Phylogenetics and environmental distribution of nitric oxide-forming nitrite reductases reveal their distinct functional and ecological roles.

The two evolutionarily unrelated nitric oxide-producing nitrite reductases, NirK and NirS, are best known for their redundant role in denitrification. They are also often found in organisms that do not perform denitrification. To assess the functional roles of the two enzymes and to address the sequence and structural variation within each, we reconstructed robust phylogenies of both proteins with sequences recovered from 6973 isolate and metagenome-assembled genomes and identified 32 well-supported clades of structurally distinct protein lineages. We then inferred the potential niche of each clade by considering other functional genes of the organisms carrying them as well as the relative abundances of each nir gene in 4082 environmental metagenomes across diverse aquatic, terrestrial, host-associated, and engineered biomes. We demonstrate that Nir phylogenies recapitulate ecology distinctly from the corresponding organismal phylogeny. While some clades of the nitrite reductase were equally prevalent across biomes, others had more restricted ranges. Nitrifiers make up a sizeable proportion of the nitrite-reducing community, especially for NirK in marine waters and dry soils. Furthermore, the two reductases showed distinct associations with genes involved in oxidizing and reducing other compounds, indicating that the NirS and NirK activities may be linked to different elemental cycles. Accordingly, the relative abundance and diversity of NirS versus NirK vary between biomes. Our results show the divergent ecological roles NirK and NirS-encoding organisms may play in the environment and provide a phylogenetic framework to distinguish the traits associated with organisms encoding the different lineages of nitrite reductases.

High diversity, abundance, and expression of hydrogenases in groundwater.

Hydrogen may be the most important electron donor available in the subsurface. Here we analyse the diversity, abundance and expression of hydrogenases in 5 proteomes, 25 metagenomes, and 265 amplicon datasets of groundwaters with diverse geochemistry. A total of 1545 new [NiFe]-hydrogenase gene sequences were recovered, which considerably increased the number of sequences (1999) in a widely used database. [NiFe]-hydrogenases were highly abundant, as abundant as the DNA-directed RNA polymerase. The abundance of hydrogenase genes increased with depth from 0 to 129 m. Hydrogenases were present in 481 out of 1245 metagenome-assembled genomes. The relative abundance of microbes with hydrogenases accounted for ~50% of the entire community. Hydrogenases were actively expressed, making up as much as 5.9% of methanogen proteomes. Most of the newly discovered diversity of hydrogenases was in "Group 3b", which has been associated with sulfur metabolism. "Group 3d", facilitating the interconversion of electrons between hydrogen and NAD, was the most abundant and mainly observed in methanotrophs and chemoautotrophs. "Group 3a", associated with methanogenesis, was the most abundant in proteomes. Two newly discovered groups of [NiFe]-hydrogenases, observed in Methanobacteriaceae and Anaerolineaceae, further expanded diversity. Our results highlight the vast diversity, abundance and expression of hydrogenases in groundwaters, suggesting a high potential for hydrogen oxidation in subsurface habitats.

Coordinated proteome change precedes cell lysis and death in a mat-forming cyanobacterium.

Cyanobacteria form dense multicellular communities that experience transient conditions in terms of access to light and oxygen. These systems are productive but also undergo substantial biomass turnover through cell death, supplementing heightened heterotrophic respiration. Here we use metagenomics and metaproteomics to survey the molecular response of a mat-forming cyanobacterium undergoing mass cell lysis after exposure to dark and anoxic conditions. A lack of evidence for viral, bacterial, or eukaryotic antagonism contradicts commonly held beliefs on the causative agent for cyanobacterial death during dense growth. Instead, proteogenomics data indicated that lysis likely resulted from a genetically programmed response triggered by a failure to maintain osmotic pressure in the wake of severe energy limitation. Cyanobacterial DNA was rapidly degraded, yet cyanobacterial proteins remained abundant. A subset of proteins, including enzymes involved in amino acid metabolism, peptidases, toxin-antitoxin systems, and a potentially self-targeting CRISPR-Cas system, were upregulated upon lysis, indicating possible involvement in the programmed cell death response. We propose this natural form of cell death could provide new pathways for controlling harmful algal blooms and for sustainable bioproduct production.

Viral diversity and dynamics and CRISPR-Cas-mediated immunity in a robust alkaliphilic cyanobacterial consortium.

