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1. Persistent symbiont colonization leads to a maturation of hemocyte response in the Euprymna scolopes / Vibrio fischeri symbiosis

   https://doi.org/10.1002/mbo3.858  

   Rader, Bethany ; McAnulty, Sarah J. ; Nyholm, Spencer V. ( June 2019 , MicrobiologyOpen)

   The binary association between the squid,Euprymna scolopes, and its symbiont,Vibrio fischeri, serves as a model system to study interactions between beneficial bacteria and the innate immune system. Previous research demonstrated that binding of the squid's immune cells, hemocytes, toV. fischeriis altered if the symbiont is removed from the light organ, suggesting that host colonization alters hemocyte recognition ofV. fischeri. To investigate the influence of symbiosis on immune maturation during development, we characterized hemocyte binding and phagocytosis ofV. fischeriand nonsymbioticVibrio harveyifrom symbiotic (sym) and aposymbiotic (apo) juveniles, and wild‐caught and laboratory‐raised sym and apo adults. Our results demonstrate that while light organ colonization byV. fischeridid not alter juvenile hemocyte response, these cells bound a similar number ofV. fischeriandV. harveyiyet phagocytosed onlyV. harveyi. Our results also indicate that long‐term colonization altered the adult hemocyte response toV. fischeribut notV. harveyi. All hemocytes from adult squid, regardless of apo or sym state, both bound and phagocytosed a similar number ofV. harveyiwhile hemocytes from both wild‐caught and sym‐raised adults bound significantly fewerV. fischeri, although moreV. fischeriwere phagocytosed by hemocytes from wild‐caught animals. In contrast, hemocytes from apo‐raised squid bound similar numbers of bothV. fischeriandV. harveyi, although moreV. harveyicells were engulfed, suggesting that blood cells from apo‐raised adults behaved similarly to juvenile hosts. Taken together, these data suggest that persistent colonization by the light organ symbiont is required for hemocytes to differentially bind and phagocytoseV. fischeri. The cellular immune system ofE. scolopeslikely possesses multiple mechanisms at different developmental stages to promote a specific and life‐long interaction with the symbiont.

    

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2. Should they stay or should they go? Nitric oxide and the clash of regulators governing Vibrio fischeri biofilm formation

   https://doi.org/10.1111/mmi.14163  

   Stabb, Eric V. ( December 2018 , Molecular Microbiology)

   A key regulatory decision for many bacteria is the switch between biofilm formation and motile dispersal, and this dynamic is well illustrated in the light‐organ symbiosis between the bioluminescent bacteriumVibrio fischeriand the Hawaiian bobtail squid. Biofilm formation mediated by thesypgene cluster helpsV. fischeritransition from a dispersed planktonic lifestyle to a robust aggregate on the surface of the nascent symbiotic organ. However, the bacteria must then swim to pores and down into the deeper crypt tissues that they ultimately colonize. A number of positive and negative regulators controlsypexpression and biofilm formation, but until recently the environmental inputs controlling this clash between opposing regulatory mechanisms have been unclear. Thompsonet al. have now shown that Syp‐mediated biofilms can be repressed by a well‐known host‐derived molecule: nitric oxide. This regulation is accomplished by the NO sensor HnoX exerting control over the biofilm regulator HahK. The discoveries reported here by Thompsonet al. cast new light on a critical early stage of symbiotic initiation in theV. fischeri‐squid model symbiosis, and more broadly it adds to a growing understanding of the role(s) that NO and HnoX play in biofilm regulation by many bacteria.

    

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3. Nitric oxide inhibits biofilm formation by Vibrio fischeri via the nitric oxide sensor HnoX

   https://doi.org/10.1111/mmi.14147  

   Thompson, Cecilia M. ; Tischler, Alice H. ; Tarnowski, Denise A. ; Mandel, Mark J. ; Visick, Karen L. ( November 2018 , Molecular Microbiology)

   Nitric oxide (NO) is an important defense molecule secreted by the squidEuprymna scolopesand sensed by the bacterial symbiont,Vibrio fischeri, via the NO sensor HnoX. HnoX inhibits colonization through an unknown mechanism. The genomic location ofhnoXadjacent tohahK, a recently identified positive regulator of biofilm formation, suggested that HnoX may inhibit colonization by controlling biofilm formation, a key early step in colonization. Indeed, the deletion ofhnoXresulted in early biofilm formationin vitro, an effect that was dependent on HahK and its putative phosphotransfer residues. An allele ofhnoXthat encodes a protein with increased activity severely delayed wrinkled colony formation. Control occurred at the level of transcription of thesypgenes, which produce the polysaccharide matrix component. The addition of NO abrogated biofilm formation and diminishedsyptranscription, effects that required HnoX. Finally, anhnoXmutant formed larger symbiotic biofilms. This work has thus uncovered a host‐relevant signal controlling biofilm and a mechanism for the inhibition of biofilm formation byV. fischeri. The study ofV. fischeriHnoX permits us to understand not only host‐associated biofilm mechanisms, but also the function of HnoX domain proteins as regulators of important bacterial processes.

