Transcriptional regulation of Streptomyces coelicolor pathway-specific antibiotic regulators by the absA and absB loci

Abstract

The four antibiotics produced by Streptomyces coelicolor are all affected by mutations in the absA and absB loci. The absA locus encodes a putative two-component signal transduction system, and the absB locus encodes a homolog of Escherichia coli RNase III. We assessed whether these loci control synthesis of the antibiotics actinorhodin and undecylprodigiosin by regulating transcript abundance from the biosynthetic and regulatory genes specific for each antibiotic. Strains that were Abs- (for antibiotic synthesis deficient) due to mutations in absA or absB were examined. In the Abs- absA mutant strain, transcripts for the actinorhodin biosynthetic genes actVI-ORF1 and actI, and for the pathway-specific regulatory gene actII-ORF4, were substantially lower in abundance than in the parent strain. The level of the transcript for the undecylprodigiosin pathway-specific regulatory gene redD was similarly reduced in this mutant. Additionally, a strain that exhibits precocious hyperproduction of antibiotics (Pha phenotype) due to disruption of the absA locus contained elevated levels of the actVI-ORF1, actII-ORF4, and redD transcripts. In the absB mutant strain, actVI-ORF1, actI, actII-ORF4, and redD transcript levels were also substantially lower than in the parent strain. These results establish that the abs genes affect production of antibiotics through regulation of expression of the antibiotic-specific regulatory genes in S. coelicolor.

FIG. 1

Growth-phase-dependent expression of actII-ORF4 mRNA in plate-grown J1501. (A) RNA isolated from mannitol minimal medium plates at the times indicated was used in S1 nuclease protection analyses. Size markers (SM), ³²P-labeled MspI-digested pBR322. (B) The probe for the actII-ORF4 gene was end labeled at the XhoI site (indicated by a star; see Materials and Methods) and should yield a protected fragment of 390 nt (19, 25). (C) The uniquely end-labeled probe for glk was generated by PCR amplification (see Materials and Methods) and should yield two protected fragments of 267 and 217 nt that correspond to transcripts initiated at promoters p2 and p3, respectively (3).

FIG. 2

Expression of actII-ORF4 mRNA in J1501 (abs⁺), C542 (absA), C120 (absB), and C430 (absA disruption). RNA was isolated from PGA plate-grown cultures at the times indicated and used for S1 nuclease protection analyses. The size markers (SM) used were as described in the legend to Fig. 1. The probes used for actII-ORF4 and glk were the same as for Fig. 1. “FLP actII-ORF4” indicates the position of full-length probe; transcriptional readthrough from the upstream promoter for actII-ORF3 likely contributes to the signal as well (19, 25). This signal intensity varied from experiment to experiment but, when present, followed the same kinetics as the labeled actII-ORF4.

FIG. 3

Expression of actVI-ORF1 mRNA in J1501 (abs⁺), C542 (absA), C120 (absB), and C430 (absA disruption). RNA was isolated from PGA plate-grown cultures at the times indicated and used for S1 nuclease protection analyses with a probe for the actVI-ORF1 transcript that should yield a protected fragment of 191 nt (22, 25). (A) The secondary bands below the actVI-ORF1 bands (approximately 167 nt), also obtained by other researchers using this probe (25), are of unknown origin; they may represent a secondary initiation site, a degradation product, or an artifact of the procedure. The glk probe was as described in the legend to Fig. 1. The size markers (SM) were as described for Fig. 1. (B) The uniquely end-labeled probe used for the actVI-ORF1 transcript is described in Materials and Methods. ORFA has been implicated in Act synthesis, but its role is unknown (22).

FIG. 4

Expression of redD mRNA in J1501 (abs⁺), C542 (absA), and C120 (absB). RNA was isolated from PGA plate-grown cultures at the times indicated and used in S1 nuclease protection assays with a probe for the redD transcript that should yield a protected fragment of 330 nt (45, 52). (A) Bands labeled “redD” represent transcript from the redD gene. The glk probe was as described for Fig. 1. Size markers (SM) were as described for Fig. 1. (B) The uniquely end-labeled probe for the redD transcript is described in Materials and Methods.

FIG. 5

Expression of redD mRNA in J1501 (abs⁺) and C430 (absA disruption). RNA was isolated from PGA plate-grown cultures at the times indicated and used in S1 nuclease protection assays. The probes for redD and glk were as described for Fig. 4.