DEVELOPMENT OF TECHNIQUES FOR THE GENETIC MANIPULATION OF THE GLIDINGBACTERIUM CYTOPHAGA-JOHNSONAE

Cytophaga johnsonae displays many features that make it an excellent m odel of bacterial gliding motility. Unfortunately, genetic analyses of C. johnsonae, or any related gliding bacteria, were not possible beca use of a complete lack of selectable markers, cloning vectors, transpo sons, and convenient methods of gene transfer. As a first step toward a molecular analysis of gliding motility of C. johnsonae we developed these genetic techniques and tools. Common broad-host-range plasmids a nd transposons did not function in C. johnsonae. We identified one Bac teroides transposon, Tn4351, that could be introduced into C. johnsona e on plasmid R751 by conjugation from Escherichia coli. Tn4351 inserte d in the C. johnsonae genome and conferred erythromycin resistance. Tn 4351 insertions resulted in auxotrophic mutations and motility mutatio ns. We constructed novel plasmids and cosmids for genetic analyses of C. johnsonae. These cloning vectors are derived from a small cryptic p lasmid (pCP1) that we identified in the fish pathogen Cytophaga psychr ophila D12. These plasmids contain the ermF (erythromycin resistance) gene from Tn4351 and a variety of features that facilitate propagation and selection in E. coli and conjugative transfer from E. coli to C. johnsonae.