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GNPS - Symbiotic squid minimum biomass test NEG

ABSTRACT: This project is in collaboration with the McFall-Ngai and Ruby labs investigating the effects of symbiosis on the metabolome of the Hawaiian bobtail squid Euprymna scolopes. This data was collected from symbiotic (SYM; Vibrio fischeri ES114 colonized) and aposymbiotic (APO; no symbiont exposure) squid, flash frozen 24 hours post hatch. This analysis a test to determine the minimum required biomass to successfully perform untargeted LC-MS/MS on juvenile squid, as well as establish a baseline metabolome for whole squid (both SYM and APO). Untargeted LC-MS/MS acquisition was performed on a Vanquish Flex UHPLC system coupled to an Orbitrap Exploris 240 (Thermo Fisher Scientific, Bremen, Germany). Chromatographic separation was performed on a Kinetex 1.7 um 100 A pore size C18 reversed phase UHPLC column 150 x 2.1 mm (Phenomenex, Torrance, CA). LC-MS/MS data was acquired in negative mode

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Euprymna Scolopes (ncbitaxon:6613)

SUBMITTER: Margareth McFall-Ngai

PROVIDER: MSV000096632 | GNPS | Tue Dec 10 13:29:00 GMT 2024

REPOSITORIES: GNPS

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Project description:The light-organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution. Six experimental treatments of juvenile animals were performed for the microarray matrix: uncolonized (Apo); uncolonized, but supplemented with AI (Apo + AI); colonized by wild-type V. fischeri (wild type); colonized by a mutant defective in luciferase synthesis (luxA); colonized by a mutant defective in AI synthesis (luxI); and, colonized by the luxI mutant, but supplemented with AI (luxI + AI). At 18 h postinoculation, animals were anesthetized in 2% ethanol in HOSW, and the light organs were removed into RNAlater (Ambion Biosystems).

Project description:Beneficial microbial symbionts are often horizontally acquired by their animal hosts from environmental sources, requiring the symbionts to complete a lifestyle transition from free-living in the environment to association with host tissues. In the model symbiosis between the Hawaiian bobtail squid and its microbial symbiont Vibrio fischeri, one mechanism used to make this transition during host colonization is the formation of biofilm-like aggregates on host mucosa. Extensive work has previously been conducted to isolate the critical factors controlling V. fischeri biofilm formation, yet much remains unknown regarding the full breadth of the biofilm-associated regulon. Here, we probed in vitro models of biofilm formation using transcriptomics, to identify novel regulatory pathways active within biofilms of the V. fischeri type strain ES114. Through comparing the gene-sets which became differentially regulated in multiple biofilm models, we discovered a shared set of 232 genes which demonstrated similar patterns in expression relative to uninduced controls. These genes contained representatives of multiple exopolysaccharide loci, genes involved in flagellar motility, and a diverse collection of other genes. Follow-up analysis suggested that these transcriptomic changes reflected true phenotypic effects, including changes in motility and cyclic-di-GMP production in biofilm-induced backgrounds. Beyond characterizing the shared biofilm response, we additionally profiled the regulatory activity of the sensor kinase RscS. This sensor kinase has previously been characterized to function as a phospho-donor within an established biofilm-inducing phospho-relay, yet our data suggests that RscS moonlights in at least one other phospho-relay that integrates downstream signaling from a homolog of the Vibrio cholerae response regulator VpsR, without a need for its established signaling partners. Overall, this study adds to our understanding of the genes involved in V. fischeri biofilm regulation, while revealing new regulatory pathways branching from previously characterized signaling networks.

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GNPS - Symbiotic squid minimum biomass test NEG

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