PKN9, A SER THR PROTEIN-KINASE INVOLVED IN THE DEVELOPMENT OF MYXOCOCCUS-XANTHUS/

The Myxococcus xanthus gene, pkn9 encodes a protein that contains sign ificant homology with eukaryotic Ser/Thr protein kinases. The pkn9 gen e was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene ex pression during selected periods of the M. xanthus life cycle. Pkn9 wa s constitutively expressed during vegetative growth and upregulated du ring the aggregation stage of early development, It consists of 589 am ino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus, This region also shows 29, 25 and 29% id entity with myosin light-chain kinase, protein kinase C, and cAMP-depe ndent protein kinase, respectively, A 22-residue hydrophobic transmemb rane domain separates the kinase domain from the 173-residue C-termina l domain that resides on the outside of the inner membrane, The C-term inal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function, When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and th reonine residues, Disruption of the pkn9 kinase catalytic subdomains I -III by the insertion of a kanamycin-resistance gene resulted in sligh tly delayed, smaller and more-crowded fruiting bodies, while spore for mation was normal, Total deletion of the pkn9 gene caused severely red uced progression through development resulting in light loose mounds t hat become slightly more compact over time, Development progressed fur ther at the centre than at the edge of the spot, and spore formation w as significantly reduced, Two-dimensional gel analysis revealed that b oth the disruption and the deletion of pkn9 prevented the expression o f five membrane proteins (KREP9-1-4). These results suggest that the l oss of Pkn9 kinase activity caused altered fruiting-body formation, th e absence of the KREP9 proteins in the membrane, and reduced spore pro duction.