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Ocean warming endangers coastal ecosystems through increased risk of infectious disease, yet detection, surveillance, and forecasting of marine diseases remain limited. Eelgrass (Zostera marina) meadows provide essential coastal habitat and are vulnerable to a temperature-sensitive wasting disease caused by the protist Labyrinthula zosterae. We assessed wasting disease sensitivity to warming temperatures across a 3500 km study range by combining long-term satellite remote sensing of ocean temperature with field surveys from 32 meadows along the Pacific coast of North America in 2019. Between 11% and 99% of plants were infected in individual meadows, with up to 35% of plant tissue damaged. Disease prevalence was 3× higher in locations with warm temperature anomalies in summer, indicating that the risk of wasting disease will increase with climate warming throughout the geographic range for eelgrass. Large-scale surveys were made possible for the first time by the Eelgrass Lesion Image Segmentation Application, an artificial intelligence (AI) system that quantifies eelgrass wasting disease 5000× faster and with comparable accuracy to a human expert. This study highlights the value of AI in marine biological observing specifically for detecting widespread climate-driven disease outbreaks., This work was supported by the National Science Foundation (awards OCE-1829921, OCE-1829922, OCE-1829992, OCE-1829890). This is contribution 104 from the Smithsonian's MarineGEO and Tennenbaum Marine Observatories Network.

Oyster larvae reared in hatcheries on the U.S. West coast often experience severe Vibrio coralliilyticus-related mortalities early in their development. Current treatment options for these molluscs are either not available or feasible; however, for decades, probiotics have been successfully used in finfish and crustacean shellfish culture. Consequently, the objectives of this work were to 1) isolate marine bacteria from oysters and evaluate their protective activity against Vibrio coralliilyticus infection of Pacific oyster (Crassostrea gigas) larvae, and 2) to determine the long-term effects of probiotic additions on growth and metamorphosis of larval Pacific and Kumamoto oysters (C. sikamea). A combination of three probiotic strains applied once 24 hours post-fertilization was more effective in improving survival of larval C. gigas exposed to lethal concentrations of V. coralliilyticus strain RE22, compared with separate additions of individual probiotics. In addition, a single application of the probiotic combination to one-day-old larvae increased the larval metamorphosis success of C. sikamea and both the Midori and Myiagi stocks of C. gigas. These results suggest that probiotics are effective at preventing bacterial infections and can significantly improve performance of oyster larvae, using a single application early in their development.Highlights• A combination of marine bacteria improved survival of Pacific oyster larvae exposed to virulent V. coralliilyticus.• Metamorphosis was increased after adding a single dose of probiotics to one-day-old larvae.• Repetitive dosing after each water change was not superior to a single dose one day post egg-fertilization.• A single dose of the probiotic combination resulted in larger size on day 12.

Several genomes of Nostocales ADA clade members from the US Pacific Northwest were recently sequenced. Biosynthetic genes for microcystin, cylindrospermopsin or anatoxin-a were present in 7 of the 15 Dolichospermum/Anabaena strains and none of the 5 Aphanizomenon flos-aquae (AFA) strains. Toxin analyses (ELISA and LC-MS/MS) were conducted to quantitate and identify microcystin (MC) and cylindrospermopsin (CYN) congeners/analogs in samples dominated by Dolichospermum spp. of known genome sequence. MC-LR was the main congener produced by Dolichospermum spp. from Junipers Reservoir, Lake Billy Chinook and Odell Lake, while a congener provisionally identified as [Dha

