Date of Award

1-1-2025

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Department

Fisheries, Animal and Veterinary Science

First Advisor

Marta Gomez-Chiarri

Abstract

Oyster larvae aquaculture is an important industry, both economically and environmentally, helping to create sustainable food systems by supplying seed oysters to farms, and also to restoration projects aimed at building coastal resilience and improving water quality by rejuvenating wild populations. However, oyster larvae are highly susceptible to rapid mortality events, crashes, which can seriously impact the industry. One source of these crashes in hatchery systems is vibriosis, caused by pathogenic species of Vibrio bacteria that are ubiquitous in temperate coastal waters, and with rising surface water temperatures their prevalence and virulence are increasing. To prevent these larval crashes from happening in hatcheries without relying on the use of environmentally damaging solutions, probiotics are being developed and investigated for use in these situations.

Phaeobacter inhibens is a gram-negative, motile, marine bacteria in the Roseobacter clade, often found associated with marine surfaces, including algae, diatoms, docks, aquaculture gear, and other marine animals. The strain S4, originally isolated from the inner shell of a healthy eastern oyster, has been developed into a probiotic for use in oyster hatchery systems. It has been found to be effective at reducing the virulence of a common oyster pathogen, Vibrio coralliilyticus RE22 through the use of numerous tools including the production of the antimicrobial tropodithietic acid (TDA), quorum signaling/quenching molecules N-acyl homoserine lactones (AHLs), and biofilm formation. However, our knowledge of its capabilities as a probiont is limited by the incomplete genome and limited molecular evidence to corroborate other experimental data.

To address these gaps in knowledge, a long-read genome of Phaeobacter inhibens S4Sm, a naturally occurring streptomycin resistant strain of S4, was assembled and annotated, allowing for deeper analysis into S4Sm capabilities. With the completed genome, comprising a chromosome and three plasmids with a total length of approximately 4.4 Mb, a pangenome analysis of 36 other P. inhibens strains found a core genome of over 3,000 genes. AntiSMASH investigation into all strains used in the study found large conformity in the suite of secondary metabolite capabilities of the strains. Nearly every strain had at least one each of the eight biosynthetic clusters found: homoserine lactones (AHL); Type I Polyketide synthase (T1PKS); beta-lactone containing protease inhibitor; unspecified ribosomally synthesized and post-translationally modified peptide product (RiPP-like); non-ribosomal peptide synthetase (NRPS); tropodithietic acid (TDA); NRPS-like fragments; and NRPS-independent, IucA/IucC-like siderophore.

Of the biosynthetic clusters found in the AntiSMASH analysis of S4Sm, both TDA and AHLs have been found to have a large impact on reducing virulence in RE22, but their individual roles have been harder to untangle. AHLs are signaling molecules used by the bacteria to coordinate many processes, and TDA can also be used as a signaling molecule for many of the same pathways at sublethal concentrations. When making mutants within the AHL or TDA pathways, one often affects the other, or related processes, like biofilm formation, that rely on cell-cell communication. This study used a previously created S4 mutant strain, WZ10, that was deficient in TDA production but maintained AHL production and biofilm formation capabilities to untangle the relative role of TDA and AHLs on the competition between pathogen RE22 and probiont S4.

Pathogenic Vibrio coralliilyticus RE22 in competition with TDA-deficient Phaeobacter inhibens S4 WZ10 showed increased survival, as well as increased expression of genes in the T6SS and genes associated with motility, while these genes were highly down regulated in RE22 when in competition with S4Sm wild type. In turn, the TDA-deficient mutant strain S4 WZ10, in comparison to S4Smwt, showed upregulation of expression of genes in some of the other biosynthetic clusters, including AHLs, siderophore, NRPS, and beta-lactone. All of these molecules are known to confer probiotic capabilities in Phaeobacters or other marine bacteria, and it is possible that the mutant is upregulating these pathways to make up for the loss of TDA. Further investigation is needed to characterize NRPS and beta-lactone products in S4Sm, as the variation in these types of clusters is broad.

This research furthers this field of study by creating a newly completed genome of Phaeobacter inhibens S4Sm that can be used in future studies and comparisons. It also highlighted the biosynthetic potential of all fully sequenced Phaeobacter inhibens strains available on NCBI at this time, highlighting the potential for a regionally available P. inhibens strain for probiotic use against local pathogens worldwide. It also characterizes the complex effects of pathogen - probiont competition on gene expression and helps untangle the relative roles of AHL and TDA on S4 activity against RE22.

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