Early Cephamycin Biosynthetic Genes Are Expressed from a Polycistronic Transcript in Streptomyces clavuligerus

Abstract

A polycistronic transcript that is initiated at the lat promoter has been implicated in the expression of the genes involved in early steps of cephamycin C biosynthesis in Streptomyces clavuligerus. pcbC is also expressed as a monocistronic transcript from its own promoter. However, an alternative interpretation involving expression via three separate yet interdependent transcripts has also been proposed. To distinguish between these possibilities, mutants lacking the lat promoter and containing a transcription terminator within the lat gene (Δlat::tsr/term mutants) were created. This mutation eliminated the production of lysine-ɛ-aminotransferase (the lat gene product) but also affected the expression of downstream genes, indicating an operon arrangement. Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) (the pcbAB gene product) was eliminated in Δlat::tsr/term mutants, while production of isopenicillin N synthase (IPNS) (the pcbC gene product) was greatly reduced. The provision of α-aminoadipate to the Δlat::tsr/term mutants, either via exogenous feeding or via lat gene complementation, did not restore production of ACVS or IPNS. Analysis of RNA isolated from the Δlat::tsr/term mutants confirmed that the polycistronic transcript was absent but also indicated that monocistronic pcbC transcript levels were greatly decreased. In contrast, Δlat mutants created by in-frame internal deletion of lat maintained the polycistronic transcript and allowed production of wild-type levels of both ACVS and IPNS.

Lysine-ɛ-aminotransferase (LAT) is present exclusively in β-lactam-producing Streptomyces spp. and catalyzes the first step of cephamycin biosynthesis (19). Kern et al. (15) demonstrated that LAT converts lysine to 1-piperideine-6-carboxylate as the first step of a two-step reaction converting lysine to α-aminoadipate (αAA). The second step is catalyzed by piperideine-6-carboxylate dehydrogenase, which has recently been purified from Streptomyces clavuligerus (7). δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) then catalyzes the condensation of the three precursor amino acids, l-αAA, l-cysteine, and l-valine, into the linear tripeptide (21). ACV undergoes an oxidative cyclization by isopenicillin N synthase (IPNS) to close the β-lactam and thiazolidine rings and generate isopenicillin N. Six further enzymatic steps are required to catalyze the isomerization, ring expansion, and modification of the penicillin nucleus to produce cephamycin C.

All of the structural genes necessary for the biosynthesis of cephamycin C in S. clavuligerus are organized into a cluster together with regulatory and resistance genes (2). The genes encoding three of the early enzymes in the biosynthetic pathway, LAT, ACVS, and IPNS, are designated lat, pcbAB, and pcbC (21). The uniform transcriptional orientation and short intergenic regions separating lat, pcbAB, and pcbC in S. clavuligerus suggested that these genes might be organized into an operon for coordinate expression.

However, two contradictory models exist for the coordinated expression of lat, pcbAB, and pcbC (Fig. 1). The interdependence model suggests that three separate transcripts originating from the lat, putative pcbAB, and pcbC promoters are produced, with the transcription of each gene dependent upon the presence of the preceding gene product (8, 28). This model is based upon the results of DNA sequence and promoter probe analyses as well as complementation studies. Analysis of the DNA sequence from the lat-pcbAB intergenic region revealed a GC-rich stem-loop structure that was preceded by a possible antiterminator, proposed to allow conditional regulation at this site (28). As well, a region within the lat open reading frame (ORF) having two Streptomyces promoter-like sequences was proposed to be responsible for the independent transcription of pcbAB, providing a means for a small molecule such as αAA to regulate expression of pcbAB. A DNA fragment containing this putative promoter region gave strong levels of activity in the promoter probe vector pIJ487. Complementation of a lat mutant with a plasmid containing the lat gene and part of the pcbAB gene increased antibiotic production and LAT activity but also caused ACVS and IPNS activities to increase.

FIG. 1.

Proposed models for the transcriptional organization of the lat, pcbAB, and pcbC genes. Closed boxes indicate coding regions of the genes, and solid arrows represent transcripts initiated at their respective promoters. PCD, 1-piperideine-6-carboxylate dehydrogenase.

Alternatively, the cotranscription model suggests that lat, pcbAB, and pcbC are organized into an operon (23). Transcription from the lat promoter is proposed to proceed through the lat, pcbAB, and pcbC coding regions, giving rise to a 14-kb polycistronic transcript; pcbC is also expressed as a monocistronic transcript from its own promoter. Organization of biosynthetic genes into multicistronic transcripts in S. clavuligerus is not uncommon, as the cefD and cefE genes are part of a single transcript (16) that also includes the pcd gene (22). In another cephamycin C producer, Nocardia lactamdurans, the lat, pcbAB, and pcbC genes are closely spaced but the lat promoter gives rise to a monocistronic transcript and the pcbAB and pcbC genes are part of a polycistronic transcript that includes four other coding regions (10). Northern analysis of RNA from S. clavuligerus, with both lat- and pcbAB-specific probes, detected smears of high-molecular-weight mRNA only (24). The absence of a monocistronic lat mRNA suggested that lat and pcbAB were present on the same transcript despite the presence of a stem-loop structure in the lat-pcbAB intergenic region. Northern blot analysis with a pcbC-specific probe detected a monocistronic pcbC transcript primarily (25), although smears of high-molecular-weight mRNA were also evident on prolonged exposure of autoradiograms. S1 nuclease analysis showed that a transcript extends across all intergenic regions within the operon. This was interpreted to mean that the transcript which is initiated at the lat promoter is polycistronic and proceeds through pcbAB and then through pcbC. In addition, S1 nuclease analysis gave evidence of the promoter located within the 3′ end of pcbAB that is responsible for production of the monocistronic pcbC transcript. Both low- and high-resolution S1 nuclease transcript analysis of the putative pcbAB promoter region predicted by the interdependence model failed to detect a transcription start point within this region (A. S. Paradkar and S. E. Jensen, unpublished results). This suggested that the putative pcbAB promoter may not be functional in vivo.

