyces spp. producing P-lactams (22) and mapped withinthe P-lactam biosynthetic gene cluster of Streptomycesclavuligerus (18, 24, 33). Romero et al. (28) had also de-scribed mutants of S. clavuligerus that have no (nccl) orreduced (ncal) LAT activity and suggested that reduction ofcephamycin 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 thisreport. The open reading frame (ORF) was cloned in anEscherichia coli expression vector to investigate the proper-ties of the putative protein. Sequencing of the region imme-diately downstream from the lat gene identified another ORFthat has similarities to 8-(L-(x-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetases (ACVSs) encoded by pcbAB fromP. chrysogenum and C. acremonium. The regulation of,B-lactam biosynthesis in the actinomycetes by lat is consid-ered in light of the close physical linkage between lat andpcbAB.

MATERIALS AND METHODS

Bacterial strains, growth conditions, DNA manipulations,and DNA sequence determination. E. coli strains were grownin TY broth (18) supplemented with either 5 ,ug of tetracy-cline per ml or 80 jig of ampicillin per ml under standardconditions, except as noted.

Restriction endonuclease digestion, ligation, and determi-nation of DNA restriction fragments by agarose or acryl-amide gel electrophoresis followed established procedures(29). DNA fragments were subcloned into pUC or M13vectors for further analysis. DNA sequencing was per-formed by the dideoxy method with TAQ-TRACK (PromegaBiotec) and followed the supplier's recommendations. Frag-ments were sequenced either directly from the plasmid orafter subcloning into M13 bacteriophage vectors, using for-ward and reverse sequencing primers for pUC and M13templates, as well as several custom DNA oligonucleotideprimers. Custom DNA oligonucleotide primers were synthe-sized by using an Applied Biosystems DNA Synthesizer(model 380A) according to the manufacturer's instructions.Computing. DNA sequences were analyzed with the GCG

6223

JOURNAL OF BACTERIOLOGY, Oct. 1991, p. 6223-62290021-9193/91/196223-07$02.00/0Copyright X) 1991, American Society for Microbiology

6224 TOBIN ET AL.

CH2NH2

(CH2)3

HCNH2

R.CO.COOH ICOOH

R.CHNH2.COOH

1-Piperideine--6-carboxylate COOH

2H20 J

2H 4COOH

a-Amino (CH2)3adipate I

HCNH2

COOH

P-lactamantibiotics "

Lysine

Co2

CH2NH2

(CH2)3

CH2NH2

Cadaverine

R.CO.COOH2

4RCHNH2.COOH + H20

1-Piperideine

2H20

2H

CH2NH2

I.

" COOH

CO2 +H20 + NH3

FIG. 1. Catabolic pathways for lysine in StrelEnzyme activities are as follows: 1, lysine decarboxverine aminotransferase; 3, 1-piperideine-6-carboxylnase. The pathway on the right is essential forStreptomyces spp. on lysine (7); the pathway on theonly in ,-lactam-producing Streptomyces spp. Anoth4the catabolism of a-AAA (---), exists in S. lividansclavuligerus. Bracketed intermediate is hypothetical

Sequence Analysis Software Package (4). Thegram was used to identify ORFs in the DNA sindicate rare codon choices for actinomycetes,preferences based on those described by Ser(30).

S. clavuligerus lat expression vector cloning.had previously been recovered by cloning aEcoRI fragment from S. clavuligerus in a StrEcoli shuttle vector (pDQ302) and transforminglividans, a LAT- host (22). The shuttle vector c4.7-kbp fragment was used as the source ovuligerus DNA. A thermoinducible lambda dbased E. coli expression plasmid, pOW241, wa:to clone NcoI-BamHI fragments. Plasmid Iexpression vector previously described (19), wby digestion with NcoI, and the ends were filleKlenow polymerase and were ligated to f4pOW240. An -5.3-kbp BamHI-EcoRI frapOW240 containing the vector elements was

