Transcriptional analysis and regulation of carnobacteriocin production in Carnobacterium piscicola LV17.

Bacteriocin production by Carnobacterium piscicola LV17 (carnobacteriocin, Cbn) depends on the level of inoculation when grown in liquid medium. With an inoculum of > or = 10(6) colony-forming units per ml (cfu/ml), bacteriocin production is observed during exponential growth, whereas with < or = 10(4) cfu/ml no bacteriocin is detected even when the culture has reached stationary phase. Using pure bacteriocins, it was demonstrated that bacteriocin production is autoregulated. To understand how bacteriocin production is regulated at the molecular level, cell-free supernatant from a bacteriocin-producing culture was added to fresh medium at 1% (v/v) together with a non-producing inoculum (10(4) cfu/ml), to induce bacteriocin production (induced culture). Northern analysis revealed major transcripts of 0.35, 1.5 and 1 kb for carnobacteriocins A, B2 and BM1, respectively, indicating that regulation of bacteriocin production by inoculum size occurs at the transcriptional level. Primer extension demonstrated that transcription initiated from the same promoters with the induced culture as with the positive control (culture inoculated at 10(7) cfu/ml). Quantitative phosphorimager analysis of the primer extension products indicated that cbnA transcript was more abundant than cbnB2 or cbnBM1.

Transcriptional analysis and heterologous expression of the gene encoding beta-lactamase inhibitor protein (BLIP) from Streptomyces clavuligerus.

Transcription of bli, the gene encoding beta-lactamase (Bla) inhibitor protein (BLIP) of Streptomyces clavuligerus, was analyzed by promoter-probe studies, Northern hybridization and high-resolution S1 nuclease mapping. The 1-kb SalI DNA fragment immediately upstream from the bli open reading frame (ORF) showed promoter activity when tested using the xylE-based promoter-probe vector, pIJ4083. The promoter activity was approx. 36-fold higher in S. clavuligerus than in S. lividans. Northern hybridization analysis of S. clavuligerus RNA revealed that bli was expressed as a 0.7-kb monocistronic transcript. High-resolution S1 nuclease mapping identified the transcription start point as an A residue 47 bp upstream from the bli start codon. When the bli ORF, along with 111 bp of upstream sequence including the promoter, was introduced into S. lividans, the transformants produced BLIP, but in amounts approx. 12-fold lower than that produced by S. clavuligerus. Involvement of some additional regulatory element that is present in S. clavuligerus, but absent in S. lividans, could explain the difference in the promoter activities and therefore the difference in the overall expression of bli in the two hosts.

Transcriptional mapping of the genes encoding the early enzymes of the cephamycin biosynthetic pathway of Streptomyces clavuligerus.

Isopenicillin-N synthase (IPNS) of Streptomyces clavuligerus is encoded by the pcbC gene which is found within the cephamycin biosynthetic gene cluster. pcbC is located directly downstream from lat and pcbAB, which encode the enzymes, lysine epsilon-amino transferase and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, respectively. These enzymes act prior to IPNS in the biosynthetic pathway, and the three genes are transcribed in the same direction. Previous pcbC transcriptional studies involving recombinant promoter probe plasmids, Northern analysis and 5' primer extension indicated the presence of a monocistronic 1.2-kb transcript that initiated within pcbAB, 92-bp upstream from the pcbC start codon. S1 nuclease mapping studies have now shown, not only the transcript initiating 92 bp upstream from pcbC, but also a transcript initiating further upstream, possibly including the entire pcbAB gene. Promoter probe analysis and S1 nuclease mapping failed to detect promoter activity or a transcription start point (tsp) directly upstream from pcbAB, suggesting that pcbAB transcripts initiated within or upstream from lat. Northern analysis, to search for a pcbAB transcript, showed no distinct transcript and indicated severely degraded mRNA. Similar results were obtained when Northern analysis was used to search for lat transcripts. Promoter probe analysis to locate the lat promoter indicated that a sequence promoting transcription was present in a 330-bp DNA fragment that extended from 227-bp upstream from the lat structural gene to 103 bp inside the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of the genes for anthranilate synthetase from Streptomyces venezuelae ISP 5230.

Fragments of genomic DNA from Streptomyces venezuelae ISP5230 were cloned in the Escherichia coli expression vector pTZ18R and the plasmids were used to transform E. coli JA194 (trpE). The transformants included a prototrophic strain containing a recombinant plasmid, pDQ181, with an approximately 6.8-kb insert. Subcloning located the trpE-complementing DNA in a 2.4-kb segment. Transformation of E. coli ED23 (lacking both trpE and trpG functions) with plasmids containing the 2.4-kb DNA segment gave prototrophic strains exhibiting both the ASI and ASII activities of anthranilate synthetase. The results indicated that trpE and trpG are clustered in S. venezuelae. Regions hybridizing to the pDQ181 insert were present in the genomic DNA of other streptomycetes.

Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

A Streptomyces clavuligerus mutant disrupted in cas2, encoding the clavaminate synthase (CAS2) isoenzyme, was constructed by a gene replacement procedure. The resulting cas2 mutant showed no clavulanic acid production when grown in starch-asparagine medium. However, in soy medium, the cas2 mutant did produce clavulanic acid, although in amounts less than those produced by wild-type cultures. This medium-dependent leaky phenotype correlated well with the presence of the cas1 transcript, encoding the CAS1 isoenzyme, in cultures grown in soy medium and with its absence from those grown in starch-asparagine medium. This suggested that CAS1 and CAS2 both contribute to clavulanic acid production but that their production is regulated differently. Under nutritional conditions in which cas1 expression is blocked, cas2 becomes essential for clavulanic acid production. Northern (RNA) analysis revealed that while cas1 is transcribed as a 1.4-kb monocistronic transcript only, cas2 is transcribed both as a 1.2-kb monocistronic transcript and as part of a 5.3-kb polycistronic transcript. High-resolution S1 nuclease analysis located the transcription start point of the monocistronic cas2 transcript at a C residue 103 nucleotides upstream from the cas2 start codon.

