A gene, pkn2, encoding a Myxococcus xanthus protein with significant similarities to eukaryotic protein serine/threonine kinases, was cloned using the polymerase chain reaction. The open reading frame for the protein, beginning with a GUG initiation codon, consists of 830 amino acids. The amino-terminal 279 residues show 37% identity to catalytic domain of Pknl, another protein serine/threonine kinase expressed during the development at the onset of sporulation. The catalytic domain of Pkn2 contains 27% and 25% identity to rat Ca2+/calmodulin-dependent protein kinase and Bos taurus rhodopsin kinase, respectively. In the middle of the carboxy-terminal regulatory domain, there is a typical transmembrane domain consisting of 18 hydrophobic residues. The gene product, Pkn2, produced in Escherichia coil under a T7 promoter was phosphorylated at both serine and threonine residues. TEM-13-1actamase produced in E. coil was found to serve as an effective substrate for Pkn2, phosphorylated only at threonine residues, shifting its apparent molecular mass from 29 to 44 kD. The phosphorylated 13-1actamase was unable to be secreted into the periplasmic space and localized in the cytoplasmic and membrane fractions. Analysis of phoA fusions with pkn2 demonstrated that Pkn2 is a transmembrane protein with the kinase domain in the cytoplasm and the 207-residue carboxy-terminal domain outside the cytoplasmic membrane. Disruption of pkn2 showed no effect on vegetative growth but reduced the yield of myxospores by 30%-50%. On the basis of the present results, we propose that Pkn2 is a transmembrane protein serine/threonine kinase that regulates the activity of endogenous 13-1actamase or related enzymes in response to an external signal yet to be identified.[Key Words: Protein serine/threonine kinase; myxobacteria; transmembrane protein kinase; ~-lactamase] Received January 25, 1995; revised version accepted March 9, 1995.Protein kinase cascades play major roles in intracellular signal transduction regulating various cellular functions in both prokaryotes and eukaryotes. In prokaryotes, protein histidine kinases are known to be the key enzymes for the cascades, and it has been believed for a long time that protein serine/threonine kinases do not exist in prokaryotes. These kinases, together with protein tyrosine kinases, are essential in signal transduction in eukaryotes. However, recently, the existence of such eukaryotic-like protein serine/threonine kinases in Myxococcus xanthus, a Gram-negative bacterium, has been demonstrated (Munoz-Dorado et al. 1991;Zhang et al. 1992). M. xanthus living in the soil shows rather spectacular morphogenesis, including multicellular fruiting body formation upon nutrient starvation (for review, see Shimkets 1990). Within the fruiting body, a significant population of rod-shaped vegetative cells differentiate into spherical heat-resistant spores. It is believed that M. xanthus responds to a large number of signals in its natural habitat to regulate its cell-cell communication, gliding motility, aggre...