Identification of a protein Ser/Thr kinase cascade that regulates essential transcriptional activators in Myxococcus xanthus development

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Abstract
Pkn8 is a membrane-associated protein Ser/Thr kinase (PSTK) of Myxoccocus xanthus that was previously found to associate with a novel cytoplasmic kinase, Pkn14. In the present study, MrpC, an essential transcription factor for fruA expression during fruiting body development, was identified using a genomic yeast two-hybrid screen with Pkn14 as bait. Our biochemical studies demonstrated that purified Pkn8 and Pkn14 are active kinases and that Pkn8 is able to phosphorylate Pkn14 that forms a tetramer via its C-terminal 41 residues. Moreover, Pkn14 phosphorylated purified MrpC, indicating that Pkn8 is a Pkn14 kinase and Pkn14 is an MrpC kinase. The pkn8 and pkn14 deletion strains (Δpkn8 and Δpkn14) developed into fruiting bodies significantly faster than that of the parent strain, DZF1. While mrpC expression was at a low level in DZF1 during vegetative growth, it was highly elevated in Δpkn8 and Δpkn14 during vegetative growth and development. Furthermore, FruA, usually induced at 6 h of development, was instead detected at the early stationary phase and accumulated faster during development in Δpkn8 and Δpkn14. Therefore, the developmental phenotype of Δpkn8 and Δpkn14 seems to be due to untimely FruA production mediated by elevated levels of MrpC in Δpkn8 and Δpkn14 during vegetative growth. As pkn14 expression was increased at the mid- and late-log. phases in DZF1 but decreased during development, the Pkn8–Pkn14 kinase cascade appears to negatively regulate mrpC expression by phosphorylating MrpC during vegetative growth. This is the first demonstration of a functional PSTK cascade in prokaryotes. mrpC expression has been proposed to be activated by MrpA and MrpB which belong to a two-component His-Asp phosphorelay signal transduction system and that MrpC autoregulate its own expression (Sun H. and Shi W., 2001 J Bacteriol 183: 4786–4795). Therefore, M. xanthus seems to utilize both eukaryotic PSTK cascade and prokaryotic His-Asp phosphorelay system to precisely regulate mrpC expression with specific timing during development.