A 1.8-Mb-reduced Streptomyces clavuligerus genome: relevance for secondary metabolism and differentiation.

A large part (21%) of the wild-type Streptomyces clavuligerus genome is located in a 1.8-Mb megaplasmid that greatly influences secondary metabolites biosynthesis even if the secondary metabolites are chromosomally encoded. The megaplasmid copy number may change depending on the nutritional and environmental conditions. The S. clavuligerus oppA2::aph mutant described by Lorenzana et al. (2004) does not form aerial mycelium, spores, and clavulanic acid, but overproduces holomycin. Transcriptomic studies, polymerase chain reactions (PCR), qPCR, and RT-qPCR analysis showed that S. clavuligerus oppA2::aph has a drastically reduced number of copies (about 25,000-fold lower than the parental strain) of plasmids pSCL1 (10.5 kb), pSCL2 (149.4 kb), and the megaplasmid pSCL4 (1.8 Mb). To clarify the role of the linear plasmids and the function of OppA2 in S. clavuligerus oppA2::aph we constructed oppA2 mutants which contained: (1) a normal copy number of the linear plasmids, (2) completely lack of the linear plasmids, and (3) a parA-parB pSCL4 mutant that resulted in lack of pSCL4. In addition, a strain with a functional oppA2 gene was constructed lacking the megaplasmid pSCL4. The results confirmed that the oppA2 gene is essential for clavulanic acid production, independently of the presence or absence of linear plasmids, but oppA2 has little relevance on differentiation. We demonstrated that the lack of sporulation of S. clavuligerus oppA2::aph is due to the absence of linear plasmids (particularly pSCL4) and the holomycin overproduction is largely due to the lack of pSCL4 and is stimulated by the oppA2 mutation.

Characterization of DNA-binding sequences for CcaR in the cephamycin-clavulanic acid supercluster of Streptomyces clavuligerus.

RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

Transcriptomic analysis of Streptomyces clavuligerus ΔccaR::tsr: effects of the cephamycin C-clavulanic acid cluster regulator CcaR on global regulation.

Streptomyces clavuligerus ATCC 27064 and S. clavuligerus ΔccaR::tsr cultures were grown in asparagine-starch medium, and samples were taken in the exponential and stationary growth phases. Transcriptomic analysis showed that the expression of 186 genes was altered in the ccaR-deleted mutant. These genes belong to the cephamycin C gene cluster, clavulanic acid gene cluster, clavams, holomycin, differentiation, carbon, nitrogen, amino acids or phosphate metabolism and energy production. All the clavulanic acid biosynthesis genes showed Mc values in the order of -4.23. The blip gene-encoding a ß-lactamase inhibitory protein was also controlled by the cephamycin C-clavulanic acid cluster regulator (Mc -2.54). The expression of the cephamycin C biosynthesis genes was greatly reduced in the mutant (Mc values up to -7.1), while the genes involved in putative ß-lactam resistance were less affected (Mc average -0.88). Genes for holomycin biosynthesis were upregulated. In addition, the lack of clavulanic acid and cephamycin production negatively affected the expression of genes for the clavulanic acid precursor arginine and of miscellaneous genes involved in nitrogen metabolism (amtB, glnB, glnA3, glnA2, glnA1). The transcriptomic results were validated by quantative reverse transcription polymerase chain reaction and luciferase assay of luxAB-coupled promoters. Transcriptomic analysis of the homologous genes of S. coelicolor validated the results obtained for S. clavuligerus primary metabolism genes.

Transcriptional analysis and proteomics of the holomycin gene cluster in overproducer mutants of Streptomyces clavuligerus.

Expression of the holomycin biosynthesis genes (hlm) has been studied in the wild type strain Streptomyces clavuligerus ATCC 27064 and holomycin overproducer mutants. RT-PCR transcription analysis of S. clavuligerus oppA2::aph showed a higher transcription of the hlmA, B, C, D, E, F, G, H, I and hlmL genes, a slightly lower expression for hlmK and no significant differences for the transcription of the two putative regulatory genes, hlmM and hlmJ, in relation to the wild type strain. Accordingly, protein spots corresponding to HlmD, HlmF and HlmG, which were barely detectable in the wild type strain, were present in high amounts in the holomycin overproducer S. clavuligerus oppA2::aph proteome. Transcription start point analysis of the hlm genes revealed that the annotated sequences in the databases for several hlm genes were incorrect. The hlm cluster was introduced into Streptomyces coelicolor M1154 and holomycin production by S. coelicolor M1154 [pVR-hol1] was validated by bioassays and confirmed by HPLC analysis and mass spectrometry. Heterologous holomycin production by the S. coelicolor transformant is 500-fold lower than in S. clavuligerus oppA2::aph. The transformant S. coelicolor M1154 [pVR-hol1] shows holomycin sensitivity to 100 µg/ml, similar to that of the parental S. coelicolor M1154 strain, suggesting that heterologous expression in S. coelicolor might be toxic due to the lack of an holomycin resistance gene in this host strain.