IMPORTANCE: Biotechnology applications utilizing the function of microbial communities have become increasingly important solutions as we strive for sustainable applications. Although viral infections are known to have a significant impact on microbial turnover and nutrient cycling, viral dynamics have remained largely overlooked in these engineered communities. Predatory perturbations to the functional stability of these microbial biotechnology applications must be investigated in order to design more robust applications. In this study, we closely examine virus-microbe dynamics in a model microbial community used in a biotechnology application. Our findings suggest that viral dynamics change significantly with environmental conditions and that microbial immunity may play an important role in maintaining functional stability. We present this study as a comprehensive template for other researchers interested in exploring predatory dynamics in engineered microbial communities.

Frequency of change determines effectiveness of microbial response strategies.

Nature challenges microbes with change at different frequencies and demands an effective response for survival. Here, we used controlled laboratory experiments to investigate the effectiveness of different response strategies, such as post-translational modification, transcriptional regulation, and specialized versus adaptable metabolisms. For this, we inoculated replicated chemostats with an enrichment culture obtained from sulfidic stream microbiomes 16 weeks prior. The chemostats were submitted to alternatingly oxic and anoxic conditions at three frequencies, with periods of 1, 4 and 16 days. The microbial response was recorded with 16S rRNA gene amplicon sequencing, shotgun metagenomics, transcriptomics and proteomics. Metagenomics resolved provisional genomes of all abundant bacterial populations, mainly affiliated with Proteobacteria and Bacteroidetes. Almost all these populations maintained a steady growth rate under both redox conditions at all three frequencies of change. Our results supported three conclusions: (1) Oscillating oxic/anoxic conditions selected for generalistic species, rather than species specializing in only a single condition. (2) A high frequency of change selected for strong codon usage bias. (3) Alignment of transcriptomes and proteomes required multiple generations and was dependent on a low frequency of change.

Hydrogen and dark oxygen drive microbial productivity in diverse groundwater ecosystems.

Around 50% of humankind relies on groundwater as a source of drinking water. Here we investigate the age, geochemistry, and microbiology of 138 groundwater samples from 95 monitoring wells (<250 m depth) located in 14 aquifers in Canada. The geochemistry and microbiology show consistent trends suggesting large-scale aerobic and anaerobic hydrogen, methane, nitrogen, and sulfur cycling carried out by diverse microbial communities. Older groundwaters, especially in aquifers with organic carbon-rich strata, contain on average more cells (up to 1.4 × 107 mL-1) than younger groundwaters, challenging current estimates of subsurface cell abundances. We observe substantial concentrations of dissolved oxygen (0.52 ± 0.12 mg L-1 [mean ± SE]; n = 57) in older groundwaters that seem to support aerobic metabolisms in subsurface ecosystems at an unprecedented scale. Metagenomics, oxygen isotope analyses and mixing models indicate that dark oxygen is produced in situ via microbial dismutation. We show that ancient groundwaters sustain productive communities and highlight an overlooked oxygen source in present and past subsurface ecosystems of Earth.

Autofermentation of alkaline cyanobacterial biomass to enable biorefinery approach.

BACKGROUND: Carbon capture using alkaliphilic cyanobacteria can be an energy-efficient and environmentally friendly process for producing bioenergy and bioproducts. The inefficiency of current harvesting and downstream processes, however, hinders large-scale feasibility. The high alkalinity of the biomass also introduces extra challenges, such as potential corrosion, inhibitory effects, or contamination of the final products. Thus, it is critical to identify low cost and energy-efficient downstream processes. RESULTS: Autofermentation was investigated as an energy-efficient and low-cost biomass pre-treatment method to reduce pH to levels suitable for downstream processes, enabling the conversion of cyanobacterial biomass into hydrogen and organic acids using cyanobacteria's own fermentative pathways. Temperature, initial biomass concentration, and oxygen presence were found to affect yield and distribution of organic acids. Autofermentation of alkaline cyanobacterial biomass was found to be a viable approach to produce hydrogen and organic acids simultaneously, while enabling the successful conversion of biomass to biogas. Between 5.8 and 60% of the initial carbon was converted into organic acids, 8.7-25% was obtained as soluble protein, and 16-72% stayed in the biomass. Interestingly, we found that extensive dewatering is not needed to effectively process the alkaline cyanobacterial biomass. Using natural settling as the only harvesting and dewatering method resulted in a slurry with relatively low biomass concentration. Nevertheless, autofermentation of this slurry led to the maximum total organic acid yield (60% C mol/C mol biomass) and hydrogen yield (326.1 µmol/g AFDM). CONCLUSION: Autofermentation is a simple, but highly effective pretreatment that can play a significant role within a cyanobacterial-based biorefinery platform by enabling the conversion of alkaline cyanobacterial biomass into organic acids, hydrogen, and methane via anaerobic digestion without the addition of energy or chemicals.

Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes.

BACKGROUND: Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput. RESULTS: Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50-99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software ( https://sourceforge.net/projects/calis-p/ ). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using 18O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics. CONCLUSIONS: We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.

Nutrient management and medium reuse for cultivation of a cyanobacterial consortium at high pH and alkalinity.

Alkaliphilic cyanobacteria have gained significant interest due to their robustness, high productivity, and ability to convert CO2 into bioenergy and other high value products. Effective nutrient management, such as re-use of spent medium, will be essential to realize sustainable applications with minimal environmental impacts. In this study, we determined the solubility and uptake of nutrients by an alkaliphilic cyanobacterial consortium grown at high pH and alkalinity. Except for Mg, Ca, Co, and Fe, all nutrients are in fully soluble form. The cyanobacterial consortium grew well without any inhibition and an overall productivity of 0.15 g L-1 d-1 (AFDW) was achieved. Quantification of nutrient uptake during growth resulted in the empirical formula CH1.81N0.17O0.20P0.013S0.009 for the consortium biomass. We showed that spent medium can be reused for at least five growth/harvest cycles. After an adaptation period, the cyanobacterial consortium fully acclimatized to the spent medium, resulting in complete restoration of biomass productivity.

Distinct oxygen isotope fractionations driven by different electron donors during microbial nitrate reduction in lake sediments.

Microbial nitrate reduction can be driven by organic carbon oxidation, as well as by inorganic electron donors, such as reduced forms of sulfur and iron. An apparent inverse oxygen isotope fractionation effect was observed during nitrate reduction in sediment incubations from five sampling sites of a freshwater lake, Hongze Lake, China. Incubations with organic and inorganic electron donor additions were performed. Especially, the inverse oxygen isotope effect was intensified after glucose addition, whereas the incubations with sulfide and Fe2+ showed normal fractionation factors. Nitrate reductase encoding genes, napA and narG, were analysed with metagenomics. Higher napA/narG ratios were associated with higher oxygen fractionation factors. The most abundant clade (59%) of NapA in the incubation with glucose was affiliated with Rhodocyclales. In contrast, it only accounted for 8%-9% of NapA in the incubations with sulfide and Fe2+ . Differences in nitrate reductases might explain different oxygen isotope effects. Our findings also suggested that large variance of O-nitrate isotope fractionations might have to be considered in the interpretation of natural isotope records.

Pilot-scale outdoor trial of a cyanobacterial consortium at pH 11 in a photobioreactor at high latitude.

The biomass of microalgae and cyanobacteria yields a variety of products. Outdoor pilot plant trials typically grow a single species at circumneutral pH and provide CO2 by gas sparging. Here a cyanobacterial consortium was grown at high pH (beyond 11) and high dissolved carbonate concentrations (0.5 M) in an outdoor 1,150 L tubular photobioreactor for 130 days in Calgary, Canada. The aim was to assess the productivity and robustness of the consortium. Importantly, the system was designed to enable future integration of air capture of CO2. Productivity was between 3.1 and 5.8 g ash-free dry weight per square metre per day, depending on biomass density and month. 16S rRNA amplicon sequencing showed that cyanobacterium Candidatus "Phormidium alkaliphilum" made up 80% of the consortium. The consortium displayed robust growth and adapted to environmental conditions. Bicarbonate uptake pushed medium pH past 11, demonstrating the ability to achieve CO2 delivery by air capture.

Ecological Interactions of Cyanobacteria and Heterotrophs Enhances the Robustness of Cyanobacterial Consortium for Carbon Sequestration.