Biosynthesis and molecular genetics of clavulanic acid.

The biosynthesis of clavulanic acid and related clavam metabolites is only now being elucidated. Understanding of this pathway has resulted from a combination of both biochemical studies of purified biosynthetic enzymes, and molecular genetic studies of the genes encoding these enzymes. Clavulanic acid biosynthesis has been most thoroughly investigated in Streptomyces clavuligerus where the biosynthetic gene cluster resides immediately adjacent to the cluster of cephamycin biosynthetic genes. A minimum of eight structural genes have been implicated in clavulanic acid biosynthesis, although more are probably involved. While details of the early and late steps of the pathway remain unclear, synthesis proceeds from arginine and pyruvate, as the most likely primary metabolic precursors, through the monocyclic beta-lactam intermediate, proclavaminic acid, to the bicyclic intermediate, clavaminic acid, which is a branch point leading either to clavulanic acid or the other clavams. Conversion of clavaminic acid to clavulanic acid requires side chain modification as well as inversion of ring stereochemistry. This stereochemical change occurs coincident with acquisition of the beta-lactamase inhibitory activity which gives clavulanic acid its therapeutic and commercial importance. In contrast, the other clavam metabolites all arise from clavaminic acid with retention of configuration and lack beta-lactamase inhibitory activity.

A pathway-specific transcriptional activator regulates late steps of clavulanic acid biosynthesis in Streptomyces clavuligerus.

A Streptomyces clavuligerus gene (designated claR) located downstream from the gene encoding clavaminate synthase in the clavulanic acid biosynthetic gene cluster is involved in regulation of the late steps in clavulanic acid biosynthesis. Nucleotide sequence analysis and database searching of ClaR identified a significant similarity to the helix-turn-helix motif (HTH) region of LysR transcriptional regulators. A gene replacement mutant disrupted in claR was unable to produce clavulanic acid, suggesting that claR is essential for clavulanic acid biosynthesis. Furthermore, the accumulation of clavaminic acid in the claR mutant suggested that ClaR regulates the late steps in the clavulanic acid pathway, i.e. those involved in the conversion of clavaminic acid to clavulanic acid. Transcriptional analysis using RNA isolated from the wild type and the claR mutant showed that the expression of the putative late genes, but not the early genes, was regulated by ClaR. High-resolution S1 nuclease analysis of claR suggested that it is expressed as a monocistronic transcript and also as a bicistronic transcript along with the late gene orf-9. The transcription start site of the monocistronic claR transcript was identified as a C residue 155 nucleotides upstream from the claR start codon.

Location of the genes for anthranilate synthase in Streptomyces venezuelae ISP5230: genetic mapping after integration of the cloned genes.

The anthranilate synthase (trpEG) genes in Streptomyces venezuelae ISP5230 were located by allowing a segregationally unstable plasmid carrying cloned S. venezuelae trpEG DNA and a thiostrepton resistance (tsr) marker to integrate into the chromosome. The integrated tsr was mapped by conjugation and transduction to a location close to tyr-2, between arg-6 and trpA13. A genomic DNA fragment containing trpC from S. venezuelae ISP5230 was cloned by complementation of a trpC mutation in Streptomyces lividans. Evidence from restriction enzyme analysis of the cloned DNA fragments, from Southern hybridization using the cloned trp DNA as probes, and from cotransduction frequencies, placed trpEG at a distance of 12-45 kb from the trpCBA cluster. The overall arrangement of tryptophan biosynthesis genes in the S. venezuelae chromosome differs from that in other bacteria examined so far.

Molecular analysis of a beta-lactam resistance gene encoded within the cephamycin gene cluster of Streptomyces clavuligerus.

A Streptomyces clavuligerus gene (designated pcbR) which is located immediately downstream from the gene encoding isopenicillin N synthase in the cephamycin gene cluster was characterized. Nucleotide sequence analysis and database searching of PcbR identified a significant similarity between PcbR and proteins belonging to the family of high-molecular-weight group B penicillin-binding proteins (PBPs). Eight of nine boxes (motifs) conserved within this family of proteins are present in the PcbR protein sequence in the same order and with approximately the same spacing between them. When a mutant disrupted in pcbR was constructed by gene replacement, the resulting pcbR mutant exhibited a significant decrease in its resistance to benzylpenicillin and cephalosporins, indicating that pcbR is involved in beta-lactam resistance in this organism. Western blot (immunoblot) analysis of S. clavuligerus cell membranes using PcbR-specific antibodies suggested that PcbR is a membrane protein. PcbR was also present in cell membranes when expressed in Escherichia coli and was able to bind radioactive penicillin in a PBP assay, suggesting that PcbR is a PBP. When genomic DNAs from several actinomycetes were probed with pcbR, hybridization was observed to some but not all beta-lactam-producing actinomycetes.

Enzymes catalyzing the early steps of clavulanic acid biosynthesis are encoded by two sets of paralogous genes in Streptomyces clavuligerus.

Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a "supercluster" in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway.

Genes specific for the biosynthesis of clavam metabolites antipodal to clavulanic acid are clustered with the gene for clavaminate synthase 1 in Streptomyces clavuligerus.

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.

Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production.

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine epsilon-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.

Five additional genes are involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for beta-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.