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Abstract Background Proteomic stable isotope probing (SIP) is used in microbial ecology to trace a non-radioactive isotope from a labeled substrate into de novo synthesized proteins in specific populations that are actively assimilating and metabolizing the substrate in a complex microbial community. The Sipros algorithm is used in proteomic SIP to identify variably labeled proteins and quantify their isotopic enrichment levels (atom%) by performing enrichment-resolved database searching. Results In this study, Sipros was upgraded to improve the labeled protein identification, isotopic enrichment quantification, and database searching speed. The new Sipros 4 was compared with the existing Sipros 3, Calisp, and MetaProSIP in terms of the number of identifications and the accuracy and precision of atom% quantification on both the peptide and protein levels using standard E. coli cultures with 1.07 atom%, 2 atom%, 5 atom%, 25 atom%, 50 atom%, and 99 atom% 13C enrichment. Sipros 4 outperformed Calisp and MetaProSIP across all samples, especially in samples with ≥ 5 atom% 13C labeling. The computational speed on Sipros 4 was > 20 times higher than Sipros 3 and was on par with the overall speed of Calisp- and MetaProSIP-based pipelines. Sipros 4 also demonstrated higher sensitivity for the detection of labeled proteins in two 13C-SIP experiments on a real-world soil community. The labeled proteins were used to trace 13C from 13C-methanol and 13C-labeled plant exudates to the consuming soil microorganisms and their newly synthesized proteins. Conclusion Overall, Sipros 4 improved the quality of the proteomic SIP results and reduced the computational cost of SIP database searching, which will make proteomic SIP more useful and accessible to the border community. Video Abstract

IntroductionThe culture of Pacific oysters (Crassostrea gigas) is of significant socio-economic importance in the U.S. Pacific Northwest and other temperate regions worldwide, with disease outbreaks acting as significant bottlenecks to the successful production of healthy seed larvae. Therefore, the current study aims to describe the mechanisms of a probiotic combination in improving the survival of C. gigas larvae. Specifically, we investigate changes in C. gigas larval gene expression in response to V. coralliilyticus infection with or without a pre-treatment of a novel probiotic combination.MethodsTreatment groups consisted of replicates of Pacific oyster larvae exposed to a) a combination of four probiotic bacteria at a total concentration of 3.0 x 105 CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 103 CFU/mL at 48 hpf, and c) the probiotic combination at 18 hpf and V. coralliilyticus RE22 at 48 hpf. RNA was extracted from washed larvae after 72 hpf, and transcriptome sequencing was used to identify significant differentially expressed genes (DEGs) within each treatment.ResultsLarvae challenged with V. coralliilyticus showed enhanced expression of genes responsible for inhibiting immune signaling (i.e., TNFAIP3, PSMD10) and inducing apoptosis (i.e., CDIP53). However, when pre-treated with the probiotic combination, these genes were no longer differentially expressed relative to untreated control larvae. Additionally, pre-treatment with the probiotic combination increased expression of immune signaling proteins and immune effectors (i.e., IL-17, MyD88). Apparent immunomodulation in response to probiotic treatment corresponds to an increase in the survival of C. gigas larvae infected with V. coralliilyticus by up to 82%.DiscussionThese results indicate that infection with V. coralliilyticus can suppress the larval immune response while also prompting cell death. Furthermore, the results suggest that the probiotic combination treatment negates the deleterious effects of V. coralliilyticus on larval gene expression while stimulating the expression of genes involved in infection defense mechanisms.

Here, by characterizing gut metagenomes of at-risk children in the TEDDY project, the authors associate onset of autoimmunity leading to Type-1 diabetes with certain sets of microorganisms in the gut microbiota, and identify metabolic capabilities encoded in the genomes of these microorganisms that provide functional insights to the association.