Lack of robustness is a major barrier to foster a sustainable cyanobacterial biotechnology. Use of cyanobacterial consortium increases biodiversity, which provides functional redundancy and prevents invading species from disrupting the production ecosystem. Here we characterized a cyanobacterial consortium enriched from microbial mats of alkaline soda lakes in BC, Canada, at high pH and alkalinity. This consortium has been grown in open laboratory culture for 4 years without crashes. Using shotgun metagenomic sequencing, 29 heterotrophic metagenome-assembled-genomes (MAGs) were retrieved and were assigned to Bacteroidota, Alphaproteobacteria, Gammaproteobacteria, Verrucomicrobiota, Patescibacteria, Planctomycetota, and Archaea. In combination with metaproteomics, the overall stability of the consortium was determined under different cultivation conditions. Genome information from each heterotrophic population was investigated for six ecological niches created by cyanobacterial metabolism and one niche for phototrophy. Genome-resolved metaproteomics with stable isotope probing using 13C-bicarbonate (protein/SIP) showed tight coupling of carbon transfer from cyanobacteria to the heterotrophic populations, specially Wenzhouxiangella. The community structure was compared to a previously described consortium of a closely related cyanobacteria, which indicated that the results may be generalized. Productivity losses associated with heterotrophic metabolism were relatively small compared to other losses during photosynthesis.

Microbial Functional Diversity Correlates with Species Diversity along a Temperature Gradient.

Microbial community diversity is often correlated with physical environmental stresses like acidity, salinity, and temperature. For example, species diversity usually declines with increasing temperature above 20°C. However, few studies have examined whether the genetic functional diversity of community metagenomes varies in a similar way as species diversity along stress gradients. Here, we investigated bacterial communities in thermal spring sediments ranging from 21 to 88°C, representing communities of 330 to 3,800 bacterial and archaeal species based on 16S rRNA gene amplicon analysis. Metagenomes were sequenced, and Pfam abundances were used as a proxy for metagenomic functional diversity. Significant decreases in both species diversity and Pfam diversity were observed with increasing temperatures. The relationship between Pfam diversity and species diversity followed a power function with the steepest slopes in the high-temperature, low-diversity region of the gradient. Species additions to simple thermophilic communities added many new Pfams, while species additions to complex mesophilic communities added relatively fewer new Pfams, indicating that species diversity does not approach saturation as rapidly as Pfam diversity does. Many Pfams appeared to have distinct temperature ceilings of 60 to 80°C. This study suggests that temperature stress limits both taxonomic and functional diversity of microbial communities, but in a quantitatively different manner. Lower functional diversity at higher temperatures is probably due to two factors, including (i) the absence of many enzymes not adapted to thermophilic conditions, and (ii) the fact that high-temperature communities are comprised of fewer species with smaller average genomes and, therefore, contain fewer rare functions. IMPORTANCE Only recently have microbial ecologists begun to assess quantitatively how microbial species diversity correlates with environmental factors like pH, temperature, and salinity. However, still, very few studies have examined how the number of distinct biochemical functions of microbial communities, termed functional diversity, varies with the same environmental factors. Our study examined 18 microbial communities sampled across a wide temperature gradient and found that increasing temperature reduced both species and functional diversity, but in different ways. Initially, functional diversity increased sharply with increasing species diversity but eventually plateaued, following a power function. This pattern has been previously predicted in theoretical models, but our study validates this predicted power function with field metagenomic data. This study also presents a unique overview of the distribution of metabolic functions along a temperature gradient, demonstrating that many functions have temperature "ceilings" above which they are no longer found.

Denitrification and dissimilatory nitrate reduction to ammonia in long-term lake sediment microcosms with iron(II).

Nitrate is an abundant pollutant in aquatic environments. Competition between the nitrate reduction processes, denitrification, which converts nitrate into nitrogen gas, and dissimilatory nitrate reduction to ammonia (DNRA), which converts nitrate into ammonia, decides whether an ecosystem removes or retains nitrogen. The presence of iron was previously reported to stimulate DNRA while sometimes inhibiting denitrification in in-situ studies, but long-term effect of iron(II) inputs on the competition is unknown. Here we inoculated long-term microcosms with sediments from two freshwater lakes. During 540 days of incubations, the microcosms with nitrate and Fe(II) additions of both lakes were able to sustain high nitrate reduction rates. Lepidocrocite was produced as a product of iron oxidation. We found both denitrification and DNRA were stimulated by nitrate and iron in the absence of external organic carbon addition. Phylogenetic analysis of denitrification genes, nirK and nirS, and DNRA genes, nirB and nrfA, was performed with metagenomic sequencing results. Enrichment was shown for reported Fe(II)-dependent nitrate reducers associated with nirS and nirB. Most of these bacteria are affiliated with Betaproteobacteria. From 16S rRNA gene analysis, Betaproteobacteria was enriched as well. In parallel, heterotrophic denitrifiers and methanotrophic DNRA archaea increased in abundance. Our results suggested heterotrophic and Fe(II)-dependent nitrate reducers both contributed to denitrification and DNRA in long-term microcosm incubations provided with iron.