Recently, Paradkar et al. (A. S. Paradkar, R. H. Mosher, C. Anders, S. E. Jensen, and B. Barton, unpublished results) created a lat::apr mutant in which lat is disrupted by an apramycin resistance (apr) marker inserted in the opposite orientation relative to the lat gene. This lat::apr mutant was designed to investigate whether blocking cephamycin C production would affect clavulanic acid production but also to provide information about the regulation of lat, pcbAB, and pcbC. The lat::apr mutant did not produce cephamycin C or LAT, but low levels of ACVS and IPNS activities remained. The addition of exogenous αAA to the medium, which bypassed the LAT defect, restored low-level antibiotic production to the lat::apr mutant (A. S. Paradkar and S. E. Jensen, unpublished results). Assuming that transcription from the lat promoter would be blocked in the lat::apr mutant, these data suggested that pcbAB and pcbC could both be expressed independently from the lat promoter, consistent with the interdependence model. However, the reduced levels of both ACVS and IPNS activities were also indicative of a polar effect. Furthermore, S1 nuclease analysis of RNA isolated from the lat::apr mutant showed no evidence of transcripts being initiated in the putative pcbAB promoter region. This raised the alternative possibility that the low levels of ACVS and IPNS produced in the lat::apr mutant might result from a polycistronic mRNA originating at the lat promoter and traversing the apr disruption marker despite its opposite orientation within lat.

Since the lat::apr mutant did not conclusively support either the interdependence or the cotranscription model for early gene regulation, two new lat mutants were created by gene replacement. Based on analyses of these mutants, we report strong in vivo evidence for the cotranscription model.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

The bacterial strains used in this study are listed in Table 1. S. clavuligerus was maintained on ISP medium 3 (Difco, Detroit, Mich.) or MYM agar (27) and stored as spore stocks in 20% (wt/vol) glycerol at −70°C. Plasmid-containing cultures were supplemented with 5 μg of thiostrepton (Sigma Chemical Corp., St. Louis, Mo.) per ml, 25 μg of apramycin (Provel Inc., Scarborough, Ontario, Canada) per ml, or 200 μg of hygromycin (Roche Molecular Biochemicals, Laval, Quebec, Canada) per ml as appropriate.

TABLE 1.

Bacterial strains and plasmids used in this study

Bacterial strains and plasmid vectors	Relevant features	Source or referencea
S. clavuligerus
NRRL 3585	Wild-type, cephamycin and clavulanic acid producer	NRRL
lat::apr	lat disruption mutant, insertion of apr marker, Aprr	Paradkar et al. (unpublished results)
Δlat::tsr/term	lat partial deletion mutant, insertion of tsr/terminator cassette, Tsrr	This study
Δlat	lat partial deletion mutant, removal of apr marker	This study
S. lividans
TK24	Wild-type, cephamycin nonproducer	D. Hopwood
E. coli
HB101	General cloning host and protein expression strain	26
ESS	β-Lactam supersensitive, indicator organism for bioassay	A. Demain
Cloning vectors
pBluescript KS/SK	E. coli general cloning vector, Ampr	Stratagene
pBluescript KSN/SKN	MCS BamHI site mutated to NcoI site by adapter insertion, Ampr	This study
pSL1180	E. coli general cloning vector, Ampr	Pharmacia
pTZ18R	E. coli general cloning vector, Ampr	United States Biochemicals
pJOE829	Streptomyces pIJ101 replicon, Hygr	J. Altenbuchner
pSET152	E. coli replicon, Streptomyces φC31 attachment site, Aprr	NRRL
pIJ486	Streptomyces pIJ101 replicon, Tsrr	D. Hopwood
pGEX-2T	E. coli glutathione S-transferase fusion expression vector, Ampr	Pharmacia
pHM8a	E. coli-Streptomyces expression plasmid, Hygr	H. Motamedi
Intermediate constructs
pDA170	ApaI fragment containing orf11, blp, and lat inserted into pKS	This study
pDA171	ApaI fragment from pDA170 into pKS in the opposite orientation	This study
pDA177	NcoI/XbaI fragment containing the lat ORF into pDA513	This study
pDA504	BclI fragment containing tsr inserted into pSL1180	This study
pDA508	hygr, ermE*, and FKMT terminator inserted into pSL1180	This study
pDA509	BamHI/SmaI fragment containing FKMT terminator into pDA504	This study
pDA513	EcoICRI/NcoI fragment containing ermE* into pSKN	This study
pDA538	EcoRI/HindIII tsr/terminator cassette fragment into pDA537	This study
Gene targeting vectors
pDA563	Shuttle vector carrying Δlat::tsr/term construct, Hygr Tsrr	This study
pDA566	Shuttle vector carrying Δlat construct, Tsrr	This study
E. coli high-level expression construct
pGEX-LAT	lat ORF inserted into pGEX-2T	This study
Complementation constructs
pDA1000	tsr marker inserted into pSET152	This study
pDA1050	Wild-type lat gene inserted into pSET152	This study
pDA1053	ermE* promoter fused to lat ORF inserted into pSET152	This study
RNA analysis constructs
PIPS-1	pcbAB and pcbC gene fragment inserted into pUC119	9
pDA205	SmaI fragment containing pcbAB and pcbC inserted into pKS	This study

