A Genomic Comparison of 13 Symbiotic Vibrio Fischeri Isolates from the Perspective of Their Host Source and Colonization Behavior
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The ISME Journal (2016) 10, 2907–2917 © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 www.nature.com/ismej ORIGINAL ARTICLE A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior Clotilde Bongrand1,3, Eric J Koch1,3, Silvia Moriano-Gutierrez1,3, Otto X Cordero2, Margaret McFall-Ngai1,3, Martin F Polz2 and Edward G Ruby1,3 1Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA and 2Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA Newly hatched Euprymna scolopes squid obtain their specific light-organ symbionts from an array of Vibrio (Allivibrio) fischeri strains present in their environment. Two genetically distinct populations of this squid species have been identified, one in Kaneohe Bay (KB), and another in Maunaloa Bay (MB), Oahu. We asked whether symbionts isolated from squid in each of these populations outcompete isolates from the other population in mixed-infection experiments. No relationship was found between a strain’s host source (KB or MB) and its ability to competitively colonize KB or MB juveniles in a mixed inoculum. Instead, two colonization behaviors were identified among the 11 KB and MB strains tested: a ‘dominant’ outcome, in which one strain outcompetes the other for colonization, and a ‘sharing’ outcome, in which two strains co-colonize the squid. A genome-level comparison of these and other V. fischeri strains suggested that the core genomic structure of this species is both syntenous and highly conserved over time and geographical distance. We also identified ~ 250 Kb of sequence, encoding 194 dispersed orfs, that was specific to those strains that expressed the dominant colonization behavior. Taken together, the results indicate a link between the genome content of V. fischeri strains and their colonization behavior when initiating a light-organ symbiosis. The ISME Journal (2016) 10, 2907–2917; doi:10.1038/ismej.2016.69; published online 29 April 2016 Introduction Goodrich-Blair, 2010; Sudakaran et al., 2015). In the symbiosis between the squid Euprymna scolopes Understanding the relationships between environ- and the luminous bacterium Vibrio fischeri,asin mental adaptation, population dynamics and geno- other environmentally acquired, non-obligatory sym- mic diversity is a key goal of the field of microbial bionts, one can experimentally expose an aposym- ecology (McInnes et al., 2004; Cordero and Polz, biotic host to genetically distinct V. fischeri strains, 2014). The study of microbial symbioses has pro- either singly or in combination, and monitor the vided a valuable platform from which this goal can establishment and dynamics of the resulting sym- be addressed. For instance, co-evolution or co- biont population. By manipulating this highly divergence of bacteria and their hosts has been specific association, one can address fundamental extensively studied in vertically transmitted associa- questions concerning the role of strategies like host tions, and can involve events like gene sharing and sanctioning in the evolution of persistent symbiotic genome reduction within the microbial partner(s) associations (Koch et al., 2014). (Kikuchi et al., 2009; Moran and Bennett, 2014). V. fischeri is the specific light-organ symbiont of Similarly, horizontally transmitted symbioses of several families of fishes and squids, including the plants and invertebrates have offered the opportu- Hawaiian bobtail squid, E. scolopes. In exchange for nity to examine the evolution of species specificity, nutrients, the bacterium provides counterillumina- revealing evidence for strategies such as congruent tion for the night-active squid by producing biolu- evolution and partner choice (Chaston and minescence that obscures the shadow of the host from its predators (Jones and Nishiguchi, 2004). The Correspondence: EG Ruby, Kewalo Marine Laboratory, University squid hatches without V. fischeri cells, but rapidly of Hawaii-Manoa, 41 Ahui Street, Honolulu, HI 96813, USA. acquires this specific bacterium from the surround- E-mail: [email protected] ing environment (Ruby and Asato, 1993). The 3 Present address: Kewalo Marine Laboratory, University of bacteria enter the nascent light organ through six Hawaii-Manoa, Honolulu, HI, USA. Received 10 December 2015; revised 14 March 2016; accepted surface pores, three on either side of the organ. They 22 March 2016; published online 29 April 2016 then traverse several different tissue environments Genomic and ecological comparison of symbiotic V. fischeri isolates C Bongrand et al 2908 before entering the six epithelium-lined crypts deep medium (10 g Bacto-Tryptone, 