Apr 22, 1991 - PAUL L. SKATRUD,1 L. C. VINING,3 COLIN STUTTARD,2 AND JAMES R. MILLER'* ...... Simmaco, M., R. A. John, C. Barra, and F. Bossa. 1986 ...
JOURNAL OF BACTERIOLOGY, Oct. 1991, p. 6223-6229
Vol. 173, No. 19
0021-9193/91/196223-07$02.00/0 Copyright X) 1991, American Society for Microbiology
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 *
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
Corresponding author. 6223
6224
TOBIN ET AL.
J. BACTERIOL. CH2NH2
(CH2)3 Lysine HCNH2
I
R.CO.COOH
COOH
Co2
R.CHNH2.COOH
CH2NH2
1-Piperideine-6-carboxylate
2H20
J
2H
4
Cadaverine
(CH2)3
COOH
CH2NH2
R.CO.COOH 2
COOH
a-Amino
adipate
4
RCHNH2.COOH
+
H20
(CH2)3
I
1-Piperideine
HCNH2 2H20
COOH
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 M64834.
2H
P-lactam
antibiotics
RESULTS
CH2NH2
" I.
"
8-Aminovalerate
COOH CO2 + H20 + NH3
FIG. 1. Catabolic pathways for lysine in Strelptomyces spp Enzyme activities are as follows: 1, lysine decarbox ylase; 2, cadaverine aminotransferase; 3, 1-piperideine-6-carboxyl ate dehydrogenase. The pathway on the right is essential for the growth of Streptomyces spp. on lysine (7); the pathway on the left is present only in ,-lactam-producing Streptomyces spp. Anoth4 er pathway, for the catabolism of a-AAA (---), exists in S. lividans Lbut not in S. clavuligerus. Bracketed intermediate is hypothetical
Sequence Analysis Software Package (4). The Frames program was used to identify ORFs in the DNA ssequence and indicate rare codon choices for actinomycetes, using codon preferences based on those described by Serno and Baltz
(30). S. clavuligerus lat expression vector cloning. LAT activity had previously been recovered by cloning a 4.7-kbp SstIEcoRI fragment from S. clavuligerus in a StrE eptomyces-E. coli shuttle vector (pDQ302) and transforming Streptomyces lividans, a LAT- host (22). The shuttle vector c,ontaining the 4.7-kbp fragment was used as the source o)f all S. clavuligerus DNA. A thermoinducible lambda dDL promoterbased E. coli expression plasmid, pOW241, wa: s constructed to clone NcoI-BamHI fragments. Plasmid IpOW382, an expression vector previously described (19), w{as linearized by digestion with NcoI, and the ends were fille d in by using Klenow polymerase and were ligated to f4orm plasmid pOW240. An -5.3-kbp BamHI-EcoRI fra.gment from pOW240 containing the vector elements was ligated to an -1.5-kbp EcoRI-BamHI fragment isolated fromipIT353 (18), creating pOW241, which combines the A p L regulatory elements with a unique NcoI site at a positic n for proper translation initiation and a unique BamHI site. The DNA region encoding the putative LAIr polypeptide was isolated from pDQ302 on contiguous -1 .1-kbp NcoI-
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. clavuligerus. An indication that this gene encoded the amino acid sequence of the enzyme rather than serving a regulatory function might come from DNA sequence analysis. Although the exact location of the gene for LAT was unknown, spontaneous deletions of pDQ301d (22) in a pool of primary transformants and concurrent loss of LAT activity 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 4.7kbp SstI-EcoRI fragment were introduced into M13 vectors and sequenced. The DNA sequence from the EcoRI site and
