High-frequency spontaneous mutation of classical Vibrio cholerae to a nonmotile phenotype

Abstract

The species Vibrio cholerae contains within it two biotypes, classical and El Tor, both of which are motile. Phenotypic expression of motility was unaffected by type of growth medium, salt concentration, pH, or temperature of incubation. However, seven strains of classical V. cholerae produced spontaneous nonmotile mutants at an unusually high frequency (ca. 10-4), while no mutants were detected for all three El Tor strains examined. No revertants of these nonmotile mutants were detected. Four independent mutants of classical strain 395 were isolated to characterize this phenomenon. By transmission electron microscopy, one of the nonmotile mutants was found to be flagellated, while the other three were found to be aflagellate. Chromosomal DNA from the mutants and parental wild-type strain 395 was exatnined by Southern blot analysis with, as probes, V. cholerae mutagenic prophages VcA-1 and VcA-2 and six cloned motility gene regions isolated from transposon insertion motility mutants of strains 395 and N16961 (El Tor, Inaba). The parental wild-type strain and all of the mutants exhibited the same pattern of bands when probed with VcA-1 and VcA-2 DNAs. Four of the cloned motility gene regions hybridized to the same fragments of DNA in both the wild-type and mutant isolates. However, two other probes detected a new fragment for a single aflagellate mutant. The observations that spontaneous nonmotile mutants occurred at a high frequency and that these mutants did not revert at a detectable frequency suggested that a genetic event is involved. The phenomenon appears to be limited to classical V. cholerae and may explain why classical V. cholerae is only sporadically associated with disease in the current pandemic.

Roles of motility and flagellar structure in pathogenicity of Vibrio cholerae: analysis of motility mutants in three animal models

“…395 is a classical biotype strain known to spontaneously mutate to the nonmotile phenotype at a high frequency in vitro (25). We examined in soft agar the motility of 17 to 24 colonies obtained from the plates used to determine CFU associated with the mucosa and in the loop fluid.…”

Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression

“…Two flagellated Tn5-lac-induced mutants, NM CG996 and NM CG997, were included in our analyses, and sequence analysis of chromosomal DNA adjacent to one of these (NM CG996) revealed it to have an insertion in a gene homologous to the motB locus of E. coli as discussed below. NM mutants have been reported to occur spontaneously, and we too have observed the appearance of NM mutants of derivatives of strain O395 occurring at a frequency of approximately 1 ϫ 10 Ϫ4 (46). Two such spontaneous NM strains selected for our analysis included NM39, a nonflagellated strain, and NM51, a flagellated strain.…”

Cholera

“…(Classical V. cholerae strains undergo a high frequency [ca. 10 Ϫ4 ] of spontaneous mutation to a nonmotile form [303].) The results varied between biotypes and among animal models, but in general it was concluded that motility is clearly a major contributing factor to V. cholerae pathogenicity and colonization while the flagellar structure alone seemed to be less important (365).…”