Abstract
During morphological differentiation of Streptomyces coelicolor A3(2), the sporogenic aerial hyphae are transformed into a chain of more than fifty spores in a highly coordinated manner. Synthesis of the thickened spore envelope is directed by the Streptomyces spore wall synthesizing complex SSSC which resembles the elongasome of rod-shaped bacteria. The SSSC includes the eukaryotic type serine/threonine protein kinase (eSTPK) PkaI, encoded within a cluster of five independently transcribed eSTPK genes (SCO4775-4779). To understand the role of PkaI in spore wall synthesis, we screened a S. coelicolor genomic library for PkaI interaction partners by bacterial two-hybrid analyses and identified several proteins with a documented role in sporulation. We inactivated pkaI and deleted the complete SCO4775-4779 cluster. Deletion of pkaI alone delayed sporulation and produced some aberrant spores. The five-fold mutant NLΔ4775-4779 had a more severe defect and produced 18% aberrant spores affected in the integrity of the spore envelope. Moreover, overbalancing phosphorylation activity by expressing a second copy of any of these kinases caused a similar defect. Following co-expression of pkaI with either mreC or pbp2 in E. coli, phosphorylation of MreC and PBP2 was demonstrated and multiple phosphosites were identified by LC-MS/MS. Our data suggest that elaborate protein phosphorylation controls activity of the SSSC to ensure proper sporulation by suppressing premature cross-wall synthesis.
Highlights
The peptidoglycan (PG) sacculus, an elastic mesh of linear carbohydrate chains cross-linked by short peptides surrounds the bacterial cell [1,2]
Expression of pkaI is down-regulated during morphological differentiation
To elucidate a possible role in morphological differentiation, we characterized the expression profiles of pkaI and the other eukaryotic type serine/threonine protein kinase (eSTPK) genes at distinct stages of the S. coelicolor life cyle by semi-quantitative RT-PCR with gene-specific primer pairs (S1C Fig)
Summary
The peptidoglycan (PG) sacculus, an elastic mesh of linear carbohydrate chains cross-linked by short peptides surrounds the bacterial cell [1,2]. This exoskeleton is the cell shape-maintaining element and allows the cell to withstand alterations in internal turgor due to changing environments. Few bacterial species developed a life style devoid of a PG sacculus [3]. Despite high conservation of the basic PG structure, various species specific modification including different amino acids in the stem peptide or variations in the interpeptide bridge or O-acetylation occur [6]
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