Alterations in Vibrio fischeri motility correlate with a delay in symbiosis initiation and are associated with additional symbiotic colonization defects

Abstract

Motility is required for Vibrio fischeri cells to interact with and specifically colonize the light-emitting organ of their host, the squid Euprymna scolopes. To investigate the influence of motility on the expression of the symbiotic phenotype, we isolated mutants of the squid symbiont V. fischeri ES114 that had altered migration abilities. Spontaneous hyperswimmer (HS) mutants, which migrated more rapidly in soft agar and were hyperflagellated relative to the wild type, were isolated and grouped into three phenotypic classes. All of the HS strains tested, regardless of class, were delayed in symbiosis initiation. This result suggested that the hypermotile phenotype alone contributes to an inability to colonize squid normally. Class III HS strains showed the greatest colonization defect: they colonized squid to a level that was only 0.1 to 10% that achieved by ES114. In addition, class III strains were defective in two capabilities, hemagglutination and luminescence, that have been previously described as colonization factors in V. fischeri. Class II and III mutants also share a mucoid colony morphology; however, class II mutants can colonize E. scolopes to a level that was 40% of that achieved by ES114. Thus, the mucoid phenotype alone does not contribute to the greater defect exhibited by class III strains. When squid were exposed to ES114 and any one of the HS mutant strains as a coinoculation, the parent strain dominated the resulting symbiotic light-organ population. To further investigate the colonization defects of the HS strains, we used confocal laser-scanning microscopy to visualize V. fischeri cells in their initial interaction with E. scolopes tissue. Compared to ES114, HS strains from all three classes were delayed in two behaviors involved in colonization: (i) aggregation on host-derived mucus structures and (ii) migration to the crypts. These results suggest that, while motility is required to initiate colonization, the presence of multiple flagella may actually interfere with normal aggregation and attachment behavior. Furthermore, the pleiotropic nature of class III HS strains provides evidence that motility is coregulated with other symbiotic determinants in V. fischeri.

FIG. 1.

Electron micrographs of V. fischeri wild-type strain ES114 and a representative strain from each of the three HS mutant classes: DM66 (class I), DM73 (class II) and DM61 (class III). Cells were grown either in liquid media (A, C, E, and G) or on agar surfaces (B, D, F, and H).

FIG. 2.

Quantitative analysis of flagellin production by V. fischeri wild-type strain ES114 and HS mutant strains. (A) Average number of flagella per cell determined from cultures of ES114 and two representative strains from each of the three classes of HS mutants grown either in liquid media (L) or on a surface (S). At least 50 cells for each strain and condition were assayed by using TEM. Cells of class II strains displayed a flagellar density that often could not be counted but was >10 flagella per cell. (B) SDS-PAGE analysis of flagellin proteins produced by V. fischeri strain ES114 and HS strains. Purified flagellar preparations from 4 × 10⁸ cells were denatured and subjected to PAGE, followed by Coomassie brilliant blue staining. The bracket indicates the location of flagellin subunits.

FIG. 3.

Luminescence of squids infected with either V. fischeri wild-type strain ES114 or HS mutant strains. Luminescence of individual animals was determined approximately every hour over the first 20 h postinoculation. Shown are the mean values for groups of 20 animals for each strain and the respective standard errors. Animals were infected with wild-type strain ES114 (•) or one representative strain from each of the three HS classes, DM66 (class I; ▴), DM73 (class II; ▪), or DM61 (class III; ○). Control animals (□) were maintained in V. fischeri-free SW. Similar results were obtained in three separate experiments. One light unit (LU) = 11 quanta s⁻¹.

FIG. 4.

Symbiotic colonization levels of V. fischeri wild-type strain ES114 and HS mutant strains. (A) Colonization levels achieved by HS class I mutant strain DM66 at 15 and 24 h postinoculation. Similar results were obtained with a second class I mutant, DM69 (data not shown). (B) Colonization levels achieved by HS class II mutant strain DM73 at 24 h postinoculation. (C) Colonization levels achieved by HS class III mutant strains DM61 and DM70 at 24 h (similar results were obtained at 48 h). Each bar represents the mean values obtained with groups of at least 15 animals, and the error bars indicate the standard error of the mean. Similar results were obtained in three separate experiments.

FIG. 5.

Colonization of E. scolopes light organs by mixed inocula of V. fischeri wild-type strain ES114 and an HS mutant strain. Individual juvenile squid were coinoculated with a mixture (the ratio of mutant to wild-type cells was between 1.1 and 1.3) of either DM66 (A), DM73 (B), or DM61 (C) strains and their parent strain, ES114. At 24 h postinoculation, the animals were sacrificed and the numbers of the two strains present in the light organs of 33 (A), 24 (B), or 24 (C) animals were determined. The extent of dominance of one strain over the other was termed the competitive index (CI), which was calculated by dividing the number of mutant cells by the number of ES114 cells present in each animal. Each circle represents the CI of an individual animal after exposure to an inoculum containing mutant and wild-type cells at a ratio of 1.1 to 1.3. Animals with a CI of <1.0 are dominated by the wild-type strain ES114, and those with a CI of >1.0 are dominated by the mutant strain. Circles with arrows are values below the limit of detection (CI < 0.01). Results for each combination of strains are representative of three separate experiments.

FIG. 6.

Aggregation behavior of V. fischeri during colonization of squid. Confocal laser-scanning microscopy was used to compare the behavior of V. fischeri wild-type strain ES114 to HS mutant strains during the initial hours of symbiotic interaction. (A) At between 4 and 6 h postinoculation, animals exposed to wild-type cells were found to contain external aggregates of bacteria. Shown is a typical aggregate comprised of hundreds of GFP-labeled bacterial cells. (B) Animals exposed to the same concentration of HS strains contained smaller bacterial aggregates (tens of cells) that formed at a later time (between 8 and 10 h postinoculation). Shown is a typical aggregate from an animal exposed to GFP-labeled cells of the class III HS strain DM61; however, similar results were obtained for animals exposed to strains of any of the three HS mutant classes. White arrows indicate the location of the light-organ pores. Bacteria were visualized by the fluorescence of GFP (shown as green), and animal tissue (shown in grayscale) was visualized either by differential interference contrast imaging (A) or by fluorescence of CellTracker orange (B). Images are at the same magnification.