-1.5-kbp EcoRI-BamHI fragment isolated fromcreating pOW241, which combines the A p

elements with a unique NcoI site at a positictranslation initiation and a unique BamHI site.The DNA region encoding the putative LAI

was isolated from pDQ302 on contiguous -1

ScaI and -3.3-kbp ScaI-BamHI fragments and was clonedin the -5.8-kbp NcoI-BamHI fragment from pOW241, re-sulting in plasmid pOW407.Gene expression in E. coli. For LAT production, E. coli

JM109 cells containing pOW407 were grown overnight at25°C, diluted 20-fold, induced at 42°C for 6 h, and examinedby phase-contrast microscopy for the appearance of gran-ules. Cell extracts were prepared as described previously(19) and subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE).LAT enzyme assay. Cells were harvested from a culture by

centrifugation 1, 2, and 6 h after induction at 42°C. Theywere resuspended in 0.2 M potassium phosphate buffer (pH7.5) and disrupted with ultrasound (six 10-s pulses with1-min cooling intervals). The supernatant fluid from centrif-ugation (20 min, 10,000 x g, 4°C) was assayed for LATactivity by measuring the formation of 1-piperideine-6-car-boxylate with an o-aminobenzaldehyde reagent as describedpreviously (20).

Nucleotide sequence accession number. These data havebeen submitted to GenBank and have been assigned acces-sion number M64834.

RESULTS

Sequence and molecular characterization of lat. Cloning in8-Aminovalerate S. lividans, a host that does not produce 1-lactam antibiot-

ics, 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 shownto reside on a single 4.7-kbp SstI-EcoRI fragment from S.

ptomyces spp clavuligerus. An indication that this gene encoded the aminoylase; 2, cada- acid sequence of the enzyme rather than serving a regulatoryate dehydroge- function might come from DNA sequence analysis. Al-the growth of though the exact location of the gene for LAT was unknown,left is present spontaneous deletions of pDQ301d (22) in a pool of primary

erpathway, for transformants and concurrent loss of LAT activity in S.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

Frames pro- and sequenced. The DNA sequence from the EcoRI site andsequence and the derived translation products suggested by the analysisusing codon described below are shown in Fig. 2.

no and Baltz The analysis of the DNA sequence (Frames [4]) indicatedthat of six possible reading frames, two were candidates for

LAT activity protein-coding regions. These ORFs had a strong Strepto-4.7-kbp SstI- myces codon bias (2, 30) with a G+C content greater thaneptomyces-E. 90% in the third position. TestCode, a program that identifiesStreptomyces protein-coding sequences independent of codon bias or,ontaining the reading frame, gave identical results. The initiation codon)f all S. cla- was selected on the basis of this analysis and identification ofDL promoter- a putative ribosome binding site immediately upstream of thes constructed start site. An inverted repeat that may act as a transcriptionpOW382, an termination signal was also located immediately following{as linearized the translation stop codon.d in by using Comparison of sequences from a protein data bank withorm plasmid the putative protein revealed a strong similarity (50%, data.gment from not shown) to rat as well as human ornithine aminotrans-ligated to an ferase (OAT; (25, 26), an enzyme of like function (3). OneipIT353 (18), region of human OAT (Fig. 3) with no computer-assistedL regulatory gaps that shows the highest similarity to the ORF (38%n for proper identical amino acid residues; 63% related) contains the

OAT active-site lysine that binds the pyridoxal cofactorr polypeptide (Lys-292 [25, 26, 31]). This alignment places the ORF.1-kbp NcoI- Lys-304 as the candidate for cofactor binding. These data