^aPlasmids or strains were generously provided by J. Altenbuchner, University of Stuttgart, Stuttgart, Germany; D. Hopwood, John Innes Institute, Norwich, United Kingdom; A. Demain, Massachusetts Institute of Technology, Cambridge; and H. Motamedi, Merck Research Laboratories, Rahway, N.J. NRRL, Northern Regional Research Laboratory. 

S. clavuligerus production cultures were prepared as described previously (2) except that seed cultures were subcultured into either fresh Trypticase soy broth (Becton Dickinson and Company, Cockeysville, Md.) supplemented with 1% (wt/vol) soluble starch (Difco; TSBS) or TSBS plus 2 mM αAA (Sigma).

Escherichia coli cultures were maintained on 2YT agar and grown in Terrific Broth medium (26) at 37°C. Plasmid-containing cultures were supplemented with 100 μg of ampicillin (Sigma) per ml, 100 μg of apramycin per ml, or 50 μg of hygromycin per ml as appropriate.

Manipulation of recombinant DNA.

Ligation reactions, generation of blunt ends on DNA fragments with Klenow DNA polymerase, plasmid isolation, dephosphorylation of DNA with alkaline phosphatase, and E. coli transformations were all done as described in the work of Sambrook et al. (26). Restriction enzyme digestion of DNA was carried out according to the suppliers' recommendations. The GlassMax DNA isolation system (GIBCO BRL, Burlington, Ontario, Canada) was used to purify insert DNA fragments from agarose gel blocks. Protoplast generation, transformation, and selection of transformants were carried out as described in the work of Hopwood (12) and Bailey and Winstanley (3).

Plasmid cloning vectors and the plasmid constructs used for gene replacement or complementation studies are listed in Table 1. Where fragments with incompatible ends were to be ligated, they were either made blunt by treatment with Klenow DNA polymerase or passaged through intermediate vectors to pick up compatible sites. Plasmid designations shown in parentheses refer to the name assigned to the new construct.

Creation of S. clavuligerus lat mutants.

In order to prepare the tsr marker/FKMT (31-demethyl-FK506-o-methyltransferase) terminator cassette, the thiostrepton resistance (tsr) marker from pIJ486 was first inserted as a 1.1-kb BclI fragment into pSL1180 (pDA504). The hygromycin resistance marker, ermE* promoter, optimized Streptomyces Shine-Dalgarno sequence, and the FKMT transcription terminator were then removed from pHM8a (20) as a 3.4-kb SacI/BglII fragment and inserted into pSL1180 (pDA508). Finally, the FKMT terminator fragment was removed from pDA508 as a 1.3-kb BamHI/SmaI fragment and inserted into pDA504 upstream of the tsr marker (pDA509).

The Δlat::tsr/term mutant construct was prepared by inserting a 3.2-kb ApaI fragment of S. clavuligerus DNA containing part of orf11 and all of blp and lat into pBluescript KS in both orientations (pDA170 and pDA171). The EcoRI site in the pDA171 multiple cloning site (MCS) was removed by self-ligation after digestion with EcoRV and BamHI. A 2.3-kb EcoRI/HindIII fragment containing the tsr/terminator cassette from pDA509 was inserted into the lat gene after digestion with EcoRI and Eco47III to replace the lat promoter and the 5′ end of the gene. The plasmid was then linearized at the unique HindIII site within the MCS and converted into an E. coli-Streptomyces shuttle vector by inserting pJOE829 (pDA563). The resulting construct carried a contiguous stretch of S. clavuligerus DNA extending from orf11 through lat, except that the 0.7-kb EcoRI/Eco47III fragment containing the lat promoter and the 5′ end of the lat ORF was replaced with the tsr/terminator cassette.

The pDA563 plasmid was introduced into protoplasts of wild-type S. clavuligerus by transformation, and two transformants were allowed to sporulate in the absence of antibiotic selection to promote the loss of free plasmid. Mutants in which the wild-type lat gene was replaced by the Δlat::tsr/term construct through homologous recombination between the plasmid construct and the corresponding region of the chromosome were detected initially by their antibiotic resistance phenotype as described by Aidoo et al. (1). Thiostrepton-resistant isolates were screened for hygromycin sensitivity to identify putative gene replacement mutants. Southern blot analysis of restriction endonuclease-digested genomic DNA isolated from the putative gene replacement mutants gave appropriate hybridization patterns when hybridized with labeled probes containing either the lat gene or the tsr marker.