Oyster aquaculture is a growing industry that depends on production of fast-growing, healthy larvae and juveniles (spat) to be sold to farmers. Despite nearly identical genetics and environmental conditions in the early life stages of oysters, larvae and spat sizes can vary drastically. As the microbiome can influence the health and size of marine invertebrates, we analyzed the microbiomes of differently-sized juvenile Pacific oyster (Crassostrea gigas) spat of the same age to examine the relationship of their microbiomes with size variation. We used 16S sequencing of 128 animals (n = 60 large, n = 68 small) to characterize the microbiomes of each size class, comparing alpha diversity, beta diversity, and differentially abundant taxa between size classes. We observed that small spat had higher alpha diversity using measures that considered only richness, but there was no difference in alpha diversity between the two size classes using measures that incorporate compositional metrics. Additionally, large and small spat had distinct microbiomes, the separation of which was driven by more dominant bacterial taxa. Taxa that were differentially abundant in large oysters were also more abundant overall, and many appear to have roles in nutrient absorption and energy acquisition. The results of this study provide insight into how the microbiome of C. gigas may affect the early development of the animal, which can inform hatchery and nursery practices.

Abstract Declines in eelgrass, an important and widespread coastal habitat, are associated with wasting disease in recent outbreaks on the Pacific coast of North America. This study presents a novel method for mapping and predicting wasting disease using Unoccupied Aerial Vehicle (UAV) with low‐altitude autonomous imaging of visible bands. We conducted UAV mapping and sampling in intertidal eelgrass beds across multiple sites in Alaska, British Columbia, and California. We designed and implemented a UAV low‐altitude mapping protocol to detect disease prevalence and validated against in situ results. Our analysis revealed that green leaf area index derived from UAV imagery was a strong and significant (inverse) predictor of spatial distribution and severity of wasting disease measured on the ground, especially for regions with extensive disease infection. This study highlights a novel, efficient, and portable method to investigate seagrass disease at landscape scales across geographic regions and conditions.

ABSTRACT Predicting outcomes of marine disease outbreaks presents a challenge in the face of both global and local stressors. Host-associated microbiomes may play important roles in disease dynamics but remain understudied in marine ecosystems. Host–pathogen–microbiome interactions can vary across host ranges, gradients of disease, and temperature; studying these relationships may aid our ability to forecast disease dynamics. Eelgrass, Zostera marina, is impacted by outbreaks of wasting disease caused by the opportunistic pathogen Labyrinthula zosterae. We investigated how Z. marina phyllosphere microbial communities vary with rising wasting disease lesion prevalence and severity relative to plant and meadow characteristics like shoot density, longest leaf length, and temperature across 23° latitude in the Northeastern Pacific. We detected effects of geography (11%) and smaller, but distinct, effects of temperature (30-day max sea surface temperature, 4%) and disease (lesion prevalence, 3%) on microbiome composition. Declines in alpha diversity on asymptomatic tissue occurred with rising wasting disease prevalence within meadows. However, no change in microbiome variability (dispersion) was detected between asymptomatic and symptomatic tissues. Further, we identified members of Cellvibrionaceae, Colwelliaceae, and Granulosicoccaceae on asymptomatic tissue that are predictive of wasting disease prevalence across the geographic range (3,100 kilometers). Functional roles of Colwelliaceae and Granulosicoccaceae are not known. Cellvibrionaceae, degraders of plant cellulose, were also enriched in lesions and adjacent green tissue relative to nonlesioned leaves. Cellvibrionaceae may play important roles in disease progression by degrading host tissues or overwhelming plant immune responses. Thus, inclusion of microbiomes in wasting disease studies may improve our ability to understand variable rates of infection, disease progression, and plant survival. IMPORTANCE The roles of marine microbiomes in disease remain poorly understood due, in part, to the challenging nature of sampling at appropriate spatiotemporal scales and across natural gradients of disease throughout host ranges. This is especially true for marine vascular plants like eelgrass (Zostera marina) that are vital for ecosystem function and biodiversity but are susceptible to rapid decline and die-off from pathogens like eukaryotic slime-mold Labyrinthula zosterae (wasting disease). We link bacterial members of phyllosphere tissues to the prevalence of wasting disease across the broadest geographic range to date for a marine plant microbiome-disease study (3,100 km). We identify Cellvibrionaceae, plant cell wall degraders, enriched (up to 61% relative abundance) within lesion tissue, which suggests this group may be playing important roles in disease progression. These findings suggest inclusion of microbiomes in marine disease studies will improve our ability to predict ecological outcomes of infection across variable landscapes spanning thousands of kilometers.

Abstract 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.

