Phosphorylation of AfsR by multiple serine/threonine kinases in Streptomyces coelicolor A3(2).
ABSTRACT AfsK, a protein serine/threonine kinase, autophosphorylates on serine and threonine residues and phosphorylates serine and threonine residues of AfsR, a transcriptional activator for afsS involved in secondary metabolism in Streptomyces coelicolor A3(2). pkaG encoding a 592-amino-acid protein and SCD10.09 (named afsL) encoding a 580-amino-acid protein, both of which encode an AfsK-like protein, were transcribed throughout growth. PkaG with a histidine-tag and the kinase catalytic domain of PkaG, produced in Escherichia coli, autophosphorylated dominantly on threonine and slightly on serine residues. In addition, these proteins phosphorylated AfsR on threonine and serine residues. The catalytic domain of AfsL also autophosphorylated and phosphorylated AfsR, on threonine and serine residues in both cases. AfsR was thus found to be phosphorylated by multiple kinases. Disruption of the chromosomal pkaG gene resulted in slightly reduced production of the pigmented antibiotic actinorhodin. These findings, together with the presence of about 40 AfsK homologues and at least five AfsR homologues in S. coelicolor A3(2), suggest that the regulatory networks via eukaryotic-type protein phosphorylation are more diverse and versatile than we have expected.
Article: Genome-wide transcriptome analysis reveals that a pleiotropic antibiotic regulator, AfsS, modulates nutritional stress response in Streptomyces coelicolor A3(2).[show abstract] [hide abstract]
ABSTRACT: A small "sigma-like" protein, AfsS, pleiotropically regulates antibiotic biosynthesis in Streptomyces coelicolor. Overexpression of afsS in S. coelicolor and certain related species causes antibiotic stimulatory effects in the host organism. Although recent studies have uncovered some of the upstream events activating this gene, the mechanisms through which this signal is relayed downstream leading to the eventual induction of antibiotic pathways remain unclear. In this study, we employed whole-genome DNA microarrays and quantitative PCRs to examine the transcriptome of an afsS disruption mutant that is completely deficient in the production of actinorhodin, a major S. coelicolor antibiotic. The production of undecylprodigiosin, another prominent antibiotic, was, however, perturbed only marginally in the mutant. Principal component analysis of temporal gene expression profiles identified two major gene classes each exhibiting a distinct coordinate differential expression pattern. Surprisingly, nearly 70% of the >117 differentially expressed genes were conspicuously associated with nutrient starvation response, particularly those of phosphate, nitrogen and sulfate. Furthermore, expression profiles of some transcriptional regulators including at least two sigma factors were perturbed in the mutant. In almost every case, the effect of afsS disruption was not observed until the onset of stationary phase. Our data suggests a comprehensive role for S. coelicolor AfsS as a master regulator of both antibiotic synthesis and nutritional stress response, reminiscent of alternative sigma factors found in several bacteria.BMC Genomics 02/2008; 9:56. · 4.07 Impact Factor
Article: Identification and characterization of NocR as a positive transcriptional regulator of the beta-lactam nocardicin A in Nocardia uniformis.[show abstract] [hide abstract]
ABSTRACT: Nocardicin A is a monocyclic beta-lactam isolated from the actinomycete Nocardia uniformis, which shows moderate activity against a broad spectrum of gram-negative bacteria. Within the biosynthetic gene cluster of nocardicin A, nocR encodes a 583-amino-acid protein with high similarity to a class of transcriptional regulators known as streptomyces antibiotic regulatory proteins. Insertional inactivation of this gene resulted in a mutant showing morphology and growth characteristics similar to the wild type, but one that did not produce detectable levels of nocardicin A or the early precursor p-hydroxybenzoyl formate. Similar disruptions of nocD, nocE, and nocO yielded mutants that maintained production of nocardicin A at levels similar to the wild-type strain. In trans complementation of the nocR::apr mutant partially restored the wild-type phenotype. Transcriptional analysis of the nocR::apr mutant using reverse transcription-PCR found an absence of mRNA transcripts for the early-stage nocardicin A biosynthetic genes. In addition, transcription of the genes responsible for the biosynthesis of the nonproteinogenic p-hydroxyphenylglycine (pHPG) precursor was attenuated on the nocR disruption mutant. NocR was heterologously expressed and purified from Escherichia coli as an N-terminal maltose binding protein-tagged fusion protein. DNA binding assays demonstrated that NocR is a DNA binding protein, targeting the 126-bp intergenic region between nocF and nocA. Within this intergenic region is the likely binding motif, a direct hexameric repeat, TGATAA, with a 5-bp spacer. These experiments establish NocR as a positive transcriptional regulator of the nocardicin A biosynthetic pathway, coordinating the initial steps of nocardicin A biosynthesis to the production of its pHPG precursor.Journal of bacteriology 12/2008; 191(3):1066-77. · 3.94 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Genomic studies have revealed the presence of Ser/Thr kinases and phosphatases in many bacterial species, although their physiological roles have largely been unclear. Here we review bacterial Ser/Thr kinases (eSTKs) that show homology in their catalytic domains to eukaryotic Ser/Thr kinases and their partner phosphatases (eSTPs) that are homologous to eukaryotic phosphatases. We first discuss insights into the enzymatic mechanism of eSTK activation derived from structural studies on both the ligand-binding and catalytic domains. We then turn our attention to the identified substrates of eSTKs and eSTPs for a number of species and to the implications of these findings for understanding their physiological roles in these organisms.Microbiology and molecular biology reviews: MMBR 03/2011; 75(1):192-212. · 12.59 Impact Factor