Dr. Lisa A Waidner

is a type of bacterium that causes cholera, a severe diarrheal disease globally affecting hundreds of people annually. However, the effect of the toxin on oyster metabolite signatures has not been well studied. In this study, nuclear magnetic resonance (NMR) based metabolomics was applied to investigate the metabolic level response of eastern oysters (Crassostrea virginica) to cholera toxin (CT), under low concentrations. Our study demonstrated that the decrease of branched-chain amino acids (BCAAs) in oysters was a response to CT exposure at low concentrations (10 ng/mL) in gill and mantle extracts. Metabolites such as leucine and isoleucine were significantly decreased in gills with toxin exposure at 10 ng/mL, and similar but weaker changes were also observed at 1 ng/mL, indicating an early response to CT. However, the trend reversed at 20 ng/mL, with acetate and propionate significantly increased over control (p < 0.07), which is a sign of antioxidant defenses that could help the recovery of the BCAAs. In the hemolymph study, acetate and propionate levels correlated strongly with those in the tissue extracts at 20 ng/mL, suggesting that hemolymph metabolites begin contributing to gill metabolic perturbations. More importantly, a principal component analysis (PCA) also revealed a partial separation between the control and the 20 ng/mL CT group, indicating potential major perturbations in hemolymph metabolites. This study provides evidence that metabolites in oyster tissues resulting from exposure to Vibrio toxin toxin can serve as a new early warning system for predicting potential human pathogen risks in both environmental and seafood exposure.

Nitrogen fixers can enhance nitrogen availability for seagrass communities that may be nitrogen limited. However, the role of epiphytic diazotrophs, particularly cyanobacteria, in seagrass communities is not well understood. We measured nitrogen fixation rates, epiphyte biomass, and relative abundances of epiphytic diazotrophs on the leaves of Thalassia testudinum and Halodule wrightii in the northern Gulf of Mexico. Greater accumulation of epiphyte biomass and diazotrophs may occur in T. testudinum due to lower leaf turnover than found in H. wrightii , particularly during periods of seagrass dormancy. Nitrogen fixation rates were determined using the acetylene reduction assay, while quantitative polymerase chain reaction was used to measure relative abundances of three cyanobacterial diazotroph groups in epiphyte DNA. Nitrogen fixation and epiphyte biomass were higher on T. testudinum leaves than on H. wrightii leaves. The lowest average fixation rates occurred in August when leaf turnover was high. Three phylotypes of nifH genes were detected in most samples, but overall, Crocosphaera ‐like Group B cyanobacteria (UCYN‐B) were present on all leaves during all seasons. Relative abundance of this group was positively correlated with nitrogen fixation rates on both species ( r = 0.59, p = 0.02). At one of the four study sites, heterocystous cyanobacterial symbionts in the Richelia‐ like (Het‐1) and Calothrix‐ like (Het‐3) groups accounted for similar relative abundances to those observed with UCYN‐B nifH genes. Because T. testudinum and H. wrightii are dominant in shallow tropical and subtropical ecosystems, understanding the role that diazotrophic epiphytes play in providing nitrogen to these vital ecosystems is critical.

Bacteria in the Vibrio vulnificus genus are known to cause serious illness in humans, including both gastrointestinal disease and open wound infection, and even septicemia if the infections are aggressive, left unchecked, or in immunocompromised individuals. They’re found naturally in estuaries and coastal waters.
There are multiple ways humans can be exposed to these bacteria such as through open wounds when in seawater or estuary water with high (~>30 Cells/mL) abundances, or through consuming raw or undercooked seafood such as oysters.
The aim of this study was to measure and record data of the environmental factors that may influence bacterial abundances and distribution of this particular group of bacteria. Environmental factors such as water temperature, salinity, and total suspended solids, as well as meteorological conditions including wind speed, wind gust, and air temperature have been shown to have positive correlation with change in V.v abundance within the Pensacola Bay system (Potdukhe 2021).

Vibrio vulnificus bacteria are naturally found in estuaries and marine waters. These bacteria can enter human hosts through open wounds or undercooked or raw seafood, such as oysters, and cause serious illness. We have been collecting oyster samples and environmental data to determine how different factors influence Vibrio abundances in oysters in Bayou Texar and Pensacola Bay. By collecting multiple forms of data, we hope to build upon existing models with a variety of environmental factors. One of those factors is wind speed and comparing it to total suspended solids (TSS) which may give insight into the reasoning of higher values. Seagrass density is another parameter we are examining, since this is an under-researched aspect of ecosystems that may affect Vibrio densities. A variety of meteorological and hydrological data have been collected over the duration of the project including temperature, precipitation, wind, and tidal data. By analyzing all of these factors at play, we hope to paint a clearer picture of the different attributes that affect Vibrio
vulnificus abundance in oysters and the water.

Prediction and process monitoring during natural attenuation, bioremediation and biotreatment require effective strategies for detection and enumeration of the responsible bacteria. The use of 2,4-dinitroanisole (DNAN) as a component of insensitive munitions leads to environmental contamination of firing ranges and manufacturing waste streams. Nocardioides sp strain JS1661 degrades DNAN under aerobic conditions via a pathway involving an unusual DNAN demethylase. We used the deeply branched sequences of DNAN degradation functional genes as a target for development of a molecular method for detection of the bacteria. A qPCR assay was designed for the junction between dnhA and dnhB, the adjacent genes encoding DNAN demethylase. The assay allowed reproducible enumeration of JS1661 during growth in liquid media and soil slurries. Results were consistent with biodegradation of DNAN, accumulation of products and classical biomass estimates including most probable number and OD600. The results provide a sensitive and specific molecular method for prediction of degradation potential and process evaluation during degradation of DNAN.