Significance Marine dissolved organic matter, which originates from phytoplankton, holds as much carbon as Earth’s atmosphere; yet, the biological processes governing its fate are primarily studied under idealized laboratory conditions or through indirect measures such as genome sequencing. In this work, we used isotope labeling to directly quantify uptake of complex carbon pools from the two primary sources of marine organic carbon (diatoms and cyanobacteria) by a natural microbial community. Our data show that carbon pools are partitioned into distinct microbial lineages whose physiological properties and resource acquisition strategies match the chemical nature of their preferred substrates. Our results provide ecological and functional insights into the patterns of microbial community structure changes that occur during marine phytoplankton blooms., Blooms of marine phytoplankton fix complex pools of dissolved organic matter (DOM) that are thought to be partitioned among hundreds of heterotrophic microbes at the base of the food web. While the relationship between microbial consumers and phytoplankton DOM is a key component of marine carbon cycling, microbial loop metabolism is largely understood from model organisms and substrates. Here, we took an untargeted approach to measure and analyze partitioning of four distinct phytoplankton-derived DOM pools among heterotrophic populations in a natural microbial community using a combination of ecogenomics, stable isotope probing (SIP), and proteomics. Each 13C-labeled exudate or lysate from a diatom or a picocyanobacterium was preferentially assimilated by different heterotrophic taxa with specialized metabolic and physiological adaptations. Bacteroidetes populations, with their unique high-molecular-weight transporters, were superior competitors for DOM derived from diatom cell lysis, rapidly increasing growth rates and ribosomal protein expression to produce new relatively high C:N biomass. Proteobacteria responses varied, with relatively low levels of assimilation by Gammaproteobacteria populations, while copiotrophic Alphaproteobacteria such as the Roseobacter clade, with their diverse array of ABC- and TRAP-type transporters to scavenge monomers and nitrogen-rich metabolites, accounted for nearly all cyanobacteria exudate assimilation and produced new relatively low C:N biomass. Carbon assimilation rates calculated from SIP data show that exudate and lysate from two common marine phytoplankton are being used by taxonomically distinct sets of heterotrophic populations with unique metabolic adaptations, providing a deeper mechanistic understanding of consumer succession and carbon use during marine bloom events.*

Earth’s largest aquifer ecosystem resides in igneous oceanic crust, where chemosynthesis and water-rock reactions provide the carbon and energy that support an active deep biosphere. The Calvin Cycle is the predominant carbon fixation pathway in cool, oxic, crust; however, the energy and carbon metabolisms in the deep thermal basaltic aquifer are poorly understood. Anaerobic carbon fixation pathways such as the Wood-Ljungdahl pathway, which uses hydrogen (H(2)) and CO(2), may be common in thermal aquifers since water-rock reactions can produce H(2) in hydrothermal environments and bicarbonate is abundant in seawater. To test this, we reconstructed the metabolisms of eleven bacterial and archaeal metagenome-assembled genomes from an olivine biofilm obtained from a Juan de Fuca Ridge basaltic aquifer. We found that the dominant carbon fixation pathway was the Wood-Ljungdahl pathway, which was present in seven of the eight bacterial genomes. Anaerobic respiration appears to be driven by sulfate reduction, and one bacterial genome contained a complete nitrogen fixation pathway. This study reveals the potential pathways for carbon and energy flux in the deep anoxic thermal aquifer ecosystem, and suggests that ancient H(2)-based chemolithoautotrophy, which once dominated Earth’s early biosphere, may thus remain one of the dominant metabolisms in the suboceanic aquifer today.

A sample from a 2019 cyanobacterial bloom in a freshwater reservoir in eastern Oregon, USA, was used to produce a metagenome from which the complete, circular 7.3 Mbp genome of Limnoraphis sp. WC205 was assembled. The Limnoraphis sp. WC205 genome contains gas vesicle genes, genes for N

The ADA clade of Nostocales cyanobacteria, a group that is prominent in current harmful algal bloom events, now includes over 40 genome sequences with the recent addition of sixteen novel sequenced genomes (Dreher et al., Harmful Algae, 2021). Fourteen genomes are complete (closed), enabling highly detailed assessments of gene content and genome architecture. ADA genomes contain 5 rRNA operons, genes expected to support a photoautotrophic and diazotrophic lifestyle, and a varied array of genes for the synthesis of bioactive secondary metabolites. Genes for the production of the taste-and-odor compound geosmin and the four major classes of cyanotoxins — anatoxin-a, cylindrospermopsin, microcystin and saxitoxin — are represented in members of the ADA clade. Notably, the gene array for the synthesis of cylindrospermopsin by Dolichospermum sp. DET69 was located on a plasmid, raising the possibility of facile horizontal transmission. However, genes supporting independent conjugative transfer of this plasmid are lacking. Further, analysis of genomic loci containing this and other cyanotoxin gene arrays shows evidence that these arrays have long-term stability and do not appear to be genomic islands easily capable of horizontal transmission to other cells. There is considerable diversity in the gene complements of individual ADA genomes, including the variable presence of physiologically important genes: genomes in three species-level subclades lack the gas vesicle genes that facilitate a planktonic lifestyle, and, surprisingly, the genome of Cuspidothrix issatschenkoi CHARLIE-1, a reported diazotroph, lacks the genes for nitrogen fixation. Notably, phylogenetically related genomes possess limited synteny, indicating a prominent role for chromosome rearrangements during ADA strain evolution. The genomes contain abundant insertion sequences and repetitive transposase genes, which could be the main drivers of genome rearrangement through active transposition and homologous recombination. No prophages were found, and no evidence of viral infection was observed in the bloom population samples from which the genomes discussed here were derived. Phages thus seem to have a limited influence on ADA evolution.