    

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4. Genetic Manipulation of Vibrio fischeri

   https://doi.org/10.1002/cpmc.115  

   Christensen, David G. ; Tepavčević, Jovanka ; Visick, Karen L. ( September 2020 , Current Protocols in Microbiology)

   Vibrio fischeriis a nonpathogenic organism related to pathogenicVibriospecies. The bacterium has been used as a model organism to study symbiosis in the context of its association with its host, the Hawaiian bobtail squidEuprymna scolopes. The genetic tractability of this bacterium has facilitated the mapping of pathways that mediate interactions between these organisms. The protocols included here describe methods for genetic manipulation ofV. fischeri. Following these protocols, the researcher will be able to introduce linear DNA via transformation to make chromosomal mutations, to introduce plasmid DNA via conjugation and subsequently eliminate unstable plasmids, to eliminate antibiotic resistance cassettes from the chromosome, and to randomly or specifically mutagenizeV. fischeriwith transposons. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Transformation ofV. fischeriwith linear DNA

   Basic Protocol 2: Plasmid transfer intoV. fischerivia conjugation

   Support Protocol 1: Removing FRT‐flanked antibiotic resistance cassettes from theV. fischerigenome

   Support Protocol 2: Eliminating unstable plasmids fromV. fischeri

   Alternate Protocol 1: Introduction of exogenous DNA using a suicide plasmid

   Alternate Protocol 2: Site‐specific transposon insertion using a suicide plasmid

   Alternate Protocol 3: Random transposon mutagenesis using a suicide plasmid

    

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5. Interactions of Symbiotic Partners Drive the Development of a Complex Biogeography in the Squid-Vibrio Symbiosis

   https://doi.org/10.1128/mBio.00853-20  

   Essock-Burns, Tara ; Bongrand, Clotilde ; Goldman, William E. ; Ruby, Edward G. ; McFall-Ngai, Margaret J. ; Graf, Joerg ( June 2020 , mBio)

   ABSTRACT Microbes live in complex microniches within host tissues, but how symbiotic partners communicate to create such niches during development remains largely unexplored. Using confocal microscopy and symbiont genetics, we characterized the shaping of host microenvironments during light organ colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri . During embryogenesis, three pairs of invaginations form sequentially on the organ’s surface, producing pores that lead to interior compressed tubules at different stages of development. After hatching, these areas expand, allowing V. fischeri cells to enter and migrate ∼120 μm through three anatomically distinct regions before reaching blind-ended crypt spaces. A dynamic gatekeeper, or bottleneck, connects these crypts with the migration path. Once V. fischeri cells have entered the crypts, the bottlenecks narrow, and colonization by the symbiont population becomes spatially restricted. The actual timing of constriction and restriction varies with crypt maturity and with different V. fischeri strains. Subsequently, starting with the first dawn following colonization, the bottleneck controls a lifelong cycle of dawn-triggered expulsions of most of the symbionts into the environment and a subsequent regrowth in the crypts. Unlike other developmental phenotypes, bottleneck constriction is not induced by known microbe-associated molecular patterns (MAMPs) or by V. fischeri - produced bioluminescence, but it does require metabolically active symbionts. Further, while symbionts in the most mature crypts have a higher proportion of live cells and a greater likelihood of expulsion at dawn, they have a lower resistance to antibiotics. The overall dynamics of these distinct microenvironments reflect the complexity of the host-symbiont dialogue. IMPORTANCE The complexity, inaccessibility, and time scales of initial colonization of most animal microbiomes present challenges for the characterization of how the bacterial symbionts influence the form and function of tissues in the minutes to hours following the initial interaction of the partners. Here, we use the naturally occurring binary squid-vibrio association to explore this phenomenon. Imaging of the spatiotemporal landscape of this symbiosis during its onset provides a window into the impact of differences in both host-tissue maturation and symbiont strain phenotypes on the establishment of a dynamically stable symbiotic system. These data provide evidence that the symbionts shape the host-tissue landscape and that tissue maturation impacts the influence of strain-level differences on the daily rhythms of the symbiosis, the competitiveness for colonization, and antibiotic sensitivity. 

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