5 g yeast extract within the organ, where they establish a lifelong, and 10 g NaCl per liter). When necessary, LB albeit dynamic, symbiosis (Nyholm and McFall- medium was supplemented with kanamycin Ngai, 2004). In nature, mature squids contain at least (50 μgml−1), carbenicillin (100 μgml−1) or chloram- three to five strains (Wollenberg and Ruby, 2009), phenicol (25 μgml− 1). indicating the possibility of mixed-strain coloniza- All of the wild-type strains used in this study have tions occurring in the crypts. Each morning for the been previously described (Ruby et al., 2005; 8–10-month lifetime of the host, the light organ Mandel et al., 2009; Wollenberg and Ruby, 2009) expels 95% of its symbionts (Lee and Ruby, 1994b) (Supplementary Table S1), and derivatives were and, within a few hours, the organ is repopulated by constructed that chromosomally carried genes the growth of the remaining symbionts. encoding either green fluorescent protein (GFP) or Distinct populations of squid occur within two RFP to simplify their differentiation (Supplementary bays on different sides of the Hawaiian Island of Table S2). To construct plasmids pCBNR6 and Oahu (Kimbell et al., 2002): Maunaloa Bay (MB) and pCBNR7, we amplified the segments pLacZ-GFP Kaneohe Bay (KB). Using a DNA-fingerprint techni- and pLacZ-RFP, from pVSV102 and pVSV208, que (VfRep-PCR) to characterize light-organ sym- respectively (Dunn et al., 2006), and inserted them bionts obtained from the two host populations, three into pEVS107 (McCann et al., 2003). We amplified genetically and phenotypically distinct groups of pVSV102 and pVSV208 by PCR using the following V. fischeri were described (Wollenberg and Ruby, primers: (5′-GCATGCCTAGGTTTACACTTTATGCT 2009) that are found associated with hosts from both TCCGGC-3′) and (5′-CGTACGGTACCTCAGTTGTAC geographic locations. Group-A strains (Wollenberg AGTTCATCCATG-3′) for pCBNR6; and (5′-CGTACG and Ruby, 2012) exhibited an advantage when GTACCCTACAGGAACAGGTGGTGG-3′) for pCBNR7. colonizing juvenile squid from the MB population; The primers were synthetized by Integrated DNA and they were less fit than other symbionts when Technologies (Coralville, IA, USA). Subsequent PCR incubated in natural seawater in the absence of the digestion by KpnI-HF and AvrII, ligation and transfor- host, suggesting divergent ecological strategies. mation were all performed according to standard In this study, we performed both functional and methods. We then transferred the fragment containing genomic analyses of 11 symbiotic V. fischeri strains the promoter, fluorescent protein and erythromycin- that are representative of those described in resistance gene (erm) from the plasmid to the Tn7 Wollenberg and Ruby, 2012, and whose genomes insertion site of wild-type strains by tetra-parental we have recently sequenced. In the functional mating (McCann et al., 2003). An additional screening approach, we first asked whether symbionts of squid process was used to check for colony fluorescence from one bay were more effective at colonizing using a Leica MZFLIII fluorescence dissecting scope juveniles from this same bay. We next examined the (Leica Microsystems Inc., Buffalo Grove, IL, USA), relative fitness of these symbionts by determining equipped with a set of G, TXR and GFP2 filters. their behavior during an experimental co-colonization: specifically, when pairs of strains were co-inocu- lated, did one outcompete the other or did a Light-organ colonization assay co-colonization of the light organ result. Finally, we To determine the relative colonization effectiveness of compared the genomes of squid light-organ sym- different strains of light-organ isolates, inocula were bionts, together with the fish light-organ symbiont, prepared that contained either an individual strain or strain MJ11 (Mandel et al., 2009), to ask whether various pairs of competing strains (Supplementary there were genes that were always found in the squid Table S2). The inocula were prepared by growing symbionts, but not in the fish symbiont. each strain overnight in LB Salt medium, before diluting them 1:100 into seawater tryptone medium Materials and methods medium. The bacteria were then grown to mid- exponential phase (OD ~ 0.3) before diluting them in Media and strains filter-sterilized Instant Ocean (FSIO). Newly hatched All V. fischeri strains were grown at 28 °C in either squids were then transferred into the inoculated FSIO (i) seawater tryptone medium containing (per liter) containing either a single strain or, for competition 700 ml of a marine salts solution Instant Ocean assays, a pair of strains, targeting a 1:1 ratio. The pairs (Spectrum Brands, Madison, WI, USA), 300 ml were differentially labeled by their expression of deionized water, 5