Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)

Abstract

Streptomyces antibiotic regulatory proteins (SARPs) have been shown to activate transcription by binding to a tandemly arrayed set of heptameric direct repeats located around the −35 region of their cognate promoters. Experimental evidence is presented here showing that vlmI is a regulatory gene in the valanimycin biosynthetic gene cluster of Streptomyces viridifaciens and encodes a protein belonging to the SARP family. The organization of the valanimycin biosynthetic gene cluster suggests that the valanimycin biosynthetic genes are located on three potential transcripts, vlmHORBCD, vlmJKL and vlmA. Disruption of vlmI abolished valanimycin biosynthesis. Western blot analyses showed that VlmR and VlmA are absent from the vlmI mutant and that the production of VlmK is severely diminished. These results demonstrate that the expression of these genes from the three potential transcripts is under the positive control of VlmI. The vlmA–vlmH and vlmI–vlmJ intergenic regions both exhibit a pattern of heptameric direct repeats. Gel shift assays with VlmI overproduced in Escherichia coli as a C-terminal FLAG-tagged protein clearly demonstrated that VlmI binds to DNA fragments from both regions that contain these heptameric repeats. When a high-copy-number vlmI expression plasmid was introduced into Streptomyces coelicolor M512, which contains mutations in the undecylprodigiosin and actinorhodin activators redD and actII-orf4, undecylprodigiosin production was restored, showing that vlmI can complement a redD mutation. Introduction of the same vlmI expression plasmid into an S. viridifaciens vlmI mutant restored valanimycin production to wild-type levels.

Characterization of DNA‐binding sequences for CcaR in the cephamycin–clavulanic acid supercluster of Streptomyces clavuligerus

“…6B; a higher frequency of cytosine was found in the second position of the first heptameric sequence (distal from the ATG codon). Perfect spacers of 4 nt were only found in ccaR ‐I heptameric repeats; spacers of 14 nt between two of the heptameric sequences were frequent in S. clavuligerus , as occurs also in the actII , actI–orf1 , dnrD , vlmA or vlmJ heptameric sequences (Garg and Parry, 2010). Additional sequences with only two heptamer repeats were found in the ccaR and ceaS2 upstream regions ( ccaR‐II and ceaS2‐II , Fig.…”

“…6). The heptamers were quite conserved in specific positions, particularly the first heptameric sequence, as compared with SARP‐binding sequences in the actII, dnr , vlm and fdm gene cluster for actinorrhodin, daunorubicin, vanimycin and fredericamycin biosynthesis (Wietzorrek and Bibb, 1997; Garg and Parry, 2010). The sequences upstream of lat (TCGAGAG‐14‐TGGAAGA‐4‐TCCAAGA) and other S. clavuligerus heptameric sequences (Fig.…”

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

“…The specific sequences recognized by SARPs generally overlap the Ϫ35 regions of their targets, but sometimes the sequences are far from the transcriptional start point (tsp) of their target genes. Some SARP-binding sites contain three discernible heptamers, such as those upstream of actII-ORF1 and actVI-ORF1 in S. coelicolor (29), claR, cefD, and cefF in S. clavuligerus (216), vlmJ and vlmA-vlmH in Streptomyces viridifaciens (217), dnrD in S. peucetius (218), fdmD in S. griseus (219), pimM in S. natalensis (220), and so on. Other sites contain only two obvious heptamers, such as sanO-sanN in S. ansochromogenes (221), polB and polC in Streptomyces cacaoi subsp.…”

“…asoensis (222), cmcI and ceaS2-II in S. clavuligerus (223), and fdmR2 in S. griseus (219). Degeneracy of the repeats may make the structure of the targets difficult to characterize, perhaps explaining why the spacers appear to vary from 4 to 15 nt in different targets (216,217,219). Alternatively, these differences could perhaps be related to variations in the structure of SARPs.…”

“…Like ActII-ORF4 and RedD, many other SARPs are less than 300 residues long, and some of these have been shown, mainly by mutant phenotypes, to function as pathway-specific activators, including CcaR (cephamycin-clavulanic acid supercluster in S. clavuligerus) (216), DnrI (daunorubicin biosynthesis in S. peucetius) (218,224), Aur1PR2 and Aur1PR3 (auricin biosynthesis in S. aureofaciens) (225), TylS and TylT (which activate the transcription of the tylosin CSR gene tylR in S. fradiae) (226), FdmR1 (fredericamycin biosynthesis in S. griseus) (219), ThnU (cephamycin C biosynthesis in Streptomyces cattleya) (227), SrrY and SrrZ (lankamycin biosynthetic regulatory cascade in Streptomyces rochei) (228), and VlmI (valanimycin biosynthesis in Streptomyces viridifaciens) (217). These ActII-ORF4-like SARPs are referred to here as "small SARPs."…”