J. BACTERIOL.

LOCALIZATION OF lat AND pcbAB FROM S. CLAVULIGERUS 6225

1 GAATTCCCCTGAACACGAAGCTGAGCAACAGCTCGTCACGCGCTCCCGAGCTGGCCATTC

61 AGGCGCAGTTCACAAAGAGCCATCGAGAGGCGTCCGAGAGAGCTGGAAGAGGGGTCCAAGA

121 GCATGGTGGGTCATTATTGTGATCCTAAAATGTCCAGTTCACCGCCATGACAGCAGAGGC

>lat181 TGGAAAG;TCCCCCATAATTC'AGCCTGATCCCCCAGGATTCTCACCCATGGGCGAAGCAG

M G E A A

241 CACGCCACCCCGACGGCGATTTCTCGGACGTGGGAAACCTCCACGCTCAGGACGGCACCR HP DGD F SDV G NL SAQ0D V HQ

10 20

301 AGGCACTTGAGCAGCATATGCTCGTCGACGGGTACGACCTCGTTCTCGACCTCGACGCCAA L E Q H M. L V D G Y D L V L D L D A S

30 40

361 GCCGC4TGTG~AGCTACCGACGACCACTTCS G V L V DA V TQ RY L DL F SF

50 60

421 TCTGCF A SA PLG I NPP SI V E DPAF M

70 80

481 TCCGGGAGCTGCCGGCGGTACACGCACcGTTTTCGR EL A VAA VNK P S NP DL Y SVP

90 100

541 CGTACGCCCGTTCGCAAGACCTTCGCCCGGGTCCTCGGCGACcCCCCGGCTGCGGCGGCY AR FV KT FA RV LGD P RL R RL

110 120

601 TGTTCTTCGTGGACGGCGGGGCGCTGGCCGTGGAGAACGCGCTCAAGGCGGCCCTCGACTF FV D GGA L A VE NAL KAA LD W

130 140

661 GGAAGGCCCAGAAGCTGGGCCTCGCCGAGCCGGACACCatACCGGCTCCAGGTGCTGCATCK A0K L GL AEP DT D RL QV L HL

150 160

721 TGGAGCGCTCGTTCCACGGCCGCAGCGGCTACACCATGTCGCTGACGAACACCGAGCCGTER SF H GR SG YT M SLT N TEP S

170 180

781 CCAAGACCGCCCGCTTCCCCAAGTTCGGCTGGCCACGGATCTCGTCCCCCGCCCTCCAGCK TA R FP KF GWP RI S SP AL Q

190 200

841 ACCCGCCGGCCGAGCACACCGGCGCCAACCAGGAGGCCGAGCGACGGGCGCTGGAGGCCGP PAEHBT GA NQ E A E RRAL EA A

210 220

901 CCCGGGAGGCGTTCGCAGCGGCGGACGGCATGATCGCCTGCTTCATCGCGGAGCCCATCCR EA FA AA DG MI AC F IA E P I

230 240

961 AGGGCGAGGC,rGGCGACAACCACCTCAGCGCGGAG;TTcCTCCAGGCCAT'GCAGCGGCTCTG EG GD N HL SA E FL QA MQRL C

250 260

1021 GCCACGAGAACGACGCCCTGTTCGTCCTGGACGAGGTGCAGAGCGGCTGCGGCATCACCG

1081

1141

H END A LPV L DEV Q SG CG I TG270 280

GTACCGCCTGGGCCTACCA'GCAGCTCGGC~TCCAGCCCGACCTGGTGGCCTTCGGCAAGT A WA YQ QL GLQ P DL VAFPG KK

290 300

AGACCCAGGTCTGCGGGGTGATGGGCGGCGGCCGGATCGACGAGGTCCCCGAGACGTCTQ0V CG V MG GG R IDE V PEHNVV

310 320

1201 TCGCCGTCTCCTCCCGGAT'CAGCTCCACCTGGGGCGGCAACCTCGCCGACATGGTCCGCGA V S SRI S ST WGGHNL A D MVRA

330 340

60

120

240

300

360

420

480

540

600

660

1261

1321

1381

1441

1501

1561

1621

1681

1741

1801

CCACCCGGCTGCTGGAGACGATCGAGCGCACCCAGGTCTTCGACACCGTCGTCCAGCGCGT R LL E T I E R T Q V F D T V V Q R G

350 360

GCAAGTACTTCCGGGACGGCCTGGAGGACCTGGCCGCCCGCCACCCCTCCGTCGTGACCAK YF RD G LE DL AA R HP SV V TN

