Change and stasis of distinct sediment microbiomes across Port Everglades Inlet (PEI) and the adjacent coral reefs

Deep water ports are human built coastal structures that by definition welcome ship traffic and disturbance. Evidence is accumulating that enhanced port activities such as dredging or deepening have negatively affected nearby natural habitats. Port Everglades Inlet (PEI) is a large active South Florida cargo port for over two million people and lies adjacent to coral reefs, dwindling mangroves, and recreational beaches. In this study, the microbial communities of PEI and adjacent reef sediments were characterized to serve as indicators for change due to dredging and assess anthropogenic influence on these sensitive ecosystems by sequencing the V4 region of 16S rRNA ahead of a large-scale port deepening event. For the first time, this study established baseline bacterial community characterizations and their patterns of diversity prior to and after a maintenance dredging event. PEI samples were collected for two consecutive years 2020 (Phase I, before maintenance dredging) and 2021 (Phase II, after maintenance dredging) from PEI sediments and adjacent coral reef sediments. In spite of their proximity and tidal connections through the PEI, reef and PEI sediment microbial communities were distinct. Changes in microbial diversity within the intracoastal waterway (ICW), a route for community exchange or transfers, were the greatest after maintenance dredging occurred. Microbial diversity in reef sediments also changed after dredging, indicating potential influence from resuspended sediments due to an associated increase in trace metals and decrease in cyanobacterial diversity. Sediments were identified as a possible source of human and coral pathogens, although dredging did not affect the relative abundances of these indicator microorganisms. This study highlighted the utility and relative ease of applying current molecular ecology methods to address macroscale questions with environmental management ramifications

Three complete mitochondrial genomes of shortfin mako sharks, Isurus oxyrinchus, from the Atlantic and Pacific Oceans

We present complete mitogenome sequences of three shortfin mako sharks (Isurus oxyrinchus) sampled from the western Pacific, and eastern and western Atlantic oceans. Mitogenome sequence lengths ranged between 16,699 bp and 16,702 bp, and all three mitogenomes contained one non-coding control region, two rRNA genes, 22 tRNA genes, and 13 protein-coding genes. Comparative assessment of five mitogenomes from globally distributed shortfin makos (the current three and two previously published mitogenomes) yielded 98.4% identity, with the protein-coding genes ATP8, ATP6, and ND5 as the most variable regions (sequence identities of 96.4%, 96.5%, and 97.6%, respectively). These mitogenome sequences contribute resources for assessing the genetic population dynamics of this endangered oceanic apex predator

Urea Is Both a Carbon and Nitrogen Source for Microcystis aeruginosa: Tracking 13C Incorporation at Bloom pH Conditions

The use of urea as a nitrogenous fertilizer has increased over the past two decades, with urea itself being readily detected at high concentrations in many lakes. Urea has been linked to cyanobacterial blooms as it is a readily assimilated nitrogen (N) - source for cyanobacteria that possess the enzyme urease. We tested the hypothesis that urea may also act as a carbon (C) source to supplemental growth requirements during the alkaline conditions created by dense cyanobacterial blooms, when concentrations of dissolved CO2 are vanishingly low. High rates of photosynthesis markedly reduce dissolved CO2 concentrations and drive up pH. This was observed in Lake Erie during the largest bloom on record (2015) over long periods (months) and short periods (days) of time, suggesting blooms experience periods of CO2-limitation on a seasonal and daily basis. We used 13C-urea to demonstrate that axenic cultures of the model toxic cyanobacterium, Microcystis aeruginosaNIES843, assimilated C at varying environmentally relevant pH conditions directly into a spectrum of metabolic pools during urea hydrolysis. Primarily, 13C from urea was assimilated into central C metabolism and amino acid biosynthesis pathways, including those important for the production of the hepatotoxin, microcystin, and incorporation into these pathways was at a higher percentage during growth at higher pH. This corresponded to increased growth rates on urea as the sole N source with increasing pH. We propose this ability to incorporate C from urea represents yet another competitive advantage for this cyanobacterium during dense algal blooms

Insight Into the Molecular Mechanisms for Microcystin Biodegradation in Lake Erie and Lake Taihu

