Genetic and functional analysis of the Treponema denticola chemotaxis system

Treponema denticola is a Gram-negative, oral spirochete identified as one of the predominant groups of microorganisms in periodontal diseases. The chemotaxis signaling pathway of T. denticola, composed of the cheA, chew, cheX and cheY genes and twenty putative chemoreceptors, the MCPs (methyl-accepting chemotaxis proteins), regulates motility through a complex series of conserved protein interactions that modulate flagellar motor function in response to environmental changes. Homologues to CheX, originally described as a novel spirochete chemotaxis protein of unknown function, have recently been demonstrated to exhibit phosphatase activity.;The potential interactions of CheX with other components of the chemotaxis pathway as well as well-established interactions were investigated with the yeast-two hybrid system. CheX was found to interact with CheA and with itself. In agreement with homologous systems in other species, CheA forms a complex with the MCPs, and CheW. Interactions of CheA with itself and its cognate response regulator were also confirmed.;Development of allelic replacement mutagenesis techniques for T. denticola has permitted targeted construction of gene inactivation mutants. Inactivation of cheA resulted in defective chemotaxis behavior. Mutants lacking cheA exhibited coordinated non-reversing cell movements confirming that CheA is involved in regulating cellular reversal frequency.;In the last part of this dissertation cheX and cheY genes were inactivated individually and in combination by insertion of an ermF-ermAM resistance cassette. Reduced expression of the cheY gene in the cheX mutant resulted in the construction of an additional cheX mutant with a chemotaxis promoter in between the ermF-ermAM cassette and the cheY gene but could not remedy the problem. cheX cheY and cheAY deletion mutants exhibited chemotaxis defects and altered motility behavior. Interestingly, majority of the cheX and cheXY mutants failed to change direction of rotation whereas about half of the cheY mutants still reversed their direction of movement albeit at a reduced frequency compared to wild-type. The other half of the observed mutant cells moved in one direction but stopped frequently. Additionally, all chemotaxis mutant strains tested in this study showed reduced tissue penetration, a feature considered important for pathogenesis of T. denticola.