DNA metabarcoding of cloacal swabs provides insight into diets of highly migratory sharks in the Mid-Atlantic Bight.

The abundances of migratory shark species observed throughout the Mid-Atlantic Bight (MAB) during productive summer months suggest that this region provides critical habitat and prey resources to these taxa. However, the principal prey assemblages sustaining migratory shark biomass in this region are poorly defined. We applied high-throughput DNA metabarcoding to shark feces derived from cloacal swabs across nine species of Carcharhinid and Lamnid sharks to (1) quantify the contribution of broad taxa (e.g., invertebrates, fishes) supporting shark biomass during seasonal residency in the MAB and (2) determine whether the species displayed distinct dietary preference indicative of resource partitioning. DNA metabarcoding resulted in high taxonomic (species-level) resolution of shark diets with actinopterygian and elasmobranch fishes as the dominant prey categories across the species. DNA metabarcoding identified several key prey groups consistent across shark taxa that are likely integral for sustaining their biomass in this region, including Atlantic menhaden (Brevoortia tyrannus), Atlantic mackerel (Scomber scombrus), and benthic elasmobranchs, including skates. Our results are consistent with previously published stomach content data for the shark species of similar size range in the Northwest Atlantic Ocean, supporting the efficacy of cloacal swab DNA metabarcoding as a minimally invasive diet reconstruction technique. The high reliance of several shark species on Atlantic menhaden could imply wasp-waist food-web conditions during the summer months, whereby high abundances of forage fishes sustain a diverse suite of migratory sharks within a complex, seasonal food web.

Nitrosomonas supralitoralis sp. nov., an ammonia-oxidizing bacterium from beach sand in a supralittoral zone.

A betaproteobacterial chemolithotrophic ammonia-oxidizing bacterium designated APG5T was isolated from supralittoral sand of the Edmonds City Beach, WA, USA. Growth was observed at 10-35 °C (optimum, 30 °C), pH 5-9 (optimum, pH 8) and ammonia concentrations as high as 100 mM (optimum, 1-30 mM NH4Cl). The strain grows optimally in a freshwater medium but tolerates up to 400 mM NaCl. It is most closely related to 'Nitrosomonas ureae' (96.7% 16S rRNA and 92.4% amoA sequence identity). The 3.75-Mbp of AGP5T draft genome contained a single rRNA operon and all necessary tRNA genes and has the lowest G+C content (43.5%) when compared to the previously reported genomes of reference strains in cluster 6 Nitrosomonas. Based on an average nucleotide identity of 82% with its closest relative ('N. ureae' Nm10T) and the suggested species boundary of 95-96%, a new species Nitrosomonas supralitoralis sp. nov. is proposed. The type strain of Nitrosomonas supralitoralis is APG5T (= NCIMB 14870T = ATCC TSD-116T).

Ecological response of nitrification to oil spills and its impact on the nitrogen cycle.

Marine oil spills are catastrophic events that cause massive damage to ecosystems at all trophic levels. While most of the research has focused on carbon-degrading microorganisms, the potential impacts of hydrocarbons on microbes responsible for nitrification have received far less attention. Nitrifiers are sensitive to hydrocarbon toxicity: ammonia-oxidizing bacteria and archaea being 100 and 1000 times more sensitive than typical heterotrophs respectively. Field studies have demonstrated the response of nitrifiers to hydrocarbons is highly variable and the loss of nitrification activity in coastal ecosystems can be restored within 1-2 years, which is much shorter than the typical recovery time of whole ecosystems (e.g., up to 20 years). Since the denitrification process is mainly driven by heterotrophs, which are more resistant to hydrocarbon toxicity than nitrifiers, the inhibition of nitrification may slow down the nitrogen turnover and increase ammonia availability, which supports the growth of oil-degrading heterotrophs and possibly various phototrophs. A better understanding of the ecological response of nitrification is paramount in predicting impacts of oil spills on the nitrogen cycle under oil spill conditions, and in improving current bioremediation practices.

Nitrosopumilus maritimus gen. nov., sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., four marine ammonia-oxidizing archaea of the phylum Thaumarchaeota.

