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. 1999 May;43(5):1215–1224. doi: 10.1128/aac.43.5.1215

Genes Specific for the Biosynthesis of Clavam Metabolites Antipodal to Clavulanic Acid Are Clustered with the Gene for Clavaminate Synthase 1 in Streptomyces clavuligerus

Roy H Mosher 1,†, Ashish S Paradkar 1,‡, Cecilia Anders 1, Barry Barton 2, Susan E Jensen 1,*
PMCID: PMC89136  PMID: 10223939

Abstract

Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames, cvm4 and cvm5, located directly downstream of cas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 and cas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis.

Clavulanic acid is a potent beta-lactamase inhibitor produced by the gram-positive actinomycete Streptomyces clavuligerus (27). During growth in soy fermentation medium, S. clavuligerus produces clavulanic acid and a group of structurally related but antipodal clavam metabolites, as well as conventional penicillin-type and cephamycin-type beta-lactam antibiotics. Some of the clavams possess antibacterial and antifungal activities, but they lack the beta-lactamase inhibitory activity of clavulanic acid (24). Unlike the penicillins and cephamycins, all clavam metabolites including clavulanic acid possess an oxygen-containing oxazolidine ring, and feeding studies have established that they are biosynthetically distinct from the sulfur-containing beta-lactam antibiotics (11, 15).

Clavulanic acid biosynthesis is thought to begin with the joining of arginine to a three-carbon glycolytic intermediate, likely pyruvate (25, 31, 34). The product of this condensation, N2-(2-carboxyethyl)-arginine, is then cyclized via β-lactam synthetase to form the monocyclic β-lactam product, deoxyguanidinoproclavaminic acid (2, 21). Deoxyguanidinoproclavaminic acid is hydroxylated by the multifunctional enzyme CAS to form guanidinoproclavaminic acid (14), which is then converted to proclavaminic acid via proclavaminate amidinohydrolase (37). This is followed by the CAS-mediated oxidative cyclization of proclavaminic acid, which forms clavaminic acid (9). The final steps of the biosynthetic pathway are poorly understood, but they most likely involve the oxidative deamination of clavaminic acid and stereochemical inversion (epimerization) at C-3 and C-5 to form the highly reactive clavulanate-9-aldehyde (22). This penultimate intermediate is then reduced by way of an NADPH-dependent dehydrogenase to clavulanic acid.

Clavaminate synthase is the best characterized of the clavulanic acid biosynthetic enzymes. It exists in S. clavuligerus as two isoenzymes, CAS1 and CAS2, that are encoded by two similar but distinct genes, cas1 and cas2, respectively (20). Sequence analysis, hybridization studies, and insertional inactivation experiments have placed the clavulanic acid biosynthetic genes in a cluster directly adjacent to the genes required for penicillin and cephamycin biosynthesis on the S. clavuligerus chromosome (1, 16, 36). In addition, these studies have shown that cas2 is located within the cluster immediately downstream of the proclavaminate amidinohydrolase-encoding gene, pah (1, 16, 37). The relative position of cas1 on the S. clavuligerus chromosome has yet to be determined, but hybridization studies have suggested that cas1 is separated from cas2 by at least 20 to 30 kb (3, 20).

The biological rationale for the possession of two CAS isozymes is unclear. One possibility is that clavulanic acid and the clavam metabolites are synthesized by separate pathways, each requiring its own CAS isozyme (20). However, a number of studies have strongly suggested that clavulanic acid and the clavams share a common pathway up to and including the clavaminic acid step (8). Another possibility is that one of the CAS-mediated biosynthetic steps is rate limiting. Duplication of an ancestral cas gene may, therefore, have been an evolutionary means of relieving the bottleneck at this point in the biosynthetic pathway (20).

Recent work has shown that the expression of cas1 is nutritionally regulated (23). When wild-type S. clavuligerus was grown in a complex soy fermentation medium, it produced both clavulanic acid and clavams, and Northern analysis revealed that transcripts of both cas1 and cas2 were present. However, in a defined starch-asparagine (SA) medium, S. clavuligerus produced clavulanic acid but failed to produce detectable levels of clavams. Northern analysis showed that only cas2 was transcribed under these conditions; no cas1-specific transcripts were detected. When a cas2 mutant of S. clavuligerus was created by targeted gene disruption, it failed to produce clavulanic acid when it was grown on SA medium. In contrast, the cas2 mutant produced both clavulanic acid and clavams when it was grown in soy medium.

These findings indicate that both cas1 and cas2 contribute to clavulanic acid biosynthesis in soy medium. However, in SA medium, in which cas1 is not expressed, cas2 becomes essential for clavulanic acid production (23). Since the cas2 mutant was capable of producing clavulanic acid and clavams in soy medium, the clavaminic acid generated through the action of CAS1 must have been freely available for the synthesis of both clavulanic acid and clavams. This strongly supports the proposal that clavaminic acid serves as a branch point precursor for both clavulanic acid and the clavams (8). The observed inability of wild-type S. clavuligerus to produce clavams when it is grown on SA medium (despite the presence of a functional cas2) suggests that additional genes specific for the biosynthesis of the clavams are under regulatory controls similar to those that govern the expression of cas1 (23).

In this paper, we report on the cloning, nucleotide sequencing, identification, and insertional inactivation of clavam-specific biosynthetic genes flanking cas1 on the S. clavuligerus chromosome. In addition, mutants disrupted either in cas1 alone or in both cas1 and cas2 were constructed by targeted gene disruption and replacement. The characteristics of these mutants with respect to clavulanic acid and clavam production are described.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