the derived translation products suggested by the analysis described below are shown in Fig. 2. The analysis of the DNA sequence (Frames [4]) indicated that of six possible reading frames, two were candidates for protein-coding regions. These ORFs had a strong Streptomyces codon bias (2, 30) with a G+C content greater than 90% in the third position. TestCode, a program that identifies protein-coding sequences independent of codon bias or reading frame, gave identical results. The initiation codon was selected on the basis of this analysis and identification of a putative ribosome binding site immediately upstream of the start site. An inverted repeat that may act as a transcription termination signal was also located immediately following the translation stop codon. Comparison of sequences from a protein data bank with the putative protein revealed a strong similarity (50%, data not shown) to rat as well as human ornithine aminotransferase (OAT; (25, 26), an enzyme of like function (3). One region of human OAT (Fig. 3) with no computer-assisted gaps that shows the highest similarity to the ORF (38% identical amino acid residues; 63% related) contains the OAT active-site lysine that binds the pyridoxal cofactor (Lys-292 [25, 26, 31]). This alignment places the ORF Lys-304 as the candidate for cofactor binding. These data
VOL. 173, 1991
LOCALIZATION
1
GAATTCCCCTGAACACGAAGCTGAGCAACAGCTCGTCACGCGCTCCCGAGCTGGCCATTC
60
61
AGGCGCAGTTCACAAAGAGCCATCGAGAGGCGTCCGAGAGAGCTGGAAGAGGGGTCCAAGA
120
121
GCATGGTGGGTCATTATTGTGATCCTAAAATGTCCAGTTCACCGCCATGACAGCAGAGGC
181
1321
>lat
TGGAAAG;TCCCCCATAATTC'AGCCTGATCCCCCAGGATTCTCACCCATGGGCGAAGCAG M G
2 41
1261
E
CACGCCACCCCGACGGCGATTTCTCGGACGTGGGAAACCTCCACGCTCAGGACGGCACC R HP DGD F SDV G NL
10
SAQ0D
240
A A
1381 300
V HQ
20
1441
301
AGGCACTTGAGCAGCATATGCTCGTCGACGGGTACGACCTCGTTCTCGACCTCGACGCCA A L E Q H M. L V D G Y D L V L D L D A S 30 40
361
GCCGC4TGTG~AGCTACCGACGACCACTTC V L V DA V RY L DL S G
TQ
50
360
1501 420
F SF
60 1561
421
TCTGC F A SA PLG I NPP SI V E DPAF M 70 80
481
TCCGGGAGCTGCCGGCGGTACACGCACcGTTTTCG
CGTACGCCCGTTCGCAAGACCTTCGCCCGGGTCCTCGGCGACcCCCCGGCTGCGGCGGC
601
TGTTCTTCGTGGACGGCGGGGCGCTGGCCGTGGAGAACGCGCTCAAGGCGGCCCTCGACT
660
TGGAGCGCTCGTTCCACGGCCGCAGCGGCTACACCATGTCGCTGACGAACACCGAGCCGT CCAAGACCGCCCGCTTCCCCAAGTTCGGCTGGCCACGGATCTCGTCCCCCGCCCTCCAGC K TA R FP KF GWP RI S SP AL Q 190 200
841
ACCCGCCGGCCGAGCACACCGGCGCCAACCAGGAGGCCGAGCGACGGGCGCTGGAGGCCG P PAEHBT GA NQ E A E RRAL EA A 210 220
ACACGCTCTCCGGGCGGCTCAACCGCGCCACCCCCACGGCACGTTCGCCTTCACCCGCAC
1740
>acv
1801
901
CCCGGGAGGCGTTCGCAGCGGCGGACGGCATGATCGCCTGCTTCATCGCGGAGCCCATCC
780
1981
840
2041
2161
960 2221
2281
AGACCCAGGTCTGCGGGGTGATGGGCGGCGGCCGGATCGACGAGGTCCCCGAGACGTC
1200
MG
GG
R
IDE
1201
V
S
SRI
S
ST
WGGHNL
330
FIG.
2.
