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

Clotilde Bongrand,Philip Arevalo,Margaret Mcfall-Ngai,Silvia Moriano-Gutierrez,Edward G Ruby,Karen L Visick,Martin Polz,Joerg Graf

doi:10.1128/mbio.03407-19

Clotilde Bongrand, Philip Arevalo + Show 6 more

Open Access

https://doi.org/10.1128/mbio.03407-19

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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.

Highlights

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
Almost all of the light organ’s symbiont population is expelled from the crypts into the surrounding seawater: the remaining bacteria multiply and repopulate the organ, while those released into the ambient environment serve as an inoculum for juvenile squid that hatch on subsequent nights [6]
One might expect that this colonization advantage would allow D strains to ecologically “sweep” a symbiont population, but several factors work against that outcome. (i) The evolution of V. fischeri is unlikely to be governed solely by symbiotic behavior because only a vanishingly small percentage of the billions of symbionts released into the bacterioplankton each day ever encounter a host again [13]. (ii) The D strains appear to be less fit at surviving in the bacterioplankton than the S strains [7]. (iii) If the newly hatched squid encounters an S strain sufficiently long before a D strain, both strains can establish in the light organ [12]

Summary

Introduction

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. 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. Vibrio (Aliivibrio) fischeri is the light-emitting, bacterial symbiont of several fish and squid species [1] These bacteria become established within the host animal’s light organ and provide bioluminescence in exchange for nutrients. When juvenile E. scolopes hatch, they harvest V. fischeri cells from the surrounding environment using ciliated fields present on surface appendages of the light organ [4] These bacteria pass through pores on the organ’s surface and establish themselves in the deep crypts of the light organ [5, 6]. One might expect that this colonization advantage would allow D strains to ecologically “sweep” a symbiont population, but several factors work against that outcome. (i) The evolution of V. fischeri is unlikely to be governed solely by symbiotic behavior because only a vanishingly small percentage of the billions of symbionts released into the bacterioplankton each day ever encounter a host again [13]. (ii) The D strains appear to be less fit at surviving in the bacterioplankton than the S strains [7]. (iii) If the newly hatched squid encounters an S strain sufficiently long before a D strain, both strains can establish in the light organ [12]

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