All bacterial strains and plasmids used in this study are described in Table 1. Plasmid pFDNEO-S was generously provided by R. Brzezinski (4). Cultures of Escherichia coli were maintained as described by Sambrook et al. (28). Cultures of S. clavuligerus and Streptomyces lividans were maintained as described by Paradkar and Jensen (23). For the selection and maintenance of plasmid-containing cultures, agar-based media were supplemented with ampicillin (100 μg/ml), kanamycin (100 μg/ml), thiostrepton (5 μg/ml for S. clavuligerus and 50 μg/ml for S. lividans), apramycin (25 μg/ml), or neomycin (55 μg/ml), as appropriate. For the production of clavulanic acid and/or clavam metabolites, a seed culture of S. clavuligerus was grown in Trypticase soy broth supplemented with 1% maltose on a rotary shaker (220 rpm) at 28°C for 48 h and was used to deliver a standardized inoculum to either SA medium or soy medium (23). Production cultures were grown under conditions similar to those used for seed cultures.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid Description Reference or source
Strains
 Bacillus sp. ATCC 27860 Indicator strain; sensitive to alanylclavam on minimal medium 26
 E. coli ER1447 ara-14 F−dam-13::Tn9 (Cmr) dcm6 fhuA31 galK2 galT22 hisG4 hsdR2 (rk− mk+) lacY1 leuB6 mcrA mcrB1 mtl-1 rpsL136 (Strr) supE44 thi-1 tsx-78 xyl-5 J. McCormick, Harvard University, Cambridge, Mass.
 E. coli ESS Beta-lactam-sensitive indicator strain A. L. Demain, Massachusetts Institute of Technology, Cambridge
 E. coli MV1193 endA hsdR4 · (lac-proAB) rpsL spcB15 · (srl-recA) thi-306::Tn10 (Tetr) 38
 E. coli XL1-Blue endA1 gyrA96 hsdR17 lac [F′ proAB lacI9Z · M15 Tn10 (Tetr) Amy Camr] recA1 relA1 supE44 thi-1 Stratagene
 K. pneumoniae ATCC 15380 Thiostrepton-resistant indicator strain for clavulanate bioassay 27
 S. clavuligerus NRRL3585 Wild type Northern Regional Research Laboratory
 S. clavuligerus AP5 cas2::apr 23
 S. lividans TK24 str-6; plasmidless cloning host (SLP2− SLP3−) D. A. Hopwood, John Innes Institute, Norwich, United Kingdom
Plasmids
 10D7 Cosmid isolated from a S. clavuligerus genomic DNA library; hybridizes to the cas1-specific probe RMO1 This study
 pBluescript II SK+ Phagemid; Ampr Stratagene
 pCEC007 pBluescript II SK+ containing a 7-kb SacI fragment of S. clavuligerus DNA from cosmid 10D7 encoding cas1 This study
 pCEC018 pBluescript II SK+ containing a 4.3-kb EcoRI-KpnI fragment from cosmid 10D7 This study
 pCEC023 pSL1180 containing a 3.6-kb SacI fragment from pCEC018 This study
 pCEC024 Same as pCEC023 but with insert oriented in the opposite direction This study
 pCEC026 pUC120 containing a 2.9-kb NcoI fragment from pCEC007 This study
 pCEC027 Same as pCEC026 but with insert oriented in the opposite direction This study
 pCEC029 pSL1180 containing a 0.9-kb NcoI fragment from pCEC023 This study
 pFDNEO-S pUC18 containing modified bifunctional neo from Tn5 4
 pIJ486 High-copy-number Streptomyces promoter-probe plasmid; Thior 35
 pIJ702 Streptomyces cloning vector; mel Thior 17
 pSKNeo pBluescript II SK+ containing neo from pFDNEO-S This study
 pSL1180 Phagemid; Ampr Pharmacia
 pUC120 Phagemid; Ampr 23
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Chemicals.

Thiostrepton was obtained from Sigma Chemical Co., St. Louis, Mo. Apramycin was generously provided by E. Seno, Eli Lilly Co., Indianapolis, Ind. Samples of clavulanic acid, clavaminic acid, and clavam-2-carboxylate for use as high-performance liquid chromatography (HPLC) standards were obtained from SmithKline Beecham, Worthing, United Kingdom.

Recombinant DNA techniques.

Genomic and plasmid DNAs were extracted from Streptomyces spp. by standard techniques (12). Preparation and transformation of S. clavuligerus protoplasts were as described by Paradkar and Jensen (23). S. lividans protoplasts were prepared and transformed by standard procedures (12). For DNA sequencing, plasmid DNA was extracted from E. coli with a QIAGEN Plasmid Mini Kit (Qiagen Inc., Santa Clarita, Calif.). For the routine screening of plasmids from E. coli, standard procedures were used (28). All DNA manipulations and the transformation of competent E. coli cells were as described by Sambrook et al. (28). DNA fragments separated by agarose gel electrophoresis were eluted and purified with a QIAquick Gel Extraction Kit (Qiagen Inc.).

Southern hybridization analysis and labelling of double-stranded DNA probes with [α-32P]dATP or [α-32P]dCTP by nick translation were carried out by standard procedures (12). Radiolabelled probes were incubated with DNA fragments immobilized on nylon membranes (Hybond-N; Amersham, Arlington Heights, Ill.) in a solution of 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M trisodium citrate), 5× Denhardt’s solution (1× Denhardt’s solution is 0.02% bovine serum albumin, 0.02% Ficoll, and 0.02% polyvinylpyrrolidone), and 0.5% sodium dodecyl sulfate (SDS) at 60°C overnight. The blots were then washed at high stringency (once with 2× SSC–0.1% SDS at 65°C for 30 min and once with 0.2×SSC–0.1% SDS at 65°C for 30 min) and were exposed to X-ray film.

Isolation of cas1.

A chromosomal DNA fragment carrying the cas1 open reading frame from S. clavuligerus was isolated with a 36-mer oligonucleotide, RMO1 (5′-GCGCGCAGCTCGGGGCCGTACGCGGTGCAGTCCACT). The RMO1 sequence, based on the noncoding strand of cas1 at nucleotides (nt) +9 to +44 relative to the start codon, was chosen as the probe because it possessed only 11% sequence identity with the corresponding region of the cas2 gene (20, 23).

RMO1 was radiolabelled with [γ-32P]ATP by standard techniques for the end labelling of DNA oligonucleotides (28) and was used to screen a cosmid bank of S. clavuligerus genomic DNA by Southern hybridization as described by Stahl and Amann (30). The genomic bank of S. clavuligerus DNA in cosmid pLAFR3 was available from a previous study (7).

