Using Colonization Assays and Comparative Genomics To Discover Symbiosis Behaviors and Factors in Vibrio fischeri

Abstract

The luminous marine Gram-negative bacterium Vibrio (Aliivibrio) fischeri is the natural light organ symbiont of several squid species, including the Hawaiian bobtail squid, Euprymna scolopes, and the Japanese bobtail squid, Euprymna morsei Work with E. scolopes has shown how the bacteria establish their niche in the light organ of the newly hatched host. Two types of V. fischeri strains have been distinguished based upon their behavior in cocolonization competition assays in juvenile E. scolopes, i.e., (i) niche-sharing or (ii) niche-dominant behavior. This study aimed to determine whether these behaviors are observed with other V. fischeri strains or whether they are specific to those isolated from E. scolopes light organs. Cocolonization competition assays between V. fischeri strains isolated from the congeneric squid E. morsei or from other marine animals revealed the same sharing or dominant behaviors. In addition, whole-genome sequencing of these strains showed that the dominant behavior is polyphyletic and not associated with the presence or absence of a single gene or genes. Comparative genomics of 44 squid light organ isolates from around the globe led to the identification of symbiosis-specific candidates in the genomes of these strains. Colonization assays using genetic derivatives with deletions of these candidates established the importance of two such genes in colonization. This study has allowed us to expand the concept of distinct colonization behaviors to strains isolated from a number of squid and fish hosts.IMPORTANCE There is an increasing recognition of the importance of strain differences in the ecology of a symbiotic bacterial species and, in particular, how these differences underlie crucial interactions with their host. Nevertheless, little is known about the genetic bases for these differences, how they manifest themselves in specific behaviors, and their distribution among symbionts of different host species. In this study, we sequenced the genomes of Vibrio fischeri isolated from the tissues of squids and fishes and applied comparative genomics approaches to look for patterns between symbiont lineages and host colonization behavior. In addition, we identified the only two genes that were exclusively present in all V. fischeri strains isolated from the light organs of sepiolid squid species. Mutational studies of these genes indicated that they both played a role in colonization of the squid light organ, emphasizing the value of applying a comparative genomics approach in the study of symbioses.

Keywords: Aliivibrio; Vibrio (Aliivibrio) fischeri; Vibrio fischeri; dominance; genome analysis; genomes; intraspecific; symbiosis.

FIG 1

Locations and species from which Vibrio fischeri strains involved in this study were isolated. The number of isolates from each host species is shown in parentheses in the figure. Twenty-nine of these strains (the E. morsei, E. tasmanica, L. pealei, and C. multispinulosus isolates) were sequenced in this study.

FIG 2

State of colonization of juvenile E. scolopes squid coinoculated with two strains. Squid were inoculated for 3 h with strain ES114 and a second strain of V. fischeri (“Strain 2”; strains listed across the x axis), and the percentages of squid that were cocolonized, colonized only by strain ES114, or colonized only by strain 2 were determined after 24 h. Each bar represents data collected from between 26 and 34 animals, analyzed in three replicates. The graph indicates the mean percentages for each result, and the error bars give the 95% confidence intervals. Strains were isolated from the light organs of the fish C. multispinulosus (CM), the squid accessory nidamental gland of L. pealei (LP), or the squid light organs of E. morsei (EM), E. scolopes (ES) (VLS2), E. tasmanica (ET), or S. robusta (SR).

FIG 3

Colonization success of squid that were inoculated with a single V. fischeri strain. Squids were inoculated for 3 h with an individual strain from the 14 strains outcompeted by strain ES114 in Fig. 2 (right). Each bar represents the percentage of colonized squid based on their bioluminescence at 24 h postinoculation. For each condition, between 30 and 41 animals were analyzed in three replicates. The graph indicates the mean percentages for each result, and the error bars give the 95% confidence intervals.

FIG 4

State of colonization of coinoculated juvenile E. scolopes squid. Squids were inoculated for 3 h with both the dominant (D) strain MB13B2 and a second strain (“Strain 2”; listed across the x axis) that were the 9 most dominant strains in Fig. 2 (middle). The percentages of squid that were cocolonized , colonized only by strain MB13B2 or only by Strain 2 were determined after 24 h. For each condition, between 30 and 32 animals were analyzed in three replicates. The graph indicates the mean percentages for each result, and the error bars give the 95% confidence intervals.

FIG 5

Colonization success of squid that were inoculated with a single V. fischeri strain for 30 min and rinsed three times. The V. fischeri strains are shown on the x axis. Each bar represents the percentage of colonized squid, based on their bioluminescence at 24 h postinoculation. Between 29 and 30 animals were analyzed for each of the strains in three replicates. The graph indicates the mean percentages for each result, and the error bars give the 95% confidence intervals.

FIG 6

Phylogenetic relationships between the V. fischeri strains reported either in this study or in the study of Bongrand et al. (9), based on core genes from whole-genome sequences. Shewanella (Shewanella oneidensis) served as the outgroup. The scale bar indicates an evolutionary distance of 2 nucleotide substitutions per position in the sequence. Node numbers indicate the bootstrap support values. Most of the symbionts carry the initials of their host in their name. Because designations of E. scolopes symbionts use several different initials, we added a black circle to help identify them. The color code refers to the colonization effectiveness of the strains. The strains were either D strains (red), S strains (blue), outcompeted by strain ES114 (green), or ineffective colonizers of E. scolopes juveniles (gray).

FIG 7

Relative competitive index of pairs of strains coincubated with juvenile squid for 3 h. The relative competitive index (RCI) was calculated as the ratio of the two strains in the light organ after 24 h, divided by their ratio in the inoculum. Those RCIs that are significantly different from zero by t test are indicated by red asterisks (*, P < 0.05; ****, P < 0.0001). The two strains competed were the parent ES114 strain and either a derived deletion mutant (Δ) or its genetic complement (+). Each dot represents the log RCI determined for an individual squid. For each condition, a total of between 26 and 29 animals were analyzed in three replicates. The error bars indicate the standard errors of the means (SEM).