D
DP
G
VRA
TRQ0RDDI
CGTCGTCGACGGGCGGGAGTACACCGCTCTGAAGGACGCGCTGCGCGCCGCCGACGGGGT V V DGR E Y TA L KD A L G RAADA
CTCGGCGGGCGCGCTCGCGCTGGCCTCCCTGCACAGCGTGATGCGCGCGTACGGCCATGG S A GA LA LAS L HSV MR A YG H G
1980
2040
CGAGCAGACGGTGGCGGCG'TTCGTCGACGC.GACGGCGAC'GGCGGAGCTGAAGACGGCCGC
2100
CGTGCTGCCGGTGATCGTCGACCATATCGAGCACACCCGGCTGACCTGCGCCGAGGCGAT
2160
LP
V
IV
D
HI
E
H
T
RL
T
CA
EA
I
CCGGGAGCTGGACGAGACGCTGCGCCGCAAGGACTCCTACACCCGTGCGGACGAGGTGCT L D ET
L RRKD
S
Y
2220
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
2280
2340
CGAGCTGGCCTCGCGGGAGCAGCGCGAGCGGCTTCACGTGGACGCGACCGATGGCG E LA SR EQ RE R LQR W TD GD
2520
2521
CTCCG6ACGG'TACACGTGGCGCTCGGTGCG F P ADQ0R L ND L VE AA VR RS P D
2580
2581
CCGTGAGGCGGTCGTCTTCGGGACACAGCGGCTGACCTATCGGGAGGTCGAC
2632
NA
320
TCGCCGTCTCCTCCCGGAT'CAGCTCCACCTGGGGCGGCAACCTCGCCGACATGGTCCGCG A
RD
24 60
2461
PEHNVV
V
IAV
GG;TCGTCCGGG-AGGTGCTC'GGGCAGTACGCCGGGCGGCCGGGACCGGGCGCCGAGAT V V RE VL GQYA G RP GD R VA E I
300
V
1920
R
2400
2401
1140
310
CAACAGGATCGCGGTACGGGACGACGACCCCGGTGTCCGTGCGACGAGGCAGCGGGACAT
CTGGACGATGGCGTACGCGGGCGAGCTGTTCGGACACGACGGTCGCCGGTGTGCTGGA W T MA YAG EL FE DT T VAG VL E
1080
GTACCGCCTGGGCCTACCA'GCAGCTCGGC~TCCAGCCCGACCTGGTGGCCTTCGGCAAG T A WA YQ QL GLQ P DL VAFPG KK CG
1860
R E
2341
290
CATTCAAGGAGTGTCGAGCGAGCGTTGCG'ATCTTGAAAT'GCTGCTGAAG'GACGAGTGGCG
V
900
1020
1800
AR YP RT A AE WT TR
EQ0T VA A FV DA TA TA EL KTA A
QA MQRL C 260
GCCACGAGAACGACGCCCTGTTCGTCCTGGACGAGGTGCAGAGCGGCTGCGGCATCACCG H END A LPV L DEV Q SG CG I TG 270 280
TQ0V
GGAGGAGCCCACGAATGATGTCAGCACGGTACCCGAGGACCGCAGCGGAGTGGACCACTCG
N
1921
R EA FA AA DG MI AC F IA E P I 230 240
AGGGCGAGGC,rGGCGACAACCACCTCAGCGCGGAG;TTcCTCCAGGCCAT'GCAGCGGCTCT G EG GD N HL SA E FL
synthetase.
IQ0G VS SE R C DLE MLL K DE WR
2101
1141
1620
SV
1681
720
160
781
1081
1560
1680
186.1
GGAAGGCCCAGAAGCTGGGCCTCGCCGAGCCGGACACCatACCGGCTCCAGGTGCTGCATC K A0K L GL AEP DT D RL QV L HL
250
TP VA
S
M M S
ER SF H GR SG YT M SLT N TEP S 170 180
1021
CGCTGGCGAGCAGTGTCACGCCGTCGCC'GAGAGCGTCTGACGCCCGGCCGGCCGG-GTGC V E
1500
CCCAGCGGGGCCCGGCCGGCCGCACCGCGGAACGGAACACCCCTGGCGCGTCGCCCGGTG
1741
140
150
961
GCCTCCGGTTCCGCCCCGCGCTGACGATCGCGGAGCACGAGATCGACCAGGCCCTTCAGG L.LRF RP A LT IA E HE I DQ0ALQ0A 430 440
120
130
721
AGGTGCTGCGGCTCATGTACACGGAGCACCAGGTCATCGCCCTGCCCTGCGGCGGGCGCA V L R LMY TE H QV I A LP CG GR S 420 410
1440
1621
F FV D GGA L A VE NAL KAA LD W
661
ACGCCCGCGGCCGGGGCCTGATGTGCGCGGTCGACCTGCCGGACACCCGGACCCGCAATG AR G RG L MCA V DLP DT R TR NE 400 390
1380
450
600
Y AR FV KT FA RV LGD P RL R RL
110
GCAAGTACTTCCGGGACGGCCTGGAGGACCTGGCCGCCCGCCACCCCTCCGTCGTGACCA K YF RD G LE DL AA R HP SV V TN 370 380
1320
540
R EL A VAA VNK P S NP DL Y SVP 90 100
541
6225
CCACCCGGCTGCTGGAGACGATCGAGCGCACCCAGGTCTTCGACACCGTCGTCCAGCGCG E T I E R T Q V F D T V V Q R G T R LL 350 360
L AS
480
pcbAB FROM S. CLAVULIGERUS
OF lat AND
D
A
R
1260
E
A
V
V
F
G
T
Q
R
L
T
Y
R
E
V
D
MVRA
340
Nucleotide and derived amino acid sequence of S.
