Identification of flagellar motility genes in Yersinia ruckeri by transposon mutagenesis

Abstract

Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype 2 Y. ruckeri through the mutational loss of flagellar secretion.

FIG. 1.

Motility and lipase phenotypes of Y. ruckeri CSF07-82 and flhA mutant derivative BTF1. (A and B) Motility agar plates (A) and Tween 80 plates (B) show the loss of motility and lipase production in flhA mutant strain BTF1 and complementation with plasmid pJE10. Plates were incubated at 28°C for 24 h. (C) The secretion of lipase activity was confirmed by examining concentrated culture supernatants by means of a radial diffusion assay utilizing Tween 80 as a substrate (27). The development of a precipitate diffusing from the sample well indicates the presence of lipase activity. Concentrated supernatants were prepared from cells grown for 24 h at 28°C in T-medium.

FIG. 2.

Schematic map of the flhBAE-flgNMABC gene cluster identified by transposon-directed cloning of mutant BTF1 and the corresponding regions in related Yersinia species. The point of transposon insertion in BTF1 is indicated with a lollipop symbol. The IS insertion in the invasion gene of Y. pestis Kim is also indicated.

FIG. 3.

Genetic complementation of mutant strain BTF1 (flhA::Tn5). (A) Motility and lipase phenotypes for flhAE clones used in the complementation analysis. Clones were generated by PCR cloning into the pBAD vector and verified by DNA sequencing. (B) Western sodium dodecyl sulfate-polyacrylamide gel electrophoresis detection of flagellin from whole-cell extracts. The following strains were analyzed: lane 1, CSF07-82 (wild type); lane 2, BTF1 (flhA::Tn5); lane 3, BTF1/pJE08 (flhA::Tn5/flhA clone); lane 4, BTF1/pJE09 (flhA::Tn5/flhE clone); lane 5, BTF1/pJE10 (flhA::Tn5/flhAE clone); lane 6, blank; lane 7, E. coli.

FIG. 4.

Survival curves for rainbow trout challenged by immersion exposure to CSF07-82 (wild type; closed circles), BTF1 (flhA::Tn5; open circles), and BTF1/pJE10 (flhA::Tn5/flhAE clone; closed diamonds). The results from two independent experiments are presented. Kaplan-Meier survival analysis indicated no significant effect of flhA::Tn5 insertion on survival after challenge.