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Abstract TheVibrio fischeri—Euprymna scolopessymbiosis has become a powerful animal—microbe model system to examine the genetic underpinnings of symbiont development and regulation. Although there has been a number of elegant bacterial genetic technologies developed to examine this symbiosis, there is still a need to develop more sophisticated methodologies to better understand complex regulatory pathways that lie within the association. Therefore, we have developed a suite of CRISPR interference (CRISPRi) vectors for inducible repression of specific V. fischeri genes associated with symbiotic competence. The suite utilizes both Tn7-integrating and shuttle vector plasmids that allow for inducible expression of CRISPRi dCas9 protein along with single-guide RNAs (sgRNA) modules. We validated this CRISPRi tool suite by targeting both exogenous (an introduced mRFP reporter) and endogenous genes (luxCin the bioluminescence producingluxoperon, and flrA, the major regulatory gene controlling flagella production). The suite includes shuttle vectors expressing both single and multiple sgRNAs complementary to the non-template strand of multiple targeted genetic loci, which were effective in inducible gene repression, with significant reductions in targeted gene expression levels.V. fischericells harboring a version of this system targeting theluxCgene and suppressing the production of luminescence were used to experimentally validate the hypothesis that continuous luminescence must be produced by the symbiont in order to maintain the symbiosis at time points longer than the known 24-h limit. This robust new CRISPRi genetic toolset has broad utility and will enhance the study of V. fischerigenes, bypassing the need for gene disruptions by standard techniques of allelic knockout-complementation-exchange and the ability to visualize symbiotic regulation in vivo. 

ABSTRACT Symbiotic marine bacteria that are transmitted through the environment are susceptible to abiotic factors (salinity, temperature, physical barriers) that can influence their ability to colonize their specific hosts. Given that many symbioses are driven by host specificity, environmentally transmitted symbionts are more susceptible to extrinsic factors depending on conditions over space and time. In order to determine whether the population structure of environmentally transmitted symbionts reflects host specificity or biogeography, we analysed the genetic structure ofSepiola atlantica(Cephalopoda: Sepiolidae) and theirVibriosymbionts (V. fischeriandV. logei) in four Galician Rías (Spain). This geographical location is characterized by a jagged coastline with a deep‐sea entrance into the land, ideal for testing whether such population barriers exist due to genetic isolation. We used haplotype estimates combined with nested clade analysis to determine the genetic relatedness for bothS. atlanticaandVibriobacteria. Analyses of molecular variance (AMOVA) were used to estimate variation within and between populations for both host and symbiont genetic data. Our analyses reveal a low percentage of variation among and between host populations, suggesting that these populations are panmictic. In contrast,Vibriosymbiont populations show certain degree of genetic structure, demonstrating that the hydrology of the rías is driving bacterial distribution (and not host specificity). Thus, for environmentally transmitted symbioses such as the sepiolid squid‐Vibrioassociation, abiotic factors can be a major selective force for determining population structure for one of the partners. 

Abstract Life on Earth comprises prokaryotes and a broad assemblage of endosymbioses. The pages of Molecular Biology and Evolution and Genome Biology and Evolution have provided an essential window into how these endosymbiotic interactions have evolved and shaped biological diversity. Here, we provide a current perspective on this knowledge by drawing on decades of revelatory research published in Molecular Biology and Evolution and Genome Biology and Evolution, and insights from the field at large. The accumulated work illustrates how endosymbioses provide hosts with novel phenotypes that allow them to transition between adaptive landscapes to access environmental resources. Such endosymbiotic relationships have shaped and reshaped life on Earth. The early serial establishment of mitochondria and chloroplasts through endosymbioses permitted massive upscaling of cellular energetics, multicellularity, and terrestrial planetary greening. These endosymbioses are also the foundation upon which all later ones are built, including everything from land–plant endosymbioses with fungi and bacteria to nutritional endosymbioses found in invertebrate animals. Common evolutionary mechanisms have shaped this broad range of interactions. Endosymbionts generally experience adaptive and stochastic genome streamlining, the extent of which depends on several key factors (e.g. mode of transmission). Hosts, in contrast, adapt complex mechanisms of resource exchange, cellular integration and regulation, and genetic support mechanisms to prop up degraded symbionts. However, there are significant differences between endosymbiotic interactions not only in how partners have evolved with each other but also in the scope of their influence on biological diversity. These differences are important considerations for predicting how endosymbioses will persist and adapt to a changing planet. 

Biofilm formation is important for microbial survival, adaptation, and persistence within mutualistic and pathogenic systems in the Vibironaceae. Biofilms offer protection against environmental stressors, immune responses, and antimicrobial treatments by increasing host colonization and resilience. This review examines the mechanisms of biofilm formation in Vibrio species, focusing on quorum sensing, cyclic-di-GMP signaling, and host-specific adaptations that influence biofilm structure and function. We discuss how biofilms differ between mutualistic and pathogenic species based on environmental and host signals. Recent advances in omics technologies such as transcriptomics and metabolomics have enhanced research in biofilm regulation under different conditions. Horizontal gene transfer and phase variation promote the greater fitness of bacterial biofilms due to the diversity of environmental isolates that utilize biofilms to colonize host species. Despite progress, questions remain regarding the long-term effects of biofilm formation and persistence on host physiology and biofilm community dynamics. Research integrating multidisciplinary approaches will help advance our understanding of biofilms and their implications for influencing microbial adaptation, symbiosis, and disease. These findings have applications in biotechnology and medicine, where the genetic manipulation of biofilm regulation can enhance or disrupt microbiome stability and pathogen resistance, eventually leading to targeted therapeutic strategies. 

Just as a phylogeny encodes the evolutionary relationships among a group of organisms, a cophylogeny represents the coevolutionary relationships among symbiotic partners. Both are primarily reconstructed using computational analysis of biomolecular sequence data. The most widely used cophylogenetic reconstruction methods utilize an important simplifying assumption: species phylogenies for each set of coevolved taxa are required as input and assumed to be correct. Many studies have shown that this assumption is rarely – if ever – satisfied, and the consequences for cophylogenetic studies are poorly understood. To address this gap, we conduct a comprehensive performance study that quantifies the relationship between species tree estimation error and downstream cophylogenetic estimation accuracy. We study the performance of state-of-the-art methods for cophylogenetic reconstruction using in silico model-based simulations. Our investigation also assessed cophylogenetic reproducibility using genomic sequence data from two important models of symbiosis: soil-associated fungi and their endosymbiotic bacteria, and bobtail squid and their bioluminescent bacterial symbionts. Our findings conclusively demonstrate the major impact that upstream phylogenetic estimation error has on downstream cophylogenetic reconstruction. Relative to other experimental factors such as cophylogenetic estimation method choice and coevolutionary event costs, phylogenetic estimation error ranked highest in importance based on a random forest-based variable importance assessment. We conclude with practical guidance and future research directions. Among the many considerations needed for accurate cophylogenetic reconstruction – choice of computational method, method settings, sampling design, and others – just as much attention must be paid to careful species phylogeny estimation using modern best practices.