Biology subjects

Ruby, E. G.

Publications and source records attributed to Ruby, E. G..

6 recordsLinked to original sources

bioRxiv2025

Nanoscale imaging of native symbiotic animal tissue using amultimodal large volume imaging pipeline for cryo-electrontomography

The field of cryo-EM offers the possibility to gain high-resolution structural information of biomolecules in their native state. Advances in sample thinning of cryo-EM samples allows the study of proteins inside intact cells using tomography, opening the door for visual proteomics. However, thicker samples such as tissues or entire organisms are still largely unsuitable for cryo-electron tomography (cryo-ET). Therefore, significant efforts are directed toward developing and improving preparation methods to enable cryo-ET of such complex samples. We focused on the binary association between the Hawaiian bobtail squid Euprymna scolopes and the luminous bacteria Vibrio fischeri. The Squid-Vibrio system has long been studied to understand host-symbiont interactions. Our goal is to study the bacterial-host interface using cryo-ET, at a resolution previously unattainable by conventional EM methods. Here, we present a multi-modal preparation and correlative imaging workflow--including cryo- fluorescence microscopy, microCT, freeze-substitution electron tomography (FS-ET), and serial blockface SEM--to localize and prepare specific regions of the dissected symbiotic light organs for cryo-ET. This approach enabled us to directly visualize symbiotic V. fischeri within the internal host crypts at macromolecular resolution, revealing spatial organization, physical contact, and putative exchange interfaces between host and microbe. Our findings provide structural insights into a foundational model of host-microbe symbiosis and demonstrate the feasibility of cryo-ET for investigating intact tissues at the nanoscale.

microbiology↗

bioRxiv2024

Developmental and transcriptomic responses of Hawaiian bobtail squid early stages to ocean warming and acidification

1Cephalopods play a central ecological role across all oceans and realms. However, under the current climate crisis, their physiology and behaviour are impacted, and we are beginning to comprehend the effects of environmental stressors at a molecular level. Here, we study the Hawaiian bobtail squid (Euprymna scolopes), known for its specific binary symbiosis with the bioluminescent bacterium Vibrio fischeri acquired post-hatching. We aim to understand the response (i.e., developmental and molecular) of E. scolopes after the embryogenetic exposure to different conditions: i) standard conditions (control), ii) increased CO2 ({Delta}pH 0.4 units), iii) warming (+3{o}C), or iv) a combination of the two treatments. We observed a decrease in hatching success across all treatments relative to the control. Using transcriptomics, we identified a potential trade-off in favour of metabolism and energy production, at the expense of development under increased CO2. In contrast, elevated temperature shortened the developmental time and, at a molecular level, showed signs of alternative splicing and the potential for RNA editing. The data also suggest that the initiation of the symbiosis may be negatively affected by these environmental drivers of change in the biosphere, although coping mechanisms by the animal may occur.

ecology↗

bioRxiv2024

Application of hsp60 amplicon sequencing to characterize microbial communities associated with juvenile and adult Euprymna scolopes squid

The symbiotic relationship between Vibrio (Aliivibrio) fischeri and the Hawaiian bobtail squid, Euprymna scolopes, serves as a key model for understanding host-microbe interactions. Traditional culture-based methods have primarily isolated V. fischeri from the light organs of wild-caught squid, yet culture-independent analyses of this symbiotic microbiome remain limited. This study aims to enhance species-level resolution of bacterial communities associated with E. scolopes using hsp60 amplicon sequencing. We validated our hsp60 sequencing approach using pure cultures and mixed bacterial populations, demonstrating its ability to distinguish V. fischeri from other closely-related vibrios and the possibility of using this approach for strain-level diversity with further optimization. This approach was applied to whole-animal juvenile squid exposed to either seawater or a clonal V. fischeri inoculum, as well as ventate samples and light organ cores from wild-caught adults. V. fischeri accounted for the majority of the identifiable taxa for whole-animal juvenile samples and comprised 94%-99% of amplicon sequence variants (ASVs) for adult light organ core samples, confirming that V. fischeri is the dominant, if not sole, symbiont typically associated with E. scolopes light organs. In one ventate sample, V. fischeri comprised 82% of reads, indicating the potential for non-invasive community assessments using this approach. Analysis of non-V. fischeri ASVs revealed that Bradyrhizobium spp. and other members of the Rhodobacterales order are conserved across juvenile and adult samples. These findings provide insight into the presence of additional microbial associations with the squid host tissue outside of the light organ that have not been previously detected through traditional culture methods.

