The model squid-vibrio symbiosis provides a window into the impact of strain- and species-level differences during the initial stages of symbiont engagement

Abstract

Among horizontally acquired symbioses, the mechanisms underlying microbial strain- and species-level specificity remain poorly understood. Here, confocal-microscopy analyses and genetic manipulation of the squid-vibrio association revealed quantitative differences in a symbiont's capacity to interact with the host during initial engagement. Specifically, dominant strains of Vibrio fischeri, 'D-type', previously named for their dominant, single-strain colonization of the squid's bioluminescent organ, were compared with 'S-type', or 'sharing', strains, which can co-colonize the organ. These D-type strains typically: (i) formed aggregations of 100s-1000s of cells on the light-organ surface, up to 3 orders of magnitude larger than those of S-type strains; (ii) showed dominance in co-aggregation experiments, independent of inoculum size or strain proportion; (iii) perturbed larger areas of the organ's ciliated surface; and, (iv) appeared at the pore of the organ approximately 4×s more quickly than S-type strains. At least in part, genes responsible for biofilm synthesis control the hyperaggregation phenotype of a D-type strain. Other marine vibrios produced relatively small aggregations, while an array of marine Gram-positive and -negative species outside of the Vibrionaceae did not attach to the organ's surface. These studies provide insight into the impact of strain variation on early events leading to establishment of an environmentally acquired symbiosis.

Fig. 1.

The onset of the squid-vibrio symbiosis. (A) A newly hatched Hawaiian bobtail squid, Euprymna scolopes, with its bioluminescent light organ (black square) in the center of the body cavity; scale bar = 500 μm. (B) An illustration of the external (left side) and internal (right side) features of the nascent organ, as viewed ventrally: aa, anterior appendage; cr, crypts; p, pores; pa, posterior appendage. (C) Events in the recruitment of the specific symbiont, Vibrio fischeri, into the organ’s crypts, depicting the stages at which selectivity occurs. The gray shading (right) shows the internal portions of the organ, regions specific to V. fischeri. PGN, peptidoglycan released from bacteria in the ambient seawater.

Fig. 2.

Differences in aggregation behavior between strains of V. fischeri. (A) Two pairs of high and low magnification confocal-microscopy images illustrating the difference in the extent of bacterial aggregation on the surface of the squid’s light organ. Images captured at 3 h post-inoculation with GFP-expressing cells of either the V. fischeri D-type strain MB13B2 (left) or S-type strain ES114 (right); aa, anterior appendage; p, pores. Red, Cell Tracker Orange; blue, WGA (wheat-germ agglutinin) Alexa 633. (B) D-type V. fischeri strains generally had higher maxima as well as a large variation in average aggregation size; Kruskal-Wallis Test: Chi²=192, df=7, p<0.001 (for N values, see Table 1); the letters above the columns signify that those values are statistically significantly different from columns with another letter; bold horizontal lines represent medians; boxes comprise the interquartile ranges; bars indicate minimum and maximum values. (C) The size of aggregates produced by MB13B2 and ES114 cells was independent of the inoculum concentration; left panel, 10³ CFU/ml, Mann-Whitney-U=7, Z=−3.13, p=0.002 (**); middle panel, 10⁴ CFU/ml, Mann-Whitney-U=20, Z=−3.24, p=0.001 (***); right panel, 10⁵ CFU/ml, Mann-Whitney-U=10, Z=−3.56, p<0.001 (***); N >5 for all treatments. (D) The relative aggregation effectiveness of MB13B2 and ES114 cells was independent of the co-inoculum ratio; left panel, ratio 1:1 = 10³ CFU/ml each strain, Wilcoxon-signed-ranks test: Z=−4.11, n=22, p<0.001 (***); right panel, ratio 1:100 = 10³ CFU/ml MB13B2 and 10⁵ CFU/ml ES114: Wilcoxon-signed-ranks test: Z=−3.41, n=15, p=0.001 (***). Shaded boxes = D-type strains

Fig. 3.