The Δlat mutant construct was prepared by inserting the 1.6-kb EcoRI/ApaI fragment containing the entire lat gene into pTZ18R. The plasmid was then digested with Acc65I and Eco47III and self-ligated, resulting in deletion of an internal portion of the lat gene. The mutated lat gene carrying the internal deletion was excised as a 1.2-kb EcoRI/XbaI fragment and inserted into pDA170 to replace the wild-type lat gene. The plasmid was linearized at the unique BamHI site in the MCS and converted to an E. coli-Streptomyces shuttle by ligation with BglII-digested pIJ486 (pDA566). The resulting construct carried a contiguous stretch of S. clavuligerus DNA extending from orf11 through lat, except that the lat gene contained an internal 354-bp in-frame deletion within the ORF.

The pDA566 plasmid was introduced into protoplasts of the S. clavuligerus lat::apr mutant (A. S. Paradkar and S. E. Jensen, unpublished results) by transformation, and two transformants were allowed to sporulate in the absence of antibiotic selection. Mutants in which the apr-disrupted lat gene had been replaced with the internally deleted lat gene by homologous recombination between the plasmid construct and the corresponding region of the chromosome (Δlat) were detected initially by their loss of apramycin resistance. The apramycin-sensitive isolates were screened for thiostrepton sensitivity to identify putative gene replacement mutants. Southern blot analysis of restriction endonuclease-digested genomic DNA isolated from the putative gene replacement mutants gave appropriate hybridization patterns when hybridized with labeled probes containing either the lat gene or the apr marker.

Creation of lat complementation constructs.

The ermE* expression cassette was prepared by removing the ermE* promoter and optimized Shine-Dalgarno sequence from pDA508 as a 0.3-kb EcoICRI/NcoI fragment and inserting it into pBluescript SKN digested with NcoI and SmaI (pDA513). The plasmid was digested with NdeI/XbaI, and a 1.6-kb NcoI/XbaI fragment of S. clavuligerus DNA containing the lat ORF was inserted (pDA177).

Three complementation constructs were prepared in the integrating vector pSET152. The first carried a 1.8-kb EcoRI/BamHI fragment of S. clavuligerus DNA containing the entire lat gene (pDA1050). The second carried a 2.0-kb EcoRI/XbaI fragment from pDA177 containing the lat ORF under the control of the ermE* promoter (pDA1053). The third carried the tsr marker from pDA504 inserted into pSET152 to act as a negative control (pDA1000).

Antibiotic quantitation and cell extract preparation.

Cultures of S. clavuligerus were analyzed for total antibiotic production by bioassay (13). The cell extracts for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were prepared as described previously (2), except that immediately after sonication samples were diluted 1:1 in 2× SDS-PAGE sample buffer to limit ACVS and LAT degradation before freezing at −20°C. Cell extracts for ACVS activity measurement were prepared and assayed as previously described (14).

SDS-PAGE and Western blot analysis.

Seventy-five-microgram amounts of cell extract proteins were separated on SDS–10% PAGs for ACVS protein analysis. Discontinuous 10% protein gels were cast and run with the Mini-Protean II gel apparatus (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada) as described previously (5). Gels were electrophoresed at 50 V at 4°C until the bromophenol blue dye migrated to the bottom of the gel.

Five-microgram amounts of cell extract protein were subjected to Western blot analysis as previously described (2). The primary antibodies were used at the dilutions indicated: IPNS, 1:10,000, and desacetoxycephalosporin C synthase (DAOCS), 1:10,000; and the purified LAT antiserum (see below) was used at a 1:400 dilution. DAOCS antibodies were generously provided by C. Reeves, Panlabs Inc.

Immunoaffinity purification of polyclonal antibodies to LAT.

Glutathione S-transferase–LAT protein inclusion bodies were purified from pGEX-LAT-containing E. coli cultures that had been induced by adding IPTG (isopropyl-β-d-thiogalactopyranoside) to a final concentration of 0.5 mM and then incubated at 37°C for 4 h. Repeated sonication, centrifugation, and washing with STE (200 mM NaCl, 1 mM EDTA, and 20 mM Tris, pH 7.4) and STE–1.0% (vol/vol) Tween 20 (three washes each) yielded a preparation of glutathione S-transferase–LAT inclusion bodies that was quite pure as judged by SDS-PAGE. The inclusion body preparation was mixed with sample buffer and electrophoresed on four separate SDS–10% PAGs, with approximately 200 μg of protein per gel, and transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated with primary antibody according to the manufacturer's description (ECL Western blotting protocols; Amersham Life Science, Inc., Oakville, Ontario, Canada) except that the LAT antiserum was used at a 1:50 dilution. Vertical strips were removed from each end, incubated with secondary antibody, developed, and exposed to film. After alignment, the undeveloped portions of the membranes containing the antigen and primary antibody complex were removed with a razor blade. The membrane pieces were incubated with 1 ml of 100 mM glycine (pH 2.5) for 10 min before addition of a 1/10 volume of 1 M Tris-HCl (pH 8.0) to neutralize (11). The immunoaffinity-purified antibody solutions were pooled and filter sterilized.