ABSTRACT A growing body of research has established that the microbiome can mediate the dynamics and functional capacities of diverse biological systems. Yet, we understand little about what governs the response of these microbial communities to host or environmental changes. Most efforts to model microbiomes focus on defining the relationships between the microbiome, host, and environmental features within a specified study system and therefore fail to capture those that may be evident across multiple systems. In parallel with these developments in microbiome research, computer scientists have developed a variety of machine learning tools that can identify subtle, but informative, patterns from complex data. Here, we recommend using deep transfer learning to resolve microbiome patterns that transcend study systems. By leveraging diverse public data sets in an unsupervised way, such models can learn contextual relationships between features and build on those patterns to perform subsequent tasks (e.g., classification) within specific biological contexts.

Eukaryotic viruses and bacteriophage have been implicated in disease and bleaching in corals, but the compositional and functional diversity of these viruses in healthy and compromised hosts remains underexplored. To investigate whether viral assemblages differ in concert with coral bleaching, we collected bleached and non-bleached conspecific pairs of corals during a minor bleaching event in 2016 from reefs on the island of Mo’orea, French Polynesia. Using electron microscopy (EM), we identified several viral particle types, all reminiscent of medium to large-sized nucleocytoplasmic large DNA viruses (NCLDV). We also found that viral metagenomes from bleached corals have significantly more eukaryotic virus sequences, whereas bacteriophage sequences are significantly more abundant in viral metagenomes from non-bleached colonies. In this study, we also initiated the assembly of the first eukaryotic dsDNA coral virus genome. Based on our EM imagery and our taxonomic annotations of viral metagenome sequences, we hypothesize that this genome represents a novel, phylogenetically distinct member of the NCLDVs, with its closest sequenced relative being a distant marine flagellate-associated virus. We also showed that this NCLDV is abundant in bleached corals, but it is also present in apparently healthy corals, suggesting it plays a role in the onset and/or severity of coral bleaching.

Proteinaceous compounds are abundant forms of organic nitrogen in soil and aquatic ecosystems, and the rate of protein depolymerization, which is accomplished by a diverse range of microbial secreted peptidases, often limits nitrogen turnover in the environment. To determine if the distribution of secreted peptidases reflects the ecological and evolutionary histories of different taxa, we analyzed their distribution across prokaryotic lineages. Peptidase gene sequences of 147 archaeal and 2,191 bacterial genomes from the MEROPS database were screened for secretion signals, resulting in 55,072 secreted peptidases belonging to 148 peptidase families. These data, along with their corresponding 16S rRNA sequences, were used in our analysis. Overall, Bacteria had a much wider collection of secreted peptidases, higher average numbers of secreted peptidases per genome, and more unique peptidase families than Archaea. We found that the distribution of secreted peptidases corresponded to phylogenetic relationships among Bacteria and Archaea and often segregated according to microbial lifestyles, suggesting that the secreted peptidase complements of microbial taxa are optimized for the environmental microhabitats they occupy. Our analyses provide the groundwork for examining the specific functional role of families of secreted peptidases in relationship to the organisms and the corresponding environments in which they function.

Thirty-two 14-month old steers died during a period of four days (19–23 June 2017) after drinking from Junipers Reservoir (southeastern Oregon, USA) during a cyanobacterial bloom. Clinical and histopathological findings were consistent with acute liver disease, and microcystin-LR was present at 3000 μg/L in a reservoir water sample and at 7100 μg/L in the rumen contents of one of the mortalities. Serum biochemistry and histological examination indicated severe liver damage consistent with microcystin toxicosis. Microscopic observation of reservoir water samples, limited to frozen or poorly stored and partially degraded samples, indicated the presence of abundant Anabaena/Dolichospermum, but the presence of other toxic cyanobacteria such as Microcystis could not be excluded. Metagenomic analysis showed the presence in these samples of a single cyanobacterium whose cpcBA, rpoB and rbcL genes indicated membership in the Anabaena/Dolichospermum genus. The sequence of a complete mcy gene cluster with homology to previously identified Anabaena mcy genes was recovered. These results emphasize the capacity for Anabaena/Dolichospermum blooms to produce lethal levels of microcystin, posing a danger to public health and livestock. Further, our findings indicate that such occurrences can occur outside the far-northern latitudes in which microcystin-producing Anabaena have typically been found. Keywords: Anabaena sp. JUN03, Cyanobacteria, Cyanotoxin, Cattle mortalities, Microcystin, Mcy genes