Seagrasses can form mutualisms with their microbiomes that facilitate the exchange of energy sources, nutrients, and hormones and ultimately impact plant stress resistance. Little is known about community succession within the belowground seagrass microbiome after disturbance and its potential role in the plant’s recovery after transplantation. We transplanted Zostera marina shoots with and without an intact rhizosphere and cultivated plants for 4 weeks while characterizing microbiome recovery and effects on plant traits. Rhizosphere and root microbiomes were compositionally distinct, likely representing discrete microbial niches. Furthermore, microbiomes of washed transplants were initially different from those of sod transplants and recovered to resemble an undisturbed state within 14 days. Conspicuously, changes in the microbial communities of washed transplants corresponded with changes in the rhizosphere sediment mass and root biomass, highlighting the strength and responsive nature of the relationship between plants, their microbiome, and the environment. Potential mutualistic microbes that were enriched over time include those that function in the cycling and turnover of sulfur, nitrogen, and plant-derived carbon in the rhizosphere environment. These findings highlight the importance and resilience of the seagrass microbiome after disturbance. Consideration of the microbiome will have meaningful implications for habitat restoration practices. IMPORTANCE Seagrasses are important coastal species that are declining globally, and transplantation can be used to combat these declines. However, the bacterial communities associated with seagrass rhizospheres and roots (the microbiome) are often disturbed or removed completely prior to transplantation. The seagrass microbiome benefits seagrasses through metabolite, nutrient, and phytohormone exchange and contributes to the ecosystem services of seagrass meadows by cycling sulfur, nitrogen, and carbon. This experiment aimed to characterize the importance and resilience of the seagrass belowground microbiome by transplanting Zostera marina with and without intact rhizospheres and tracking microbiome and plant morphological recovery over 4 weeks. We found the seagrass microbiome to be resilient to transplantation disturbance, recovering after 14 days. Additionally, microbiome recovery was linked with seagrass morphology, coinciding with increases in the rhizosphere sediment mass and root biomass. The results of this study can be used to include microbiome responses in informing future restoration work.

Seagrasses are vital coastal ecosystem engineers, which are mutualistically associated with microbial communities that contribute to the ecosystem services provided by meadows. The seagrass microbiome and sediment microbiota play vital roles in belowground biogeochemical and carbon cycling. These activities are influenced by nutrient, carbon and oxygen availability, all of which are modulated by environmental factors and plant physiology. Seagrass meadows are increasingly threatened by nutrient pollution, and it is unknown how the seagrass microbiome will respond to this stressor. We investigated the effects of fertilization on the physiology, morphology and microbiome of eelgrass (Zostera marina) cultivated over 4 weeks in mesocosms. We analyzed the community structure associated with eelgrass leaf, root and rhizosphere microbiomes, and of communities from water column and bulk sediment using 16S rRNA amplicon sequencing. Fertilization led to a higher number of leaves compared with that of eelgrass kept under ambient conditions. Additionally, fertilization led to enrichment of sulfur and nitrogen bacteria in belowground communities. These results suggest nutrient enrichment can stimulate belowground biogeochemical cycling, potentially exacerbating sulfide toxicity in sediments and decreasing future carbon sequestration stocks., This work was supported by the Department of Microbiology at Oregon State University and the Salvador de Madariaga Program (PRX18/00124) (to F. Tomas).

Background Vibriosis has been implicated in major losses of larvae at shellfish hatcheries. However, the species of Vibrio responsible for disease in aquaculture settings and their associated virulence genes are often variable or undefined. Knowledge of the specific nature of these factors is essential to developing a better understanding of the environmental and biological conditions that lead to larvae mortality events in hatcheries. We tested the virulence of 51 Vibrio strains towards Pacific Oyster (Crassostreae gigas) larvae and sequenced draft genomes of 42 hatchery-associated vibrios to determine groups of orthologous genes associated with virulence and to determine the phylogenetic relationships among pathogens and non-pathogens of C. gigas larvae. Results V. coralliilyticus strains were the most prevalent pathogenic isolates. A phylogenetic logistic regression model identified over 500 protein-coding genes correlated with pathogenicity. Many of these genes had straightforward links to disease mechanisms, including predicted hemolysins, proteases, and multiple Type 3 Secretion System genes, while others appear to have possible indirect roles in pathogenesis and may be more important for general survival in the host environment. Multiple metabolism and nutrient acquisition genes were also identified to correlate with pathogenicity, highlighting specific features that may enable pathogen survival within C. gigas larvae. Conclusions These findings have important implications on the range of pathogenic Vibrio spp. found in oyster-rearing environments and the genetic determinants of virulence in these populations.

