Transcriptional analysis of the isopenicillin N synthase-encoding gene of Streptomyces clavuligerus

Petrich, Astrid; Wu, Xiaoning; Roy, Kanak; Jensen, Susan E.

doi:10.1016/0378-1119(92)90605-o

Cited by 17 publications

(18 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…8, lanes H and I). It has been demonstrated that isopenicillin N synthase enzymatic activity also appears only after 36 h of growth in liquid media (47). .i DISCUSSION Sequence analysis has confirmed the conclusion of a previous study that inverted terminal repeats =900 bp long exist in pSCL1 (23).…”

Section: Cgcgg-cctg-ccg----gct-cgct-cg-g---c-ct---gc-g-gcc-c-gag--c 146supporting

confidence: 78%

Complete nucleotide sequence of a linear plasmid from Streptomyces clavuligerus and characterization of its RNA transcripts

Roy

1993

J Bacteriol

View full text Add to dashboard Cite

The complete nucleotide sequence of a small linear plasmid (pSCL1) from Streptomyces clavuligerus has been determined. This plasmid is 11,696 bp in length, has a 72% G+C content, and has =900-bp inverted terminal repeat sequences. A comparison of the inverted terminal repeats of pSCL1 with those of a linear plasmid from S. rochei shows that the two terminal sequences have a high degree of similarity (=70%) (19). One of the most notable features of this genus is that many members are capable of producing antibiotics. Because of their consequent medical and industrial importance, much attention has been focused on the genetics of this and related genera, particularly with regard to the organization and regulation of genes involved in antibiotic biosynthesis (18,56). Most of the genes directing the synthesis of antibiotics in streptomycetes appear to be chromosomal. However, pulse-field gel electrophoresis experiments have shown that a number of antibiotic-producing Streptomyces spp. harbor giant linear plasmids (27). Moreover, plasmid SCP1 in Streptomyces coelicolor A3(2), which has been demonstrated by genetic methods (29) to encode methylenomycin biosynthesis and resistance genes, is a 350-kb giant linear plasmid (26). Less convincing is the evidence suggesting that a 520-kb linear plasmid (pKSL) may be required for the production of lasalocid and echinomycin by S. lasaliensis (26,27) and that a 17-kb linear plasmid (pSLA2) may be involved in the production of the lankacidin group of antibiotics by S. rochei (15).Among the prokaryotes, linear plasmids have been found largely in Streptomyces spp. and other actinomycetes (e.g., 22), although they have also been found in a wide variety of eukaryotic organisms (12). The linear plasmids in eukaryotes are almost all double-stranded DNA molecules, with proteins covalently linked to their 5' termini. These have been completely sequenced, including two mitochondrial plasmids in Zea mays, S1 and S2 (45), and three nonmitochondrial plasmids in fungi, pGKL-1 and pGKL-2 in Kluyveromyces lactis (59, 67) and pClK-1 in Claviceps purpurea (43). Almost all linear plasmids sequenced to date have long inverted terminal repeats and include a putative DNA polymerase gene.Recently, a different type of linear plasmid, having covalently closed ends at the termini of its DNA, was reported for Escherichia coli harboring plasmid prophage N15 (63), for the bacterium Borrelia burgdorferi (2), as well as for the fungus Rhizoctonia solani (37).Although eukaryotic linear plasmids have been extensively studied, much less is known of linear plasmids in Streptomyces spp. Until recently, the only sequence information reported for Streptomyces linear plasmids was the 614-bp terminally repeated sequence of pSLA2 from S. rochei (17) and the mmr region of SCP1 (42). During the past year, portions of the terminal sequences of SCP1 have also been published (28). In a previous study, we reported the isolation and characterization of a 12-kb linear plasmid (pSCL1) in S. clavuligerus, a ,-lactam antibiot...

show abstract

Section: Cgcgg-cctg-ccg----gct-cgct-cg-g---c-ct---gc-g-gcc-c-gag--c 146supporting

confidence: 78%

Complete nucleotide sequence of a linear plasmid from Streptomyces clavuligerus and characterization of its RNA transcripts

Roy

1993

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Resultsmentioning

confidence: 97%

“…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.…”

mentioning

confidence: 98%

See 1 more Smart Citation

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

et al. 2000

Self Cite

View full text Add to dashboard Cite

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

show abstract

“…The predicted promoter region associated with TSP-1 is TGGAAT-17 bp-AAACAT while that of TSP-2 is TCCCGA-18 bp-GTTCTT. Both of these promoters show only marginal similarity to other promoters associated with cephamycin and clavulanic acid production (Kovacevic et al, 1990;Paradkar et al, 1998;Petrich et al, 1992Petrich et al, , 1994.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional and translational analysis of the ccaR gene from Streptomyces clavuligerus

et al. 2004

View full text Add to dashboard Cite

CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in Streptomyces clavuligerus. Previous sequence analyses of the ccaR gene revealed two possible start codons, an ATG, and a GTG located in-frame 18 bp downstream of the ATG. To determine the true start codon, ccaR was expressed, either from the GTG or ATG codon, in Escherichia coli. A protein product was only obtained from the ATG construct. Similarly, ccaR constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in Streptomyces species were prepared and used to complement a S. clavuligerus ccaR mutant. Bioassays showed that only the ATG construct could complement the ccaR mutant to restore cephamycin C production, and Western analysis confirmed the presence of CcaR in the mutant complemented with the ATG construct only. To ensure that expression of ccaR from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into ccaR, converting the GTG to GTC. The GTC construct still fully complemented a ccaR mutant, confirming that ATG is the true start codon. Inspection of the region upstream of ccaR by S1 nuclease protection and primer extension analyses indicated the presence of two transcript start points that mapped to residues located 74 and 173 bp upstream of the ATG codon.

show abstract

Transcriptional analysis of the isopenicillin N synthase-encoding gene of Streptomyces clavuligerus

Cited by 17 publications

References 31 publications

Complete nucleotide sequence of a linear plasmid from Streptomyces clavuligerus and characterization of its RNA transcripts

Complete nucleotide sequence of a linear plasmid from Streptomyces clavuligerus and characterization of its RNA transcripts

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

Transcriptional and translational analysis of the ccaR gene from Streptomyces clavuligerus

Contact Info

Product

Resources

About