Four mesophilic, neutrophilic, and aerobic marine ammonia-oxidizing archaea, designated strains SCM1T, HCA1T, HCE1T and PS0T, were isolated from a tropical marine fish tank, dimly lit deep coastal waters, the lower euphotic zone of coastal waters, and near-surface sediment in the Puget Sound estuary, respectively. Cells are straight or slightly curved small rods, 0.15-0.26 µm in diameter and 0.50-1.59 µm in length. Motility was not observed, although strain PS0T possesses genes associated with archaeal flagella and chemotaxis, suggesting it may be motile under some conditions. Cell membranes consist of glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, with crenarchaeol as the major component. Strain SCM1T displays a single surface layer (S-layer) with p6 symmetry, distinct from the p3-S-layer reported for the soil ammonia-oxidizing archaeon Nitrososphaera viennensis EN76T. Respiratory quinones consist of fully saturated and monounsaturated menaquinones with 6 isoprenoid units in the side chain. Cells obtain energy from ammonia oxidation and use carbon dioxide as carbon source; addition of an α-keto acid (α-ketoglutaric acid) was necessary to sustain growth of strains HCA1T, HCE1T, and PS0T. Strain PS0T uses urea as a source of ammonia for energy production and growth. All strains synthesize vitamin B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), and B12 (cobalamin). Optimal growth occurs between 25 and 32 °C, between pH 6.8 and 7.3, and between 25 and 37 ‰ salinity. All strains have a low mol% G+C content of 33.0-34.2. Strains are related by 98 % or greater 16S rRNA gene sequence identity, sharing ~85 % 16S rRNA gene sequence identity with Nitrososphaera viennensis EN76T. All four isolates are well separated by phenotypic and genotypic characteristics and are here assigned to distinct species within the genus Nitrosopumilus gen. nov. Isolates SCM1T (=ATCC TSD-97T =NCIMB 15022T), HCA1T (=ATCC TSD-96T), HCE1T (=ATCC TSD-98T), and PS0T (=ATCC TSD-99T) are type strains of the species Nitrosopumilusmaritimus sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., respectively. In addition, we propose the family Nitrosopumilaceae fam. nov. and the order Nitrosopumilales ord. nov. within the class Nitrososphaeria.

Marine ammonia-oxidizing archaeal isolates display obligate mixotrophy and wide ecotypic variation.

Ammonia-oxidizing archaea (AOA) are now implicated in exerting significant control over the form and availability of reactive nitrogen species in marine environments. Detailed studies of specific metabolic traits and physicochemical factors controlling their activities and distribution have not been well constrained in part due to the scarcity of isolated AOA strains. Here, we report the isolation of two new coastal marine AOA, strains PS0 and HCA1. Comparison of the new strains to Nitrosopumilus maritimus strain SCM1, the only marine AOA in pure culture thus far, demonstrated distinct adaptations to pH, salinity, organic carbon, temperature, and light. Strain PS0 sustained nearly 80% of ammonia oxidation activity at a pH as low as 5.9, indicating that coastal strains may be less sensitive to the ongoing reduction in ocean pH. Notably, the two novel isolates are obligate mixotrophs that rely on uptake and assimilation of organic carbon compounds, suggesting a direct coupling between chemolithotrophy and organic matter assimilation in marine food webs. All three isolates showed only minor photoinhibition at 15 µE ⋅ m(-2) ⋅ s(-1) and rapid recovery of ammonia oxidation in the dark, consistent with an AOA contribution to the primary nitrite maximum and the plausibility of a diurnal cycle of archaeal ammonia oxidation activity in the euphotic zone. Together, these findings highlight an unexpected adaptive capacity within closely related marine group I Archaea and provide new understanding of the physiological basis of the remarkable ecological success reflected by their generally high abundance in marine environments.

The production of nitric oxide by marine ammonia-oxidizing archaea and inhibition of archaeal ammonia oxidation by a nitric oxide scavenger.

