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.

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.