Identification and characterization of eukaryotic-like protein Ser/Thr kinases in Myxococcus xanthus, a developmental bacterium.

摘要

A multi-gene family of eukaryotic-like protein Ser/Thr kinases was found in Myxococcus xanthus by Southern blot analysis of chromosomal DNA using a consensus sequence of the kinase catalytic domain.; To date, a total of 13 kinase genes have been identified in M. xanthus. Out of these 13 genes, I characterized two genes, pkn5 and pkn6. These two genes are divergently located on the chromosome sharing a 46-bp promoter region. Pkn5, consisting of 380 amino acid residues, is a soluble protein in the cytoplasm, while Pkn6, consisting of 710 amino acid residues, is a transmembrane protein. The membrane topology of Pkn6 was determined using Pkn6-PhoA fusion protein in Escherichia coli having a single transmembrane domain with the N-terminal domain in the cytoplasm and the C-terminal domain outside the cytoplasmic membrane. Both proteins, when expressed in E. coli, were autophosphorylated; Pkn5 only at Ser and Pkn6 at both Ser and Thr.; In order to examine a possible interaction between Pkn5 and Pkn6, recombinant His-tagged Pkn5 and Pkn6 proteins were expressed in E. coli and purified to near homogeneity by affinity chromatography using Ni-NTA resin. Both purified proteins were shown to have autophosphorylation activity. Pkn6 kinase activity required Ca{dollar}sp{lcub}2+{rcub}{dollar}, while Pkn5 is active in the absence of Ca{dollar}sp{lcub}2+{rcub}.{dollar} Kinase-negative mutant Pkn5 and Pkn6 were also purified and used as substrates for Pkn6 and Pkn5, respectively. However, neither mutant Pkn5 nor mutant Pkn6 were found to be phosphorylated; suggesting that these two kinases may not be directly interacting with each other in a single signal transduction pathway.; In M. xanthus, both genes are expressed constitutively throughout the life cycle with slight increases at an early stage of development. A pkn5-deletion strain forms fruiting bodies much faster than the wild-type strain, while a pkn6-deletion strain develops slower than the wild-type strain. These results, together with the fact that the pkn5-deletion strain grow slower in rich media, suggest that Pkn5 and Pkn6 have reciprocal roles in M. xanthus growth and development. It could be hypothesized that Pkn6 may be a transmembrane sensor of external signals for development, while Pkn5 is a kinase negatively regulating M. xanthus development.