Seagrasses can form mutualisms with their microbiomes that facilitate the exchange of energy sources, nutrients, and hormones and ultimately impact plant stress resistance. Little is known about community succession within the belowground seagrass microbiome after disturbance and its potential role in the plant's recovery after transplantation. We transplanted Zostera marina shoots with and without an intact rhizosphere and cultivated plants for 4 weeks while characterizing microbiome recovery and effects on plant traits. Rhizosphere and root microbiomes were compositionally distinct, likely representing discrete microbial niches. Furthermore, microbiomes of washed transplants were initially different from those of sod transplants and recovered to resemble an undisturbed state within 14 days. Conspicuously, changes in the microbial communities of washed transplants corresponded with changes in the rhizosphere sediment mass and root biomass, highlighting the strength and responsive nature of the relationship between plants, their microbiome, and the environment. Potential mutualistic microbes that were enriched over time include those that function in the cycling and turnover of sulfur, nitrogen, and plant-derived carbon in the rhizosphere environment. These findings highlight the importance and resilience of the seagrass microbiome after disturbance. Consideration of the microbiome will have meaningful implications for habitat restoration practices.IMPORTANCE Seagrasses are important coastal species that are declining globally, and transplantation can be used to combat these declines. However, the bacterial communities associated with seagrass rhizospheres and roots (the microbiome) are often disturbed or removed completely prior to transplantation. The seagrass microbiome benefits seagrasses through metabolite, nutrient, and phytohormone exchange and contributes to the ecosystem services of seagrass meadows by cycling sulfur, nitrogen, and carbon. This experiment aimed to characterize the importance and resilience of the seagrass belowground microbiome by transplanting Zostera marina with and without intact rhizospheres and tracking microbiome and plant morphological recovery over 4 weeks. We found the seagrass microbiome to be resilient to transplantation disturbance, recovering after 14 days. Additionally, microbiome recovery was linked with seagrass morphology, coinciding with increases in the rhizosphere sediment mass and root biomass. The results of this study can be used to include microbiome responses in informing future restoration work., The authors acknowledges Oregon State University’s Summer Undergraduate Research Experience program, HMSC’s Mamie Markham Research Award, and the Joan Countryman Suit Scholarship for financial support.

Motivation Complex microbial communities can be characterized by metagenomics and metaproteomics. However, metagenome assemblies often generate enormous, and yet incomplete, protein databases, which undermines the identification of peptides and proteins in metaproteomics. This challenge calls for increased discrimination of true identifications from false identifications by database searching and filtering algorithms in metaproteomics. Results Sipros Ensemble was developed here for metaproteomics using an ensemble approach. Three diverse scoring functions from MyriMatch, Comet and the original Sipros were incorporated within a single database searching engine. Supervised classification with logistic regression was used to filter database searching results. Benchmarking with soil and marine microbial communities demonstrated a higher number of peptide and protein identifications by Sipros Ensemble than MyriMatch/Percolator, Comet/Percolator, MS-GF+/Percolator, Comet & MyriMatch/iProphet and Comet & MyriMatch & MS-GF+/iProphet. Sipros Ensemble was computationally efficient and scalable on supercomputers. Availability and implementation Freely available under the GNU GPL license at http://sipros.omicsbio.org. Supplementary information Supplementary data are available at Bioinformatics online.

