The genetic organization of the region upstream of the car gene of the clavulanic acid biosynthetic gene cluster of Streptomyces clavuligerus has been determined. Sequence analysis of a 121 kb region revealed the presence of 10 ORFs whose putative functions, according to database searches, are discussed. Three co-transcriptional units are proposed : ORF10-11, ORF12-13 and ORF15-16-17-18. Potential transcriptional terminators were identified downstream of ORF11 (fd ) and ORF15. Targeted disruption of ORF10 (cyp) gave rise to transformants unable to produce clavulanic acid, but with a considerably higher production of cephamycin C. Transformants inactivated at ORF14 had a remarkably lower production of clavulanic acid and similar production of cephamycin C. Significant improvements of clavulanic acid production, associated with a drop in cephamycin C biosynthesis, were obtained with transformants of S. clavuligerus harbouring multiple copies of plasmids carrying different constructions from the ORF10-14 region. This information can be used to guide strain improvement programs, blending random mutagenesis and molecular cloning, to optimize the yield of clavulanic acid.

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....

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.

In the article by Santamarta et al . (2005) Molecular Microbiology , 56: 824-835, some errors appeared in the published article.Errors appeared in the summary and the correct summary is printed below. SummaryCell-free extracts from Streptomyces clavuligerus , purified by elution from heparin-agarose with an AREcontaining DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (ARE ccaR ). This sequence is located upstream of the ccaR gene, encoding the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus . Sixty-one nucleotides upstream of brp another ARE sequence (ARE brp ) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp . A brp -deleted mutant, S. clavuligerus D brp::neo 1, produced 150-300% clavulanic acid and 120-220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus D brp::neo 1 lacked a high-mobility band-shift due to Brp but still showed a slow-mobility band-shift observed in the wild-type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin C biosynthesis by its action on ccaR gene expression.

SummaryA protein highly overrepresented in the proteome of Streptomyces clavuligerus oppA2::aph was characterized by MS/MS as a rhodanese‐like enzyme. The rhlA gene, encoding this protein, was deleted from strains S. clavuligerus ATCC 27064 and S. clavuligerus oppA2::aph to characterized the RhlA enzyme activity, growth on different sulfur sources and antibiotic production by the mutants. Whereas total thiosulfate sulfurtransferase activity in cell extracts was not affected by the rhlA deletion, growth, cephamycin C and clavulanic acid production were impaired in the rhlA mutants. Holomycin production was drastically reduced (66–90%) in the rhlA mutants even when using S. clavuligerusΔrhlA pregrown cells, suggesting that this enzyme might be involved in the formation of the cysteine precursor for this sulfur‐containing antibiotic. While growth on thiosulfate as the sole sulfur source was particularly low the volumetric and specific antibiotic production of the three antibiotics increased in all the strains in the presence of thiosulfate. This stimulatory effect of thiosulfate on antibiotic production was confirmed by addition of thiosulfate to pre‐grown cells and appears to be a general effect of thiosulfate on oxidative stress as was also evident in the production of staurosporin by S. clavuligerus.