Nitrification is a critical process for the balance of reduced and oxidized nitrogen pools in nature, linking mineralization to the nitrogen loss processes of denitrification and anammox. Recent studies indicate a significant contribution of ammonia-oxidizing archaea (AOA) to nitrification. However, quantification of the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to in situ ammonia oxidation remains challenging. We show here the production of nitric oxide (NO) by Nitrosopumilus maritimus SCM1. Activity of SCM1 was always associated with the release of NO with quasi-steady state concentrations between 0.05 and 0.08 µM. NO production and metabolic activity were inhibited by the nitrogen free radical scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Comparison of marine and terrestrial AOB strains with SCM1 and the recently isolated marine AOA strain HCA1 demonstrated a differential sensitivity of AOB and AOA to PTIO and allylthiourea (ATU). Similar to the investigated AOA strains, bulk water column nitrification at coastal and open ocean sites with sub-micromolar ammonia/ammonium concentrations was inhibited by PTIO and insensitive to ATU. These experiments support predictions from kinetic, molecular and biogeochemical studies, indicating that marine nitrification at low ammonia/ammonium concentrations is largely driven by archaea and suggest an important role of NO in the archaeal metabolism.

Nitrosospira lacus sp. nov., a psychrotolerant, ammonia-oxidizing bacterium from sandy lake sediment.

A Gram-negative, spiral-shaped, chemolithotrophic, ammonia-oxidizing bacterium, designated APG3(T), was isolated into pure culture from sandy lake sediment collected from Green Lake, Seattle, WA, USA. Phylogenetic analyses based on the 16S rRNA gene sequence showed that strain APG3(T) belongs to cluster 0 of the genus Nitrosospira, which is presently not represented by described species, with Nitrosospira multiformis (cluster 3) as the closest species with a validly published name (identity of 98.6 % to the type strain). Strain APG3(T) grew at 4 °C but could not grow at 35 °C, indicating that this bacterium is psychrotolerant. Remarkably, the strain was able to grow over a wide range of pH (pH 5-9), which was greater than the pH range of any studied ammonia-oxidizing bacteria in pure culture. The DNA G+C content of the APG3(T) genome is 53.5 %, which is similar to that of Nitrosospira multiformis ATCC 25196(T) (53.9 %) but higher than that of Nitrosomonas europaea ATCC 19718 (50.7 %) and Nitrosomonas eutropha C71 (48.5 %). The average nucleotide identity (ANI) calculated for the genomes of strain APG3(T) and Nitrosospira multiformis ATCC 25196(T) was 75.45 %, significantly lower than the value of 95 % ANI that corresponds to the 70 % species-level cut-off based on DNA-DNA hybridization. Overall polyphasic taxonomy study indicated that strain APG3(T) represents a novel species in the genus Nitrosospira, for which the name Nitrosospira lacus sp. nov. is proposed (type strain APG3(T) = NCIMB 14869(T) = LMG 27536(T) = ATCC BAA-2542(T)).

Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria.

The discovery of ammonia oxidation by mesophilic and thermophilic Crenarchaeota and the widespread distribution of these organisms in marine and terrestrial environments indicated an important role for them in the global nitrogen cycle. However, very little is known about their physiology or their contribution to nitrification. Here we report oligotrophic ammonia oxidation kinetics and cellular characteristics of the mesophilic crenarchaeon 'Candidatus Nitrosopumilus maritimus' strain SCM1. Unlike characterized ammonia-oxidizing bacteria, SCM1 is adapted to life under extreme nutrient limitation, sustaining high specific oxidation rates at ammonium concentrations found in open oceans. Its half-saturation constant (K(m) = 133 nM total ammonium) and substrate threshold (

Influence of freshwater discharge on the microbial degradation processes of dissolved organic nitrogen in a subtropical estuary.