Proteome and strain analysis of cyanobacterium Candidatus "Phormidium alkaliphilum" reveals traits for success in biotechnology.

Cyanobacteria encompass a diverse group of photoautotrophic bacteria with important roles in nature and biotechnology. Here we characterized Candidatus "Phormidium alkaliphilum," an abundant member in alkaline soda lake microbial communities globally. The complete, circular whole-genome sequence of Ca. "P. alkaliphilum" was obtained using combined Nanopore and Illumina sequencing of a Ca. "P. alkaliphilum" consortium. Strain-level diversity of Ca. "P. alkaliphilum" was shown to contribute to photobioreactor robustness under different operational conditions. Comparative genomics of closely related species showed that adaptation to high pH was not attributed to specific genes. Proteomics at high and low pH showed only minimal changes in gene expression, but higher productivity in high pH. Diverse photosystem antennae proteins, and high-affinity terminal oxidase, compared with other soda lake cyanobacteria, appear to contribute to the success of Ca. "P. alkaliphilum" in photobioreactors and biotechnology applications.

Operation of Laboratory Photobioreactors with Online Growth Measurements and Customizable Light Regimes.

The laboratory study of microalgae can be experimentally challenging. In addition to the cultivation requirements of non-photosynthetic microorganisms, phototrophs also require illumination. Routinely, researchers seek to provide custom light supplies, i.e., vary the light intensity and time over which it is delivered. Such flexibility is difficult with standard benchtop lights. Usually, cultivation studies also require growth comparisons between experimental treatments. Frequently, growth is assessed over an extended duration, e.g., multiple times a day over a week-long trial. Manual measurements can be time-consuming and lack data resolution. Therefore, photobioreactors (PBRs) with automatic growth monitoring and customizable light supply are useful for replicated experiments with multiple treatments. The current work presents the design, construction, and operation of laboratory PBRs. The materials are easily sourced and relatively inexpensive. The design could be duplicated with moderate skill. Each structure has a footprint of ~40 cm2 and hosts three 1 L glass bottles for triplicate replication. Bottles rest upon platforms containing magnetic stirrers and are arranged vertically within a 1 m high and 15 cm diameter polyvinyl chloride (PVC) pipe. The pipe interior is lined with light-emitting diodes (LEDs). These LEDs produce continuous light intensities from 0-2400 µmol photons m-2 s-1 of photosynthetically active radiation (PAR). Users design a custom lighting program. The light intensity can be adjusted each second or held constant for longer durations. Oxygen produced from photosynthesis exits each bottle via a one-way volumetric gas sensor. Software is used to record gas sensor data. The amount of oxygen produced can be correlated to biomass growth. If biomass samples are required, a syringe can be used to extract culture. The method is suited for microalgae grown with bicarbonate as the carbon source. These PBRs are valuable to a laboratory that requires replicated experiments, light regime flexibility, and continuous high-resolution growth data.

Methane-dependent selenate reduction by a bacterial consortium.

Methanotrophic microorganisms play a critical role in controlling the flux of methane from natural sediments into the atmosphere. Methanotrophs have been shown to couple the oxidation of methane to the reduction of diverse electron acceptors (e.g., oxygen, sulfate, nitrate, and metal oxides), either independently or in consortia with other microbial partners. Although several studies have reported the phenomenon of methane oxidation linked to selenate reduction, neither the microorganisms involved nor the underlying trophic interaction has been clearly identified. Here, we provide the first detailed evidence for interspecies electron transfer between bacterial populations in a bioreactor community where the reduction of selenate is linked to methane oxidation. Metagenomic and metaproteomic analyses of the community revealed a novel species of Methylocystis as the most abundant methanotroph, which actively expressed proteins for oxygen-dependent methane oxidation and fermentation pathways, but lacked the genetic potential for selenate reduction. Pseudoxanthomonas, Piscinibacter, and Rhodocyclaceae populations appeared to be responsible for the observed selenate reduction using proteins initially annotated as periplasmic nitrate reductases, with fermentation by-products released by the methanotrophs as electron donors. The ability for the annotated nitrate reductases to reduce selenate was confirmed by gene knockout studies in an isolate of Pseudoxanthomonas. Overall, this study provides novel insights into the metabolic flexibility of the aerobic methanotrophs that likely allows them to thrive across natural oxygen gradients, and highlights the potential role for similar microbial consortia in linking methane and other biogeochemical cycles in environments where oxygen is limited.

