Electron Transport Chain Is Biochemically Linked to Pilus Assembly Required for Polymicrobial Interactions and Biofilm Formation in the Gram-Positive Actinobacterium .

The Gram-positive actinobacteria spp. are key colonizers in the development of oral biofilms due to the inherent ability of to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about -acting factors contributing to these processes. We report here a large-scale Tn transposon screen for mutants defective in coaggregation with We obtained 33 independent clones, 13 of which completely failed to aggregate with , and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn insertions in , , or , as expected; the latter were mapped to genes coding for uncharacterized proteins and various genes encoding the NADH dehydrogenase subunits. Electron microscopy and biochemical analyses of mutants with nonpolar deletions of genes and , a menaquinone C-methyltransferase-encoding gene downstream of the locus, confirmed the pilus and coaggregation defects. Both and mutants were defective in oxidation of MdbA, the major oxidoreductase required for oxidative folding of pilus proteins. Furthermore, supplementation of the mutant with exogenous menaquinone-4 rescued the cell growth and pilus defects. Altogether, we propose that the electron transport chain is biochemically linked to pilus assembly via oxidative protein folding. The Gram-positive actinobacterium expresses adhesive pili, or fimbriae, that are essential to biofilm formation and interactions with other bacteria, termed coaggregation. While the critical role of the conserved sortase machine in pilus assembly and the disulfide bond-forming catalyst MdbA in oxidative folding of pilins has been established, little is known about other -acting factors involved in these processes. Using a Tn transposon screen for mutants defective in coaggregation with , we found that genetic disruption of the NADH dehydrogenase and menaquinone biosynthesis detrimentally alters pilus assembly. Further biochemical characterizations determined that menaquinone is important for reactivation of MdbA. This study supports the notion that the electron transport chain is biochemically linked to pilus assembly in via oxidative folding of pilin precursors.