Analysis of serine/threonine protein kinases of Myxococcus xanthus.

摘要

Myxococcus xanthus, one of the well studied Myxobacteria, is a gram negative soil dwelling bacterium which moves by gliding motility on semi-solid surface preying on other microorganisms. Evolutionarily, myxobacteria are probably the oldest living organisms that are able to form elaborate multicellular structures called fruiting bodies upon nutrient starvation. Inside a fruiting body many cells undergo autolysis while some differentiate to form spores. Biochemically, M. xanthus shows many eukaryotic like features, e.g., presence of a reverse transcriptase and presence of eukaryotic like serine/threonine protein kinases and therefore is very fascinating to study.; A family of thirteen eukaryotic like protein serine/threonine protein kinases was discovered in our lab by previous scientists. Amino acid sequence analysis of these protein kinases reveals that some of them have certain unique features and their mode of action might be different from the eukaryotic protein kinases. Pkn11 is one such protein kinase. Biochemical characterization of this kinase revealed that when expressed in E. coli it appears as a 120 kDa, transmembrane protein, it is autophosphorylated on both serine and threonine residues and requires Ca{dollar}sp{lcub}2+{rcub}{dollar} for its catalytic activity. It was found to be expressed at very low basal levels during vegetative growth of M. xanthus, and the expression was found to be enhanced five fold in defined medium.; It was found that when overexpressed in E. coli, Pkn11 is toxic for the cell. This fact was exploited to design a genetic approach to identify proteins that can suppress the toxicity of Pkn11. By co-transformation of E. coli genomic library into a Pkn11 overexpressing strain, a DNA fragment was obtained that contained two open reading frames. ORF2 was found to be more important for suppression of Pkn11 toxicity but was not phosphorylated directly by Pkn11 in vitro. During the course of in vitro experiments an 18 kDa protein from E. coli was found to be phosphorylated on histidine or aspartate residue in presence of Pkn11. In addition it was also found that when Pkn11 was overexpressed in E. coli, rate of DNA synthesis was inhibited within 1h of induction of expression of Pkn11. Together these results seem to indicate that there exists a His-Asp pathway in E. coli to control cell growth that can be hampered by the kinase activity of Pkn11. Consequently, it appears that Pkn11 is a growth inhibitory protein kinase of M. xanthus, which might arrest cell growth in response to some specific signal.; When the effects of a number of inhibitors of eukaryotic protein serine/threonine and tyrosine kinases were examined during development, they could inhibit fruiting body formation and sporulation to some degree. However none of the inhibitors tested had any effect on vegetative growth. Most of them seemed to act during the early stage of development. These results suggest that a group of specific kinases are involved only in fruiting body formation and sporulation, but not during vegetative growth of M. xanthus.