Resource availability influences marine microbial community structure, suggesting that population-specific resource partitioning defines discrete niches. Identifying how resources are partitioned among populations, thereby characterizing functional guilds within the communities, remains a challenge for microbial ecologists. We used proteomic stable isotope probing (SIP) and NanoSIMS analysis of phylogenetic microarrays (Chip-SIP) along with 16S rRNA gene amplicon and metagenomic sequencing to characterize the assimilation of six 13C-labeled common metabolic substrates and changes in the microbial community structure within surface water collected from Monterey Bay, CA. Both sequencing approaches indicated distinct substrate-specific community shifts. However, observed changes in relative abundance for individual populations did not correlate well with directly measured substrate assimilation. The complementary SIP techniques identified assimilation of all six substrates by diverse taxa, but also revealed differential assimilation of substrates into protein and ribonucleotide biomass between taxa. Substrate assimilation trends indicated significantly conserved resource partitioning among populations within the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria classes, suggesting that functional guilds within marine microbial communities are phylogenetically cohesive. However, populations within these classes exhibited heterogeneity in biosynthetic activity, which distinguished high-activity copiotrophs from low-activity oligotrophs. These results indicate distinct growth responses between populations that is not apparent by genome sequencing alone.

The genome sequences of 16 Nostocales cyanobacteria have been determined. Most of them are complete or near-complete genome sequences derived by long-read metagenome sequencing of recent harmful algal blooms (HABs) in freshwater lakes without the potential bias of culture isolation. The genomes are all members of the recently recognized ADA clade (Driscoll et al., Harmful Algae, 77:93, 2018), which we argue represents a genus. We identify 10 putative species-level branches within the clade, on the basis of 91-gene phylogenomic and average nucleotide identity analyses. The assembled genomes each correspond to a single morphotype in the original sample, but distinct genomes from different HABs in some cases correspond to similar morphotypes. We present data indicating that the ADA clade is a highly significant component of current cyanobacterial HABs, including members assigned to the prevalent Dolichospermum and Aphanizomenon genera, as well as Cuspidothrix and Anabaena. In general, currently used genus and species names within the ADA clade are not monophyletic. We infer that the morphological characters routinely used in taxonomic assignments are not reliable for discriminating species within the ADA clade. Taxonomic revisions will be needed to create a genus with a single name (we recommend Anabaena) and to adopt species names that do not depend on morphological traits that lack sufficient discrimination and specificity, while recognizing the utility of some easily observable and distinct morphologies.

The Chloroflexi CL500-11 clade contributes a large proportion of the bacterial biomass in the oxygenated hypolimnia of deep lakes worldwide, including the world's largest freshwater system, the Laurentian Great Lakes. Traits that allow CL500-11 to thrive and its biogeochemical role in these environments are currently unknown. Here, we found that a CL500-11 population was present mostly in offshore waters along a transect in ultraoligotrophic Lake Michigan (a Laurentian Great Lake). It occurred throughout the water column in spring and only in the hypolimnion during summer stratification, contributing up to 18.1% of all cells. Genome reconstruction from metagenomic data suggested an aerobic, motile, heterotrophic lifestyle, with additional energy being gained through carboxidovory and methylovory. Comparisons to other available streamlined freshwater genomes revealed that the CL500-11 genome contained a disproportionate number of cell wall/capsule biosynthesis genes and the most diverse spectrum of genes involved in the uptake of dissolved organic matter (DOM) substrates, particularly peptides. In situ expression patterns indicated the importance of DOM uptake and protein/peptide turnover, as well as type I and type II carbon monoxide dehydrogenase and flagellar motility. Its location in the water column influenced its gene expression patterns the most. We observed increased bacteriorhodopsin gene expression and a response to oxidative stress in surface waters compared to its response in deep waters. While CL500-11 carries multiple adaptations to an oligotrophic lifestyle, its investment in motility, its large cell size, and its distribution in both oligotrophic and mesotrophic lakes indicate its ability to thrive under conditions where resources are more plentiful. Our data indicate that CL500-11 plays an important role in nitrogen-rich DOM mineralization in the extensive deep-lake hypolimnion habitat.

Terrestrial plants benefit from many well-understood mutualistic relationships with root- and leaf-associated microbiomes, but relatively little is known about these relationships for seagrass and other aquatic plants. We used 16S rRNA gene amplicon sequencing and metatranscriptomics to assess potential mutualisms between microorganisms and the seagrasses Zostera marina and Zostera japonica collected from mixed beds in Netarts Bay, OR, United States. The phylogenetic composition of leaf-, root-, and water column-associated bacterial communities were strikingly different, but these communities were not significantly different between plant species. Many taxa present on leaves were related to organisms capable of consuming the common plant metabolic waste product methanol, and of producing agarases, which can limit the growth of epiphytic algae. Taxa present on roots were related to organisms capable of oxidizing toxic sulfur compounds and of fixing nitrogen. Metatranscriptomic sequencing identified expression of genes involved in all of these microbial metabolic processes at levels greater than typical water column bacterioplankton, and also identified expression of genes involved in denitrification and in bacterial synthesis of the plant growth hormone indole-3-acetate. These results provide the first evidence using metatranscriptomics that seagrass microbiomes carry out a broad range of functions that may benefit their hosts, and imply that microbe–plant mutualisms support the health and growth of aquatic plants.