370 380

ACGCCCGCGGCCGGGGCCTGATGTGCGCGGTCGACCTGCCGGACACCCGGACCCGCAATGAR G RG L MCA V DLP DT R TR NE

390 400

AGGTGCTGCGGCTCATGTACACGGAGCACCAGGTCATCGCCCTGCCCTGCGGCGGGCGCAV L R LMY TE H QV I A LP CG GR S

410 420

GCCTCCGGTTCCGCCCCGCGCTGACGATCGCGGAGCACGAGATCGACCAGGCCCTTCAGGL.LRF RP A LT IA E HE I DQ0ALQ0A

430 440

CGCTGGCGAGCAGTGTCACGCCGTCGCC'GAGAGCGTCTGACGCCCGGCCGGCCGG-GTGCL AS S V TP VA E SV

450

CCCAGCGGGGCCCGGCCGGCCGCACCGCGGAACGGAACACCCCTGGCGCGTCGCCCGGTG

ACACGCTCTCCGGGCGGCTCAACCGCGCCACCCCCACGGCACGTTCGCCTTCACCCGCAC

>acv synthetase.GGAGGAGCCCACGAATGATGTCAGCACGGTACCCGAGGACCGCAGCGGAGTGGACCACTCG

M M S AR YP RT A AE WT TR

CATTCAAGGAGTGTCGAGCGAGCGTTGCG'ATCTTGAAAT'GCTGCTGAAG'GACGAGTGGCGIQ0G VS SE R C DLE MLL K DE WR

186.1 CAACAGGATCGCGGTACGGGACGACGACCCCGGTGTCCGTGCGACGAGGCAGCGGGACAT

720 N R IAV RD D DP G VRA TRQ0RDDI

1921 CGTCGTCGACGGGCGGGAGTACACCGCTCTGAAGGACGCGCTGCGCGCCGCCGACGGGGTV V DGR E Y TA L KD A L RAADA G

780

840

900

1981 CTCGGCGGGCGCGCTCGCGCTGGCCTCCCTGCACAGCGTGATGCGCGCGTACGGCCATGGS A GA LA LAS L HSV MR A YG H G

2041 CGAGCAGACGGTGGCGGCG'TTCGTCGACGC.GACGGCGAC'GGCGGAGCTGAAGACGGCCGCEQ0T VA A FV DA TA TA EL KTA A

1320

1380

1440

1500

1560

1620

1680

1740

1800

1860

1920

1980

2040

2100

2101 CGTGCTGCCGGTGATCGTCGACCATATCGAGCACACCCGGCTGACCTGCGCCGAGGCGAT 2160

V LP V IV D HI E H T RL T CA EA I

2161

960

2221

1020 2281

2341

1080

2401

1140 2461

1200 2521

CCGGGAGCTGGACGAGACGCTGCGCCGCAAGGACTCCTACACCCGTGCGGACGAGGTGCTR E L D ET L RRKD S Y T RA DE VL

CCGGG6TTCA'CCGTGGTGCACCAGGCCGCGQR GL FDA LL VL AE R E VAL S E

GCTGCCGTCGGCCCCGCTGG6TCATGGTCG'TCCGGGCGACGCGGCCCGGGGCCGGCT~GTLP0 S AP L VM V V RD DAA R GRL C

CTGGACGATGGCGTACGCGGGCGAGCTGTTCGGACACGACGGTCGCCGGTGTGCTGGAW T MA YAG EL FE DT T VAG VL E

GG;TCGTCCGGG-AGGTGCTC'GGGCAGTACGCCGGGCGGCCGGGACCGGGCGCCGAGATV V RE VL GQYA G RP GD R VA E I

CGAGCTGGCCTCGCGGGAGCAGCGCGAGCGGCTTCACGTGGACGCGACCGATGGCGE LA SR EQ RE R LQR W NA TD GD

CTCCG6ACGG'TACACGTGGCGCTCGGTGCGF P ADQ0R L ND L VE AA VR RS P D

2581 CCGTGAGGCGGTCGTCTTCGGGACACAGCGGCTGACCTATCGGGAGGTCGAC1260 R E A V V F G T Q R L T Y R E V D

2220

2280

2340

2400

2460

2520

2580

2632

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 + 1754, 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 ami-

notransferases and may be a structural gene (lat) for LAT

activity.