RNA analysis.

The 587-bp SmaI fragment containing the pcbAB-pcbC intergenic region was removed from PIPS-1 (9) and inserted into pBluescript KS. The resulting plasmid (pDA205) was sequenced to confirm the orientation of the insert. The pDA205 plasmid was linearized with PstI, and in vitro transcription was carried out with the MAXIscript in vitro transcription kit (Ambion, Inc., Austin, Tex.) and [α-³²P]UTP (New England Nuclear, Guelph, Ontario, Canada). The riboprobe (ca. 703 nucleotides) contained 110 bases of nonhomologous RNA from the vector MCS, 287 bases of the pcbC gene, the 31 bases of the pcbAB-pcbC intergenic region, and 275 bases from the 3′ end of the pcbAB gene. The full-length probe was purified by elution from a 5% PAG gel containing 8 M urea. The macerated gel slice containing the riboprobe was incubated in 0.5 M ammonium acetate–0.1 mM EDTA (pH 8.0) buffer containing RNAguard (Pharmacia Biotech, Baie d'Urfé, Quebec).

Cultures for RNA extraction were harvested after 48 h of growth, and total RNA was isolated and purified according to the method of Hopwood (12). Purified total RNA was quantitated spectrophotometrically at 260 nm (26), and quality was judged by agarose gel electrophoresis. Aliquots of total RNA isolated from Streptomyces lividans (5 μg), wild-type S. clavuligerus (5 μg), and the Δlat::tsr/term mutant (50 μg) were hybridized overnight at 42°C with 2.5 × 10⁴ cpm of labeled riboprobe by using the RPA II RNase protection assay (RPA) kit (Ambion, Inc.). Following hybridization, the reaction mixtures were digested with RNase A and RNase T₁ according to the manufacturers' recommendations. Digested samples were precipitated, redissolved in gel loading buffer, and briefly heated to 90°C prior to being loaded on a 5% PAG containing 8 M urea. Molecular size markers were generated by a Klenow polymerase reaction to fill in the 5′ overhangs of a BamHI-digested unrelated plasmid with [α-³²P]dCTP (New England Nuclear) and dATP, dGTP, and dTTP. The single-stranded DNA molecular size markers (5.9 kilobases, 460 bases, and 120 bases) were generated by boiling the labeled DNA prior to loading it on the gel.

RESULTS

lat was disrupted by homologous recombination.

A lat::apr mutant created by A. S. Paradkar et al. (unpublished results) was devoid of LAT activity but also showed significantly reduced levels of both ACVS and IPNS activity, suggestive of a polar effect. To investigate this situation more fully, two new lat mutants were generated by homologous recombination (Fig. 2). The first lat mutant, Δlat::tsr/term, was created with the pDA563 plasmid, which carries a stretch of S. clavuligerus DNA from the cephamycin gene cluster in which the lat promoter and the 5′ end of the lat ORF were deleted and replaced with a cassette containing the tsr marker and the FKMT transcription terminator (see Materials and Methods). Putative Δlat::tsr/term mutants were identified by their antibiotic resistance phenotype (thiostrepton resistant and hygromycin sensitive) and confirmed by Southern blot analysis. In the Δlat::tsr/term mutants, the point of insertion of the tsr/term cassette is upstream of the putative pcbAB promoter region. Therefore, in the Δlat::tsr/term mutants transcription from the pcbAB promoter should be detectable if the promoter is functional, but transcription from the lat promoter should be blocked.

FIG. 2.

lat mutants generated by homologous recombination. The darkly shaded boxes represent the 5′ end of pcbAB, and the lightly shaded boxes represent the lat target gene. The open boxes represent the tsr or apr antibiotic resistance markers, while the hatched box represents the FKMT transcription terminator. Asterisks mark the locations of the putative pcbAB promoters, and the dashed arrows represent possible transcripts originating from this region.

The second lat mutant, Δlat, was created with the pDA566 plasmid, which carries a stretch of S. clavuligerus DNA containing a mutated version of the lat gene in which an internal DNA fragment was deleted (see Materials and Methods). These Δlat mutants were created by carrying out a second gene replacement procedure on a lat::apr mutant in which the apr-disrupted lat gene was replaced with a defective lat gene containing an in-frame internal deletion but no selectable marker. Deletion of this internal fragment should block LAT production without greatly affecting production or stability of the 14-kb lat-pcbAB-pcbC transcript. In the Δlat mutants, the putative pcbAB promoter region is still present, since it is located downstream of the deleted fragment. Again, putative mutants were identified by their antibiotic resistance phenotype (thiostrepton sensitive and apramycin sensitive) and confirmed by Southern blot analysis.

lat mutants have lost the ability to produce cephamycin C.