Algae generate hydrogen from sunlight and water utilizing high-energy electrons generated during photosynthesis. The amount of hydrogen produced in heterologous expression of the wild-type hydrogenase is currently insufficient for industrial applications. One approach to improve hydrogen yields is through directed evolution of the DNA of the native hydrogenase. Here, we created 113 chimeric algal hydrogenase gene variants derived from combining segments of three parent hydrogenases, two from Chlamydomonas reinhardtii (CrHydA1 and CrHydA2) and one from Scenedesmus obliquus (HydA1). To generate chimeras, there were seven segments into which each of the parent hydrogenase genes was divided and recombined in a variety of combinations. The chimeric and parental hydrogenase sequences were cloned for heterologous expression in Escherichia coli, and 40 of the resultant enzymes expressed were assayed for H2 production. Chimeric clones that resulted in equal or greater production obtained with the cloned CrHydA1 parent hydrogenase were those comprised of CrHydA1 sequence in segments #1, 2, 3, and/or 4. These best-performing chimeras all contained one common region, segment #2, the part of the sequence known to contain important amino acids involved in proton transfer or hydrogen cluster coordination. The amino acid sequence distances among all chimeric clones to that of the CrHydA1 parent were determined, and the relationship between sequence distances and experimentally-derived H2 production was evaluated. An additional model determined the correlation between electrostatic potential energy surface area ratios and H2 production. The model yielded several algal mutants with predicted hydrogen productions in a range of two to three times that of the wild-type hydrogenase. The mutant data and the model can now be used to predict which specific mutant sequences may result in even higher hydrogen yields. Overall, results provide more precise details in planning future directed evolution to functionally improve algal hydrogenases.
•Chimeric algal hydrogenase gene mutants were created from three genes of two microalgal species
•Swapping gene segments resulted in some constructs with improved hydrogen production
•Sequence and electrostatic potential models may help predict improved function with mutation

The 2010 Deepwater Horizon (DwH) Oil spill released an enormous volume of oil into the Gulf of Mexico (GoM), prompting the widespread use of chemical dispersants like Corexit ® EC9500A. The ecological consequences of this treatment, especially when combined with natural factors such as sunlight, remain unexplored in the context of marine bacterial communities’ dynamics. To address this knowledge gap, our study employed a unique metaproteomic approach, investigating the combined effects of sunlight, crude Macondo surrogate oil, and Corexit on GoM microbiome across different mesocosms. Exposure to oil and/or Corexit caused a marked change in community composition, with a decrease in taxonomic diversity relative to controls in only 24 hours. Hydrocarbon (HC) degraders, particularly those more tolerant to Corexit and phototoxic properties of crude oil and/or Corexit, proliferated at the expense of more sensitive taxa. Solar radiation exacerbated these effects in most taxa. We demonstrated that sunlight increased the dispersant’s toxicity, impacting on community structure and functioning. These functional changes were primarily directed by oxidative stress with upregulated proteins and enzymes involved in protein turnover, general stress response, DNA replication and repair, chromosome condensation, and cell division. These factors were more abundant in chemically treated conditions, especially in the presence of Corexit compared to controls. Oil treatment significantly enhanced the relative abundance of Alteromonas , an oil-degrading Gammaproteobacteria . In combined oil-Corexit treatments, the majority of identified protein functions were assigned to Alteromonas , with strongly expressed proteins involved in membrane transport, motility, carbon and amino acid metabolism and cellular defense mechanisms. Marinomonas , one of the most active genera in dark conditions, was absent from the light treatment. Numerous metabolic pathways and HC-degrading genes provided insights into bacterial community adaptation to oil spills. Key enzymes of the glyoxylate bypass, enriched in contaminant-containing treatments, were predominantly associated with Rhodobacterales and Alteromonadales. Several proteins related to outer membrane transport, photosynthesis, and nutrient metabolisms were characterized, allowing predictions of the various treatments on biogeochemical cycles. The study also presents novel perspectives for future oil spill clean-up processes.

Excerpt - The project provided key baseline information about the shallow continental shelf environment in the northeastern Gulf of Mexico. This area is poorly studied compared to other continental shelves such as the South Atlantic Bight. Colonization of benthic invertebrates increased secondary production. This in turn supports higher trophic levels, particularly the commercially important reef fishes such as snapper, triggerfish and sheepshead. Results from this work represent one of the few studies of primary production, biogeochemical cycling, and the implications to fisheries production in the northeastern Gulf of Mexico. With the increasing deployment of artificial reefs in Florida, this provides key information for management of these habitats.

Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are water- and foodborne bacteria that can cause several distinct human diseases, collectively called vibriosis. The success of oyster aquaculture is negatively impacted by high Vibrio abundances. Myriad environmental factors affect the distribution of pathogenic Vibrio, including temperature, salinity, eutrophication, extreme weather events, and plankton loads, including harmful algal blooms. In this paper, we synthesize the current understanding of ecological drivers of Vv and Vp and provide a summary of various tools used to enumerate Vv and Vp in a variety of environments and environmental samples. We also highlight the limitations and benefits of each of the measurement tools and propose example alternative tools for more specific enumeration of pathogenic Vv and Vp. Improvement of molecular methods can tighten better predictive models that are potentially important for mitigation in more controlled environments such as aquaculture.