Colony blots of the S. clavuligerus cosmid bank were incubated overnight with radiolabelled RMO1 at 60°C. The blots were then washed at 68°C for 30 min in a solution of 0.5× SSC–0.1% SDS. One cosmid clone, 10D7, was isolated. Clone 10D7 hybridized strongly to RMQ1 and upon digestion with restriction endonucleases SacI and EcoRI gave hybridization signals that were consistent with the hybridization signals detected in similar experiments with digests of S. clavuligerus genomic DNA (data not shown). The identity of cosmid 10D7 was confirmed by probing digests of cosmid DNA with a radiolabelled 27-mer oligonucleotide, RMO4 (5′-GAGGTCACGGAGGCGGTGTATCTGGAG), the sequence of which is based on nt +769 to +795 of cas1 (20).

DNA sequencing of the S. clavuligerus chromosome flanking cas1.

A partial restriction map of cosmid 10D7 was generated with restriction endonucleases SacI, NcoI, and KpnI. Southern hybridization experiments between RMO1 and various digests of 10D7 DNA indicated that cas1 was located at one end of a 7-kb SacI DNA subfragment (data not shown). This fragment consisted of cas1 and approximately 6 kb of upstream DNA. The 7-kb fragment was then subcloned in the phagemid vector pBluescript II SK+ from a SacI digest of 10D7, generating the recombinant plasmid pCEC007.

To facilitate the process of sequencing the chromosome upstream of cas1, a 3-kb NcoI subfragment of the 7-kb SacI fragment was cloned in pUC120 in both orientations, generating the recombinant plasmids pCEC026 and pCEC027. The 3-kb subfragment consisted of the 5′ end of cas1 and approximately 2.6 kb of upstream DNA.

Nested, overlapping deletions were created in both pCEC026 and pCEC027 by exonuclease III and S1 nuclease digestion (28). The DNA sequences of both strands of the 3-kb NcoI fragment were then determined (Fig. 1) by the dideoxy chain termination method (29) with a Taq dye-deoxy terminator kit (Applied Biosystems) and an Applied Biosystems 373A Sequencer. Sequence data were compiled and analyzed with DNA Strider 1.2 (19), the FRAME program (6, 33), and the GCG Wisconsin Package, version 8, DNA analysis software (5). Comparison of deduced amino acid sequences with those in the GenBank and SWISSPROT databases was accomplished by using the online BLAST program at the National Center for Biotechnology Information (NCBI) (21a) and the online FASTA program at the European Bioinformatics Institute (11a).

FIG. 1.

FIG. 1

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Nucleotide and deduced amino acid sequences of the 5′ end of cas1 and 2.6 kb of upstream DNA. The first nucleotide of each potential translational start codon is marked above, with an arrowhead indicating the direction of the open reading frame. Potential translational stop codons are indicated with an asterisk beneath the codon. The location of the BsaBI site used to create the cvm1 disruption mutation is shown above the nucleotide sequence.

To determine the DNA sequence of the chromosome immediately downstream of cas1, a 4.3-kb KpnI-EcoRI DNA fragment from 10D7 was subcloned in pBluescript II SK+, generating pCEC018. The 4.3-kb fragment consisted of 0.65 kb from the 3′ end of cas1 and 3.6 kb of downstream DNA. From pCEC018 a 3.6-kb SacI subfragment was cloned in pSL1180. One of the SacI termini of this fragment partially overlapped the TGA stop codon of cas1; the other was vector encoded. Both orientations of the 3.6-kb fragment were obtained during subcloning, and the resulting recombinant plasmids were designated pCEC023 and pCEC024. Nested, overlapping deletions were created in both plasmids, and the DNA sequences of both strands of the 3.6-kb fragment were determined (Fig. 2). The location of the SacI site at the 3′ end of cas1 was confirmed by sequencing across the junction by using pCEC018 as the template and RM04 as the primer.

FIG. 2.

FIG. 2

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Nucleotide and deduced amino acid sequences of the 3′ end of cas1 and 3.6-kb of downstream DNA. The cas1-encoding portion of the presented sequence was determined for one strand only, but it agreed exactly with that previously reported by Marsh et al. (20). The sequences of both strands of the remaining 3.6 kb of DNA were determined as described in Materials and Methods. Potential translational start and stop codons are marked as indicated in the legend to Fig. 1. The locations of the two NcoI sites used to create the cvm4 and cvm5 deletion mutation are indicated above the nucleotide sequence.

Insertional inactivation of cvm1 by gene replacement.

Gene disruption and replacement of cvm1 were accomplished basically as described by Paradkar and Jensen (23) with a bifunctional apramycin resistance gene (apr) cassette. The apr cassette was inserted into pCEC026 at the unique BsaBI site located within cvm1 636 bp from the translational start codon (Fig. 3). Constructs carrying the apr cassette inserted in the same orientation relative to cvm1 were isolated, ligated to the high-copy-number Streptomyces vector pIJ486 (35), and then introduced by transformation into S. lividans and from there into S. clavuligerus. The transformants were allowed to sporulate on nonselective medium and were then replica plated to identify putative mutants.

FIG. 3.

FIG. 3

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Partial restriction endonuclease map of cas1 and flanking regions from the S. clavuligerus chromosome. The thick bar at the top of the diagram represents the S. clavuligerus chromosome, and the arrows immediately below the bar represent the predicted open reading frames and the proposed direction of their transcription. The lower portion of the diagram depicts the DNA inserts from the plasmid constructs used to create the cvm1, cvm4 and cvm5, and cas1 mutants. Arrows immediately above the rectangular boxes indicate the orientation of the resistance cassette. Brackets surrounding the EcoRI site indicate that the site is vector derived and does not exist in the S. clavuligerus genome. Abbreviations: B, BsaBI; E, EcoRI; K, KpnI; N, NcoI; S, SacI.

To confirm that gene disruption and replacement had occurred, SacI-digested genomic DNA from mutants and wild-type S. clavuligerus was subjected to Southern analysis by using pCEC026 as the probe. The wild-type digest showed a single hybridizing band of about 7 kb which was replaced with a band of approximately 5.3 kb in the digests of the mutants (data not shown). The results were consistent with the insertion of an apr cassette at the cvm1 locus on the chromosome.

Creation of a deletion mutation in cvm4 and cvm5 by gene replacement.

