INTRODUCTION

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Lysine--aminotransferase (LAT) is present exclusively in -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 -aminoadipate (AA). The second step is catalyzed by piperideine-6-carboxylate dehydrogenase, which has recently been purified from Streptomyces clavuligerus (7). -(L--Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) then catalyzes the condensation of the three precursor amino acids, L-AA, L-cysteine, and L-valine, into the linear tripeptide (21). ACV undergoes an oxidative cyclization by isopenicillin N synthase (IPNS) to close the -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 AA 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.

View larger version (22K): [in this window] [in a new window]   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 AA 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

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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.

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).

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).

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).

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.

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--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 [-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).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

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, lat::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 lat::tsr/term mutants were identified by their antibiotic resistance phenotype (thiostrepton resistant and hygromycin sensitive) and confirmed by Southern blot analysis. In the lat::tsr/term mutants, the point of insertion of the tsr/term cassette is upstream of the putative pcbAB promoter region. Therefore, in the lat::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.

View larger version (18K): [in this window] [in a new window]   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.

lat mutants have lost the ability to produce cephamycin C. Four independently created lat::tsr/term mutants and lat mutants were grown on TSBS medium or TSBS supplemented with 2 mM AA. 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 lat mutants, the antibiotic-negative phenotype was shown to be due solely to mutation of the lat gene, since supplementation of cultures with AA restored antibiotic production levels to an average of 18 and 33% of wild-type levels after 48 and 72 h, respectively. However, in the lat::tsr/term mutants supplementation with AA 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, lat mutants, and lat::tsr/term mutants, all grown in TSBS or TSBS plus 2 mM AA. Western blots were developed with antibodies specific for the LAT, IPNS, and DAOCS biosynthetic enzymes. The lat 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 lat mutants made approximately equivalent amounts of DAOCS and IPNS protein. The addition of AA restored low-level production of antibiotic to the lat mutants but had no stimulatory effect on IPNS production. The observation of wild-type levels of IPNS protein in the lat mutants suggests that neither a functional lat gene nor AA 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 lat mutant) restored IPNS protein production to wild-type levels.

View larger version (45K): [in this window] [in a new window]   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 lat mutant strains harvested after 72 h of growth in TSBS or TSBS plus 2 mM AA. (B) Western blot analysis of 5 µg of cell extract protein from wild-type, lat::apr, and lat::tsr/term mutant strains harvested after 72 h of growth in TSBS or TSBS plus 2 mM AA. 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 AA. (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.

lat::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 lat cell extracts, while ACVS was greatly reduced in lat::apr cell extracts and was absent from lat::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 lat mutant was at 115% of wild-type levels. The lat::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 AA 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 AA to TSBS medium-grown cultures restored low-level antibiotic production to the lat mutants but did not stimulate increased production of IPNS protein in any of the mutants. In an effort to increase the intracellular concentration of AA, 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 C31 attB site within the Streptomyces genome (4), and therefore the lat gene would have to function in trans to give complementation.

View larger version (59K): [in this window] [in a new window]   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, lat mutants, and lat 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, lat::tsr/term mutants, and lat::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.

lat::tsr/term mutants produce no pcbC polycistronic transcript and reduced levels of pcbC monocistronic transcript. Analysis of the lat mutants demonstrated that high-level production of ACVS and IPNS is not dependent upon the presence of AA or ACV, thus contradicting the interdependence model. The lat::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 lat::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 lat::tsr/term mutants and would result in production of a reasonable amount of IPNS.

View larger version (31K): [in this window] [in a new window]   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, lat::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.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

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 lat 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, lat mutants were unable to produce LAT but high levels of ACVS, IPNS, and DAOCS proteins remained present. This observation confirmed that production of AA 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 AA to the lat 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 lat 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 lat::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 AA nor complementation with lat gene constructs was able to stimulate ACVS and IPNS production. Since the putative pcbAB promoter region was present in the lat::tsr/term mutants, it appears that under these conditions the pcbAB promoter, in the presence or absence of AA, 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 lat::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 lat::tsr/term mutants. However, RPA of the pcbAB-pcbC intergenic region confirmed the absence of the pcbC-containing polycistronic transcript in lat::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 lat::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 lat::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 lat::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 lat mutants produce wild-type levels of IPNS even in the absence of AA and therefore ACV. As well, the pcbC monocistronic transcript was easily detected by RPA in RNA isolated from the lat mutants (data not shown).

The decreased transcription from the pcbC promoter seen in lat::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.