River bacterioplankton communities, influenced by watershed usage, are responsible for water purification. Bacterioplankton may be critical in the degradation of dissolved organic nitrogen (DON), the major nitrogen pool in the Caloosahatchee River, Florida. We investigated how freshwater discharge influences estuarine bacterioplankton and how the freshwater-originated DON is utilized by estuarine bacterioplankton. Microcosm experiments were conducted during low and high discharge using two upstream freshwater samples: one site primarily influenced by Lake Okeechobee and the other site moderately influenced by an agricultural watershed. These freshwater samples were filtered to eliminate indigenous microbial populations, then mixed with estuarine bacterioplankton. High-throughput sequencing revealed that bacterioplankton differed between low and high discharge and were influenced by salinity. Alphaproteobacteria and Bacteroidetes dominated in low discharge while Bacteroidetes and Cyanobacteria dominated during high discharge. In the microcosm experiment, DON concentration decreased with increasing cell densities, suggesting that the DON was utilized as a carbon and nitrogen source. Band signals in denaturing gradient gel electrophoresis corresponding to Alphaproteobacteria and Actinobacteria decreased while Gammaproteobacteria increased during the 1 month incubation. This data suggests that estuarine bacterioplankton communities are influenced by variations in discharge patterns and use freshwater-originated DON as demonstrated by a shift in community structure.

Bacterial community shifts induced by high concentration hydrogen peroxide treatment of Microcystis bloom in a mesocosm study.

Hydrogen peroxide has gained popularity as an environmentally friendly treatment for cyanobacterial harmful algal blooms (cHABs) that takes advantage of oxidative stress sensitivity in cyanobacteria at controlled concentrations. Higher concentrations of hydrogen peroxide treatments may seem appealing for more severe cHABs but there is currently little understanding of the environmental impacts of this approach. Of specific concern is the associated microbial community, which may play key roles in the succession/recovery process post-treatment. To better understand impacts of a high concentration treatment on non-target microbial communities, we applied a hydrogen peroxide spray equating to a total volume concentration of 14 mM (473 mg/L, 0.04%) to 250 L mesocosms containing Microcystis bloom biomass, monitoring treatment and control mesocosms for 4 days. Cyanobacteria dominated control mesocosms throughout the experiment while treatment mesocosms experienced a 99% reduction, as determined by bacterial amplicon sequencing, and a 92% reduction in bacterial cell density within 1 day post-treatment. Only the bacterial community exhibited signs of regrowth, with a fold change of 9.2 bacterial cell density from day 1 to day 2. Recovery consisted of succession by Planctomycetota (47%) and Gammaproteobacteria (17%), which were likely resilient due to passive cell component compartmentalization and rapid upregulation of dnaK and groEL oxidative stress genes, respectively. The altered microbiome retained beneficial functionality of microcystin degradation through a currently recognized but unidentified pathway in Gammaproteobacteria, resulting in a 70% reduction coinciding with bacterial regrowth. There was also an 81% reduction of both total nitrogen and phosphorus, as compared to 91 and 93% in the control, respectively, due to high expressions of genes related to nitrogen (argH, carB, glts, glnA) and phosphorus (pntAB, phoB, pstSCB) cycling. Overall, we found a portion of the bacterial community was resilient to the high-concentration hydrogen peroxide treatment, resulting in Planctomycetota and Gammaproteobacteria dominance. This high-concentration treatment may be suitable to rapidly end cHABs which have already negatively impacted the aquatic environment rather than allow them to persist.

The synchronicity of bloom-forming cyanobacteria transcription patterns and hydrogen peroxide dynamics.

Hydrogen peroxide is a reactive oxygen species (ROS) naturally occurring at low levels in aquatic environments and production varies widely across different ecosystems. Oxygenic photosynthesis generates hydrogen peroxide as a byproduct, of which some portion can be released to ambient water. However, few studies have examined hydrogen peroxide dynamics in relation to cyanobacterial harmful algal blooms (cHABs). A year-long investigation of algal succession and hydrogen peroxide dynamics was conducted at the Caloosahatchee River, Florida, USA. We aimed to identify potential biological mechanisms responsible for elevated hydrogen peroxide production during cHAB events through the exploration of the freshwater microbial metatranscriptome. Hydrogen peroxide concentrations were elevated from February to September of 2021 when cyanobacteria were active and abundant. We observed one Microcystis cHAB event in spring and one in winter. Both had distinct nutrient uptake and cyanotoxin gene expression patterns. While meaningful levels of microcystin were only detected during periods of elevated hydrogen peroxide, cyanopeptolin was by far the most expressed cyanotoxin during the spring bloom when hydrogen peroxide was at its yearly maxima. Gene expressions of five microbial enzymes (Rubisco, superoxide dismutase, cytochrome b559, pyruvate oxidase, and NADH dehydrogenase) positively correlated to hydrogen peroxide concentrations. Additionally, there was higher nitrogen-fixing gene (nifDKH) expression by filamentous cyanobacteria after the spring bloom but no secondary bloom formation occurred. Overall, elevated environmental hydrogen peroxide concentrations were linked to cyanobacterial dominance and greater expression of specific enzymes in the photosynthesis of cyanobacteria. This implicates cyanobacterial photosynthesis and growth results in increased hydrogen peroxide generation as reflected in measured environmental concentrations.