A deletion mutant in cvm4 and cvm5 was created by digesting pCEC018 with NcoI to liberate a 1-kb subfragment containing most of cvm4 and a portion of cvm5 (see Fig. 3). The deleted fragment was replaced with the apr cassette oriented in the direction opposite that of cvm4 and cvm5. The plasmid was then fused with pIJ486 and was introduced by transformation into wild-type S. clavuligerus via S. lividans. The resulting transformants were allowed to sporulate under nonselective conditions and were then replica plated to identify putative mutants. Southern analysis of BstEII-digested genomic DNA from wild-type S. clavuligerus and from the mutant with pCEC029 (Table 1) as the probe showed a 4.5-kb hybridizing band in the wild type and a 2.3-kb hybridizing band in the mutants (data not shown). These results were consistent with the deletion from the S. clavuligerus chromosome of a 1-kb DNA fragment that included portions of both cvm4 and cvm5 and its replacement with the apr cassette.

Insertional inactivation of cas1 by gene replacement.

A neomycin and kanamycin resistance gene (neo) cassette was subcloned on a 1-kb SacI-HindIII fragment from pFDNEO-S into pBluescript II SK+, creating pSKNeo. The neo cassette was then excised from pSKNeo as a 1-kb KpnI fragment and was inserted into pCEC007 at the KpnI site within cas1 (Fig. 3). Clones possessing recombinant plasmids carrying the neo cassette in both possible orientations within cas1 were fused to pIJ702, and these fusions were then introduced by transformation into S. lividans and from there into wild-type S. clavuligerus. Putative mutants identified after sporulation on nonselective medium were subjected to Southern analysis.

NcoI-digested genomic DNA from wild-type S. clavuligerus gave a 1.4-kb hybridizing band which was replaced by a 1.8-kb band or a 2.2-kb band in the mutants, depending on the orientation of neo (data not shown). These results were consistent with the insertion of neo in cas1 on the chromosomes of all mutants.

Creation of a cas1 and cas2 mutant by gene replacement.

The same plasmid constructs used to generate the cas1 mutant (see above) were introduced by transformation into S. clavuligerus AP5, a mutant strain carrying the cas2 gene disrupted by insertion of apr (23). Gene disruption and replacement were confirmed by Southern analysis as described above.

HPLC.

The levels of clavulanic acid and clavam metabolites were analyzed by HPLC as described by Paradkar and Jensen (23), except that the running buffer that was used consisted of 0.1 M NaH2PO4 plus 6% methanol (pH 3.68; adjusted with glacial acetic acid). Peaks representing clavulanic acid, clavaminic acid, and clavam-2-carboxylate in culture broths of S. clavuligerus were identified by comparison with authentic standards. 2-Hydroxymethylclavam was identified by its known chromatographic properties relative to those of clavulanic acid and clavam-2-carboxylate (3).

Estimation of beta-lactam antibiotic, clavulanic acid, and alanylclavam production.

Bioassays for bioactive clavam compounds were carried out as described by Paradkar and Jensen (23), except that Klebsiella pneumoniae ATCC 29665 instead of Staphylococcus aureus N2 was used as the indicator organism in the bioassay for clavulanic acid.

Nucleotide sequence accession numbers.

The nucleotide sequence of the region upstream of cas1 reported here has been deposited in the GenBank and EMBL databases under accession no. AF124928, while the region downstream of cas1 has been deposited under accession no. AF124929.

RESULTS

Sequence analysis of open reading frames flanking cas1.

CODONPREFERENCE and FRAME analysis (data not shown) of the sequenced DNA from the region of the S. clavuligerus chromosome flanking cas1 predicted the presence of two complete open reading frames and one incomplete open reading frame upstream of cas1. All of the open reading frames were located on the opposite DNA strand relative to cas1 and were thus oppositely oriented (Fig. 3). The first open reading frame, cvm1, began at an ATG codon located 579 bp upstream of cas1 and encodes a putative 344-amino-acid polypeptide (Fig. 1). cvm1 was preceded by a potential ribosome-binding site at nt 2075 to 2079. Further examination of the DNA sequence immediately upstream of the cas1 and cvm1 open reading frames revealed a number of direct repeats, the complexity and number of which were most pronounced in the region 100 to 200 bp upstream of cas1 (data not shown).

When the derived amino acid sequence of cvm1 was compared to those in the current protein sequence databases, the polypeptide showed a high degree of similarity (>50% identity) to the derived amino acid sequence of an auxin-induced protein (PCNT115; NCBI accession no. 728744) recovered from the tobacco plant Nicotiana tabacum and to the derived amino acid sequence (IN2-2; NCBI accession no. 1352461) of a cDNA recovered from the corn plant Zea mays after induction with a substituted benzenesulfonamide. Both of these proteins are thought to belong to the aldo and keto reductase family 2 enzymes. Additionally, the sequence of the CVM1 protein showed good similarity (>40% identity) to the sequences of a number of putative aldo and keto reductase and dehydrogenase enzymes.

Another open reading frame, cvm2, was located 437 bp beyond the 3′ end of cvm1. It encodes a putative 151-amino-acid polypeptide that showed no significant similarity to any of the sequences in the protein sequence databases. The start codon of cvm2 was preceded by a potential ribosome-binding site at nt 605 to 609. A third open reading frame, cvm3, was located still further beyond the 3′ end of cvm2. The start codon of cvm3 overlapped the translational stop codon of cvm2, which suggested that the two open reading frames might be translationally coupled. No translational stop for cvm3 was located on the 3-kb NcoI-NcoI fragment, and the deduced polypeptide sequence of cvm3 showed no significant similarity to sequences in the protein sequence databases.

When the 3.7-kb cas1 downstream sequence was analyzed by the CODONPREFERENCE and FRAME programs, the presence of two complete open reading frames and one incomplete open reading frame was predicted (data not shown). Two of the open reading frames (cvm4 and cvm5) were located on the opposite DNA strand relative to cas1 and were thus oppositely oriented (Fig. 3). The third open reading frame (cvm6) was located on the same DNA strand as cas1 and would therefore be transcribed in the same sense as cas1. An inverted repeat (nt 424 to 461; Fig. 2) was identified downstream of cas1 and cvm4. The putative stem-loop structure formed from the inverted repeat (ΔG = −51.6 kcal/mol) might function as a bidirectional transcriptional terminator for both cas1 and cvm4. Recent evidence has suggested that cas1 gives rise to a monocistronic transcript (23), and this would be consistent with the location of the inverted repeat and its putative function as a transcriptional terminator.

