Expression and Characterization of theStreptomyces coelicolorSerine/Threonine Protein Kinase PkaD

Abstract

We identified and characterized the gene encoding a new eukaryotic-type protein kinase from Streptomyces coelicolor A3(2) M145. PkaD, consisting of 598 amino acid residues, contained the catalytic domain of eukaryotic protein kinases in the N-terminal region. A hydrophobicity plot indicated the presence of a putative transmembrane spanning sequence downstream of the catalytic domain, suggesting that PkaD is a transmembrane protein kinase. The recombinant PkaD was found to be phosphorylated at the threonine and tyrosine residues. In S. coelicolor A3(2), pkaD was transcribed as a monocistronic mRNA, and it was expressed constitutively throughout the life cycle. Disruption of chromosomal pkaD resulted in a significant loss of actinorhodin production. This result implies the involvement of pkaD in the regulation of secondary metabolism.

Analysis of the phosphoproteome of the multicellular bacterium Streptomyces coelicolor A3(2) by protein/peptide fractionation, phosphopeptide enrichment and high‐accuracy mass spectrometry

“…Through homology searching using the standard BLAST program, the completed genome sequence of S. coelicolor revealed 34 putative eukaryotic‐like STPKs 26, 29. Of these, only ten of the STPKs of S. coelicolor have been partially characterised through a number of studies 30–36, with the pleiotropic regulatory protein AfsR (SCO4426) the only confirmed STPK target substrate 33. To date, very few phosphoproteins have been identified in S. coelicolor through in vitro and in vivo studies and include elongation factor Tuf‐1 37–38, the single‐stranded DNA‐binding protein SCO3907 39, the STPK AfsK (SCO4423) 40 and AfsR 33.…”

Control of Morphological Differentiation of Streptomyces coelicolor A3(2) by Phosphorylation of MreC and PBP2

“…PkaI is encoded in a cluster of five putative eSTPK genes, SCO4775 (pkaH) , SCO4776 , SCO4777 (pkaD) , SCO4778 (pkaI) , SCO4779 (pkaJ) in S . coelicolor M145 [ 31 ]; S1A Fig ). The genomic organization of the eSTPK genes, with intergenic sequences between 93 bp and 173 bp, indicates that each gene is transcribed separately.…”

“…But if the purpose of phosphorylation is to keep the synthesized proteins inactive, the topology of the phosphosites is of minor relevance. Moreover, the phosphosites of the penicillin binding protein PBPA of Mycobacterium tuberculosis , phosphorylated by the eSTPK PknB are also in the (extracellular) transpeptidase domain [ 31 ]. Here it was shown that phosphorylation affected positioning of PBPA at the septum, thereby regulating septal peptidoglycan biosynthesis.…”

“…For example, S . coelicolor contains more than 30 eSTPKs [ 30 , 31 ] and more than 50 eukaryotic-type protein phosphatases [ 32 ]. The catalytic domains of many bacterial eSTPKs are linked to additional domains.…”

Repression of Antibiotic Production and Sporulation inStreptomyces coelicolorby Overexpression of a TetR Family Transcriptional Regulator

“…The genes responsible for the biosynthesis of these secondary metabolites are clustered in the genome and coordinately regulated by pathway-specific transcriptional regulators (1,4,18,19,62,70). The expression of these specific regulators linked to the biosynthetic pathways is directly or indirectly controlled either by positive pleiotropic regulators, such as AfsR2 (69), AfsS (29), AtrA (65), PtpA (67), PkaD (68), and the two-component systems AfsK/AfsR (66) and EcrA1/EcrA2 (39), by negative regulators, including the two-component systems AbsA1/AbsA2 (44), PhoR/PhoP (55), and CutR/CutS (9), or by enzymatic systems, such as Ppk (16). The pleiotropic regulators, thought to sense a variety of extracellular or intracellular signals related to nutriment availability, cell crowding, or energy shortage, are necessary to trigger the necessary metabolic adjustments to adapt to these conditions (27,43), whereas the ppk gene is thought to act as an ATP-regenerating enzyme (54).…”