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 (Deltalat::tsr/term mutants) were created. This mutation eliminated the production of lysine-epsilon-aminotransferase (the lat gene product) but also affected the expression of downstream genes, indicating an operon arrangement. Production of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) (the pcbAB gene product) was eliminated in Deltalat::tsr/term mutants, while production of isopenicillin N synthase (IPNS) (the pcbC gene product) was greatly reduced. The provision of alpha-aminoadipate to the Deltalat::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 Deltalat::tsr/term mutants confirmed that the polycistronic transcript was absent but also indicated that monocistronic pcbC transcript levels were greatly decreased. In contrast, Deltalat 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.

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.

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.

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.

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.

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.