The translational start for cvm4 was predicted to be a GTG codon at nt 1558 to 1560 (Fig. 2) and was preceded by a potential ribosome-binding site at nt 1567 to 1571. The open reading frame encoded a putative 328-amino-acid polypeptide that showed a low level of but significant degree of similarity (25.3% identity in a 348-amino-acid overlap) to the cefG-encoded deacetylcephalosporin C acetyltransferase (DCPC-ATF; NCBI accession no. 729098) from Cephalosporium acremonium. The CVM4 sequence also showed some similarity to known or putative homoserine o-acetyltransferases from a number of sources, with the highest degree of similarity (27.473% identity in a 364-amino-acid overlap) being to the enzyme (NCBI accession no. 2326681) from Mycobacterium leprae.

cvm5 is located 55 bp upstream of the start codon for cvm4. The translational start for cvm5 was predicted to be a GTG codon at nt 2797 to 2799 that was preceded by a potential ribosome-binding site at nt 2805 to 2811. The cvm5 open reading frame encoded a putative 394-amino-acid polypeptide that showed limited but significant similarity to the amino-terminal regions of various bacterial luciferases such as that encoded by the luxA gene of Photobacterium leiognathi (28.5% identity in a 165-amino-acid overlap). Luciferases are alkanal monooxygenases that, in the presence of reduced flavin mononucleotide, catalyze the oxidation of long-chain aliphatic aldehydes to fatty acids and, in the process, generate light and water.

A third open reading frame, cvm6, is located 315 bp upstream of cvm5 and on the strand opposite to that on which cvm5 is located. Because no stop codon for cvm6 was observed on the 3.7-kb fragment, it encoded a putative incomplete polypeptide of 219 amino acids. Although cvm6 is not preceded by a typical ribosome-binding site, CODONPREFERENCE analysis (data not shown) suggested that cvm6 might be preceded by at least two small open reading frames, the first of which was located at nt 2835 to 2885 and the second of which was located at nt 2917 to 3071. However, an analysis of this region for base composition revealed that the G+C composition in all three open reading frames exceeded 90%, a pattern not usually observed in the open reading frames of Streptomyces spp. The deduced polypeptides of these small open reading frames showed no similarity to sequences in the protein sequence databases but are relatively rich in arginine residues. In contrast, the deduced sequence of cvm6 showed good similarity to a number of class III pyridoxal phosphate-dependent aminotransferases, such as diaminopimelic acid aminotransferase (EC 2.6.1.62; 31.053% identity in a 190-amino-acid overlap) from Bacillus subtilis, acetylornithine aminotransferase (EC 2.6.1.11; 31.325% identity in a 166-amino-acid overlap) from E. coli, and the bifunctional, pyridoxal phosphate-dependent dialkylglycine decarboxylase (EC 4.1.1.64; 32.057% identity in a 209-amino-acid overlap) from Pseudomonas cepacia.

Characterization of the cvm1 mutant.

Although sequence information was obtained for part or all of the six open reading frames surrounding cas1, it was not evident from the deduced protein sequences whether any of these open reading frames might play a role in clavam biosynthesis. However, since the details of clavam biosynthesis beyond the level of clavaminic acid are essentially unknown, there was also no basis to eliminate these open reading frames as potential clavam biosynthetic genes. To gain further insights into the possible involvement of cvm1 in clavam biosynthesis, mutants were prepared by a gene replacement procedure. A cloned copy of cvm1 was disrupted by the introduction of apr oriented in the same direction as cvm1. The cvm1 disruption construct was then introduced by transformation into S. clavuligerus, where the disrupted gene exchanged with the corresponding wild-type region of the chromosome by a homologous recombination procedure. The mutants that arose from four different primary transformants (apparent frequency of mutation, 2 to 25%) were characterized.

HPLC analysis of culture supernatants from each of the four cvm1 disruptants grown in soy medium and sampled at 70 and 93 h after inoculation revealed that none of the strains produced detectable levels of clavam-2-carboxylate, clavaminic acid, or 2-hydroxymethylclavam, but clavulanic acid production was observed (Fig. 4A and B). Furthermore, a bioassay with Bacillus sp. ATCC 27860 indicated that none of the isolates produced alanylclavam. All of the strains, however, appeared to produce wild-type levels of cephamycin C.

FIG. 4.

FIG. 4

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HPLC analysis of culture filtrates from cvm1 and cvm4/cvm5 mutants of S. clavuligerus. (A) Wild-type culture filtrate. (B) cvm1 mutant culture filtrate. (C) cvm4 and cvm5 mutant culture filtrate. Peak 1, clavaminic acid; peak 2, clavam-2-carboxylic acid; peak 3, 2-hydroxymethylclavam; peak 4, clavulanic acid.

Characterization of the cvm4 and cvm5 deletion mutant.

To explore the possibility that cvm4 and cvm5 were also clavam biosynthetic genes, a cvm4 and cvm5 double mutant was created. In that mutant portions of both genes were deleted and replaced with apr oriented in the direction opposite those of the cdo genes. The cvm4 and cvm5 deletion construct was introduced by transformation into S. clavuligerus, in which it exchanged with the corresponding wild-type region of the chromosome by a homologous recombination procedure. Mutants were isolated from three independent primary transformants (apparent frequency of mutation, 1 to 6%).

Cultures of the three mutant strains were cultivated in soy medium, and culture filtrates were examined by HPLC. All three produced clavulanic acid, but once again, none of the mutants produced clavam-2-carboxylate, clavaminic acid, or 2-hydroxymethylclavam (Fig. 4C). Likewise, a bioassay with Bacillus sp. strain ATCC 27860 failed to detect alanylclavam in the culture supernatants of the three mutants. However, bioassays with E. coli ESS showed that all of the isolates produced wild-type levels of cephamycin C.

Characterization of the S. clavuligerus cas1 mutant.

In previous studies, a cas2 disruption mutant had been shown to be conditionally defective in clavulanic acid production, with production occurring on soy medium but not on SA medium. Since this nutritional regulation was associated with cas1, a cas1 disruption mutant was prepared to examine the effect of the mutation on clavam and clavulanic acid production. The cloned cas1 gene, which was disrupted by insertion of a neo cassette in both orientations relative to the orientation of cas1, was introduced by transformation into S. clavuligerus, in which it exchanged with the resident wild-type gene at a low frequency to generate cas1 disruption mutants. Four mutants arising from two primary transformants were subjected to further analysis.