Expression of lat in E. coli. Most aminotransferases (trans-

aminases), e.g., OAT and aspartate transaminase, are ho-

mopolymeric 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

aod looked for the production of LAT activity. The putativeinitiation 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 tem-

porature induction of the PL promoter, crude cell extracts

wf,re analyzed for foreign protein; supernatants from clari-

fied 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 (isopenicil-lin 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 mnightbe expressed in E. coli.

VOL. 173, 1991

6226 TOBIN ET AL.

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

230

304LAT L D V S C G I T T A W j Y Q Q L G L Q f L A F[TOAT A I T L A R T R W L V D Y E N V RLH I L L

292FIG. 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 inboxes.

Location of ACVS (pcbAB) N terminus. The Frames anal-ysis and other parameters (G+C content in the third posi-tion; streptomycete codon bias; TestCode program) indi-cated another ORF immediately downstream from lat. Smithet al. (33) reported that S. clavuligerus pcbAB coding forACVS mapped between cefE and pcbC. Comparison of theamino acid sequence encoded by DNA immediately down-stream from lat with the amino acid sequences encoded bypcbAB of P. chrysogenum (5, 34) and C. acremonium (9-11)revealed a region of identity between the streptomyceteORF and the corresponding fungal ORFs (Fig. 5A). That thisidentity continued further downstream was shown by se-quencing a contiguous restriction fragment (Fig. 5B). Also, apreviously reported ORF (6) located just upstream of pcbCin S. clavuligerus is similar to the C terminus of the fungalACVS (13). Since P. chrysogenum pcbAB is approximately12 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 thefungal protein. Furthermore, the direction of transcriptionfor pcbAB is toward pcbC in S. clavuligerus.

DISCUSSIONThe gene for LAT (lat), previously shown to be closely

linked to other genes of the P-lactam biosynthetic pathway inS. clavuligerus (22), was characterized at the molecular level

A.

BamHI

from the DNA sequence. Our data establish that lat is theLAT structural gene and confirm that it maps approximatelymidway between pcbC and cefE (19, 22, 24) (Fig. 6).Moreover, cloning ofDNA leading to the production ofLATactivity 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 isactive as either a monomer or a homopolymer, as are most ofthe aminotransferases (3). This contrasts with the best-characterized LAT protein (from Flavobacterium lutes-cens), which is composed of four nonidentical subunits andhas a molecular weight of about 110,000 (38). Other LATactivities have been identified in Pseudomonas spp. thatmetabolize lysine (7), but these have yet to be characterizedbiochemically.

In actinomycetes, LAT is found only in the P-lactam-producing species and is not needed for sustaining growth onL-lysine (21). The gram-negative bacterium Flavobacteriumsp. strain SC 12,154 can presumably express LAT activitybecause it produces the ,-lactam deacetoxycephalosporin C(32). However, although many P-lactam biosynthetic genesare known to be physically linked in this organism (33), LATactivity has not been investigated and it is not knownwhether lat is included in this cluster. It is noteworthy thatonly 1 kbp ofDNA remains uncharacterized in the P-lactamcluster of Flavobacterium sp. strain SC 12,154. This would

B.CL

al42 °25' 42°25 °42° 25°

.Kpn9

66.2-

4 LAT45-.....

Ncol - IPNS

31 -

21.5-

FIG. 4. Expression vector pOW407 and production of LAT polypeptide in E. coli. (A) Restriction map of plasmid pOW407. cI857, theconstitutively expressed gene for the temperature-sensitive lambda repressor, tet, the tetracycline resistance gene, are shown ascross-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°Cfor 6 h. The proteins induced in the expression system and their positions in the gel are indicated at the right. Standard proteins are in lane3 (sizes on the left in kilodaltons).