microbiology↗

bioRxiv2021

Transitioning to confined spaces impacts bacterial swimming and escape response

Symbiotic bacteria often navigate complex environments before colonizing privileged sites in their host organism. Chemical gradients are known to facilitate directional taxis of these bacteria, guiding them towards their eventual destination. However, less is known about the role of physical features in shaping the path the bacteria take and defining how they traverse a given space. The flagellated marine bacterium Vibrio fischeri,which forms a binary symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, must navigate tight physical confinement, squeezing through a bottleneck constricting to ~2 m in width on the way to its eventual home. Using microfluidic in vitro experiments, we discovered that V. fischeri cells alter their behavior upon entry into confined space, straightening their swimming paths and promoting escape from confinement. Using a computational model, we attributed this escape response to two factors: reduced directional fluctuation and a refractory period between reversals. Additional experiments in asymmetric capillary tubes confirmed that V. fischeri quickly escape from tapered ends, even when drawn into the ends by chemoattraction. This avoidance was apparent down to a limit of confinement approaching the diameter of the cell itself, resulting in a balance between chemoattraction and evasion of physical confinement. Our findings demonstrate that non-trivial distributions of swimming bacteria can emerge from simple physical gradients in the level of confinement. Tight spaces may serve as an additional, crucial cue for bacteria while they navigate complex environments to enter specific habitats. Significance StatementSymbiotic bacteria that navigate to and through specific host tissues often face tight physical confinement. This work reveals that confinement-associated changes in swimming can dramatically alter taxis, shaping bacterial localization in conjuncture with other motility-directing cues. This work helps explain how bacteria can avoid getting stuck in confined areas while transiting to privileged spaces, adding confinement as an environmental cue that symbiotic bacteria use to shape their motility behavior.

biophysics↗

bioRxiv2021

Independent host- and bacterium-based determinants protect a model symbiosis from phage predation

Summary/AbstractBacteriophages (phages) are diverse and abundant constituents of microbial communities worldwide, and are capable of modulating bacterial populations in diverse ways. Here we describe a novel phage, {phi}HNL01, which infects the marine bacterium Vibrio fischeri. We use culture-based approaches to demonstrate that mutations in the exopolysaccharide locus of V. fischeri render this bacterium resistant to infection by {phi}HNL01, highlighting the extracellular matrix as a key determinant of phage tropism in this interaction. Additionally, using the natural symbiosis between V. fischeri and the squid Euprymna scolopes, we show that during colonization, V. fischeri is protected from phage present in the ambient seawater. Taken together, these findings shed light on independent yet synergistic host- and bacterium-based strategies for resisting symbiosis-disrupting phage predation, and present important implications for understanding these strategies in the context of host-associated microbial ecosystems.

microbiology↗

bioRxiv2020

HbtR, a heterofunctional homolog of the virulence regulator TcpP, facilitates the transition between symbiotic and planktonic lifestyles in Vibrio fischeri

The bioluminescent bacterium Vibrio fischeri forms a mutually beneficial symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, in which the bacteria, housed inside a specialized light organ, produce light used by the squid in its nocturnal activities. Upon hatching, E. scolopes juveniles acquire V. fischeri from the seawater through a complex process that requires, among other factors, chemotaxis by the bacteria along a gradient of N-acetylated sugars into the crypts of the light organ, the niche in which the bacteria reside. Once inside the light organ, V. fischeri transitions into a symbiotic, sessile state in which the quorum-signaling regulator LitR induces luminescence. In this work we show that expression of litR and luminescence are repressed by a homolog of the V. cholerae virulence factor TcpP, which we have named HbtR. Further, we demonstrate that LitR represses genes involved in motility and chemotaxis into the light organ and activates genes required for exopolysaccharide production. ImportanceTcpP homologs are widespread throughout the Vibrio genus; however, the only protein in this family described thus far is a V. cholerae virulence regulator. Here we show that HbtR, the TcpP homolog in V. fischeri, has both a biological role and regulatory pathway completely unlike that in V. cholerae. Through its repression of the quorum-signaling regulator LitR, HbtR affects the expression of genes important for colonization of the E. scolopes light organ. While LitR becomes activated within the crypts, and upregulates luminescence and exopolysaccharide genes and downregulates chemotaxis and motility genes, it appears that HbtR, upon expulsion of V. fischeri cells into seawater, reverses this process to aid the switch from a symbiotic to a planktonic state. The possible importance of HbtR to the survival of V. fischeri outside of its animal host may have broader implications for the ways in which bacteria transition between often vastly different environmental niches.

microbiology↗