The bkdR gene of Streptomyces coelicolor is required for morphogenesis and antibiotic production and encodes a transcriptional regulator of a branched-chain amino acid dehydrogenase complex

Abstract

Products from the degradation of the branched-chain amino acids valine, leucine, and isoleucine contribute to the production of a number of important cellular metabolites, including branched-chain fatty acids, ATP and other energy production, cell-cell signaling for morphological development, and the synthesis of precursors for polyketide antibiotics. The first nonreversible reactions in the degradation of all three amino acids are catalyzed by the same branched-chain alpha-keto acid dehydrogenase (BCDH) complex. Actinomycetes are apparently unique among bacteria in that they contain two separate gene clusters, each of which encodes a BCDH enzyme complex. Here, we show that one of these clusters in Streptomyces coelicolor is regulated, at least in part, at the level of transcription by the product of the bkdR gene. The predicted product of this gene is a protein with similarity to a family of proteins that respond to leucine and serve to activate transcription of amino acid utilization operons. Unlike most other members of this class, however, the S. coelicolor bkdR gene product serves to repress transcription, suggesting that the branched-chain amino acids act as inducers rather than coactivators of transcription. BkdR likely responds to the presence of branched-chain amino acids. Its role in transcriptional regulation may be rationalized by the fact that transition from vegetative growth to aerial mycelium production, the first stage of morphological development in these complex bacteria, is coincident with extensive cellular lysis generating abundant amounts of protein that likely serve as the predominant source of carbon and nitrogen for metabolism. We suggest that bkdR plays a key role in the ability of Streptomyces species to sense nutrient availability and redirect metabolism for the utilization of branched-chain amino acids for energy, carbon, and perhaps even morphogen synthesis. A null mutant of bkdR is itself defective in morphogenesis and antibiotic production, suggesting that the role of the bkdR gene product may be more global than specific nutrient utilization.

FIG. 1.

Organization of the S. coelicolor branched-chain amino acid dehydrogenase gene clusters. Gene designations are based on the nomenclature used by the Sanger Centre Genome Sequencing Projects (www.sanger.ac.uk/Projects/S_coelicolor). The numbers above the diagram indicate the base pair positions of the genes in the cosmid DNA sequence. The function of each open reading frame is shown below the arrow, indicating its position.

FIG. 2.

End-to-end alignment of conserved (black background) and functionally similar (grey background) amino acids in the sequences of inferred and known proteins with similarity to the predicted protein encoded by S. coelicolor BkdR. Abbreviations: Sc, S. coelicolor; Sa, S. avermitilis; Pa, Pseudomonas aeruginosa; Pp, P. putida; Ec, E. coli; At, Agrobacterium tumeofaciens. Protein alignment was performed with the ClustalW program, and the graphic was generated with MacVector 7.0.

FIG. 3.

Construction of plasmid pSETΔbkdR::aadA used to generate a deletion of the bkdR open reading frame in the S. coelicolor genome. Abbreviations: E, EcoRI; Sm, SmaI; B, BamHI; H, HindIII; Sp, SphI; Xh, XhoI; Nt, NotI; Xb, XbaI.

FIG. 4.

Comparison of the bkdR deletion strain (ΔbkdR::aadA) with the wild type (M145), the deletion mutant containing a wild-type copy of bkdR under the control of the tipA promoter (ΔbkdR::aadA ptipA-bkdR), and the deletion mutant containing the pIJ8600 vector without the bkdR wild-type allele (ΔbkdR::aadA pIJ8600) grown on MYM agar plates (A and B) or on MYM agar with the addition of 2 μg of thiostrepton/ml (C and D). Photographed from the top of the plate (A) to visualize morphological development or from the bottom of the plate (B) to visualize the pigments associated with antibiotic production.

FIG. 5.

A comparison of the growth of the bkdR mutant (squares) with wild-type cells (circles and diamonds) grown in minimal (NMMP) medium with either glucose (closed symbols) or leucine (open symbols) as the sole carbon source. One-liter cultures were sampled (each sample, 30 ml) at 24-h time intervals, and changes in cell dry weight were measured.

FIG. 6.

S1 nuclease mapping of transcripts originating upstream of bkdA2 (A) and bkdA1 (B) in wild-type and bkdR mutant cells, harvested at 16, 24, 36, 48, and 60 h of growth on MYM agar plates. The hrdB transcript (C) was used as a control for RNA. The sequence of the predicted bkdA2 promoter region (D) with the putative RNA polymerase-binding site (underlined and in boldface type) and 14-bp inverted repeat sequence (boldface type with arrows above the sequence) are shown.