Linking microbial community structure to ecological processes requires understanding of the functional roles among individual populations and the factors that influence their distributions. These structure–function relationships are particularly difficult to disentangle in estuaries, due to highly variable physico-chemical conditions. Yet, examining microbe-mediated turnover of resources in these “bioreactor” ecosystems is critical for understanding estuarine ecology. In this study, a combined metagenomics and metaproteomics approach was used to show that the unequal distribution of microbial populations across the Yaquina Bay estuary led to a habitat-specific taxonomic and functional structure and a clear spatial distribution in microbe-mediated capacities for cycling of carbon and nitrogen. For example, size-fractionation revealed that communities inhabiting suspended particulate material encoded more diverse types of metabolisms (e.g., fermentation and denitrification) than those with a planktonic lifestyle, suggesting that the metabolic reactions can differ between size fractions of the same parcel of an estuarine water column. Similarly, communities inhabiting oligotrophic conditions in the lower estuary were enriched in genes involved in central carbon metabolism (e.g., TCA cycle), while communities in the upper estuary were enriched in genes typical of copiotrophic populations (e.g., cell growth, cell division). Integrating gene and protein data revealed that abundant populations of Flavobacteriales and Rhodobacterales encoded similar genomic functions, yet differed significantly in protein expression, dedicating a large proportion of their respective proteomes to rapid growth and division versus metabolic versatility and resource acquisition. This suggested potentially distinct life-strategies between these two co-occurring lineages and was concomitant with differing patterns of positive evolutionary selection on their encoded genes. Microbial communities and their functions across Yaquina Bay appear to be structured by population-level habitat preferences, resulting in spatially distinct elemental cycling, while within each community, forces such as competitive exclusion and evolutionary selection influence species life-strategies and may help maintain microbial diversity.

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.

ABSTRACT 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.

While there has been growing interest in the gut microbiome in recent years, it remains unclear whether closely related species and strains have similar or distinct functional roles and if organisms capable of both aerobic and anaerobic growth do so simultaneously. To investigate these questions, we implemented a high-throughput mass spectrometry-based proteomics approach to identify proteins in fecal samples collected on days of life 13-21 from an infant born at 28 weeks gestation. No prior studies have coupled strain-resolved community metagenomics to proteomics for such a purpose. Sequences were manually curated to resolve the genomes of two strains of Citrobacter that were present during the later stage of colonization. Proteome extracts from fecal samples were processed via a nano-2D-LC-MS/MS and peptides were identified based on information predicted from the genome sequences for the dominant organisms, Serratia and the two Citrobacter strains. These organisms are facultative anaerobes, and proteomic information indicates the utilization of both aerobic and anaerobic metabolisms throughout the time series. This may indicate growth in distinct niches within the gastrointestinal tract. We uncovered differences in the physiology of coexisting Citrobacter strains, including differences in motility and chemotaxis functions. Additionally, for both Citrobacter strains we resolved a community-essential role in vitamin metabolism and a predominant role in propionate production. Finally, in this case study we detected differences between genome abundance and activity levels for the dominant populations. This underlines the value in layering proteomic information over genetic potential.

ABSTRACT 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.

Abstract An important challenge in microbial ecology is developing methods that simultaneously examine the physiology of organisms at the molecular level and their ecosystem level interactions in complex natural systems. We integrated extensive proteomic, geochemical, and biological information from 28 microbial communities collected from an acid mine drainage environment and representing a range of biofilm development stages and geochemical conditions to evaluate how the physiologies of the dominant and less abundant organisms change along environmental gradients. The initial colonist dominates across all environments, but its proteome changes between two stable states as communities diversify, implying that interspecies interactions affect this organism's metabolism. Its overall physiology is robust to abiotic environmental factors, but strong correlations exist between these factors and certain subsets of proteins, possibly accounting for its wide environmental distribution. Lower abundance populations are patchier in their distribution, and proteomic data indicate that their environmental niches may be constrained by specific sets of abiotic environmental factors. This research establishes an effective strategy to investigate ecological relationships between microbial physiology and the environment for whole communities in situ.