Microcystins are potent hepatotoxins that are frequently detected in fresh water lakes plagued by toxic cyanobacteria. Microbial biodegradation has been referred to as the most important avenue for removal of microcystin from aquatic environments. The biochemical pathway most commonly associated with the degradation of microcystin is encoded by the mlrABCD (mlr) cassette. The ecological significance of this pathway remains unclear as no studies have examined the expression of these genes in natural environments. Six metatranscriptomes were generated from microcystin-producing Microcystis blooms and analyzed to assess the activity of this pathway in environmental samples. Seventy-eight samples were collected from Lake Erie, United States/Canada and Lake Tai (Taihu), China, and screened for the presence of mlr gene transcripts. Read mapping to the mlrcassette indicated transcripts for these genes were absent, with only 77 of the collective 3.7 billion reads mapping to any part of the mlr cassette. Analysis of the assembled metatranscriptomes supported this, with only distantly related sequences identified as mlrABC-like. These observations were made despite the presence of microcystin and over 500,000 reads mapping to the mcy cassette for microcystin production. Glutathione S-transferases and alkaline proteases have been previously hypothesized to be alternative pathways for microcystin biodegradation, and expression of these genes was detected across space and time in both lakes. While the activity of these alternative pathways needs to be experimentally confirmed, they may be individually or collectively more important than mlr genes in the natural environment. Importantly, the lack of mlr expression could indicate microcystin biodegradation was not occurring in the analyzed samples. This study raises interesting questions about the ubiquity, specificity and locality of microcystin biodegradation, and highlights the need for the characterization of relevant mechanisms in natural communities to understand the fate of microcystin in the environment and risk to public health

Molecular prediction of lytic vs lysogenic states for Microcystis phage: Metatranscriptomic evidence of lysogeny during large bloom events

Microcystis aeruginosa is a freshwater bloom-forming cyanobacterium capable of producing the potent hepatotoxin, microcystin. Despite increased interest in this organism, little is known about the viruses that infect it and drive nutrient mobilization and transfer of genetic material between organisms. The genomic complement of sequenced phage suggests these viruses are capable of integrating into the host genome, though this activity has not been observed in the laboratory. While analyzing RNA-sequence data obtained from Microcystis blooms in Lake Tai (Taihu, China), we observed that a series of lysogeny-associated genes were highly expressed when genes involved in lytic infection were down-regulated. This pattern was consistent, though not always statistically significant, across multiple spatial and temporally distinct samples. For example, samples from Lake Tai (2014) showed a predominance of lytic virus activity from late July through October, while genes associated with lysogeny were strongly expressed in the early months (June–July) and toward the end of bloom season (October). Analyses of whole phage genome expression shows that transcription patterns are shared across sampling locations and that genes consistently clustered by co-expression into lytic and lysogenic groups. Expression of lytic-cycle associated genes was positively correlated to total dissolved nitrogen, ammonium concentration, and salinity. Lysogeny-associated gene expression was positively correlated with pH and total dissolved phosphorous. Our results suggest that lysogeny may be prevalent in Microcystis blooms and support the hypothesis that environmental conditions drive switching between temperate and lytic life cycles during bloom proliferation

Change and stasis of distinct sediment microbiomes across Port Everglades Inlet (PEI) and the adjacent coral reefs

Deep water ports are human built coastal structures that by definition welcome ship traffic and disturbance. Evidence is accumulating that enhanced port activities such as dredging or deepening have negatively affected nearby natural habitats. Port Everglades Inlet (PEI) is a large active South Florida cargo port for over two million people and lies adjacent to coral reefs, dwindling mangroves, and recreational beaches. In this study, the microbial communities of PEI and adjacent reef sediments were characterized to serve as indicators for change due to dredging and assess anthropogenic influence on these sensitive ecosystems by sequencing the V4 region of 16S rRNA ahead of a large-scale port deepening event. For the first time, this study established baseline bacterial community characterizations and their patterns of diversity prior to and after a maintenance dredging event. PEI samples were collected for two consecutive years 2020 (Phase I, before maintenance dredging) and 2021 (Phase II, after maintenance dredging) from PEI sediments and adjacent coral reef sediments. In spite of their proximity and tidal connections through the PEI, reef and PEI sediment microbial communities were distinct. Changes in microbial diversity within the intracoastal waterway (ICW), a route for community exchange or transfers, were the greatest after maintenance dredging occurred. Microbial diversity in reef sediments also changed after dredging, indicating potential influence from resuspended sediments due to an associated increase in trace metals and decrease in cyanobacterial diversity. Sediments were identified as a possible source of human and coral pathogens, although dredging did not affect the relative abundances of these indicator microorganisms. This study highlighted the utility and relative ease of applying current molecular ecology methods to address macroscale questions with environmental management ramifications