J. BACTERIOL.

LOCALIZATION OF lat AND pcbAB FROM S. CLAVULIGERUS

A.

Strep R E A T Q R ETY R P R H WL H G P G V R RPen 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 VCeph 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 PPen LF L D K S L IVTILGWKSGAAYVP I D P T Y PDERVRFVLCeph [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 KAPen 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 GPen 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[DA 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 LCeph 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 onthe C-terminal portion of the A-domain core (residues 768 to 884).

be insufficient to encode LAT if the enzyme is a heteropoly-mer similar to that of F. lutescens. Therefore, lat in theFlavobacterium spp. may not be within the ,-lactam genecluster; it may be unrelated to the actinomycete lat gene, theorigin of which is uncertain.The nucleotide sequence in contiguous S. clavuligerus

restriction fragments containing DNA immediately down-stream from lat encodes amino acid sequences similar tothose of the N and C termini of the "core" polypeptidefound as three repeated domains in both C. acremonium andP. chrysogenum ACVS (5, 10, 34). The C. acremoniumpebAB sequence is near completion (11; see Addendumbelow); in the region encoding the N terminus, it contains asequence for one of the three repeated domains that issimilar to the streptomycete ORF reported here, as well as totyrocidine A and gramicidin S synthetases. The report byDoran et al. (6) that an ORF immediately upstream of pcbCis similar to a sequence at the C terminus of the fungalACVSs (13) also supports the close relationship between S.clavuligerus ACVS and this class of peptide synthetases.

Intergenic hybridization indicates a close evolutionaryrelationship between the fungal ACVSs of P. chrysogenumand A. nidulans, even though the Mr of P. chrysogenumACVS derived from the DNA sequence is >420,000 and the

EJ -.,-.

size by gel chromatography of purified A. nidulans ACVS isonly 230,000 Da (35). Smith et al. (34) have pointed out thatthe Mr of ACVSs may be wrongly estimated by twofold. TheC. acremonium ACVS may also be closely related to these insize and functional organization (1, 9-11; see Addendumbelow). Results reported by two different laboratories indi-cate that the ACVS from S. clavuligerus has a subunit withan Mr of approximately 300,000 that shows aggregation (1,14), and Zhang and Demain (40) have reported a subunit sizeof 360,000 Da. Both Zhang and Demain (40) and Baldwin etal. (1) report identical migration patterns on SDS-PAGE forCephalosporium and Streptomyces ACVSs. Jensen et al.(14) have also shown that ACVS may be multimeric with twolarge (identical?) subunits but may also contain one smallsubunit per complex. Because our DNA sequence is incom-plete, we cannot rule out that ACVS from S. clavuligerus iscomposed of subunits with individual enzyme activities.However, we anticipate that it will prove to be closelyrelated in size to the fungal enzyme, and therefore, wepropose that the region between lat and pcbC in S. cla-vuligerus represents a single ORF coding for a multifunc-tional ACVS. This does not preclude the possibility that theACVS is multimeric with a small thioesterase subunit (Mr-32,000) as proposed by Jensen et al. (14).

-_ .,_

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 codingregion. 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 oftranscription and derived amino acid sequence. The transcript for ACVS is represented as a dashed line in the region where the DNAsequence is not available but is proposed to code for a single polypeptide. The dashed region downstream from deacetoxycephalosporin Csynthetase (cejE) has been reported to be part of a large transcript and contains an ORF of unknown function (18). The region betweendeacetylcephalosporin C synthetase and isopenicillin N epimerase has been reported to contain the gene for O-carbamoyl deacetylcephalos-porin C hydroxylase (33), but the DNA sequence was not reported.

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6228 TOBIN ET AL.