sites for LAT and ACVS sequences -10
are
bp upstream
+228 and + 1754,
of both ORFs
are
clavuligerus lat and partial sequence of pcbAB. The putative translation start respectively. The asterisk following LAT amino acid 457 denotes a stop codon. Overlined potential ribosome binding sites, and the overlined sequences following lat represent an
inverted repeat.
suggested that
the ORF
was
notransferases and may be
a
related to other known amistructural 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
OV$F p
was
'-driven
part of an NcoI site, and the entire 4.4-kbp NcoI-BamHI fragment in a expression vector (pOW407; Fig. 4A). After tem-
inserted
porature induction wf,re analyzed for
was
as a
of the
PL promoter, crude cell
extracts
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
be
could not exclude the presence of other ORFs that
expressed
in E.
coli.
mnight
6226
TOBIN ET AL.
J. BACTERIOL. 242 LAT E P I Q G E G OAT E P I Q G E A 230
J
D N H L S A E F
Q A M Q R L CTH E N
V V V P D P G Y
M G V R E
D
ALF
T R H Q V
V
I
304 LAT L D OAT A
V I
S T
C G I T L A R T
T A Wj R W L
Y Q Q L G L Qf V D Y E N V RLH
L I
A F[T 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
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
A. CL
B.
al
.Kpn
42 °25'
42°25 °42° 25°
9
66.245
-.....
Ncol
-
31
BamHI
4
LAT IPNS
-
21.5-
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).
LOCALIZATION OF lat AND pcbAB FROM S. CLAVULIGERUS
VOL. 173, 1991
6227
A. Strep
Pen
TTYQ
R E A
K I A V V CE RE L T
Ceph Q H V
Pen
G
LF L D K S
E
R P
R
H W
H G P
G V R R
G E L N AIQ G NL AY L R I G I. . L P E Q L V T Y E E L N AM A N R L AHIH L V S G. . Q TE Q L V
D K
Strep
LR
A
A
P
L IVTILGWKSGAAYVP I D P T Y PDERVRFVL M S1TILGIWKSGAAF P I D P G YPDER1
Ceph [EL F L D K Tn B.
Strep Pen
Ceph Strep Pen Ceph
L L S F
LER I S
.
V[DV
V N V N G KA I P V N V N G K A D L R A L P A A
LG|L[V
P DWG G GA[1GI .
.
.
R
I T T T[G V R G E R[E S A| DT EI A L GE I W A D NSTERR JDLR L E S D L A Ai G N
L
H K Q D G E R G N Q
A P
Strep
Pen Ceph
R
L L S F L E K K L P M P T R L V Q L S L L S F L E K K L P R Y M V P T R L V Q L A
L
G
LI
V|F
RQRL NFFR L G G H S TC I VS F R L G G H S I AC I Q L I A RG
V
S RIGD G lA R Q R S V S R N A Q D G S E
V[D
G|VE D V F T L|R L R T L E D V F A t QT
SV jSI
TL
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
EJ IPNS
-.,-.
ACVS
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).
-_
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.
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. This work was supported by the Lilly Research Laboratories (a division of Eli Lilly & Co.) and the Natural Sciences and Engineering Research Council of Canada.