Four independently created Δlat::tsr/term mutants and Δlat mutants were grown on TSBS medium or TSBS supplemented with 2 mM αAA. Culture supernatants were assayed for total antibiotic production after 48 and 72 h. None of the lat mutants showed any production of antibiotic in TSBS medium, whereas wild-type S. clavuligerus gave large zones of inhibition in an antibiotic bioassay. In the Δlat mutants, the antibiotic-negative phenotype was shown to be due solely to mutation of the lat gene, since supplementation of cultures with αAA restored antibiotic production levels to an average of 18 and 33% of wild-type levels after 48 and 72 h, respectively. However, in the Δlat::tsr/term mutants supplementation with αAA was not able to restore antibiotic production, suggesting that the antibiotic-negative phenotype was not due solely to the loss of the lat gene.

lat mutants were analyzed for LAT, IPNS, and DAOCS proteins by Western analysis.

Cell extracts were prepared for Western analysis from wild-type S. clavuligerus, lat::apr mutants, Δlat mutants, and Δlat::tsr/term mutants, all grown in TSBS or TSBS plus 2 mM αAA. Western blots were developed with antibodies specific for the LAT, IPNS, and DAOCS biosynthetic enzymes. The Δlat mutants, as expected, did not make any LAT protein when examined by Western analysis, whereas LAT protein was observed as a strongly reacting band in wild-type S. clavuligerus (Fig. 3A). In contrast, both wild-type S. clavuligerus and the Δlat mutants made approximately equivalent amounts of DAOCS and IPNS protein. The addition of αAA restored low-level production of antibiotic to the Δlat mutants but had no stimulatory effect on IPNS production. The observation of wild-type levels of IPNS protein in the Δlat mutants suggests that neither a functional lat gene nor αAA was required for production of IPNS protein. The lat::apr mutant produced small amounts of IPNS protein compared to the wild type, but the removal of the apr marker from the lat gene (converting the lat::apr mutant into a Δlat mutant) restored IPNS protein production to wild-type levels.

FIG. 3.

Presence of the LAT, ACVS, IPNS, and DAOCS biosynthetic enzymes in the lat mutant strains. (A) Western blot analysis of 5 μg of cell extract protein from wild-type, lat::apr, and Δlat mutant strains harvested after 72 h of growth in TSBS or TSBS plus 2 mM αAA. (B) Western blot analysis of 5 μg of cell extract protein from wild-type, lat::apr, and Δlat::tsr/term mutant strains harvested after 72 h of growth in TSBS or TSBS plus 2 mM αAA. The ability of each strain to produce antibiotic (Ceph C) was determined by bioassay, and the results were scored as follows: +, production; −, no production. The asterisks denote cultures that were supplemented with 2 mM αAA. (C) SDS–10% PAGE analysis of 75 μg of cell extract protein from each strain harvested after 48 h of growth. The arrow indicates the location of the ACVS protein band.

The Δlat::tsr/term mutants, like the lat::apr mutant, did not make any LAT protein when examined by Western analysis under conditions where the LAT protein was easily detected in wild-type S. clavuligerus (Fig. 3B). The Δlat::tsr/term mutants made near-wild-type amounts of DAOCS protein, but they did not appear to produce IPNS protein. IPNS enzyme activity was also not detected in the Δlat::tsr/term mutant cell extracts, but prolonged exposure of Western blots allowed the detection of very small amounts of IPNS protein, estimated to be less than 1% of wild-type levels (data not shown). Just as addition of αAA did not stimulate antibiotic production in the Δlat::tsr/term mutants, it also did not stimulate production of the IPNS protein.

Δlat::tsr/term mutants do not produce ACVS protein.

Although antibodies are not available to allow detection of ACVS by Western blot analysis, ACVS can be resolved from the other proteins in cell extracts by electrophoresis on an SDS–10% PAG because of its very high molecular mass (>400,000 Da). This provides a means to estimate qualitatively the amount of ACVS protein present in samples. Strong protein bands due to ACVS were present in both wild-type and Δlat cell extracts, while ACVS was greatly reduced in lat::apr cell extracts and was absent from Δlat::tsr/term extracts (Fig. 3C). Consistent with these observations, when cell extracts from the lat::apr mutant were assayed for ACVS activity, they showed specific activities at approximately 15% of wild-type levels. Specific ACVS activity in extracts from Δlat mutant was at 115% of wild-type levels. The Δlat::tsr/term mutant, which did not produce any detectable ACVS protein when analyzed by SDS-PAGE, also had no detectable ACVS activity. These results suggested that αAA is not required for ACVS production but that transcription from the lat promoter is essential.

Complementation of lat mutants does not restore ACVS or IPNS production.

The addition of αAA to TSBS medium-grown cultures restored low-level antibiotic production to the Δlat mutants but did not stimulate increased production of IPNS protein in any of the mutants. In an effort to increase the intracellular concentration of αAA, the lat mutants were transformed with pSET152-based vectors containing either the wild-type lat gene (pDA1050), a similar construct carrying the lat gene with its native promoter replaced by the strong constitutive ermE*-based expression cassette (pDA1053), or a negative control construct containing only the tsr marker (pDA1000). The pSET152-based vectors integrate at the φC31 attB site within the Streptomyces genome (4), and therefore the lat gene would have to function in trans to give complementation.