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

Periodically Disturbing the Spatial Structure of Biofilms Can Affect the Production of an Essential Virulence Factor in Pseudomonas aeruginosa

Understanding the environmental factors that affect the production of virulence factors has major implications in evolution and medicine. While spatial structure is important in virulence factor production, observations of this relationship have occurred in undisturbed or continuously disturbed environments. However, natural environments are subject to periodic fluctuations, including changes in physical forces, which could alter the spatial structure of bacterial populations and impact virulence factor production. Using Pseudomonas aeruginosa PA14, we periodically applied a physical force to biofilms and examined production of pyoverdine. Intermediate frequencies of disturbance reduced the amount of pyoverdine produced compared to undisturbed or frequently disturbed conditions. To explore the generality of this finding, we examined how an intermediate disturbance frequency affected pyoverdine production in 21 different strains of P. aeruginosa. Periodic disturbance increased, decreased, or did not change the amount of pyoverdine produced relative to undisturbed populations. Mathematical modeling predicts that interactions between pyoverdine synthesis rate and biofilm density determine the amount of pyoverdine synthesized. When the pyoverdine synthesis rates are high, depletion of the biofilm due to disturbance reduces the accumulation of pyoverdine. At intermediate synthesis rates, production of pyoverdine increases during disturbance as bacteria dispersed into the planktonic state enjoy increased growth and pyoverdine production rates. At low synthesis rates, disturbance does not alter the amount of pyoverdine produced since disturbance-driven access to nutrients does not augment pyoverdine synthesis. Our results suggest that environmental conditions shape robustness in the production of virulence factors and may lead to novel approaches to treat infections

Urea Is Both a Carbon and Nitrogen Source for Microcystis aeruginosa: Tracking 13C Incorporation at Bloom pH Conditions

The use of urea as a nitrogenous fertilizer has increased over the past two decades, with urea itself being readily detected at high concentrations in many lakes. Urea has been linked to cyanobacterial blooms as it is a readily assimilated nitrogen (N) - source for cyanobacteria that possess the enzyme urease. We tested the hypothesis that urea may also act as a carbon (C) source to supplemental growth requirements during the alkaline conditions created by dense cyanobacterial blooms, when concentrations of dissolved CO2 are vanishingly low. High rates of photosynthesis markedly reduce dissolved CO2 concentrations and drive up pH. This was observed in Lake Erie during the largest bloom on record (2015) over long periods (months) and short periods (days) of time, suggesting blooms experience periods of CO2-limitation on a seasonal and daily basis. We used 13C-urea to demonstrate that axenic cultures of the model toxic cyanobacterium, Microcystis aeruginosa NIES843, assimilated C at varying environmentally relevant pH conditions directly into a spectrum of metabolic pools during urea hydrolysis. Primarily, 13C from urea was assimilated into central C metabolism and amino acid biosynthesis pathways, including those important for the production of the hepatotoxin, microcystin, and incorporation into these pathways was at a higher percentage during growth at higher pH. This corresponded to increased growth rates on urea as the sole N source with increasing pH. We propose this ability to incorporate C from urea represents yet another competitive advantage for this cyanobacterium during dense algal blooms

Metatranscriptomic Analyses of Diel Metabolic Functions During a Microcystis Bloom in Western Lake Erie (United States)

This study examined diel shifts in metabolic functions of spp. during a 48-h Lagrangian survey of a toxin-producing cyanobacterial bloom in western Lake Erie in the aftermath of the 2014 Toledo Water Crisis. Transcripts mapped to the genomes of recently sequenced lower Great Lakes isolates showed distinct patterns of gene expression between samples collected across day (10:00 h, 16:00 h) and night (22:00 h, 04:00 h). Daytime transcripts were enriched in functions related to Photosystem II (e.g., ), nitrogen and phosphate acquisition, cell division (), heat shock response (, ), and uptake of inorganic carbon (, ). Genes transcribed during nighttime included those involved in phycobilisome protein synthesis and Photosystem I core subunits. Hierarchical clustering and principal component analysis (PCA) showed a tightly clustered group of nighttime expressed genes, whereas daytime transcripts were separated from each other over the 48-h duration. Lack of uniform clustering within the daytime transcripts suggested that the partitioning of gene expression in is dependent on both circadian regulation and physicochemical changes within the environment