Identification of a Transcriptomic Network Underlying the Wrinkly and Smooth Phenotypes of Vibrio fischeri.

Vibrio fischeri is a cosmopolitan marine bacterium that oftentimes displays different colony morphologies, switching from a smooth to a wrinkly phenotype in order to adapt to changes in the environment. This wrinkly phenotype has also been associated with increased biofilm formation, an essential characteristic for V. fischeri to adhere to substrates, to suspended debris, and within the light organs of sepiolid squids. Elevated levels of biofilm formation are correlated with increased microbial survival of exposure to environmental stressors and the ability to expand niche breadth. Since V. fischeri has a biphasic life history strategy between its free-living and symbiotic states, we were interested in whether the wrinkly morphotype demonstrated differences in its expression profile in comparison to the naturally occurring and more common smooth variant. We show that genes involved in major biochemical cascades, including those involved in protein sorting, oxidative stress, and membrane transport, play a role in the wrinkly phenotype. Interestingly, only a few unique genes are specifically involved in macromolecule biosynthesis in the wrinkly phenotype, which underlies the importance of other pathways utilized for adaptation under the conditions in which Vibrio bacteria are producing this change in phenotype. These results provide the first comprehensive analysis of the complex form of genetic activation that underlies the diversity in morphologies of V. fischeri when switching between two different colony morphotypes, each representing a unique biofilm ecotype.IMPORTANCE The wrinkly bacterial colony phenotype has been associated with increased squid host colonization in V. fischeri The significance of our research is in identifying the genetic mechanisms that are responsible for heightened biofilm formation in V. fischeri This report also advances our understanding of gene regulation in V. fischeri and brings to the forefront a number of previously overlooked genetic networks. Several loci that were identified in this study were not previously known to be associated with biofilm formation in V. fischeri.

Marine Actinomycetes, New Sources of Biotechnological Products.

The Actinomycetales order is one of great genetic and functional diversity, including diversity in the production of secondary metabolites which have uses in medical, environmental rehabilitation, and industrial applications. Secondary metabolites produced by actinomycete species are an abundant source of antibiotics, antitumor agents, anthelmintics, and antifungals. These actinomycete-derived medicines are in circulation as current treatments, but actinomycetes are also being explored as potential sources of new compounds to combat multidrug resistance in pathogenic bacteria. Actinomycetes as a potential to solve environmental concerns is another area of recent investigation, particularly their utility in the bioremediation of pesticides, toxic metals, radioactive wastes, and biofouling. Other applications include biofuels, detergents, and food preservatives/additives. Exploring other unique properties of actinomycetes will allow for a deeper understanding of this interesting taxonomic group. Combined with genetic engineering, microbial experimental evolution, and other enhancement techniques, it is reasonable to assume that the use of marine actinomycetes will continue to increase. Novel products will begin to be developed for diverse applied research purposes, including zymology and enology. This paper outlines the current knowledge of actinomycete usage in applied research, focusing on marine isolates and providing direction for future research.

Adaptation to pH stress by Vibrio fischeri can affect its symbiosis with the Hawaiian bobtail squid (Euprymna scolopes).

Many microorganisms engaged in host-microbe interactions pendulate between a free-living phase and a host-affiliated stage. How adaptation to stress during the free-living phase affects host-microbe associations is unclear and understudied. To explore this topic, the symbiosis between Hawaiian bobtail squid (Euprymna scolopes) and the luminous bacterium Vibrio fischeri was leveraged for a microbial experimental evolution study. V. fischeri experienced adaptation to extreme pH while apart from the squid host. V. fischeri was serially passaged for 2000 generations to the lower and upper pH growth limits for this microorganism, which were pH 6.0 and 10.0, respectively. V. fischeri was also serially passaged for 2000 generations to vacillating pH 6.0 and 10.0. Evolution to pH stress both facilitated and impaired symbiosis. Microbial evolution to acid stress promoted squid colonization and increased bioluminescence for V. fischeri, while symbiont adaptation to alkaline stress diminished these two traits. Oscillatory selection to acid and alkaline stress also improved symbiosis for V. fischeri, but the facilitating effects were less than that provided by microbial adaptation to acid stress. In summary, microbial adaptation to harsh environments amid the free-living phase may impact the evolution of host-microbe interactions in ways that were not formerly considered.

