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.

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.

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.

The increased potential for contamination of seawater by crude oils requires studies of bacterial biodegradation potential, but little is known of the differential negative impacts of oils on bacterial growth. No two wells generate chemically identical oils; and importantly, solar exposure of crude oil may differentially affect the bacterial response. Elucidating the role that sunlight plays on the potential toxicity of spilled crude oils is imperative to understanding how oil spills might affect microbes in the tropical and subtropical waters of Florida. This study examined light exposure of six different crude oils, and subsequent microbial responses to altered oils. Marine bacterioplankton heterotrophic activities were measured via3H-leucine incorporation after the addition of oils’ water accommodated fractions (WAFs) that were created under varied solar conditions. Inhibition of production increased with higher concentrations of WAFs, but dose-response trends varied among the oils. Increased solar exposure during WAF preparation generally led to more inhibition, but trends varied among oils. WAFs were also prepared under different parts of the solar spectrum. Solar-irradiated WAFs resulted in significant but variable acute toxicity vs. dark counterparts. Solar-induced toxicity was primarily a result of visible and not ultraviolet light exposure. Results indicate responses to oil spills are highly dependent on the source of the oil and solar conditions at the time and location of the spill. The data presented here demonstrate the importance of photochemical changes and oil source in modulating microbial activity and bioremediation potential.

The chemical synthesis intermediate 3,4-dichloronitrobenzene (3,4-DCNB) is an environmental pollutant. Diaphorobacter sp. strain JS3050 utilizes 3,4-DCNB as a sole source of carbon, nitrogen and energy. However, the molecular determinants of its catabolism are poorly understood. Here, the complete genome of strain JS3050 was sequenced and key genes were expressed heterologously to establish the details of its degradation pathway. A chromosome-encoded three-component
nitroarene dioxygenase (DcnAaAbAcAd) converted 3,4-DCNB stoichiometrically to 4,5-dichlorocatechol, which was transformed to 3,4-dichloromuconate by a plasmid-borne ring-cleavage chlorocatechol 1,2-dioxygenase (DcnC). On the chromosome, there are also genes encoding enzymes (DcnDEF) responsible for the subsequent transformation of 3,4-dichloromuconate to β-ketoadipic acid. The fact that the genes responsible for the catabolic pathway are separately located on plasmid and chromosome indicates that recent assembly and ongoing evolution of the genes encoding the pathway is likely. The regiospecificity of 4,5-dichlorocatechol formation from 3,4-DCNB by DcnAaAbAcAd represents a sophisticated evolution of the nitroarene dioxygenase that avoids misrouting of toxic intermediates. The findings enhance the understanding of microbial catabolic diversity during adaptive evolution in response to xenobiotics released into the environment.

Vibriosis is the general term for human illnesses caused by infection of pathogenic Vibrio species. Vibrio vulnificus (Vv) and parahaemolyticus (Vp) are two problematic waterborne pathogens that have yet to be enumerated in northwest Florida coastal Gulf of Mexico estuaries. In this regionally novel study, we surveyed 43 locations in two subtropical estuarine systems, Perdido Bay and Pensacola Bay, over seven dates in winter 2020. Sampling included three substrate types: surface waters, sediments, and invertebrate biofilms. We determined baseline abundances of presumptive viable Vv and Vp appearing as colonies on CHROMagar (Vv, blue; Vp, purple). Vv was detected in 37 out of 43 water samples, with maximum levels of 3,556 CFU/mL. Vp was only detected in 15 water samples, with a maximum concentration of 8,919 CFU/mL. Sediments contained Vv in all but one sample, with concentrations ranging from 121 to 607,222 CFU/mL. In contrast, Vp were only detected in 33 sediment samples, where concentrations ranged from 28 to 77,333 CFU/mL. Opportunistically-sampled surface swabs (biofilms), collected from shells (either oyster or barnacle) and polychaete worms found in sediment samples, contained on average 7,735 and 1,490 CFU/mL of Vv and Vp, respectively. Surface water Vv abundances covaried with bottom water pH, maximum prior cumulative wind speeds, and tidal coefficient on the day of sampling. Vp surface water abundances negatively correlated with surface water salinity, surface water pH, and bottom water pH and positively correlated with total surface dissolved inorganic and total Kjeldahl nitrogen concentrations, and wind. Spatially, there was large variation in Vibrio densities in surface waters; abundances of both species were strongly correlated with wind, suggesting resuspension was important. Sedimentary abundances of both putative Vv and Vp shared a correlation with one parameter: salinity stratification. Due to the length of this study, temperature was not considered a major factor. This short-term (one month) study was designed not to enumerate pathogenic Vv or Vp, but rather to establish the first winter baseline of Vibrio abundances for this region. Determination of these baseline winter cultivable putative Vibrio abundances will be valuable in predicting relative risk factors in each waterbody of interest.