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BackgroundCandida parapsilosis is a common cause of invasive candidiasis, especially in newborn infants, and infections have been increasing over the past two decades. C. parapsilosis has been primarily studied in pure culture, leaving gaps in understanding of its function in a microbiome context.ResultsHere, we compare five unique C. parapsilosis genomes assembled from premature infant fecal samples, three of which are newly reconstructed, and analyze their genome structure, population diversity, and in situ activity relative to reference strains in pure culture. All five genomes contain hotspots of single nucleotide variants, some of which are shared by strains from multiple hospitals. A subset of environmental and hospital-derived genomes share variants within these hotspots suggesting derivation of that region from a common ancestor. Four of the newly reconstructed C. parapsilosis genomes have 4 to 16 copies of the gene RTA3, which encodes a lipid translocase and is implicated in antifungal resistance, potentially indicating adaptation to hospital antifungal use. Time course metatranscriptomics and metaproteomics on fecal samples from a premature infant with a C. parapsilosis blood infection revealed highly variable in situ expression patterns that are distinct from those of similar strains in pure cultures. For example, biofilm formation genes were relatively less expressed in situ, whereas genes linked to oxygen utilization were more highly expressed, indicative of growth in a relatively aerobic environment. In gut microbiome samples, C. parapsilosis co-existed with Enterococcus faecalis that shifted in relative abundance over time, accompanied by changes in bacterial and fungal gene expression and proteome composition.ConclusionsThe results reveal potentially medically relevant differences in Candida function in gut vs. laboratory environments, and constrain evolutionary processes that could contribute to hospital strain persistence and transfer into premature infant microbiomes. Video abstract.

Following publication of the original article.

Production of unconventional oil and gas continues to rise, but the effects of high-density hydraulic fracturing (HF) activity near aquatic ecosystems are not fully understood. A commonly used biocide in HF, 2,2-dibromo-3-nitrilopropionamide (DBNPA), was studied in microcosms of HF-impacted (HF+) versus HF-unimpacted (HF-) surface water streams to (i) compare the microbial community response, (ii) investigate DBNPA degradation products based on past HF exposure, and (iii) compare the microbial community response differences and similarities between the HF biocides DBNPA and glutaraldehyde. The microbial community responded to DBNPA differently in HF-impacted versus HF-unimpacted microcosms in terms of the number of 16S rRNA gene copies quantified, alpha and beta diversity, and differential abundance analyses of microbial community composition through time. The differences in microbial community changes affected degradation dynamics. HF-impacted microbial communities were more sensitive to DBNPA, causing the biocide and by-products of the degradation to persist for longer than in HF-unimpacted microcosms. A total of 17 DBNPA by-products were detected, many of them not widely known as DBNPA by-products. Many of the brominated by-products detected that are believed to be uncharacterized may pose environmental and health impacts. Similar taxa were able to tolerate glutaraldehyde and DBNPA; however, DBNPA was not as effective for microbial control, as indicated by a smaller overall decrease of 16S rRNA gene copies/ml after exposure to the biocide, and a more diverse set of taxa was able to tolerate it. These findings suggest that past HF activity in streams can affect the microbial community response to environmental perturbation such as that caused by the biocide DBNPA.IMPORTANCE Unconventional oil and gas activity can affect pH, total organic carbon, and microbial communities in surface water, altering their ability to respond to new environmental and/or anthropogenic perturbations. These findings demonstrate that 2,2-dibromo-3-nitrilopropionamide (DBNPA), a common hydraulic fracturing (HF) biocide, affects microbial communities differently as a consequence of past HF exposure, persisting longer in HF-impacted (HF+) waters. These findings also demonstrate that DBNPA has low efficacy in environmental microbial communities regardless of HF impact. These findings are of interest, as understanding microbial responses is key for formulating remediation strategies in unconventional oil and gas (UOG)-impacted environments. Moreover, some DBNPA degradation by-products are even more toxic and recalcitrant than DBNPA itself, and this work identifies novel brominated degradation by-products formed.