These strains were grown in SA and soy media, and their culture supernatants were sampled and analyzed by HPLC at 69 and 93 h. In SA medium, all of the mutants produced close to wild-type levels of clavulanic acid by 93 h. However, in soy medium, all of the mutants produced substantially less clavulanic acid than the wild type did (31 to 73% less) at 93 h. Likewise, the levels of clavam-2-carboxylate and 2-hydroxymethylclavam production were significantly reduced relative to the level of production by the wild type (reductions of 56 to 76% and 13 to 72%, respectively). Surprisingly, bioassays showed little change in the level of alanylclavam production by the mutants relative to that by the wild type, and the level of cephamycin C production was unchanged. The effect of the cas1 mutation on clavulanic acid and clavam production was the same regardless of the orientation of the disrupting neo cassette.

Characterization of the S. clavuligerus cas1 and cas2 mutant.

To complete the analysis of the cas mutants, a cas1 and cas2 double mutant was prepared by introducing the cas1 disruption construct (described above) into the cas2 mutant created previously (23). In these double mutants, each cas gene is disrupted by the insertion of an antibiotic resistance marker: neo in cas1 and apr in cas2. The cas1 and cas2 mutants were grown in SA and soy media, and samples of culture supernatants were analyzed by HPLC at 70 and 93 h for the production of clavulanic acid, clavam-2-carboxylate, and 2-hydroxymethylclavam and were analyzed for cephamycin C and alanylclavam production by bioassay. All four of the cas1 and cas2 disruptants, the wild type, and strain AP5 produced similar amounts of cephamycin C. As previously observed (23), the wild type produced clavulanic acid in both SA and soy media but produced clavam metabolites only in soy medium. As expected, all four of the cas1 and cas2 mutants failed to produce clavulanic acid or any of the other clavam metabolites, including alanylclavam, when they were grown in either medium.

DISCUSSION

Although a basic outline for the biosynthesis of clavulanic acid exists, little is known about the production of the antipodal clavams by S. clavuligerus and Streptomyces antibioticus, and even less is understood about the clavamycins produced by Streptomyces hygroscopicus (18). Feeding studies with S. clavuligerus showed that 13C-labelled ornithine was incorporated with equal efficiency into both clavulanic acid and clavam-2-carboxylate. Similar results were obtained when 13C-labelled proclavaminate was fed to S. clavuligerus (13). Feeding studies have also shown that clavaminic acid is a precursor of clavulanic acid in S. clavuligerus (10), and when doubly labelled clavaminic acid was fed to cultures of S. antibioticus, it was incorporated at low but equal efficiencies into both valclavam and 2-(2-hydroxyethyl)clavam (8). These results strongly suggested that clavulanic acid and the antipodal clavams have a common biosynthetic pathway up to and including the clavaminic acid step.

In a previous study, we provided support for the proposal that CAS1 and CAS2 contribute to a common intracellular pool of clavaminic acid that can be shunted toward the synthesis of clavulanic acid and/or the antipodal clavams (23). When cas2 was disrupted by gene replacement, the resulting mutant still produced 2-alanylclavam and clavulanic acid when it was grown in soy medium. This suggested that the cas2 mutation was complemented by cas1 and that the clavaminic acid produced was being used as a precursor for both clavulanic acid and antipodal clavam biosynthesis. A corollary to this proposal is that a cas1 mutation should be complemented by cas2.

In the present study we tested this hypothesis by constructing mutant strains of S. clavuligerus in which cas1 had been disrupted by insertional inactivation with a neo cassette. The resulting cas1 disruptants produced lower levels of clavulanic acid, clavam-2-carboxylate, and 2-hydroxymethylclavam and normal levels of 2-alanylclavam compared to the levels produced by the wild type grown in soy medium. These results demonstrate that the defect in cas1 is at least partially complemented by cas2. To confirm that all of the clavam metabolites required CAS for their synthesis and to eliminate the possibility of a third isozyme of CAS, we created S. clavuligerus double mutants that were disrupted in both cas1 and cas2. These strains failed to produce clavulanic acid or any of the clavam metabolites, including 2-alanylclavam, when they were grown in soy medium or SA medium. These results provided conclusive evidence that CAS1 and CAS2 contribute to a common pool of clavaminic acid that serves as a precursor for the biosynthesis of all clavams produced by S. clavuligerus.

Paradkar and Jensen (23) observed that wild-type S. clavuligerus efficiently produces clavulanic acid in SA medium but is unable to produce detectable levels of 2-alanylclavam. Furthermore, when a cas2 mutant of S. clavuligerus was grown in SA medium, it produced neither clavulanic acid nor 2-alanylclavam. A Northern analysis of RNA extracted from wild-type S. clavuligerus grown in SA medium failed to detect cas1 transcripts but showed high levels of the cas2 transcript. These results showed that cas2 is essential for clavulanic acid production in SA medium, in which cas1 is not expressed. More importantly, however, a correlation was observed between cas1 expression and clavam metabolite production. This correlation might be explained if cas1 and the genes for clavam biosynthesis are coordinately downregulated when S. clavuligerus is grown in SA medium. Alternatively, if the clavaminic acid produced by way of CAS2 was somehow sequestered and could be used only to synthesize clavulanic acid, a similar effect would result.

To extend the work of Paradkar and Jensen (23), we isolated a cosmid clone that contained cas1 and surrounding regions of the S. clavuligerus chromosome. Sequence analysis of the DNA flanking cas1 revealed the presence of several open reading frames, none of which appeared to be similar to the open reading frames immediately upstream or downstream of cas2. To determine if any of the cas1-associated open reading frames were involved in the biosynthesis of clavam metabolites, we generated mutants of S. clavuligerus that were disrupted in either cvm1 or cvm4 and cvm5. An HPLC analysis of the culture supernatants showed that both mutant types failed to produce detectable levels of clavam-2-carboxylate, clavaminic acid, or 2-hydroxymethylclavam in soy medium but produced wild-type amounts of clavulanic acid. Likewise, bioassays of culture supernatants revealed that the mutants were unable to produce 2-alanylclavam but produced wild-type levels of cephamycin C. Thus, the disruption of genes both upstream and downstream of cas1 completely blocked clavam biosynthesis but had no apparent effect on clavulanic acid or cephamycin C production.