ADDENDUM
At the time this report was submitted, Martin and his colleagues (9) confirmed the linkage of pcbAB to pcbC in C. acremonium reported previously (10) and reported an ORF encoding an ACVS polypeptide with a molecular weight of 414,791 with three related domains. REFERENCES 1. Baldwin, J. E., J. W. Bird, R. A. Field, N. M. O'Callaghan, C. J. Schofield, and A. C. Willis. 1991. Isolation and partial characterisation of ACV synthetase from Cephalosporium acremonium and Streptomyces clavuligerus. J. Antibiot. 44:241-248. 2. Bibb, M. J., P. R. Findlay, and M. W. Johnson. 1984. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene 30:157-166. 3. Braunstein, A. E. 1973. Amino group transfer, p. 379-481. In P. D. Boyer (ed.), The enzymes. vol. 9. Academic Press, Inc., New York. 4. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. 5. Diez, B., S. Gutierrez, J. L. Barredo, P. van Solingen, L. H. M. van der Voort, and J. F. Martin. 1990. The cluster of penicillin biosynthetic genes. Identification and characterization of the pcbAB gene encoding the a-aminoadipyl-cysteinyl-valine synthetase and linkage to the pcbC and penDE genes. J. Biol. Chem. 27:16358-16365. 6. Doran, J. L., B. K. Leskiw, A. K. Petich, D. W. S. Westlake, and S. E. Jensen. 1990. Production of Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli using two-cistron expression systems. J. Indust. Microbiol. 5:197-206. 7. Fothergill, J. C., and J. R. Guest. 1977. Catabolism of L-lysine by Pseudomonas aeruginosa. J. Gen. Microbiol. 99:139-155. 8. Garcia-Dominguez, M., P. Liras, and J. F. Martin. 1991. Cloning and characterization of the isopenicillin N synthase gene of Streptomyces griseus NRRL 3851 and studies of expression and complementation of the cephamycin pathway in Streptomyces clavuligerus. Antimicrob. Agents Chemother. 35:44-52. 9. Guti6rrez, S., B. Diez, D. Montenegro, and J. F. Martin. 1991. Characterization of the Cephalosporium acremonium pcbAB gene encoding a-aminoadipyl-cysteinyl-valine synthetase, a large multidomain peptide synthetase: linkage to the pcbC gene as a cluster of early cephalosporin biosynthetic genes and evidence of multiple functional domains. J. Bacteriol. 173:23542365. 10. Hoskins, J. A., N. O'Callaghan, S. W. Queener, C. A. Cantwell, J. S. Wood, V. Chen, and P. L. Skatrud. 1990. Gene disruption of the pcbAB gene encoding ACV synthetase in Cephalosporium acremonium. Curr. Genet. 18:523-530. 11. Hoskins, J. A., and P. L. Skatrud. Unpublished data.
VOL. 173, 1991
LOCALIZATION OF lat AND pcbAB FROM S. CLAVULIGERUS
12. Ingolia, T. D., and S. W. Queener. 1989. Beta-lactam biosynthetic genes. Med. Res. Rev. 9:245-264. 13. Jensen, S. E. Personal communication. 14. Jensen, S. E., A. Wong, M. J. Rollins, and D. W. S. Westlake. 1990. Purification and partial characterization of b-(L-ax-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus. J. Bacteriol. 172:7269-7271. 15. Kern, B. A., D. Hendlin, and E. Inamine. 1980. L-Lysine E-aminotransferase involved in cephamycin C synthesis in Streptomyces lactamdurans. Antimicrob. Agents Chemother. 17:679-685. 16. Kirkpatrick, J. R., 0. W. Godfrey, and L. E. Doolin. 1973. Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, E-74, p. 13. 17. Kovacevic, S., and J. R. Miller. 1991. Cloning and sequencing of the P-lactam hydroxylase gene (cefF) from Streptomyces clavuligerus: gene duplication may have led to separate hydroxylase and expandase activities in the actinomycetes. J. Bacteriol. 173:398-400. 18. Kovacevic, S., M. B. Tobin, and J. R. Miller. 1990. The P-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus. J. Bacteriol. 172:39523958. 19. Kovacevic, S., B. J. Weigel, M. B. Tobin, T. D. Ingolia, and J. R. Miller. 1989. Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase. J. Bacteriol. 171:754760. 20. Madduri, K., S. Shapiro, A. C. DeMarco, R. L. White, C. Stuttard, and L. C. Vining. 1991. Lysine catabolism and a-aminoadipate synthesis in Streptomyces clavuligerus. Appl. Microbiol. Biotechnol. 35:358-363. 21. Madduri, K., C. Stuttard, and L. C. Vining. 1989. Lysine catabolism in Streptomyces spp. is primarily through cadaverine: 13-lactam producers also make a-aminoadipate. J. Bacteriol. 171:299-302. 22. Madduri, K., C. Stuttard, and L. C. Vining. 1991. Cloning and location of a gene governing lysine E-aminotransferase, an enzyme initiating P-lactam biosynthesis in Streptomyces spp. J. Bacteriol. 173:985-988. 23. Martin, J. F., and Y. Aharonowitz. 1983. Regulation of biosynthesis of ,-lactam antibiotics, p. 229-255. In A. L. Demain and N. A. Solomon (ed.), Antibiotics containing the beta-lactam structure, vol. I. Springer-Verlag, New York. 24. Miller, J. R., and T. D. Ingolia. 1989. Cloning 13-lactam genes from Streptomyces spp. and fungi, p. 246-255. In C. L. Hershberger, S. W. Queener, and G. Hegeman (ed.), Genetics and molecular biology of industrial microorganisms. American Society for Microbiology, Washington, D.C. 25. Mitchell, G. A., J. E. Looney, L. C. Brody, G. Steel, M. Suchanek, J. F. Engelhardt, H. R. Willard, and D. Valle. 1988. Human ornithine-f-aminotransferase. cDNA cloning and analysis of the structural gene. J. Biol. Chem. 263:14288-14295. 26. Mueckler, M. M., and H. C. Pitot. 1985. Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone. J. Biol. Chem. 260:12993-12997.
