A tactile sensory system of Myxococcus xanthus involves an extracellular NAD(P)(+)-containing protein.

Abstract

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 3a120P-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 RlOA in the NAD(P)+ binding pocket failed to develop. Third, exogenous MalE-CsgA rescues csgA development, whereas MalE-CsgA with the amino acid substitution CsgA T6A does not. Finally, csgA spore yield increased -20% when buffer containing 100 nM of MalE-CsgA was supplemented with 10 p~ of NAD+ or NADP+. Conversely, 10 p~ of NADH or NADPH delayed development for -24 hr and depressed spore levels -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, a&ino acids conserved in the coenzyme binding pocket and catalytic site are essential for C-signaling.

Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases

“…However, the current model fails to account for many critical pieces of data that are inconsistent with it. For example, CsgA has features associated with enzymatic function, such as a Rossmann fold, shown to bind NAD + in vitro, and conserved catalytic residues typical of short-chain alcohol dehydrogenases (SCADs), shown to be essential for development in vivo (Lee et al 1995). Membrane fractionation indicated that CsgA is an inner membrane protein (Simunovic et al 2003), not an outer membrane protein as previously reported (Lobedanz and Sogaard-Andersen 2003).…”

“…As expected, p17 CsgA displays no activity in this assay, since it lacks the NAD + -binding domain. As an additional negative control, CsgA bearing the active site mutation K155R (Lee et al 1995) was examined and is inactive. Enzymatic reactions with CsgA did not take place in the presence of NADP + (Supplemental Fig.…”

C factor, a cell-surface-associated intercellular signaling protein, stimulates the cytoplasmic Frz signal transduction system in Myxococcus xanthus.

“…Fusion protein has C-factor activity measured by the standard C-factor bioassay (26). Addition of the fusion protein (specific activity, 16 C-factor units/mg of protein) also increased FrzCD methylation (Fig.…”

Role of fruA and csgA Genes in Gene Expression during Development of Myxococcus xanthus

“…All C-signal mutations were mapped to the csgA gene encoding a homologue of short chain alcohol dehydrogenase (13). A 25-kDa protein encoded by the entire csgA gene was produced at a basal level during vegetative growth and induced during development, whereas a 17-kDa protein was produced after 6 h post-starvation (14,15). The 17-kDa csgA protein is a cell surface associated-protein called C-factor and carries an activity that extracellularly complements the csgA mutations during development (16 -18).…”