A comparison of the deduced amino acid sequences of the genes immediately upstream and downstream of cas1 with sequences found in current protein sequence databases has revealed some similarities. However, the exact role and significance of the proposed products encoded by these genes are uncertain, primarily because little is known about the biochemistry of the pathway leading from clavaminic acid to the various clavam metabolites produced by S. clavuligerus. Egan et al. (8) have proposed a tentative pathway (Fig. 5) in which clavaminic acid is successively decarboxylated and deaminated to produce a branch-point intermediate that is then converted by way of a series of oxidation, hydrolysis, and condensation reactions to clavam-2-carboxylate, 2-hydroxymethylclavam, 2-formyloxymethylclavam, and 2-alanylclavam.

FIG. 5.

FIG. 5

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Pathway for the biosynthesis of clavulanic acid and the antipodal clavams beginning at proclavaminic acid (adapted from Egan et al. [8]). 1, proclavaminic acid; 2, clavaminic acid; 3, clavulanate-9-aldehyde; 4, clavulanic acid; 5, 2-formyloxymethylclavam; 6, 2-hydroxymethylclavam; 7, clavam-2-carboxylic acid; 8, 2-alanylclavam.

In this study, preliminary evidence for a common precursor that serves as a branch-point intermediate to all of the clavam metabolites is provided by the fact that cvm4 and cvm5 disruption mutants fail to produce clavam-2-carboxylate, 2-hydroxymethylclavam, and 2-alanylclavam. This hypothesis is also supported by the lack of clavam production in cvm1 mutant cultures. In the proposed biosynthetic pathway (8), clavaminic acid is decarboxylated and deaminated by way of a pyridoxal 5′-phosphate-dependent enzyme(s). The derived amino acid sequence of cvm6 possessed significant similarity to a variety of pyridoxal 5′-phosphate-dependent transaminases, as well as to the bifunctional 2,2-dialkylglycine decarboxylase from P. cepacia (32). It may therefore be that the cvm6 product plays a part in both the decarboxylation and the deamination of clavaminic acid and commits the product to the synthesis of the clavam metabolites.

ACKNOWLEDGMENTS

This work was supported by SmithKline Beecham Pharmaceuticals and by the Natural Sciences and Engineering Research Council of Canada.

