Transposon-induced non-motile mutants of Vibrio cholerae

Abstract

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or TnlOptac mini-kan in bacteriophage 1. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 TnlOptuc mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 TnlOptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of nonmotile mutants with antibody to wild-type flagellar core protein indicated that two of the nonmotile mutants made flagellar core protein. Three additional mutants redc%ed weakly with the antibodies. However, thee mutants with immunopositive reactions did not produce any structures which resembled flagella by tranhissbm electron microscopy. In addition, none of the other nonmotile mutants produced wild-type flagella.-However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest thait sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.

Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri

“…1) (15), the Mu dI 1681 appeared to insert randomly into the genome of V fischeri. This conclusion was further supported by the fact that nonmotile mutants were isolated at a frequency of about 1% among the transconjugants, a proportion similar to the 0.5% obtained in a random mutagenesis of V. cholerae (31). A complicating factor in these analyses was that at the selection temperature of 15°C, the donor plasmid was maintained in 94% of the V fischeri recipients (15).…”

A model symbiosis reveals a role for sheathed-flagellum rotation in the release of immunogenic lipopolysaccharide

“…However, no effect of flagellar motility on LPS presentation has been reported previously. We posited that, as sheath production and flagellar biogenesis may not be tightly coordinated (Richardson et al, 1990; Josenhans et al, 1995; Ferooz and Letesson, 2010), a sheathed flagellum might shed LPS due to a rotation-weakened association of the sheath with the spinning flagellar filament. This hypothesis is supported by negative-stain transmission electron microscopy (TEM) of sheathed flagella, which has revealed vesicle-like structures (Figure 2A), often at the distal tip of the flagellum, and ∼10–80 nm in diameter (Geis et al, 1993; Millikan and Ruby, 2004; Ferooz and Letesson, 2010).…”

The G‐protein FlhF has a role in polar flagellar placement and general stress response induction in Pseudomonas putida

“…To our knowledge, only one previous study impinged on the genetic basis of flagellar placement. Richardson et al (1990) isolated a mutant of Vibrio cholerae, which, like P. putida, is polarly flagellated, that possessed lateral appendages. But these appendages lacked the flagellar core and represented merely the membranous sheaths.…”