Reported here are the genome sequences of three Vibrio coralliilyticus isolates RE87, AIC-7, and 080116A. Each strain was isolated in association with oyster larvae in commercial aquaculture systems. These draft genomes will be useful for further studies in understanding the genomic features contributing to V. coralliilyticus pathogenicity.

An estimated 50 gigatons of carbon is annually fixed within marine systems, of which heterotrophic microbial populations process nearly half. These communities vary in composition and activity across spatial and temporal scales, so understanding how these changes affect global processes requires the delineation of functional roles for individual members. In a step toward ascertaining these roles, we applied proteomic stable isotope probing to quantify the assimilation of organic carbon from DFAAs into microbial protein biomass, since the turnover of DFAAs accounts for a substantial fraction of marine microbial carbon metabolism that is directed into biomass production. We conducted experiments at two coastal North Pacific locations and found taxonomically distinct responses. This approach allowed us to compare amino acid assimilation by specific bacterioplankton populations and characterize their allocation of this substrate among cellular functions., Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual primary production, yet defining how substrate utilization preferences and resource partitioning structure microbial communities remains a challenge. In this study, proteomic stable isotope probing (proteomic SIP) was used to characterize population-specific assimilation of dissolved free amino acids (DFAAs), a major source of dissolved organic carbon for bacterial secondary production in aquatic environments. Microcosms of seawater collected from Newport, Oregon, and Monterey Bay, California, were incubated with 1 µM 13C-labeled amino acids for 15 and 32 h. The taxonomic compositions of microcosm metaproteomes were highly similar to those of the sampled natural communities, with Rhodobacteriales, SAR11, and Flavobacteriales representing the dominant taxa. Analysis of 13C incorporation into protein biomass allowed for quantification of the isotopic enrichment of identified proteins and subsequent determination of differential amino acid assimilation patterns between specific bacterioplankton populations. Proteins associated with Rhodobacterales tended to have a significantly high frequency of 13C-enriched peptides, opposite the trend for Flavobacteriales and SAR11 proteins. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of 13C-enriched spectra at time point 2. Alteromonadales proteins also had a significantly high frequency of 13C-enriched peptides, particularly within ribosomal proteins, demonstrating their rapid growth during incubations. Overall, proteomic SIP facilitated quantitative comparisons of DFAA assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population level metabolic responses to resource acquisition in complex microbial communities. IMPORTANCE An estimated 50 gigatons of carbon is annually fixed within marine systems, of which heterotrophic microbial populations process nearly half. These communities vary in composition and activity across spatial and temporal scales, so understanding how these changes affect global processes requires the delineation of functional roles for individual members. In a step toward ascertaining these roles, we applied proteomic stable isotope probing to quantify the assimilation of organic carbon from DFAAs into microbial protein biomass, since the turnover of DFAAs accounts for a substantial fraction of marine microbial carbon metabolism that is directed into biomass production. We conducted experiments at two coastal North Pacific locations and found taxonomically distinct responses. This approach allowed us to compare amino acid assimilation by specific bacterioplankton populations and characterize their allocation of this substrate among cellular functions.

Archaea are widely distributed and yet are most often not the most abundant members of microbial communities. Here, we document a transition from Bacteria - to Archaea -dominated communities in microbial biofilms sampled from the Richmond Mine acid mine drainage (AMD) system (∼pH 1.0, ∼38°C) and in laboratory-cultivated biofilms. This transition occurs when chemoautotrophic microbial communities that develop at the air-solution interface sink to the sediment-solution interface and degrade under microaerobic and anaerobic conditions. The archaea identified in these sunken biofilms are from the class Thermoplasmata , and in some cases, the highly divergent ARMAN nanoarchaeal lineage. In several of the sunken biofilms, nanoarchaea comprise 10 to 25% of the community, based on fluorescent in situ hybridization and metagenomic analyses. Comparative community proteomic analyses show a persistence of bacterial proteins in sunken biofilms, but there is clear evidence for amino acid modifications due to acid hydrolysis. Given the low representation of bacterial cells in sunken biofilms based on microscopy, we infer that hydrolysis reflects proteins derived from lysed cells. For archaea, we detected ∼2,400 distinct proteins, including a subset involved in proteolysis and peptide uptake. Laboratory cultivation experiments using complex carbon substrates demonstrated anaerobic enrichment of Ferroplasma and Aplasma coupled to the reduction of ferric iron. These findings indicate dominance of acidophilic archaea in degrading biofilms and suggest that they play roles in anaerobic nutrient cycling at low pH.

Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added ∼20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as "hypothetical" were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO.

Proteomes of acid mine drainage biofilms at different stages of ecological succession were examined to understand microbial responses to changing community membership. We evaluated the degree of reproducibility of the community proteomes between samples of the same growth stage and found stable and predictable protein abundance patterns across time and sampling space, allowing for a set of 50 classifier proteins to be identified for use in predicting growth stages of undefined communities. Additionally, physiological changes in the dominant species, Leptospirillum Group II, were analysed as biofilms mature. During early growth stages, this population responds to abiotic stresses related to growth on the acid mine drainage solution. Enzymes involved in protein synthesis, cell division and utilization of 1- and 2-carbon compounds were more abundant in early growth stages, suggesting rapid growth and a reorganization of metabolism during biofilm initiation. As biofilms thicken and diversify, external stresses arise from competition for dwindling resources, which may inhibit cell division of Leptospirillum Group II through the SOS response. This population also represses translation and synthesizes more complex carbohydrates and amino acids in mature biofilms. These findings provide unprecedented insight into the physiological changes that may result from competitive interactions within communities in natural environments.

In nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the gram-negative bacterium Pseudomonas aeruginosa , an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C(12)-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C(12)-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in nature as well as significant human pathogens. The mechanism of action of C(12)-TA was also elucidated, and C(12)-TA was found to dissipate both the membrane potential and the pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C(12)-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C(12)-TA. Our results suggest that the QS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species and also to defend against host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C(12)-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics.

We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae ) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.

Indole has been proposed to act as an extracellular signal molecule influencing biofilm formation in a range of bacteria. For this study, the role of indole in Vibrio cholerae biofilm formation was examined. It was shown that indole activates genes involved in vibrio polysaccharide (VPS) production, which is essential for V. cholerae biofilm formation. In addition to activating these genes, it was determined using microarrays that indole influences the expression of many other genes, including those involved in motility, protozoan grazing resistance, iron utilization, and ion transport. A transposon mutagenesis screen revealed additional components of the indole-VPS regulatory circuitry. The indole signaling cascade includes the DksA protein along with known regulators of VPS production, VpsR and CdgA. A working model is presented in which global control of gene expression by indole is coordinated through 54 and associated transcriptional regulators. Bacterial cells synthesize myriad small organic molecules to signal and adapt to environmental, physiological, and population structure changes. These molecules include extracellular signals such as acyl homoserine lactones, butyrolactones, quinolones, a furanosyl borate diester, oligopeptides, 3-hydroxypalmitic acid methyl ester, and a hydroxyketone and intracellular signals including cyclic nucleotides and ppGpp (25; reviewed in reference 11). The phenotypic response to signaling compounds often involves traits that are beneficial under adverse conditions, such as biofilm formation, virulence, motility, bioluminescence, sporulation, competence, modification of carbon and energy utilization, and macromolecule biosynthesis.

Despite its notoriety as a human pathogen, Vibrio cholerae is an aquatic microbe suited to live in freshwater, estuarine, and marine environments where biofilm formation may provide a selective advantage. Here we report characterization of biofilms formed on abiotic and biotic surfaces by two non-O1/O139 V. cholerae strains, TP and SIO, and by the O1 V. cholerae strain N16961 in addition to the isolation of 44 transposon mutants of SIO and TP impaired in biofilm formation. During the course of characterizing the mutants, 30 loci which have not previously been associated with V. cholerae biofilms were identified. These loci code for proteins which perform a wide variety of functions, including amino acid metabolism, ion transport, and gene regulation. Also, when the plankton colonization abilities of strains N16961, SIO, and TP were examined, each strain showed increased colonization of dead plankton compared with colonization of live plankton (the dinoflagellate Lingulodinium polyedrum and the copepod Tigriopus californicus ). Surprisingly, most of the biofilm mutants were not impaired in plankton colonization. Only mutants impaired in motility or chemotaxis showed reduced colonization. These results indicate the presence of both conserved and variable genes which influence the surface colonization properties of different V. cholerae subspecies.