Symbiont evolution during the free-living phase can improve host colonization.

For micro-organisms cycling between free-living and host-associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether evolution during the free-living stage can be positively pleiotropic to microbial fitness in a host environment. To address this topic, the squid host Euprymna tasmanica and the marine bioluminescent bacterium Vibrio fischeri were utilized. Microbial ecological diversification in static liquid microcosms was used to simulate symbiont evolution during the free-living stage. Thirteen genetically distinct V. fischeri strains from a broad diversity of ecological sources (e.g. squid light organs, fish light organs and seawater) were examined to see if the results were reproducible in many different genetic settings. Genetic backgrounds that are closely related can be predisposed to considerable differences in how they respond to similar selection pressures. For all strains examined, new mutations with striking and facilitating effects on host colonization arose quickly during microbial evolution in the free-living stage, regardless of the ecological context under consideration for a strain's genetic background. Microbial evolution outside a host environment promoted host range expansion, improved host colonization for a micro-organism, and diminished the negative correlation between biofilm formation and motility.

Ecological diversification of Vibrio fischeri serially passaged for 500 generations in novel squid host Euprymna tasmanica.

Vibrio fischeri isolated from Euprymna scolopes (Cephalopoda: Sepiolidae) was used to create 24 lines that were serially passaged through the non-native host Euprymna tasmanica for 500 generations. These derived lines were characterized for biofilm formation, swarming motility, carbon source utilization, and in vitro bioluminescence. Phenotypic assays were compared between "ES" (E. scolopes) and "ET" (E. tasmanica) V. fischeri wild isolates to determine if convergent evolution was apparent between E. tasmanica evolved lines and ET V. fischeri. Ecological diversification was observed in utilization of most carbon sources examined. Convergent evolution was evident in motility, biofilm formation, and select carbon sources displaying hyperpolymorphic usage in V. fischeri. Convergence in bioluminescence (a 2.5-fold increase in brightness) was collectively evident in the derived lines relative to the ancestor. However, dramatic changes in other properties--time points and cell densities of first light emission and maximal light output and emergence of a lag phase in growth curves of derived lines--suggest that increased light intensity per se was not the only important factor. Convergent evolution implies that gnotobiotic squid light organs subject colonizing V. fischeri to similar selection pressures. Adaptation to novel hosts appears to involve flexible microbial metabolism, establishment of biofilm and swarmer V. fischeri ecotypes, and complex changes in bioluminescence. Our data demonstrate that numerous alternate fitness optima or peaks are available to V. fischeri in host adaptive landscapes, where novel host squids serve as habitat islands. Thus, V. fischeri founder flushes occur during the initiation of light organ colonization that ultimately trigger founder effect diversification.

Deployment of a Vibrio cholerae ordered transposon mutant library in a quorum-competent genetic background.

Vibrio cholerae, the causative agent of cholera, has sparked seven pandemics in recent centuries, with the current one being the most prolonged. V. cholerae's pathogenesis hinges on its ability to switch between low and high cell density gene regulatory states, enabling transmission between host and the environment. Previously, a transposon mutant library for V. cholerae was created to support investigations aimed toward uncovering the genetic determinants of its pathogenesis. However, subsequent sequencing uncovered a mutation in the gene luxO of the parent strain, rendering mutants unable to exhibit high cell density behaviors. In this study, we used chitin-independent natural transformation to move transposon insertions from these low cell density mutants into a wildtype genomic background. Library transfer was aided by a novel gDNA extraction we developed using thymol, which also showed high lysis-specificity for Vibrio. The resulting Grant Library comprises 3,102 unique transposon mutants, covering 79.8% of V. cholerae's open reading frames. Whole genome sequencing of randomly selected mutants demonstrates 100% precision in transposon transfer to cognate genomic positions of the recipient strain. Notably, in no instance did the luxO mutation transfer into the wildtype background. Our research uncovered density-dependent epistasis in growth on inosine, an immunomodulatory metabolite secreted by gut bacteria that is implicated in enhancing gut barrier functions. Additionally, Grant Library mutants retain the plasmid that enables rapid, scarless genomic editing. In summary, the Grant Library reintroduces organismal relevant genetic contexts absent in the low cell density locked library equivalent.

