Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by
            <i>Streptomyces clavuligerus</i>

Li, Rongfeng; Khaleeli, Nusrat; Townsend, Craig A.

doi:10.1128/jb.182.14.4087-4095.2000

Cited by 53 publications

(67 citation statements)

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“…The known clavulanic acid gene cluster has been extended recently by sequencing an additional region upstream of the car gene, which is essential for clavulanic acid biosynthesis (14,16). While studying this region we found an open reading frame (ORF), orf15, which is similar to orf7, a gene previously described (7).…”

mentioning

confidence: 76%

Two Oligopeptide-Permease-Encoding Genes in the Clavulanic Acid Cluster of Streptomyces clavuligerus Are Essential for Production of the β-Lactamase Inhibitor

et al. 2004

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orf7 (oppA1) and orf15 (oppA2) are located 8 kb apart in the clavulanic acid gene cluster of Streptomyces clavuligerus and encode proteins which are 48.0% identical. These proteins show sequence similarity to periplasmic oligopeptide-binding proteins. Mutant S. clavuligerus oppA1::acc, disrupted in oppA1, lacks clavulanic acid production. Clavulanic acid production is restored by transformation with plasmid pIJ699-oppA1, which carries oppA1, but not with the multicopy plasmid pIJ699-oppA2, which carries oppA2. The mutant S. clavuligerus oppA2::aph also lacks clavulanic acid production, shows a bald phenotype, and overproduces holomycin (5). Clavulanic acid production at low levels is restored in the oppA2-disrupted mutants by transformation with plasmid pIJ699-oppA2, but it is not complemented by the multicopy plasmid pIJ699-oppA1. Both genes encode oligopeptide permeases with different substrate specificities. The disrupted S. clavuligerus oppA2::aph is not able to grow on RPPGFSPFR (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; bradykinin), but both mutants grow on VAPG (Val-Ala-Pro-Gly) as the only nitrogen source, indicating differences in the peptide bound by the proteins encoded by both genes. The null S. clavuligerus oppA1::acc and S. clavuligerus oppA2::aph mutants are more resistant to the toxic tripeptide phosphinothricyl-alanyl-alanine (also named bialaphos) than the wild-type strain, suggesting that this peptide might be transported by these peptidebinding proteins.

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mentioning

confidence: 76%

Two Oligopeptide-Permease-Encoding Genes in the Clavulanic Acid Cluster of Streptomyces clavuligerus Are Essential for Production of the β-Lactamase Inhibitor

et al. 2004

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“…Very little is known about the cyp, ORF12, or ORF15 genes, disruption of which results in a large accumulation of holomycin. It has been suggested that the cyp-fd gene and probably the protein encoded by ORF12 are involved in the poorly characterized oxidation steps between clavaminic and clavulanic acid (14,23). The ClaR regulatory protein appears to control the expression of late genes of the pathway: i.e., car and cyp (23).…”

Section: Discussionmentioning

confidence: 99%

“…2 and Table 1) obtained by replacement with either the aph gene (conferring kanamycin resistance) or the apr gene (conferring apramycin resistance) were used for this study. They include mutants with deletions of the ceaS (pyc) gene, which encodes the carboxyethylarginine synthase (27); the bls gene, which encodes the ␤-lactam synthetase (2); the cla-ORF6 gene, which encodes a protein with ornithine acetyltransferase activity (7,11,28); the car gene, which encodes clavaldehyde reductase (26); the cyp gene, which encodes the cytochrome P450 involved in clavulanic acid formation (14,17); the two genes cla-ORF12 and cla-ORF15, which are located downstream of cyp (16,17); as well as mutants blocked in genes coding for the two ␤-lactam biosynthesis regulatory proteins, ccaR (25) and claR (26). In addition, the holomycin production of double mutants with disruptions in the ccaR and ORF6 genes was also studied.…”

Section: Formation Of Holomycin By Different Strains Of S Clavuligerusmentioning

confidence: 99%

“…Both S. clavuligerus car::aph and claR::aph strains produced holomycin at levels 7-and 16-fold higher, respectively, than those of the S. clavuligerus ccaR::aph strain, in which holomycin was first detected and purified. A third group of clavulanic acid-nonproducing mutants, with the genotypes cyp:: aph, ORF12::aph, and ORF15::aph (14,16), showed very high levels of holomycin production, which reached up to 1,200 g of holomycin per mg of DNA, or about 112-fold in relation to the wild-type strain and 45-fold in relation to the S. clavuligerus ccaR::aph strain at 96 h. The production of holomycin by the S. clavuligerus ORF15::aph strain reached levels similar to those of the S. clavuligerus cyp::aph strain at late times during the fermentation.…”