Background Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture. Results The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90. Conclusion Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2738-7) contains supplementary material, which is available to authorized users.

Heterotrophic microbes are critical components of aquatic food webs. Linkages between populations and the substrates they utilize are not well defined. We present the metagenome of microbial communities from the coastal Pacific Ocean exposed to various nutrient additions in order to better understand substrate utilization and partitioning in this environment.

Metaproteomic studies of whole microbial communities from environmental samples (e.g., soil, sediments, freshwater, seawater, etc.) have rapidly increased in recent years due to many technological advances in mass spectrometry (MS). A single 24-h liquid chromatograph-tandem mass spectrometry (LC-MS/MS) measurement can potentially detect and quantify thousands of proteins from many dominant and subdominant naturally occurring microbial populations. Importantly, amino acid sequences and relative abundance information for detected peptides are determined, which allows for the characterization of expressed protein functions within communities and specific matches to be made to microbial lineages, with potential subspecies resolution. Continued optimization of protein extraction and fractionation protocols, development of quantification methods, and advances in mass spectrometry instrumentation are enabling more accurate and comprehensive peptide detection within samples, leading to wider research applicability, greater ease of use, and overall accessibility. This chapter provides a brief overview of metaproteomics experimental options, including a general protocol for sample handling and LC-MS/MS measurement.

Metaproteomic studies of whole microbial communities from environmental samples (e.g., soil, sediments, freshwater, seawater, etc.) have rapidly increased in recent years due to many technological advances in mass spectrometry (MS). A single 24-h liquid chromatograph-tandem mass spectrometry (LC-MS/MS) measurement can potentially detect and quantify thousands of proteins from many dominant and subdominant naturally occurring microbial populations. Importantly, amino acid sequences and relative abundance information for detected peptides are determined, which allows for the characterization of expressed protein functions within communities and specific matches to be made to microbial lineages, with potential subspecies resolution. Continued optimization of protein extraction and fractionation protocols, development of quantification methods, and advances in mass spectrometry instrumentation are enabling more accurate and comprehensive peptide detection within samples, leading to wider research applicability, greater ease of use, and overall accessibility. This chapter provides a brief overview of metaproteomics experimental options, including a general protocol for sample handling and LC-MS/MS measurement.

Proteomes of acid mine drainage biofilms at different stages of ecological succession were examined to understand microbial responses to changing community membership. We evaluated the degree of reproducibility of the community proteomes between samples of the same growth stage and found stable and predictable protein abundance patterns across time and sampling space, allowing for a set of 50 classifier proteins to be identified for use in predicting growth stages of undefined communities. Additionally, physiological changes in the dominant species, Leptospirillum Group II, were analysed as biofilms mature. During early growth stages, this population responds to abiotic stresses related to growth on the acid mine drainage solution. Enzymes involved in protein synthesis, cell division and utilization of 1- and 2-carbon compounds were more abundant in early growth stages, suggesting rapid growth and a reorganization of metabolism during biofilm initiation. As biofilms thicken and diversify, external stresses arise from competition for dwindling resources, which may inhibit cell division of Leptospirillum Group II through the SOS response. This population also represses translation and synthesizes more complex carbohydrates and amino acids in mature biofilms. These findings provide unprecedented insight into the physiological changes that may result from competitive interactions within communities in natural environments.

Tandem high-throughput proteomics and metabolomics were employed to functionally characterize natural microbial biofilm communities. Distinct molecular signatures exist for each analyzed sample. Deconvolution of the high-resolution molecular data demonstrates that identified proteins and detected metabolites exhibit organism-specific correlation patterns. These patterns are reflective of the functional differentiation of two bacterial species that share the same genus and that co-occur in the sampled microbial communities. Our analyses indicate that the two species have similar niche breadths and are not in strong competition with one another., IMPORTANCE Natural microbial assemblages represent dynamic consortia that exhibit extensive complexity at all levels. In the present study, we demonstrate that correlations between protein and metabolite abundances allow the deconvolution of complex molecular data sets into shared and organism-specific contingents. We demonstrate that evolutionary divergence is associated with the restructuring of cellular metabolic networks, which in turn allows bacterial species to occupy distinct ecological niches. The apparent lack of interspecific competition may explain the extensive population-level genetic heterogeneity observed extensively within microbial communities. The reported findings have broad implications for the in-depth investigation of the ecology and evolution of distinct microbial community members and for leveraging the solution of cryptic metabolic processes in the future.