MUTATIONS IN THE CONSENSUS ATP-BINDING SITES OF XCPR AND PILB ELIMINATE EXTRACELLULAR PROTEIN SECRETION AND PILUS BIOGENESIS IN PSEUDOMONAS-AERUGINOSA

The process of extracellular secretion in Pseudomonas aeruginosa requi res specialized machinery which is widely distributed among bacteria t hat actively secrete proteins to the extracellular medium. One of the components of this machinery is the product of the xcpR gene, which is homologous to pilB, a gene encoding a protein essential for the bioge nesis of type IV pili. Both XcpR and PilB are characterized by the pre sence of a conserved ATP-binding motif (Walker sequence). ne codons of highly conserved glycine residues within the Walker sequences of xcpR and pilB were altered to encode a serine, and the effects of these su bstitutions were examined. Bacteria expressing mutant XcpR or PilB wer e unable to secrete exotoxin A or assemble pili, respectively. In addi tion, high-level expression of mutant XcpR in wild-type P. aeruginosa led to a pleiotropic extracellular secretion defect, resulting in the periplasmic accumulation of enzymes that are normally secreted from th e cell. These studies show that the putative ATP-binding sites of XcpR and PilB are essential for their functions in protein secretion and a ssembly of pili, respectively. Moreover, the observed dominant negativ e phenotype of mutant XcpR suggests that this protein functions as a m ultimer or, alternatively, interacts with another essential component of the extracellular protein secretion machinery.