Section: Formation Of Holomycin By Different Strains Of S Clavuligerusmentioning

confidence: 99%

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Mutants of Streptomyces clavuligerus with Disruptions in Different Genes for Clavulanic Acid Biosynthesis Produce Large Amounts of Holomycin: Possible Cross-Regulation of Two Unrelated Secondary Metabolic Pathways

et al. 2002

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A Streptomyces clavuligerus ccaR::aph strain, which has a disruption in the regulatory gene ccaR, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry. S. clavuligerus strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis. (i) Mutants with mutations in the early steps of the pathway blocked in the gene ceaS (pyc) (encoding carboxyethylarginine synthase), bls (encoding a ␤-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers. (ii) Mutants blocked in the regulatory gene ccaR or claR or blocked in the last gene of the pathway encoding clavulanic acid reductase (car) produce holomycin at higher levels than the wild-type strain. (iii) Mutants with disruption in cyp (coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain. An assay for holomycin synthetase was developed. This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor. The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin.Streptomyces clavuligerus produces several secondary metabolites with interesting pharmacological activities. It synthesizes the ␤-lactam antibiotic cephamycin C, the ␤-lactamase inhibitor clavulanic acid, and several antifungal compounds with a clavam structure (for reviews, see references 3 and 15). The clavulanic acid biosynthesis pathway has several steps in common with the pathway for clavam biosynthesis (18,19). In addition to the compounds indicated above, S. clavuligerus produces the antibiotics holomycin and tunicamycin (10). Holomycin is a compound with pyrrothine structure, while tunicamycin is a glucosamine-containing antibiotic. This wealth of genetic information for the biosynthesis of secondary metabolites is characteristic of some Streptomyces species (4, 22).S. clavuligerus is an excellent model for the study of the relationships between the regulatory mechanisms controlling the biosynthesis of the different secondary metabolites produced by these microorganisms. Formation of clavulanic acid is controlled by a LysR-type regulatory protein encoded by the claR gene. Formation of both clavulanic acid and cephamycin C in S. clavuligerus is controlled by the positive autoregulatory protein CcaR (25, 32). Mutant strains with disruption in ccaR do not express the claR gene (26), although this control is not exerted directly by the CcaR regulatory protein and appears to involve a cascade mechanism (32). The control of the formation of cephamycin C and/or clavulanic acid by CcaR or ClaR is exerted at the transcription level (1, 25).However, the ccaR::aph S....

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“…The clavulanic acid gene cluster is situated immediately downstream of the cephamycin gene cluster, and together they form a larger gene cluster often referred to as the ␤-lactam supercluster (1,13,44). This clavulanic acid gene cluster contains genes encoding enzymes involved in the early shared stages of the clavulanic acid and 5S clavam pathway (the early genes), as well as genes encoding proteins involved in the later stages of clavulanic acid biosynthesis only (the late genes) (15,17,21,24). The clavam gene cluster encompasses the gene encoding clavaminate synthase 1 (cas1), a paralogue of the cas2 gene from the clavulanic acid gene cluster and one of the genes involved in the biosynthesis of both clavulanic acid and the 5S clavams.…”

mentioning

confidence: 99%

The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus

2004

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Carboxyethylarginine synthase, encoded by the paralogous ceaS1 and ceaS2 genes, catalyzes the first reaction in the shared biosynthetic pathway leading to clavulanic acid and the other clavam metabolites in Streptomyces clavuligerus. The nutritional regulation of ceaS1 and ceaS2 expression was analyzed by reverse transcriptase PCR and by the use of the enhanced green fluorescent protein-encoding gene (egfp) as a reporter. ceaS1 was transcribed in complex soy medium only, whereas ceaS2 was transcribed in both soy and defined starchasparagine (SA) media. The transcriptional start points of the two genes were also mapped to a C residue 98 bp upstream of ceaS1 and a G residue 51 bp upstream of the ceaS2 start codon by S1 nuclease protection and primer extension analyses. Furthermore, transcriptional mapping of the genes encoding the beta-lactam synthetase (bls1) and proclavaminate amidinohydrolase (pah1) isoenzymes from the paralogue gene cluster indicated that a single polycistronic transcript of ϳ4.9 kb includes ceaS1, bls1, and pah1. The expression of ceaS1 and ceaS2 in a mutant strain defective in the regulatory protein CcaR was also examined. ceaS1 transcription was not affected in the ccaR mutant, whereas that of ceaS2 was greatly reduced compared to the wild-type strain. Overall, our results suggest that different mechanisms are involved in regulating the expression of ceaS1 and ceaS2, and presumably also of other paralogous genes that encode proteins involved in the early stages of clavulanic acid and clavam metabolite biosynthesis.

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Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

Cited by 53 publications

References 59 publications

Two Oligopeptide-Permease-Encoding Genes in the Clavulanic Acid Cluster of Streptomyces clavuligerus Are Essential for Production of the β-Lactamase Inhibitor

Two Oligopeptide-Permease-Encoding Genes in the Clavulanic Acid Cluster of Streptomyces clavuligerus Are Essential for Production of the β-Lactamase Inhibitor

Mutants of Streptomyces clavuligerus with Disruptions in Different Genes for Clavulanic Acid Biosynthesis Produce Large Amounts of Holomycin: Possible Cross-Regulation of Two Unrelated Secondary Metabolic Pathways

The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus

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