6229
27. Piret, J., B. Resendiz, B. Mahro, J.-Y. Zhang, E. Serpe, J. Romero, N. Connors, and A. L. Demain. 1990. Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585. Appl. Microbiol. Biotechnol. 32:560-567. 28. Romero, J., P. Liras, and J. F. Martin. 1988. Isolation and biochemical characterization of Streptomyces clavuligerus mutants in the biosynthesis of clavulanic acid and cephamycin C. Appl. Microbiol. Biotechnol. 28:510-516. 29. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 30. Seno, E. T., and R. H. Baltz. 1989. Structural organization and regulation of antibiotic biosynthesis and resistance genes in Actinomycetes, p. 1-48. In S. Shapiro (ed.), Regulation of secondary metabolism in Actinomycetes. CRC Press, Inc., Boca Raton, Fla. 31. Simmaco, M., R. A. John, C. Barra, and F. Bossa. 1986. The primary structure of ornithine aminotransferase. Identification of active-site sequence and site of post-translational proteolysis. FEBS Lett. 199:39-42. 32. Singh, P. D., P. C. Ward, J. S. Wells, C. M. Ricca, W. H. Trejo, P. A. Principe, and R. B. Sykes. 1982. Bacterial production of deacetoxycephalosporin C. J. Antibiot. 35:1397-1399. 33. Smith, D. J., M. K. R. Burnham, J. H. Bull, J. E. Hodgson, J. M. Ward, P. Browne, J. Brown, B. Barton, A. J. Earl, and G. Turner. 1990. ,B-Lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes. EMBO J. 9:741-747. 34. Smith, D. J., A. J. Earl, and G. Turner. 1990. The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421 073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases. EMBO J. 9:2743-2750. 35. van Liempt, H., H. von Dohren, and H. Kleinkauf. 1989. b-(L-a-Aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. J. Biol. Chem. 264:3680-3684. 36. Vining, L. C., S. Shapiro, K. Madduri, and C. Studdard. 1990. Biosynthesis and control of 3-lactam antibiotics: the early steps in the "classical" tripeptide pathway. Biotechnol. Adv. 8:159183. 37. Whitney, J. G., D. R. Brannon, J. A. Mabe, and K. J. Wicker. 1972. Incorporation of labeled precursors into A16886B, a novel 1-lactam antibiotic produced by Streptomyces clavuligerus. Antimicrob. Agents Chemother. 1:247-251. 38. Yagi, T., H. Misono, N. Kurihara, T. Yamamoto, and K. Soda. 1980. L-Lysine: 2-oxoglutarate 6-aminotransferase. Subunit structure composed of non-identical polypeptides and pyridoxal 5'-phosphate-binding subunit. J. Biochem. 87:1395-1402. 39. Zhang, J., and A. L. Demain. 1990. Purification from Cephalosporium acremonium of the initial enzyme unique to the biosynthesis of penicillins and cephalosporins. Biochem. Biophys. Res. Commun. 169:1145-1152. 40. Zhang, J., and A. L. Demain. 1990. Purification of ACV synthetase from Streptomyces clavuligerus. Biotechnol. Lett. 12: 649-654.
