title: Development and use of a gene deletion strategy for Flavobacterium johnsoniae to identify the redundant gliding motility genes remF, remG, remH, and remI.
abstract: Cells of Flavobacterium johnsoniae exhibit rapid gliding motility over surfaces. Cell movement is thought to involve motor complexes comprised of Gld proteins that propel the cell surface adhesin SprB. The four distal genes of the sprB operon (sprC, sprD, sprB, and sprF) are required for normal motility and for formation of spreading colonies, but the roles of the remaining three genes (remF, remG, and fjoh_0982) are unclear. A gene deletion strategy was developed to determine whether these genes are involved in gliding. A spontaneous streptomycin-resistant rpsL mutant of F. johnsoniae was isolated. Introduction of wild-type rpsL on a plasmid restored streptomycin sensitivity, demonstrating that wild-type rpsL is dominant to the mutant allele. The gene deletion strategy employed a suicide vector carrying wild-type rpsL and used streptomycin for counterselection. This approach was used to delete the region spanning remF, remG, and fjoh_0982. The mutant cells formed spreading colonies, demonstrating that these genes are not required for normal motility. Analysis of the genome revealed a paralog of remF (remH) and a paralog of remG (remI). Deletion of remH and remI had no effect on motility of wild-type cells, but cells lacking remF and remH, or cells lacking remG and remI, formed nonspreading colonies. The motility defects resulting from the combination of mutations suggest that the paralogous proteins perform redundant functions in motility. The rpsL counterselection strategy allows construction of unmarked mutations to determine the functions of individual motility proteins or to analyze other aspects of F. johnsoniae physiology.
authors.affiliation: Department of Biological Sciences, University of Wisconsin-Milwaukee, P.O. Box 413, Milwaukee, WI 53201, USA.
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Rhodes
authors.forename: Halley G
authors.initials: HG
authors.lastname: Pucker
authors.forename: Mark J
authors.initials: MJ
authors.lastname: McBride
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D024881:Drug Resistance, Bacterial; D005417:Flavobacterium / Q000235:genetics* / Q000502:physiology; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008124:Locomotion; D010957:Plasmids; D011995:Recombination, Genetic; D012269:Ribosomal Proteins / Q000235:genetics; D012641:Selection, Genetic; D017384:Sequence Deletion; D013307:Streptomycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D012269:Ribosomal Proteins; D013307:Streptomycin