Characterization ofrrdA, a TetR Family Protein Gene Involved in the Regulation of Secondary Metabolism inStreptomyces coelicolor

Abstract

Streptomyces not only exhibits complex morphological differentiation but also produces a plethora of secondary metabolites, particularly antibiotics. To improve our general understanding of the complex network of undecylprodigiosin (Red) biosynthesis regulation, we used an in vivo transposition system to identify novel regulators that influence Red production in Streptomyces coelicolor M145. Using this screening system, we obtained 25 Red-deficient mutants. Twenty-four of these mutants had a transposon inserted in the previously described Red biosynthetic gene cluster and produced different amounts of another secondary metabolite, actinorhodin (Act). One mutant was shown to have an insertion in a different region of the chromosome upstream of the previously uncharacterized gene rrdA (regulator of redD, sco1104), which encodes a putative TetR family transcription factor. Compared with wild-type strain M145, the rrdA null mutant exhibited increased Red production and decreased Act production. A high level of rrdA expression resulted in a severe reduction in Red production and Act overproduction. Reverse transcription-PCR analysis showed that RrdA negatively regulated Red production by controlling redD mRNA abundance, while no change was observed at the transcript level of the Act-specific activator gene, actII-orf4. The effects on Act biosynthesis might arise from competition for precursors that are common to both pathways.In addition to its complex morphological differentiation, the gram-positive genus Streptomyces is notable for its ability to produce a wide range of secondary metabolites during its life cycle. These metabolites include the majority of known pharmaceutically important secondary metabolites that exhibit antibacterial, anticancer, and immunosuppressive activities (6, 7). The genes responsible for the biosynthesis of secondary metabolites are often physically clustered in the genome and coordinately regulated by pathway-specific transcriptional activators (1,4,10,12,29,33). These specific regulators are controlled by various higher-level pleiotropic regulators, and their expression is typically affected by a variety of environmental and physiological cues, including the nature and levels of carbon and nitrogen sources and the availability of phosphate and small signaling molecules, such as ppGpp and ␥-butyrolactone (5).Streptomyces coelicolor A3 (2) has been used for many years as a model organism in morphological and physiological differentiation studies, particularly in studies of the regulation of antibiotic biosynthesis (7). S. coelicolor produces four antibiotics: actinorhodin (Act), undecylprodigiosin (Red), methylenomycin, and calcium-dependent antibiotic. It has been shown that certain regulators are involved in the pleiotropic control of antibiotic production in S. coelicolor, including AbsA1/A2, AfsR/K, AtrA, and PhoR/P (18, 24, 31, 32). Recently, mutational analysis and adventitious overexpression of key regulators in S. coelicolor revealed cross-regulation at the transcriptional level a...

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

“…Tn316 integration sites were determined via plasmid rescue experiments followed by DNA sequencing and were found to be randomly distributed on the U32 chromosome. Based on our previous studies, this transposon system also works efficiently in Streptomyces (32,33), which therefore suggests that this in vivo transposon system may also be applied in functional genomics study of other actinomycetes.…”

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

“…4D), each with more than 10 inserts, were two clusters of genes, i.e., cmdB to -F and the two-component regulatory genes SCO3008, SCO3012, and SCO3013, and two single genes, i.e., the NAD ϩ -dependent glutamate dehydrogenase gene SCO2999 and the ATP/GTP binding protein gene SCO5677. Other reliable down-modulators included 42 genes with more than one RED-increasing insert, 13 of which have been reported previously (wblA [49], bldM [50], bldN [51], rmdA [52], rrdA [18], relA [53], ohkA [54], cutRS [55], nsdB [56], SCO4174 [21], SCO5745 [19], and adpA [57]) (Table S5). A further 140 potential RED down-modulators were identified by single insertions (Data Set S1).…”

“…RED is generated via a hybrid nonribosomal peptide synthase (NRPS)-type I polyketide synthase (PKS) pathway from L-serine, L-proline, glycine, and acetyl-coenzyme A (acetylCoA). Global transcriptional regulatory genes, such as afsRS, wblA, dasR, abaA, and rrdA, regulate the production of RED (14)(15)(16)(17)(18). Many other genes also modulate RED production, such as the RNase J gene SCO5745 (19) and the membrane protein genes sarA and SCO4174 (20,21).…”

Bacterial Competition Reveals Differential Regulation of the pks Genes by Bacillus subtilis

“…Many loci that encode assembly-line enzyme complexes also encode transcription factors that control the expression of the biosynthetic genes (12,36,37). The pksA gene, located adjacent to the pks gene cluster, encodes a putative TetR family regulatory protein that is predicted to function as the associated regulator of the pks genes (14-16).…”