The Δlat mutant was complemented to produce antibiotic at 47% of wild-type levels when the wild-type lat construct was added and to 97% of wild-type levels when the ermE*-lat construct was added. The increase in antibiotic production levels in the complemented strains suggested that low intracellular αAA levels limited production in previous experiments where αAA was added exogenously. LAT protein was not detected by Western blot analysis in strains that were transformed with the negative control plasmid pDA1000 but was easily detected in strains transformed with either pDA1050 or pDA1053 (Fig. 4A). Production of DAOCS and IPNS proteins by the Δlat mutants was not affected by the presence of a functional lat gene.

FIG. 4.

Western blot analysis of lat mutants complemented with pSET152-based recombinant plasmids containing the lat gene. (A) Western blot analysis of 5 μg of cell extract protein harvested after 48 h of growth in TSBS from the wild type, Δlat mutants, and Δlat mutants transformed with lat gene constructs. (B) Western blot analysis of 5 μg of cell extract protein harvested after 48 h of growth in TSBS from the wild type, Δlat::tsr/term mutants, and Δlat::tsr/term mutants transformed with lat gene constructs. The ability of each strain to produce antibiotic (Ceph C) was determined by bioassay, and the results were scored as follows: +, production; −, no production. The A and B designations represent two independent transformants generated during the same transformation.

The Δlat::tsr/term mutants were not complemented to produce antibiotic by any of the pSET152-based constructs. Western blot analysis confirmed the return of LAT protein production when mutants were transformed with either pDA1050 or pDA1053, but IPNS protein production was not stimulated (Fig. 4B). Similarly, the Δlat::tsr/term mutant transformed with either of the functional lat genes also did not produce any ACVS protein when analyzed by SDS-PAGE (data not shown). Therefore, even in the presence of intracellular αAA levels capable of supporting wild-type antibiotic production, no evidence for a putative pcbAB promoter was seen in the Δlat::tsr/term mutant.

Δlat::tsr/term mutants produce no pcbC polycistronic transcript and reduced levels of pcbC monocistronic transcript.

Analysis of the Δlat mutants demonstrated that high-level production of ACVS and IPNS is not dependent upon the presence of αAA or ACV, thus contradicting the interdependence model. The Δlat::tsr/term mutants confirmed the prediction of the cotranscription model that premature termination of the lat-pcbAB-pcbC polycistronic message eliminates the production of ACVS. However, the severe reduction in IPNS in the Δlat::tsr/term mutants was unexpected since Petrich et al. (25) detected a monocistronic pcbC transcript in wild-type S. clavuligerus. It was presumed that the monocistronic transcript would still be present in the Δlat::tsr/term mutants and would result in production of a reasonable amount of IPNS.

In order to investigate this phenomenon more fully, total RNA was isolated from wild-type S. clavuligerus and two Δlat::tsr/term mutants to determine if the monocistronic pcbC transcript was still present in the Δlat::tsr/term mutants. RPAs were carried out with a 703-nucleotide riboprobe which spans the pcbAB-pcbC intergenic region and also includes 110 nucleotides of nonhomologous vector-derived sequence (see Materials and Methods). The presence of a pcbC-containing polycistronic transcript, originating at the lat promoter or the putative pcbAB promoter, would give full-length protection over the homologous region of the riboprobe, resulting in a 593-nucleotide protected fragment, whereas the monocistronic pcbC transcript would protect a 376-nucleotide fragment (Fig. 5A).

FIG. 5.

Analysis of RNA for pcbC-containing transcripts. (A) Riboprobe used to characterize the pcbAB-pcbC intergenic region. pcbC and the 3′ end of pcbAB are represented as gray boxes. The expected sizes of the RNase-protected fragments are shown. (B) RPA of RNA isolated from wild-type S. clavuligerus, Δlat::tsr/term mutant, and S. lividans. The RNA samples were hybridized with the labeled riboprobe, and the resulting hybrids were treated with RNase A and RNase T₁. Hybridization signals due to full-length protection of the probe (minus the nonhomologous sequence) and the pcbC start protected fragment are indicated with arrows. Numbers at left indicate sizes in nucleotides.

RPA of RNA isolated from wild-type S. clavuligerus confirmed the presence of both the polycistronic transcript (full-length protected product) and the pcbC monocistronic transcript (Fig. 5B), originally observed by Petrich et al. (24) by S1 nuclease transcript analysis. Analysis of RNA from the Δlat::tsr/term mutant showed no evidence of full-length protected product but also showed greatly reduced production of the pcbC monocistronic transcript. To confirm the absence of any transcription from the putative pcbAB promoter, a 10-fold-greater amount of Δlat::tsr/term RNA than of wild-type RNA was used in the RPA (Fig. 5B). Even with this 10-fold-increased amount of RNA, the pcbC-containing polycistronic transcript was not detected in the Δlat::tsr/term mutant, and the hybridizing band due to the pcbC monocistronic transcript was still less intense than that seen with the wild type. No hybridizing transcripts were detected in the S. lividans control RNA.

