title: Transcriptional analysis and proteomics of the holomycin gene cluster in overproducer mutants of Streptomyces clavuligerus.
abstract: 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.
authors.affiliation: Área de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain.
authors.forename: V
authors.initials: V
authors.lastname: Robles-Reglero
authors.forename: I
authors.initials: I
authors.lastname: Santamarta
authors.forename: R
authors.initials: R
authors.lastname: Álvarez-Álvarez
authors.forename: J F
authors.initials: JF
authors.lastname: Martín
authors.forename: P
authors.initials: P
authors.lastname: Liras
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D007769:Lactams / Q000378:metabolism*; D005810:Multigene Family; D009154:Mutation; D040901:Proteomics / Q000379:methods; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D001426:Bacterial Proteins; D007769:Lactams; C015265:holomycin