Degradation of biological macromolecules supports uncultured microbial populations in Guaymas Basin hydrothermal sediments.

Hydrothermal sediments contain large numbers of uncultured heterotrophic microbial lineages. Here, we amended Guaymas Basin sediments with proteins, polysaccharides, nucleic acids or lipids under different redox conditions and cultivated heterotrophic thermophiles with the genomic potential for macromolecule degradation. We reconstructed 20 metagenome-assembled genomes (MAGs) of uncultured lineages affiliating with known archaeal and bacterial phyla, including endospore-forming Bacilli and candidate phylum Marinisomatota. One Marinisomatota MAG had 35 different glycoside hydrolases often in multiple copies, seven extracellular CAZymes, six polysaccharide lyases, and multiple sugar transporters. This population has the potential to degrade a broad spectrum of polysaccharides including chitin, cellulose, pectin, alginate, chondroitin, and carrageenan. We also describe thermophiles affiliating with the genera Thermosyntropha, Thermovirga, and Kosmotoga with the capability to make a living on nucleic acids, lipids, or multiple macromolecule classes, respectively. Several populations seemed to lack extracellular enzyme machinery and thus likely scavenged oligo- or monomers (e.g., MAGs affiliating with Archaeoglobus) or metabolic products like hydrogen (e.g., MAGs affiliating with Thermodesulfobacterium or Desulforudaceae). The growth of methanogens or the production of methane was not observed in any condition, indicating that the tested macromolecules are not degraded into substrates for methanogenesis in hydrothermal sediments. We provide new insights into the niches, and genomes of microorganisms that actively degrade abundant necromass macromolecules under oxic, sulfate-reducing, and fermentative thermophilic conditions. These findings improve our understanding of the carbon flow across trophic levels and indicate how primary produced biomass sustains complex and productive ecosystems.

CANT-HYD: A Curated Database of Phylogeny-Derived Hidden Markov Models for Annotation of Marker Genes Involved in Hydrocarbon Degradation.

Many pathways for hydrocarbon degradation have been discovered, yet there are no dedicated tools to identify and predict the hydrocarbon degradation potential of microbial genomes and metagenomes. Here we present the Calgary approach to ANnoTating HYDrocarbon degradation genes (CANT-HYD), a database of 37 HMMs of marker genes involved in anaerobic and aerobic degradation pathways of aliphatic and aromatic hydrocarbons. Using this database, we identify understudied or overlooked hydrocarbon degradation potential in many phyla. We also demonstrate its application in analyzing high-throughput sequence data by predicting hydrocarbon utilization in large metagenomic datasets from diverse environments. CANT-HYD is available at https://github.com/dgittins/CANT-HYD-HydrocarbonBiodegradation.

Wenzhouxiangella Strain AB-CW3, a Proteolytic Bacterium From Hypersaline Soda Lakes That Preys on Cells of Gram-Positive Bacteria.

A new haloalkaliphilic species of Wenzhouxiangella, strain AB-CW3, was isolated from a system of hypersaline alkaline soda lakes in the Kulunda Steppe using cells of Staphylococcus aureus as growth substrate. AB-CW3's complete, circular genome was assembled from combined nanopore and Illumina sequencing and its proteome was determined for three different experimental conditions. AB-CW3 is an aerobic gammaproteobacterium feeding mainly on proteins and peptides. Unique among Wenzhouxiangella, it uses a flagellum for motility, fimbria for cell attachment and is capable of complete denitrification. AB-CW3 can use proteins derived from living or dead cells of Staphylococcus and other Gram-positive bacteria as the carbon and energy source. It encodes and expresses production of a novel Lantibiotic, a class of antimicrobial peptides which have so far only been found to be produced by Gram-positive bacteria. AB-CW3 likely excretes this peptide via a type I secretion system encoded upstream of the genes for production of the Lanthipeptide. Comparison of AB-CW3's genome to 18 other Wenzhouxiangella genomes from marine, hypersaline, and soda lake habitats indicated one or two transitions from marine to soda lake environments followed by a transition of W. marina back to the oceans. Only 19 genes appear to set haloalkaliphilic Wenzhouxiangella apart from their neutrophilic relatives. As strain AB-CW3 is only distantly related to other members of the genus, we propose to provisionally name it "Wenzhouxiangella alkaliphila".