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. 2009 Aug;191(15):4786-97.
doi: 10.1128/JB.00437-09. Epub 2009 May 22.

Characterization of gamma-butyrolactone autoregulatory signaling gene homologs in the angucyclinone polyketide WS5995B producer Streptomyces acidiscabies

Frank G Healy  1 , Kevin P Eaton, Prajit Limsirichai, Joel F Aldrich, Alaina K Plowman, Russell R King
Affiliations

Characterization of gamma-butyrolactone autoregulatory signaling gene homologs in the angucyclinone polyketide WS5995B producer Streptomyces acidiscabies

Frank G Healy et al. J Bacteriol. 2009 Aug.

Abstract

Organisms belonging to the genus Streptomyces produce numerous important secondary metabolites and undergo a sophisticated morphological differentiation program. In many instances these processes are under the control of gamma-butyrolactone (GBL) autoregulatory systems. Streptomyces acidiscabies strain 84.104 produces the secondary metabolite aromatic angucyclinone polyketide WS5995B. In order to explore the role of GBL regulatory circuitry in WS5995B production and morphogenesis in S. acidiscabies, a gene cluster encoding GBL autoregulatory signaling homologs was identified and characterized. Two GBL receptor homologs, sabR and sabS, were found flanking a GBL synthase homolog sabA. Strains carrying mutations in sabS produced elevated levels of WS5995B and displayed conditional morphological defects reminiscent of defects seen in Streptomyces bldA mutants. Notably, sabS possesses a TTA codon predicted to be recognized by tRNA(leu). sabA mutants produced higher levels of WS5995B than the wild-type strain but to a lesser extent than the levels of WS5995B seen in sabS mutants. Purified recombinant SabR and SabS were tested for their abilities to bind predicted AT-rich autoregulatory element (ARE) boxes within the sabRAS region. SabS did not bind any DNA sequences in this region, while SabR bound an ARE box in the region upstream of sabS. Quantitative reverse transcription-PCR analysis revealed higher levels of sabS transcript in sabR mutants than in the wild-type strain, suggesting that sabS expression is repressed by SabR. Based on these data, we propose that the S. acidiscabies sabRAS genes encode components of a signaling pathway which participates in the regulation of WS5995B production and morphogenesis.

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Figures

FIG. 1.
FIG. 1.
Structure of WS5995B.
FIG. 2.
FIG. 2.
Genetic organization of the sabRAS region in S. acidiscabies 84.104. GBL receptor gene homologs sabR and sabS and GBL synthase homolog sabA are indicated by arrows below scale, given in kilobases. ARE on the diagram represents a 26-bp ARE DNA sequence (not drawn to scale) bound by SabR (Fig. 7). For comparative purposes, a partial list of characterized receptor/synthase partners from other streptomycetes is given below the sabRAS gene map although these gene pairs are not necessarily organized in the same manner as shown in figure.
FIG. 3.
FIG. 3.
Phylogenetic analysis of sabRAS gene products. (A) Phylogram showing relationships between Streptomyces GBL synthases and SabA. (B) Phylogram showing relationships between Streptomyces GBL receptors and S. acidiscabies SabS and SabR proteins. Proteins are represented by gene identification (gi) numbers and names in the NCBI/GenBank protein sequence database; SabR_S.ansochromogenes represents GBL receptor SabR found in Streptomyces ansochromogenes. Bootstrap values are indicated at branches.
FIG. 4.
FIG. 4.
Phenotypic properties of sabR, sabA, and sabS mutants. (A) Pigment production characteristics of sabR, sabA, and sabS deletion mutants on SGM. wt, wild-type strain S. acidiscabies 84.104. (B) Complementation of sabS deletion mutant on SGM with plasmid pIJ86::sabS. (C) Complementation of sabA deletion mutant on SGM with plasmid pIJ86::sabA. (D) Complementation of conditional morphological defect of sabS mutant on ISP2 medium with plasmid pIJ86::sabS.
FIG. 5.
FIG. 5.
Growth and WS5995B production characteristics of S. acidiscabies 84.104 wild type and a ΔsabS deletion mutant. Cell weight and WS5995B production of the wild type and the ΔsabS mutant are given, as indicated on the figure. Given large differences in WS5995B production for the wild-type and mutant strains, note that the right-hand y axis is segmented to allow inspection of WS5995B production rates for both strains. Over the 12-h interval from 36 h to 48 h, the rate of WS5995B production was 1.36 μM·h−1.
FIG. 6.
FIG. 6.
S. acidiscabies 84.104 ARE sequence and ARE consensus. (A) Position of the ARE (boxed) relative to possible SabS translation initiation sites and the upstream region of sabS. Double-stranded DNA sequence is shown. Two possible alternative start codons and Shine-Dalgarno sequences are underlined, and the N terminus of the deduced SabS peptide sequence is shown below the DNA, with leucine specified by a TTA codon indicated by an asterisk. (B) Nucleotide sequence alignment of the predicted ARE sequence from S. acidiscabies (sabS-ARE) with previously characterized Streptomyces ARE boxes. (C) Sequence logo illustrating conservation of bases within aligned ARE sequences.
FIG. 7.
FIG. 7.
SabR-ARE DNA mobility shift assay. A 32P-labeled 209-bp DNA fragment encompassing the ARE box was incubated with various amounts of recombinant SabR, and binding reaction products were electrophoresed through a 5% native PAGE gel in 0.5× Tris-borate-EDTA buffer. Lane 1, no SabR; lane 2, 1.5 nM SabR; lane 3, 3 nM SabR; lane 4, 6 nM SabR; lane 5, 12 nM SabR; lane 6, 18 nM SabR; lane 7, 24 nM SabR; lane 8, 24 nM SabR plus 150 nM unlabeled 34-bp ARE duplex competitor DNA.
FIG. 8.
FIG. 8.
Reverse transcriptase PCR analysis of sabRAS gene expression. Strains used for RNA extraction are given across four vertical columns, as indicated at top; PCR amplicons analyzed from cDNA samples are given over four horizontal rows, as indicated at right. hrdB represents a principal vegetative sigma factor. No bands are seen for an sabR amplicon from sabR RNA samples or for an sabS amplicon from sabS RNA samples because amplicon regions were removed in construction of these deletion mutants. The sabA amplicon is seen from sabA RNA samples because the amplicon region was retained in the creation of an sabA deletion strain.
FIG. 9.
FIG. 9.
qRT-PCR analysis of sabS expression in wild-type 84.104 and sabR mutants. Graph shows average relative increase in sabS expression in the wild type (wt) and sabR deletion mutant from three independent experiments. Expression differences were computed using the ΔΔCT method. Significance was calculated by analysis of variance, and error bars represent standard error.
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