A TACTILE SENSORY SYSTEM OF MYXOCOCCUS-XANTHUS INVOLVES AN EXTRACELLULAR NAD(P)(+)-CONTAINING PROTEIN

CsgA is a cell surface protein that plays an essential role in tactile responses during Myxococcus xanthus fruiting body formation by producing the morphogenic C-signal. The primary amino acid sequence of CsgA exhibits homology with members of the short-chain alcohol dehydrogenase (SCAD) family and several lines of evidence suggest that NAD(P)(+) binding is essential for biological activity. First, the predicted CsgA. secondary structure based on the 3 alpha/20 beta-hydroxysteroid dehydrogenase crystal structure suggests that the amino-terminal portion of the protein contains an NAD(P)(+) binding pocket. Second, strains with csgA alleles encoding amino acid substitutions T6A and R10A in the NAD(P)(+) binding pocket failed to develop. Third, exogenous MalE-CsgA rescues csgA development, whereas MalG-CsgA with the amino acid substitution CsgA T6A does not. Finally, csgA spore yield increased similar to 20% when buffer containing 100 nM of MalG-CsgA was supplemented with 10 mu M of NAD(+) or NADP(+). Conversely, 10 mu M of NADH or NADPH delayed development for similar to 24 hr and depressed spore levels similar to 10%. Together, these results argue that NAD(P)(+) binding is critical for C-signaling. S135 and K155 are conserved amino acids in the catalytic domain of SCAD members. Strains with csgA alleles encoding the amino acid substitutions S135T or K155R failed to develop. Furthermore, a MalE-CsgA protein containing CsgA S135T was not able to restore development to csgA cells. In conclusion, amino acids conserved in the coenzyme binding pocket and catalytic site are essential for C-signaling.