The autoregulator receptor homologue AvaR3 plays a regulatory role in antibiotic production, mycelial aggregation and colony development of Streptomyces avermitilis

Abstract

The c-butyrolactone autoregulator receptor has been shown to control secondary metabolism and/or morphological differentiation across many Streptomyces species. Streptomyces avermitilis produces an important anthelmintic agent (avermectin) and two further polyketide antibiotics, filipin and oligomycin. Genomic analysis of S. avermitilis revealed that this micro-organism has the clustered putative autoregulator receptor genes distant from the antibiotic biosynthetic gene clusters. Here, we describe the characterization of avaR3, one of the clustered receptor genes, which encodes a protein containing an extra stretch of amino acid residues that has not been found in the family of autoregulator receptors. Disruption of avaR3 resulted in markedly decreased production of avermectins, with delayed expression of avermectin biosynthetic genes, suggesting that AvaR3 positively controls the avermectin biosynthetic genes. Moreover, the disruption caused increased production of filipin without any changes in the transcriptional profile of the filipin biosynthetic genes, suggesting that filipin production is indirectly controlled by AvaR3. The avaR3 disruptant displayed fragmented growth in liquid culture and conditional morphological defects on solid medium. These findings demonstrated that AvaR3 acts as a global regulator that controls antibiotic production and cell morphology.

SdrA, a New DeoR Family Regulator Involved in Streptomyces avermitilis Morphological Development and Antibiotic Production

“…To confirm that the lowered sporulation resulted solely from the sdrA disruption, we reintroduced the entire sdrA gene with its upstream region into the sdrA disruptant. Complementation of the sdrA disruptant with pLT123 (⌬sdrA/sdrA) increased the number of spores by 5.6-fold relative to the parental strain (⌬sdrA/pSET152), in which integration of an empty vector alone impaired sporulation severely as reported previously (14). These findings, taken together with phenotypic data from the analysis by a gene knockdown, clearly indicated that SdrA functions as a positive regulator of morphological development.…”

“…Because the biosynthetic pathways of the three antibiotics require malonyl-coenzyme A (CoA) and methylmalonyl-CoA as a common extender unit (22,23), the increase and decrease of antibiotic production may be explained by precursor competition between these biosynthetic pathways. In fact, a mutation of the avermectin biosynthetic gene or a disruption of the positive regulatory gene for avermectin production leads to increased production of oligomycin and filipin, whereas deletion of biosynthetic gene clusters of oligomycin or filipin increases avermectin production (14,24). Thus, SdrA appears to have a regulatory function as an activator of the avermectin biosynthetic pathway or as a repressor of biosynthetic pathways for other antibiotics or as both concomitantly.…”

“…E. coli strains were grown in 2ϫ yeast extract-tryptone (YT) medium, with appropriate antibiotics when necessary. The plasmids used were pUC19 for general cloning, pKC1132 (12) for gene disruption, pLT106 (13), a derivative of pSET152 (12) containing a strong and constitutive promoter (ermEp*) and a tfd terminator with a Gateway cloning cassette (Life Technologies), for overexpression screening, pLT113 (14) for gene complementation, and pIJ8781 (15), a Streptomyces multicopy conjugatable plasmid containing the thiostrepton-inducible promoter tipAp, for gene knockdown. The medium conditions and general E. coli and Streptomyces manipulations were as described previously (16).…”

“…The agar culture was diced and extracted with 2 volumes of methanol. The methanol extract was collected by centrifugation and analyzed by using a high-pressure liquid chromatography system as described previously (13,14).…”

Pleiotropic Control of Secondary Metabolism and Morphological Development by KsbC, a Butyrolactone Autoregulator Receptor Homologue in Kitasatospora setae

“…E. coli BL21(DE3)/pLysS and the plasmid pET-15b, which were used for the expression of recombinant KsbC (rKsbC), were obtained from Novagen. The plasmids used were pUC19 for general cloning, and pKU451, pKU474, and pKU250 were used for gene disruption (21,24). The genome-integrated vectors, pKU492Aaac(3)IV (see Fig.…”