Synechococcus dominance induced after hydrogen peroxide treatment of Microcystis bloom in the Caloosahatchee River, Florida.

Few field trials examining hydrogen peroxide as a cyanobacterial harmful algal bloom (cHAB) treatment have been conducted in subtropical and tropical regions. None have been tested in Florida, home to Lake Okeechobee and downstream waterways which periodically experience Microcystis bloom events. To investigate treatment effects in Florida, we applied a 490 µM (16.7 mg/L; 0.0015%) hydrogen peroxide spray to a minor bloom of Microcystis aeruginosa on the downstream side of Franklin Lock and Dam in the Caloosahatchee River. Although hydrogen peroxide decreased to background level one day post-treatment, succession was observed in phytoplankton community amplicon sequencing. The relative abundance of Microcystis decreased on day 3 by 86%, whereas the picocyanobacteria Synechococcus became dominant, increasing by 77% on day 3 and by 173% on day 14 to 57% of the phytoplankton community. Metatranscriptomics revealed Synechococcus likely benefitted from the antioxidant defense of upregulated peroxiredoxin, peroxidase/catalase, and rubrerythrin expressions immediately after treatment, and upregulated nitrate transport and urease to take advantage of available nitrogen. Our results indicated hydrogen peroxide induces succession of the phytoplankton community from Microcystis to non-toxic picocyanobacteria and could be used for selective suppression of harmful cyanobacteria.

Microbial diversity, genomics, and phage-host interactions of cyanobacterial harmful algal blooms.

The occurrence of cyanobacterial harmful algal blooms (cyanoHABs) is related to their physical and chemical environment. However, less is known about their associated microbial interactions and processes. In this study, cyanoHABs were analyzed as a microbial ecosystem, using 1 year of 16S rRNA sequencing and 70 metagenomes collected during the bloom season from Lake Okeechobee (Florida, USA). Biogeographical patterns observed in microbial community composition and function reflected ecological zones distinct in their physical and chemical parameters that resulted in bloom "hotspots" near major lake inflows. Changes in relative abundances of taxa within multiple phyla followed increasing bloom severity. Functional pathways that correlated with increasing bloom severity encoded organic nitrogen and phosphorus utilization, storage of nutrients, exchange of genetic material, phage defense, and protection against oxidative stress, suggesting that microbial interactions may promote cyanoHAB resilience. Cyanobacterial communities were highly diverse, with picocyanobacteria ubiquitous and oftentimes most abundant, especially in the absence of blooms. The identification of novel bloom-forming cyanobacteria and genomic comparisons indicated a functionally diverse cyanobacterial community with differences in its capability to store nitrogen using cyanophycin and to defend against phage using CRISPR and restriction-modification systems. Considering blooms in the context of a microbial ecosystem and their interactions in nature, physiologies and interactions supporting the proliferation and stability of cyanoHABs are proposed, including a role for phage infection of picocyanobacteria. This study displayed the power of "-omics" to reveal important biological processes that could support the effective management and prediction of cyanoHABs. IMPORTANCE: Cyanobacterial harmful algal blooms pose a significant threat to aquatic ecosystems and human health. Although physical and chemical conditions in aquatic systems that facilitate bloom development are well studied, there are fundamental gaps in the biological understanding of the microbial ecosystem that makes a cyanobacterial bloom. High-throughput sequencing was used to determine the drivers of cyanobacteria blooms in nature. Multiple functions and interactions important to consider in cyanobacterial bloom ecology were identified. The microbial biodiversity of blooms revealed microbial functions, genomic characteristics, and interactions between cyanobacterial populations that could be involved in bloom stability and more coherently define cyanobacteria blooms. Our results highlight the importance of considering cyanobacterial blooms as a microbial ecosystem to predict, prevent, and mitigate them.

Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences.

Ammonia-oxidizing microorganisms (AOM) contribute to one of the largest nitrogen fluxes in the global nitrogen budget. Four distinct lineages of AOM: ammonia-oxidizing archaea (AOA), beta- and gamma-proteobacterial ammonia-oxidizing bacteria (ß-AOB and γ-AOB) and complete ammonia oxidizers (comammox), are thought to compete for ammonia as their primary nitrogen substrate. In addition, many AOM species can utilize urea as an alternative energy and nitrogen source through hydrolysis to ammonia. How the coordination of ammonia and urea metabolism in AOM influences their ecology remains poorly understood. Here we use stable isotope tracing, kinetics and transcriptomics experiments to show that representatives of the AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct substrate competition. The tested AOA and comammox species preferentially used ammonia over urea, while ß-AOB favoured urea utilization, repressed ammonia transport in the presence of urea and showed higher affinity for urea than for ammonia. Characterized γ-AOB co-utilized both substrates. These results reveal contrasting niche adaptation and coexistence patterns among the major AOM lineages.

Interaction among spring phytoplankton succession, water discharge patterns, and hydrogen peroxide dynamics in the Caloosahatchee River in southwest Florida.

Phytoplankton communities are major primary producers in the aquatic realm and are responsible for shaping aquatic ecosystems. The dynamics of algal blooms could be determined by a succession of variable taxonomic groups, which are altered based on complex environmental factors such as nutrient availability and hydraulic factors. In-river structures potentially increase the occurrence of harmful algal blooms (HABs) by increasing water residence time and deteriorating water quality. How flowing water stimulates cell growth and affects the population dynamics of phytoplankton communities is a prioritized question that needs to be addressed for water management tactics. The goal of this study was to determine if an interaction between water flow and water chemistry is present, furthermore, to determine the relationship among phytoplankton community successions in the Caloosahatchee River, a subtropical river strongly influenced by human-controlled water discharge patterns from Lake Okeechobee. Particularly we focused on how phytoplankton community shifts influence the natural abundance of hydrogen peroxide, the most stable reactive oxygen species and a byproduct of oxidative photosynthesis. High-throughput amplicon sequencing using universal primers amplify 23S rRNA gene in cyanobacteria and eukaryotic algal plastids revealed that Synechococcus and Cyanobium were the dominant cyanobacterial genera and their relative abundance ranged between 19.5 and 95.3% of the whole community throughout the monitoring period. Their relative abundance declined when the water discharge increased. On the contrary, the relative abundance of eukaryotic algae sharply increased after water discharge increased. As water temperature increased in May, initially dominant Dolichospermum decreased as Microcystis increased. When Microcystis declined other filamentous cyanobacteria such as Geitlerinema, Pseudanabaena, and Prochlorothreix increased in their relative abundances. Interestingly, a peak of extracellular hydrogen peroxide was observed when Dolichospermum dominance was ended, and M. aeruginosa numbers increased. Overall, phytoplankton communities were strongly impacted by human-induced water discharge patterns.

QT-AMP: Sequencing PCR amplicons from Quanti-Tray wells to analyze enterococci communities.

Enterococcus is ubiquitous in human feces and has been adopted as a useful indicator of human fecal pollution in water. Although regular enterococci monitoring only examines their numbers, identifying human-specific Enterococcus species or genotypes could help discriminate human fecal contamination from other environmental sources. We documented a new approach to characterize enterococci using a high-throughput 16S rRNA gene amplicon sequencing platform from Quanti Trays after following the counting of the most probable numbers of enterococci. We named this method QT-AMP (Quanti-Tray-based amplicon sequencing). We tested surface water samples collected from three rivers in southwest Florida. We detected 11 Enterococcus species from 45 samples in 1.1 million sequence reads. The method detected three rare species and eight cosmopolitan species (Enterococcus faecalis, E. faecium, E. casseliflavus, E. hirae, E. mundtii, E. gallinarum, E. avium, and E. durans) which have been commonly documented in previous studies. The approximate detection level of QT-AMP was four orders of magnitude higher than regular 16S rRNA gene amplicon sequencing. The current Enterolert MPN method only provides quantitative information but now we can look into the relative abundance of Enterococci species composition by accompanying Illumina sequencing. This QT-AMP could be a useful tool to streamline the quantification and identification of enterococci and could be used in various water management projects and human health risk assessment.