Emerging Research Topics in the Vibrionaceae and the Squid-Vibrio Symbiosis.

The Vibrionaceae encompasses a cosmopolitan group that is mostly aquatic and possesses tremendous metabolic and genetic diversity. Given the importance of this taxon, it deserves continued and deeper research in a multitude of areas. This review outlines emerging topics of interest within the Vibrionaceae. Moreover, previously understudied research areas are highlighted that merit further exploration, including affiliations with marine plants (seagrasses), microbial predators, intracellular niches, and resistance to heavy metal toxicity. Agarases, phototrophy, phage shock protein response, and microbial experimental evolution are also fields discussed. The squid-Vibrio symbiosis is a stellar model system, which can be a useful guiding light on deeper expeditions and voyages traversing these "seas of interest". Where appropriate, the squid-Vibrio mutualism is mentioned in how it has or could facilitate the illumination of these various subjects. Additional research is warranted on the topics specified herein, since they have critical relevance for biomedical science, pharmaceuticals, and health care. There are also practical applications in agriculture, zymology, food science, and culinary use. The tractability of microbial experimental evolution is explained. Examples are given of how microbial selection studies can be used to examine the roles of chance, contingency, and determinism (natural selection) in shaping Earth's natural history.

Adaptation to temperature stress by Vibrio fischeri facilitates this microbe's symbiosis with the Hawaiian bobtail squid (Euprymna scolopes).

For microorganisms cycling between free-living and host-associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether adaptation to stress during the free-living stage can impact microbial fitness in the host. To address this topic, the mutualism between the Hawaiian bobtail squid (Euprymna scolopes) and the marine bioluminescent bacterium Vibrio fischeri was utilized. Using microbial experimental evolution, V. fischeri was selected to low (8°C), high (34°C), and fluctuating temperature stress (8°C/34°C) for 2000 generations. The temperatures 8°C and 34°C were the lower and upper growth limits, respectively. V. fischeri was also selected to benign temperatures (21°C and 28°C) for 2000 generations, which served as controls. V. fischeri demonstrated significant adaptation to low, high, and fluctuating temperature stress. V. fischeri did not display significant adaptation to the benign temperatures. Adaptation to stressful temperatures facilitated V. fischeri's ability to colonize the squid host relative to the ancestral lines. Bioluminescence levels also increased. Evolution to benign temperatures did not manifest these results. In summary, microbial adaptation to stress during the free-living stage can promote coevolution between hosts and microorganisms.

Microbial experimental evolution as a novel research approach in the Vibrionaceae and squid-Vibrio symbiosis.

The Vibrionaceae are a genetically and metabolically diverse family living in aquatic habitats with a great propensity toward developing interactions with eukaryotic microbial and multicellular hosts (as either commensals, pathogens, and mutualists). The Vibrionaceae frequently possess a life history cycle where bacteria are attached to a host in one phase and then another where they are free from their host as either part of the bacterioplankton or adhered to solid substrates such as marine sediment, riverbeds, lakebeds, or floating particulate debris. These two stages in their life history exert quite distinct and separate selection pressures. When bound to solid substrates or to host cells, the Vibrionaceae can also exist as complex biofilms. The association between bioluminescent Vibrio spp. and sepiolid squids (Cephalopoda: Sepiolidae) is an experimentally tractable model to study bacteria and animal host interactions, since the symbionts and squid hosts can be maintained in the laboratory independently of one another. The bacteria can be grown in pure culture and the squid hosts raised gnotobiotically with sterile light organs. The partnership between free-living Vibrio symbionts and axenic squid hatchlings emerging from eggs must be renewed every generation of the cephalopod host. Thus, symbiotic bacteria and animal host can each be studied alone and together in union. Despite virtues provided by the Vibrionaceae and sepiolid squid-Vibrio symbiosis, these assets to evolutionary biology have yet to be fully utilized for microbial experimental evolution. Experimental evolution studies already completed are reviewed, along with exploratory topics for future study.