Abstract
Streptomyces clavuligerus ATCC 27064, the industrial producer of the β-lactamase inhibitor clavulanic acid, carries 49 putative secondary metabolite gene clusters. These secondary metabolite gene clusters are distributed between its linear chromosome and the 1.8 Mb-plasmid pSCL4, a rich reservoir of bioactive compound gene clusters. The transcriptome and metabolome of S. clavuligerus ATCC 27064 and the pSCL4− derived strain, S. clavuligerus pSCL4−, were analysed. Construction of the S. clavuligerus pSCL4− strain resulted in the excision of a 303 kb stretch in the right arm of the chromosome and its translocation to pSCL4, producing a 2.1 Mb plasmid named pSCL4* . The absence of pSCL4* results in changes in the transcription level of genes encoding regulatory proteins or proteins with various functions. Lack of pSCL4* results in upregulation of three chromosomal gene clusters for secondary metabolites (SMC), SMC18, for holomycin and N-propionylholothin biosynthesis, SMC11b for tunicamycin biosynthesis (located between SMC10 and SMC11), and SMC5. The SMC10, SMC11 and SMC6 gene clusters were downregulated, resulting in lower production of clavulanic acid, cephamycin C and desferrioxamine E, respectively. Clusters SMC8, SMC12, SMC13 and SMC19 were also downregulated. Production levels of bioactive compounds, such as alkylresorcinol or thiol-derived compounds, were affected in the plasmid-less strain. The excision and translocation to pSCL4 of 303 kb from the right arm of the chromosome confirms that the ends of the chromosome arms are regions of high instability and supports the hypothesis that pSCL4 might have been excised from S. clavuligerus chromosomal right arm end. Cysteine and methionine metabolism in S. clavuligerus lacking pSCL4* may differ from that of the wild type strain, given the absence of sulfur metabolism genes located either in pSCL4 or at the right end of the chromosome, which led to levels of dithiolopyrrolones (holomycin, N-propionylholothin) and acetylhomocysteine thiolactone (citiolone) higher than those of the wild type strain. S. clavuligerus pSCL4− shows strong differences in its transcriptome and metabolome; however, the loss of 2.1 Mb DNA is dispensable in this strain.
Highlights
Streptomyces clavuligerus ATCC 27064, the industrial producer of the β-lactamase inhibitor clavulanic acid, carries 49 putative secondary metabolite gene clusters
To determine the origin of this downregulation, the following genes were analyzed by qPCR in both strains: i) 10 genes located in the underexpressed region of the right end of the chromosome, ii) genes located in the central region of the chromosome (SCLAV_5146, SCLAV_5308) or located in plasmid pSCL2, and iii) genes, such as parBpSCL4, located in pSCL4, and absent in S. clavuligerus pSCL4− (Fig. 1b)
In order to determine when the translocation occurred, strains S. clavuligerus ATCC 27064, S. clavuligerus parAB::aac, derived from the wild type strain, and S. clavuligerus pSCL4− constructed from S. clavuligerus parAB::aac, were analyzed by final time PCR to test every gene between SCLAV_5487 and SCLAV_5491 (Fig. 2)
Summary
Streptomyces clavuligerus ATCC 27064, the industrial producer of the β-lactamase inhibitor clavulanic acid, carries 49 putative secondary metabolite gene clusters. Charusanti et al [3] exploited adaptative laboratory evolution by co-cultivation of S. clavuligerus with a methicillin-resistant Staphylococcus aureus strain; in this way they obtained fourteen S. clavuligerus isolates producing holomycin, a bioactive compound whose gene cluster is silent in the wild type strain in tested laboratory conditions. Two of these strains lack the whole pSCL4, and a third one lacks the leftmost stretch of pSCL4. The holomycin producer strains S. clavuligerus oppA2::aph [4] and S. clavuligerus claR::aph [5] showed a lower copy number of pSCL4 than the wild type strain, suggesting that pSCL4 might be involved in the activation of some secondary metabolite gene clusters
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