REFERENCES

  • 1.Aidoo K A, Wong A, Alexander D C, Rittammer R A R, Jensen S E. Cloning, sequencing and disruption of a gene from Streptomyces clavuligerus involved in clavulanic acid biosynthesis. Gene. 1994;147:41–46. doi: 10.1016/0378-1119(94)90036-1. [DOI] [PubMed] [Google Scholar]
  • 2.Bachmann B O, Li R, Townsend C A. β-Lactam synthetase: a new biosynthetic enzyme. Proc Natl Acad Sci USA. 1998;95:9082–9086. doi: 10.1073/pnas.95.16.9082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Barton, B. 1996. Unpublished data.
  • 4.Denis F, Brzezinski R. An improved aminoglycoside resistance gene cassette for use in gram-negative bacteria and Streptomyces. FEMS Microbiol Lett. 1991;81:261–264. doi: 10.1016/0378-1097(91)90224-x. [DOI] [PubMed] [Google Scholar]
  • 5.Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1988;12:387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Doran J L, Leskiw B K, Petrich A K, Westlake D W S, Jensen S E. Production of Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli using two-cistron expression systems. J Ind Microbiol. 1990;5:197–206. doi: 10.1007/BF01569677. [DOI] [PubMed] [Google Scholar]
  • 7.Doran J L, Leskiw B K, Aippersbach S, Jensen S E. Isolation and characterization of a beta-lactamase-inhibitory protein from Streptomyces clavuligerus and cloning and analysis of the corresponding gene. J Bacteriol. 1990;172:4909–4918. doi: 10.1128/jb.172.9.4909-4918.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Egan L A, Busby R W, Iwata-Reuyl D, Townsend C A. Probable role of clavaminic acid as the terminal intermediate in the common pathway to clavulanic acid and the antipodal clavam metabolites. J Am Chem Soc. 1997;119:2348–2355. [Google Scholar]
  • 9.Elson S W, Baggaley K H, Gillett J, Holland S, Nicholson N H, Sime J T, Woroniecki S R. Isolation of two novel intracellular β-lactams and a novel dioxygenase cyclising enzyme from Streptomyces clavuligerus. J Chem Soc Chem Commun. 1987;1987:1736–1738. [Google Scholar]
  • 10.Elson S W, Baggaley K H, Gillett J, Holland S, Nicholson N H, Sime J T, Woroniecki S R. The roles of clavaminic acid and proclavaminic acid in clavulanic acid biosynthesis. J Chem Soc Chem Commun. 1987;1987:1739–1740. [Google Scholar]
  • 11.Elson S W, Oliver R S, Bycroft B W, Faruk E A. Studies on the biosynthesis of clavulanic acid. III. Incorporation of dl-[3,4-13C2]glutamic acid. J Antibiot. 1982;35:81–86. doi: 10.7164/antibiotics.35.81. [DOI] [PubMed] [Google Scholar]
  • 11a.European Bioinformaties Institute. 18 March 1999, revision date. [Online.] Fasta 3 database searches. http://www.ebi.ac.uk/htbin/fasta.py?request. [28 January 1999, last date accessed.]
  • 12.Hopwood D A, Bibb M J, Chater K F, Kieser T, Bruton C J, Kieser H M, Lydiate D J, Smith C P, Ward J M, Schrempf H. Genetic manipulation of Streptomyces: a laboratory manual. Norwich, United Kingdom: The John Innes Foundation; 1985. [Google Scholar]
  • 13.Iwata-Reuyl D, Townsend C A. Common origin of clavulanic acid and other clavam metabolites in Streptomyces. J Am Chem Soc. 1992;114:2762–2763. [Google Scholar]
  • 14.Janc J W, Egan L A, Townsend C A. Purification and characterization of clavaminate synthase from Streptomyces antibioticus: a multifunctional enzyme of clavam biosynthesis. J Biol Chem. 1995;270:5399–5404. doi: 10.1074/jbc.270.10.5399. [DOI] [PubMed] [Google Scholar]
  • 15.Jensen S E, Demain A L. Beta-lactams. In: Vining L C, Stuttard C, editors. Genetics and biochemistry of antibiotic production. Toronto, Ontario, Canada: Butterworth-Heinemann; 1995. pp. 239–268. [Google Scholar]
  • 16.Jensen S E, Alexander D C, Paradkar A S, Aidoo K A. Extending the β-lactam biosynthetic gene cluster in Streptomyces clavuligerus. In: Baltz R H, Hegeman G D, Skatrud P L, editors. Industrial microorganisms: basic and applied molecular genetics. Washington, D.C: American Society for Microbiology; 1993. pp. 169–176. [Google Scholar]
  • 17.Katz E, Thompson C J, Hopwood D A. Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol. 1983;129:2703–2714. doi: 10.1099/00221287-129-9-2703. [DOI] [PubMed] [Google Scholar]
  • 18.King H D, Langhärig J, Sanglier J J. Clavamycins, new clavam antibiotics from two variants of Streptomyces hygroscopicus. I. Taxonomy of the producing organisms, fermentation, and biological activities. J Antibiot. 1986;39:510–515. doi: 10.7164/antibiotics.39.510. [DOI] [PubMed] [Google Scholar]
  • 19.Marck C. ’DNA Strider’: a ’C’ program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988;16:1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Marsh E N, Chang M D-T, Townsend C A. Two isozymes of clavaminate synthase central to clavulanic acid formation: cloning and sequencing of both genes from Streptomyces clavuligerus. Biochemistry. 1992;31:12648–12657. doi: 10.1021/bi00165a015. [DOI] [PubMed] [Google Scholar]
  • 21.McNaughton H J, Thirkettle J E, Zhang Z, Schofield C J, Jensen S E, Barton B, Greaves P. Beta-lactam synthetase: implications for beta-lactamase evolution. J Chem Soc Chem Commun. 1998;1998:2325–2326. [Google Scholar]
  • 21a.National Center for Biotechnology Information. 19 March 1999 revision date. [Online.] The National Center for Biotechnology Information. http://www.ncbi.nih.gov/BLAST/. [28 January 1999, last date accessed.]
  • 22.Nicholson N H, Baggaley K H, Cassels R, Davison M, Elson S W, Fulston M, Tyler J W, Woroniecki S R. Evidence that the immediate biosynthetic precursor of clavulanic acid is its N-aldehyde analogue. J Chem Soc Chem Commun. 1994;1994:1281–1282. [Google Scholar]
  • 23.Paradkar A, Jensen S E. Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus. J Bacteriol. 1995;177:1307–1314. doi: 10.1128/jb.177.5.1307-1314.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Paradkar A S, Jensen S E, Mosher R H. Comparative genetics and molecular biology of β-lactam biosynthesis. In: Strohl W R, editor. Biotechnology of antibiotics, 2nd ed., revised and expanded. New York, N.Y: Marcel Dekker, Inc.; 1997. pp. 241–277. [Google Scholar]
  • 25.Pitlik J, Townsend C A. The fate of [2,3,3-2H3, 1,2-13C2]-d,l-glycerate in clavulanic acid biosynthesis. J Chem Soc Chem Commun. 1997;1997:225–226. [Google Scholar]
  • 26.Pruess D L, Kellett M. Ro-22-5417, a new clavam antibiotic from Streptomyces clavuligerus. I. Discovery and biological activity. J Antibiot. 1983;36:208–212. doi: 10.7164/antibiotics.36.208. [DOI] [PubMed] [Google Scholar]
  • 27.Reading C, Cole M. Clavulanic acid: a beta-lactamase-inhibiting beta-lactam from Streptomyces clavuligerus. Antimicrob Agents Chemother. 1977;11:852–857. doi: 10.1128/aac.11.5.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
  • 29.Sanger F, Nicklen S, Coulson A R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977;74:5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Stahl D A, Amann R. Development and application of nucleic acid probes. In: Stackebrandt E, Goodfellow M, editors. Nucleic acid techniques in bacterial systematics. Toronto, Ontario, Canada: John Wiley & Sons Ltd.; 1991. pp. 205–248. [Google Scholar]
  • 31.Thirkettle J E, Baldwin J E, Edwards J, Griffin J P, Schofield C J. The origin of the β-lactam carbons of clavulanic acid. J Chem Soc Chem Commun. 1997;1997:1025–1026. [Google Scholar]
  • 32.Toney M D, Hohenester E, Cowen S W, Jansonius J N. Dialkylglycine decarboxylase structure: bifunctional active site and alkali metal sites. Science. 1993;261:756–759. doi: 10.1126/science.8342040. [DOI] [PubMed] [Google Scholar]
  • 33.Uchiyama H, Weisblum B. N-Methyl transferase of Streptomyces erythraeus that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics: amino acid sequence and its homology to cognate R-factor enzymes from pathogenic bacilli and cocci. Gene. 1985;38:103–110. doi: 10.1016/0378-1119(85)90208-2. [DOI] [PubMed] [Google Scholar]
  • 34.Valentine B P, Bailey C R, Doherty A, Morris J, Elson S W, Baggaley K H, Nicholson N H. Evidence that arginine is a later metabolic intermediate than ornithine in the biosynthesis of clavulanic acid by Streptomyces clavuligerus. J Chem Soc Chem Commun. 1993;1993:1210–1211. [Google Scholar]
  • 35.Ward J M, Janssen G R, Kieser T, Bibb M J, Buttner M J, Bibb M J. Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol Gen Genet. 1986;203:468–478. doi: 10.1007/BF00422072. [DOI] [PubMed] [Google Scholar]
  • 36.Ward J M, Hodgson J E. The biosynthetic genes for clavulanic acid and cephamycin production occur as a ’super-cluster’ in three Streptomyces. FEMS Microbiol Lett. 1993;110:239–242. doi: 10.1111/j.1574-6968.1993.tb06326.x. [DOI] [PubMed] [Google Scholar]
  • 37.Wu T-K, Busby R W, Houston T A, McIlwaine D B, Egan L A, Townsend C A. Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis. J Bacteriol. 1995;177:3714–3720. doi: 10.1128/jb.177.13.3714-3720.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Zoller M J, Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. Methods Enzymol. 1987;154:329–350. doi: 10.1016/0076-6879(87)54083-6. [DOI] [PubMed] [Google Scholar]

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