Succession pattern and phylotype analysis of microphytobenthic communities in a simulated oil spill seagrass mesocosm experiment.

Microphytobenthic communities play a significant role in nutrient modulation, sediment stabilization, and primary production in seagrass beds, which provide various ecosystem services. We hypothesized that microphytobenthic communities in sediments of chronically oil-exposed seagrass beds will exhibit increased resiliency to stressors associated with oil exposure as opposed to seagrass beds never exposed to oil spills. We prepared 14-liter seawater mesocosms, each containing a submersed macrophyte Ruppia maritima collected from the Chandeleur Islands, Louisiana, and Estero Bay, Florida. Mesocosms were initially exposed to 50% water-accommodated oil fractions (WAF) and subsequently diluted by 50% with daily artificial seawater exchanges over 8 days to simulate tidal dilution. High-throughput amplicon sequencing based on 23S rRNA gene targeting cyanobacteria and chloroplasts of eukaryotic microphytobenthos was conducted to assess the impact of oiling on microphytobenthic communities with additional assessment via microscopy. High-throughput sequencing in combination with traditional microscopic analysis provided a robust examination in which both methods roughly complemented each other. Distinct succession patterns were detected in benthic algal communities of chronically oil-exposed (Louisiana) versus unexposed (Florida) seagrass bed sediments. The impact of oiling in microphytobenthos across all samples showed that benthic diatoms dominated all algal communities with sample percentages ranging from 42 to 97%, followed by cyanobacteria (2 to 50%). It is noteworthy that drastic changes in microphytobenthic community structure in terms of the larger taxonomic level were not observed, rather change occurred at the phylotype level. These results were also confirmed by microscopy. Similarity percentages (SIMPER) analysis identified seven phylotypes (Cyanobacteria, Bacillariophyceae, and Mediophyceae) in the Louisiana samples and one phylotype (Bacillariophyceae) in the Florida samples that increased in relative sequence abundance after oil exposure. The detailed phylotype analysis identifying sentinel microphytobenthic indicators provides a base for future research on benthic microalgae response to ecosystem disturbance.

High abundance of ammonia-oxidizing Archaea in coastal waters, determined using a modified DNA extraction method.

Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community.

Complete Genome Sequence of Microcystis aeruginosa FD4, Isolated from a Subtropical River in Southwest Florida.

We report the first complete genome of Microcystis aeruginosa from North America. A harmful bloom that occurred in the Caloosahatchee River in 2018 led to a state of emergency declaration in Florida. Although strain FD4 was isolated from this toxic bloom, the genome did not have a microcystin biosynthetic gene cluster.

Alternative strategies of nutrient acquisition and energy conservation map to the biogeography of marine ammonia-oxidizing archaea.

Ammonia-oxidizing archaea (AOA) are among the most abundant and ubiquitous microorganisms in the ocean, exerting primary control on nitrification and nitrogen oxides emission. Although united by a common physiology of chemoautotrophic growth on ammonia, a corresponding high genomic and habitat variability suggests tremendous adaptive capacity. Here, we compared 44 diverse AOA genomes, 37 from species cultivated from samples collected across diverse geographic locations and seven assembled from metagenomic sequences from the mesopelagic to hadopelagic zones of the deep ocean. Comparative analysis identified seven major marine AOA genotypic groups having gene content correlated with their distinctive biogeographies. Phosphorus and ammonia availabilities as well as hydrostatic pressure were identified as selective forces driving marine AOA genotypic and gene content variability in different oceanic regions. Notably, AOA methylphosphonate biosynthetic genes span diverse oceanic provinces, reinforcing their importance for methane production in the ocean. Together, our combined comparative physiological, genomic, and metagenomic analyses provide a comprehensive view of the biogeography of globally abundant AOA and their adaptive radiation into a vast range of marine and terrestrial habitats.