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. 2003 Dec;185(24):7202-12.
doi: 10.1128/JB.185.24.7202-7212.2003.

GacA regulates symbiotic colonization traits of Vibrio fischeri and facilitates a beneficial association with an animal host

Cheryl A Whistler  1 , Edward G Ruby
Affiliations

GacA regulates symbiotic colonization traits of Vibrio fischeri and facilitates a beneficial association with an animal host

Cheryl A Whistler et al. J Bacteriol. 2003 Dec.

Abstract

The GacS/GacA two-component system regulates the expression of bacterial traits during host association. Although the importance of GacS/GacA as a regulator of virulence is well established, its role in benign associations is not clear, as mutations in either the gacS or gacA gene have little impact on the success of colonization in nonpathogenic associations studied thus far. Using as a model the symbiotic association of the bioluminescent marine bacterium Vibrio fischeri with its animal host, the Hawaiian bobtail squid, Euprymna scolopes, we investigated the role of GacA in this beneficial animal-microbe interaction. When grown in culture, gacA mutants were defective in several traits important for symbiosis, including luminescence, growth in defined media, growth yield, siderophore activity, and motility. However, gacA mutants were not deficient in production of acylated homoserine lactone signals or catalase activity. The ability of the gacA mutants to initiate squid colonization was impaired but not abolished, and they reached lower-than-wild-type population densities within the host light organ. In contrast to their dark phenotype in culture, gacA mutants that reached population densities above the luminescence detection limit had normal levels of luminescence per bacterial cell in squid light organs, indicating that GacA is not required for light production within the host. The gacA mutants were impaired at competitive colonization and could only successfully cocolonize squid light organs when present in the seawater at higher inoculum densities than wild-type bacteria. Although severely impaired during colonization initiation, gacA mutants were not displaced by the wild-type strain in light organs that were colonized with both strains. This study establishes the role of GacA as a regulator of a beneficial animal-microbe association and indicates that GacA regulates utilization of growth substrates as well as other colonization traits.

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Figures

FIG. 1.
FIG. 1.
Growth of wild-type V. fischeri and derivatives in culture. The optical densities (OD600) of wild-type (▪), luxI (▴), and ΔgacA (•) cultures grown in SWT were determined throughout the growth cycle. One representative experiment is presented.
FIG. 2.
FIG. 2.
Growth yield of wild-type V. fischeri (black bars) and the ΔgacA mutant (gray bars) in various diluted and amended complex media (SWT) after 18 h of incubation. Conditioned broth was prepared by mixing SWT with cell-free supernatants of either the wild type or the ΔgacA mutant at a ratio of 1:1, or by combining SWT with cell-free supernatants of both the wild type and the ΔgacA mutant at a ratio of 2:1:1. Bars indicate the SE.
FIG. 3.
FIG. 3.
Luminescence of wild-type V. fischeri and derivatives in culture. The luminescence of wild-type (▪), luxI (▴), and ΔgacA (•) cultures grown in SWT was determined throughout the growth cycle. One representative experiment is presented. Bars indicate the SE and are sometimes obscured by the symbols.
FIG. 4.
FIG. 4.
Effect of medium viscosity on the motility of wild-type V. fischeri and derivatives. The extent of movement of duplicate samples of wild-type (gray bars), ΔgacA (black bars), gacA::EZ::TN<KAN> (white bars), and hyperswimmer strain DM66 (hatched bars) cells was measured over time. Average values (± 1 standard deviation) were normalized to the wild-type rates at each viscosity. The absence of error bars indicates no variability within treatment.
FIG. 5.
FIG. 5.
Patterns of chemotaxis in soft agar by hyperswimmer derivatives of V. fischeri. The relative migration rates towards serine (inner ring) or nucleosides (outer ring) are indicated by the ring diameters. Shown are the patterns of strain DM66 (A and C), which is the same as the wild-type pattern, and the ΔgacA mutant (B and D) in medium without (A and B) or with (C and D) the addition of 1.6 mM serine.
FIG. 6.
FIG. 6.
Colonization levels of wild-type V. fischeri and its ΔgacA derivative. The mean number (± SE) of symbiotic bacteria per colonized squid for each treatment was determined by plating light organ contents at different times following colonization with the wild type (gray bars) or the ΔgacA mutant (black bars). The colonization levels of the subsets of ΔgacA mutant-colonized animals that were either detectably luminous (white bars) or not (hatched bars) are also plotted separately. The dashed line represents the average CFU at the luminescence detection limit for wild-type-colonized squid. The mean CFU level in nonluminous wild-type-colonized squid was below this detection limit.
FIG. 7.
FIG. 7.
Colonization of squid by wild-type V. fischeri and its ΔgacA derivative in mixed bacterial inoculations. (A and B) The proportion of wild-type-colonized (light gray), ΔgacA mutant-colonized (black), cocolonized (dark gray), or uncolonized (white) squid after inoculation with either 6,000 CFU of each strain by itself or 12,000 CFU of both strains combined at a 1:1 ratio (wild type/ΔgacA mutant) (n = 20 for each treatment) (A), or with a total of 110 CFU of the wild type by itself (n = 20), 2,500 CFU of the ΔgacA mutant by itself (n = 20), or 2,600 CFU of both strains combined at a 1:23 ratio (wild type/ΔgacA) (n = 70) (B). (C) The mean number (± SE) of wild-type (light gray) and ΔgacA (black) CFU per light organ at 48 h postinoculation is shown from the three subgroups that resulted from the combined treatment presented in panel B (n = 46). For wild-type-colonized or cocolonized squid a minimum of 200 colonies were identified, but for ΔgacA mutant-colonized squid frequently fewer than 100 bacterial colonies were available for assessment due to this mutant's lower colonization level.
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