Association of V. fischeri cells with cilia on the surface of the light organ. (A, B) TEM images of cells of V. fischeri strain MB13B2, demonstrating structurally altered cilia in areas where bacteria directly interact with the ciliated surface of the light organ. (A) Upper panel: low magnification, showing the relationship of aggregating V. fischeri cells to the host’s ciliated epithelium; b, bacteria; c, structurally altered cilia. Lower panel: boxed area (rotated 90˚ clockwise) at higher magnification; altered cilia with swollen, paddle-shaped tips (green arrows) in association with V. fischeri cells. (B) High-magnification image, showing typical features of distorted cilia tip. (C-E) Confocal images at increasing magnification reveal host-cilia interactions with MB13B2 cells. (C) Low magnification, showing whole light organ. (D) Three-fold higher magnification of boxed area in C, showing aggregating bacteria. Yellow area indicates the co-localization of cilia (green) and V. fischeri cells (red). (E) Five-fold higher magnification than D, with only the green laser channel active to show the structurally altered cilia (green threads with terminal paddles) that form in areas of bacterial association (white arrows). The upper sector of this image shows structurally unaltered cilia for comparison (F) High-magnification confocal image of the ciliated surface of an aposymbiotic animal, where only an occasional cilium has an altered tip (inset, lower left); aa, anterior appendage; pa, posterior appendage; p, pore. Green,Tubulin Tracker; blue, WGA (wheat-germ agglutinin) Alexa 633.

Fig. 4.

Role of bacterial biofilm-formation genes in a hyperaggregating strain. Left panel: Genetic manipulation of strain MB13B2 by deletion of the gene encoding RscS, a positive regulator of Syp-biofilm formation, only partially reduces the level of bacterial aggregation as compared to its wild-type parent. In contrast, a deletion of an essential Syp structural gene (sypQ) almost completely eliminated the aggregating phenotype of MB13B2 (Kruskal-Wallis Test: Chi²=20.6, df=3, p<0.001). The defect could be complemented by carriage of a wild-type copy of sypQ, in trans, on pMKF15 (Table S2), but not the vector (pKV282) alone. The letters above the columns signify that those values are statistically significantly different from columns with another letter; bold horizontal lines represent medians, boxes in this plot comprise the interquartile ranges (IQR); bars indicate 1.5 IQR of the data shown. The dots represent outliers. Right panel: confocal images showing the aggregation of mutants compared to the wild-type parent, V. fischeri strain MB13B2. aa, anterior appendage; pa, posterior appendage; p, pores. Red, Cell Tracker Orange; blue, WGA (wheat-germ agglutinin) Alexa 633.

Fig. 5.

In vitro biofilm phenotypes. (A) Development of a wrinkled-colony morphology over time. Ten microliters of a culture of each of three V. fischeri strains were spotted onto LBS agar plates. After 72 h of growth at 24 °C, colonies were disrupted with a toothpick to assess colony cohesiveness, which is an indicator of Syp-encoded polysaccharide production (Yip et al., 2005; Yip et al., 2006). (B) Pellicle formation in static liquid culture. Five strains of V. fischeri [(i) ES114, (ii) ES114 rscS** (KV4366), (iii) MB13B2, (iv) MB13B2 ΔsypQ (KV8195), and (v) MB13B2 ΔrscS (CBNR107)] were inoculated into 5 ml of LBS broth, and grown to log phase under shaking conditions. Cultures were then incubated statically at 24 °C for 10 days, and assessed for the presence of a surface pellicle.

Fig. 6.

Aggregation behavior of environmental bacteria. (A) Sizes of aggregates of bacterial species that have the ability to form aggregations on the light organ surface at inocula of 10⁵ CFU/ml (Kruskal-Wallis Test: Chi2=18.623, df=6, p=0.005); bold horizontal lines represent medians; boxes comprise the interquartile ranges; bars indicate the 1.5IQR of the data shown; dots represent outliers; asterisks indicate a significant difference in comparison to the control, S-type strain V. fischeri ES114 (V. anguillarum: Mann-Whitney-U=10.0, Wilcoxon W=20.0, Z=−2.324, p=0.018; V. nigripulchritudo: Mann-Whitney-U=9.0, Wilcoxon W=19.0, Z=−2.402, p=0.013). (B) Laser scanning-confocal micrographs show differences between the aggregation on the light-organ surface of V. fischeri and other environmental bacteria; aa, anterior appendage; pa, posterior appendage; p, pores. Environmental phylotypes (Table S1): V. camp, Vibrio campbellii; V. ang, Vibrio anguillarum; V. nigri, V. nigripulchritudo; P. leiogn, Photobacterium leiognathi. Red, Cell Tracker Orange; blue, WGA (wheat-germ agglutinin) Alexa 633.