In S. clavuligerus, pcbC and pcbAB are transcribed in thesame direction, yet in the fungi they are transcribed in anopposite orientation (9, 10). Also, lat and pcbAB mapbetween pcbC and ceJE in the gram-positive procaryote, butpcbAB lies outside this region in the gram-negative Flavo-bacterium sp. strain SC 12,154 (33), and the location of lat isunknown. If the pathway genes from actinomycetes (specif-ically S. clavuligerus), Flavobacterium sp. strain SC 12,154,and the fungi are related evolutionarily by horizontal transferof the DNA as suggested (12, 24, 33), then they haveundergone rearrangement yet remained tightly linked. Theposition of genes and the placement of promoter sequencesmay be a way to regulate the synthesis of the blocks ofenzymes during antibiotic formation. We have alreadyshown that the expression of cefD and cefE as a singlemessage may coordinately control the enzymes necessaryfor cephalosporin (cephamycin) biosynthesis after growthhas 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 cephamycinbiosynthesis was due to a deficiency of LAT, yet themutations may not be within lat since these mutants werealso pleiotropic with respect to the production of otherenzymes in the cephamycin C pathway or the clavulanic acidpathway. For example, arginase, a proposed enzyme of theclavulanic acid pathway (an oxycephem) was also reduced innccl, the mutant completely blocked in cephamycin Cproduction. Because the levels of epimerase, expandase,and IPNS were also reduced in this mutant, they alsosuggested that these enzymes might be regulated in a con-certed manner. Yet pcbAB, which maps between lat andpcbC, was seemingly not regulated because the level ofACV(and therefore the level of ACVS?) did not differ from that ofthe cephamycin-producing parent strain. Although an in-verted repeat sequence indicative of a transcription termina-tor is present between lat and pcbAB (Fig. 1), transcriptionanalysis with specific gene probes is needed to determinewhether pcbAB and lat can be coordinately expressed.Moreover, complementation of the blocked mutants mayhelp determine the regions of biosynthetic control.

Piret et al. (27) reported that S. clavuligerus NP1, a mutantseverely impaired in cephalosporin synthesis and with lower(but not absent) ACVS and IPNS activity, was comple-mented by DNA that mapped close to pcbC. Interestingly,although pcbC from Streptomyces griseus (8) and S. cla-vuligerus (13) is transcribed as a monocistronic message,IPNS is down-regulated in the mutant and activity is onlypartially restored in the transformants. We judge from thepublished restriction fragment analysis that the complement-ing DNA fragment contained lat as well as one-third of theputative pcbAB region; therefore, the lesion in S. cla-vuligerus NP1 may have been present in the lat codingregion or within the lat promoter. While further sequence,transcriptional, and biochemical analyses are needed toestablish whether lat has a role in the regulation of antibioticsynthesis, cloning lat on a multicopy plasmid without sup-plying exogenous lysine to the host may lead to lysineauxotrophy (22) resulting from excessive lysine catabolism.This may explain the anomalous plasmid copy number effectseen with pNBR1 reported by Piret et al. (27).LAT activity, and its gene, are specific to P-lactam-

producing actinomycetes (20-22), and LAT now representsthe first enzyme directed toward cephamycin C synthesis.Like ACVS and IPNS, two other early enzymes in the

biosynthetic pathway, LAT in N. lactamdurans (15) and S.clavuligerus (20, 21) peaks in activity before exponentialgrowth ends (prior to cephamycin production) and thendeclines. Other activities, epimerase and expandase, aresubsequently produced and sustained during antibiotic pro-duction. As pointed out by Piret et al. (27), ACVS is a majortarget for nutritional control of cephalosporin biosynthesis,but it now seems that regulation of LAT and the possiblecoordinated regulation of LAT and ACVS should be consid-ered in the overall control system.

ACKNOWLEDGMENTS

We thank Susan E. Jensen for valuable discussions and disclosingresults before publication.

This work was supported by the Lilly Research Laboratories (adivision of Eli Lilly & Co.) and the Natural Sciences and Engineer-ing Research Council of Canada.

ADDENDUM

At the time this report was submitted, Martin and hiscolleagues (9) confirmed the linkage ofpcbAB to pcbC in C.acremonium reported previously (10) and reported an ORFencoding an ACVS polypeptide with a molecular weight of414,791 with three related domains.

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