DISCUSSION

Our results establish that the cotranscription model more accurately describes the transcriptional regulation of early cephamycin biosynthetic genes in S. clavuligerus than does the interdependence model. Polar effects on downstream genes resulting from insertional disruption of an ORF often confuse the assignment of a mutant phenotype (17). The Δlat mutants were designed to be defective in LAT specifically, while having little or no effect on other genes that might be part of a polycistronic transcript. As expected, Δlat mutants were unable to produce LAT but high levels of ACVS, IPNS, and DAOCS proteins remained present. This observation confirmed that production of αAA was not necessary for the expression of pcbAB and pcbC to wild-type levels, which is contrary to the prediction of the interdependence model. As well, the provision of αAA to the Δlat mutants by exogenous supplementation or via lat gene complementation did not cause any further increase in the production of ACVS or IPNS.

The removal of the apr marker from the lat::apr mutant to generate the Δlat mutants had a marked stimulatory effect on the levels of ACVS and IPNS produced. This result further supports the cotranscription model, suggesting that the apr marker was interfering with transcription of the lat-pcbAB-pcbC polycistronic transcript and that its removal resulted in a return to wild-type levels of ACVS and IPNS production.

SDS-PAGE or Western blot analysis of the Δlat::tsr/term mutants demonstrated the absence of the LAT and ACVS proteins and the production of only trace amounts of IPNS protein. These mutants were capable of producing near-wild-type levels of DAOCS, a cephamycin biosynthetic enzyme arising from an unrelated transcript. Neither exogenous supplementation of αAA nor complementation with lat gene constructs was able to stimulate ACVS and IPNS production. Since the putative pcbAB promoter region was present in the Δlat::tsr/term mutants, it appears that under these conditions the pcbAB promoter, in the presence or absence of αAA, was unable to direct pcbAB transcription.

Conversely, this also suggests that in the wild-type organism, the stem-loop structure located between lat and pcbAB does not prevent transcription from proceeding into pcbAB and pcbC under normal growth conditions. This raises the possibility of an antiterminator regulation relationship (28) that allows transcription to proceed through the stem-loop. Alternatively, the stem-loop may not be a terminator but may function to protect the lat coding region of the transcript from degradation by RNases.

Surprisingly, in the Δlat::tsr/term mutants production of IPNS was barely detectable. Since pcbC is transcribed as both a monocistronic and a polycistronic transcript in wild-type S. clavuligerus, IPNS derived from translation of the pcbC monocistronic transcript was expected to be present in Δlat::tsr/term mutants. However, RPA of the pcbAB-pcbC intergenic region confirmed the absence of the pcbC-containing polycistronic transcript in Δlat::tsr/term mutants but also indicated that the level of the monocistronic pcbC transcript was dramatically reduced. Therefore, the severe decrease in IPNS production seen in the Δlat::tsr/term mutants is attributable not only to the elimination of the polycistronic pcbC transcript but also to a marked reduction in the abundance of the monocistronic pcbC transcript. It is not immediately evident why a mutation which eliminates the polycistronic transcript should have any deleterious effect on production of the monocistronic pcbC transcript, especially since the lat and pcbC promoters are separated by about 13 kb of DNA.

In past studies, heterologous expression of pcbC from S. clavuligerus has proven to be difficult to achieve in both S. lividans and E. coli (9, 24). However, poor translation of the pcbC monocistronic transcript, rather than poor transcription, was presented as the most likely cause of the poor expression. Irrespective of how well the pcbC monocistronic transcript may be translated, the Δlat::tsr/term mutants demonstrated that elimination of transcription from the lat promoter decreased the production of the pcbC monocistronic transcript. This deleterious effect of the Δlat::tsr/term mutation on the production of the monocistronic pcbC transcript cannot simply be due to the failure of the mutants to produce ACV, as predicted by the interdependence model, because Δlat mutants produce wild-type levels of IPNS even in the absence of αAA and therefore ACV. As well, the pcbC monocistronic transcript was easily detected by RPA in RNA isolated from the Δlat mutants (data not shown).

The decreased transcription from the pcbC promoter seen in Δlat::tsr/term mutants suggests that transcription from the lat promoter is necessary for optimal transcription from the pcbC promoter. Interactions between promoters have been observed in other systems, where topological effects of transcription from one promoter can influence transcription from a second promoter. The twin-supercoiled domain model of Liu and Wang (18) predicts that a rotationally hindered RNA polymerase would generate positive supercoils ahead of its passage and negative supercoils in its wake. The large bulk of the transcription apparatus involved in transcription-translation of the 14-kb lat-pcbAB-pcbC polycistronic transcript could cause such a rotational hindrance and therefore could significantly affect the local level of DNA supercoiling as it passed through lat, pcbAB, and pcbC. Perhaps a transient alteration of supercoiling is necessary for transcriptional initiation from the pcbC individual promoter. However, for this pcbC regulation mechanism to function it must be assumed that DNA topoisomerases do not immediately resolve the supercoiling changes and that the presumably linear S. clavuligerus chromosome is sufficiently constrained to allow localized domains of supercoiling to occur. To test if pcbC expression is topologically coupled to lat promoter activity, a gene replacement mutant in which the lat promoter is replaced with a regulatable promoter could be created. Levels of pcbC monocistronic transcript could then be monitored to determine if addition of inducer causes an increase in expression from the pcbC promoter.