Stable isotope probing (SIP) enables tracking the nutrient flows from isotopically labeled substrates to specific microorganisms in microbial communities. In proteomic SIP, labeled proteins synthesized by the microbial consumers of labeled substrates are identified with a shotgun proteomics approach. Here, proteomic SIP was combined with targeted metagenomic binning to reconstruct metagenome-assembled genomes (MAGs) of the microorganisms producing labeled proteins. This approach was used to track carbon flows from 13CO2 to the rhizosphere communities of Zea mays, Triticum aestivum, and Arabidopsis thaliana. Rhizosphere microorganisms that assimilated plant-derived 13C were capable of metabolic and signaling interactions with their plant hosts, as shown by their MAGs containing genes for phytohormone modulation, quorum sensing, and transport and metabolism of nutrients typical of those found in root exudates. XoxF-type methanol dehydrogenases were among the most abundant proteins identified in the rhizosphere metaproteomes. 13C-methanol proteomic SIP was used to test the hypothesis that XoxF was used to metabolize and assimilate methanol in the rhizosphere. We detected 7 13C-labeled XoxF proteins and identified methylotrophic pathways in the MAGs of 8 13C-labeled microorganisms, which supported the hypothesis. These two studies demonstrated the capability of proteomic SIP for functional characterization of active microorganisms in complex microbial communities.

BackgroundCrassulacean acid metabolism (CAM) enhances plant water-use efficiency through an inverse day/night pattern of stomatal closure/opening that facilitates nocturnal CO2 uptake. CAM has evolved independently in over 35 plant lineages, accounting for ~ 6% of all higher plants. Agave species are highly heat- and drought-tolerant, and have been domesticated as model CAM crops for beverage, fiber, and biofuel production in semi-arid and arid regions. However, the genomic basis of evolutionary innovation of CAM in genus Agave is largely unknown.ResultsUsing an approach that integrated genomics, gene co-expression networks, comparative genomics and protein structure analyses, we investigated the molecular evolution of CAM as exemplified in Agave. Comparative genomics analyses among C3, C4 and CAM species revealed that core metabolic components required for CAM have ancient genomic origins traceable to non-vascular plants while regulatory proteins required for diel re-programming of metabolism have a more recent origin shared among C3, C4 and CAM species. We showed that accelerated evolution of key functional domains in proteins responsible for primary metabolism and signaling, together with a diel re-programming of the transcription of genes involved in carbon fixation, carbohydrate processing, redox homeostasis, and circadian control is required for the evolution of CAM in Agave. Furthermore, we highlighted the potential candidates contributing to the adaptation of CAM functional modules.ConclusionsThis work provides evidence of adaptive evolution of CAM related pathways. We showed that the core metabolic components required for CAM are shared by non-vascular plants, but regulatory proteins involved in re-reprogramming of carbon fixation and metabolite transportation appeared more recently. We propose that the accelerated evolution of key proteins together with a diel re-programming of gene expression were required for CAM evolution from C3 ancestors in Agave.

Background: Clostridium autoethanogenum is an acetogenic bacterium that autotrophically converts carbon monoxide (CO) and carbon dioxide (CO2) gases into bioproducts and fuels via the Wood–Ljungdahl pathway (WLP). To facilitate overall carbon capture efficiency, the reaction stoichiometry requires supplementation of hydrogen at an increased ratio of H2:CO to maximize CO2 utilization; however, the molecular details and thus the ability to understand the mechanism of this supplementation are largely unknown. Results: In order to elucidate the microbial physiology and fermentation where at least 75% of the carbon in ethanol comes from CO2, we established controlled chemostats that facilitated a novel and high (11:1) H2:CO uptake ratio. We compared and contrasted proteomic and metabolomics profiles to replicate continuous stirred tank reactors (CSTRs) at the same growth rate from a lower (5:1) H2:CO condition where ~ 50% of the carbon in ethanol is derived from CO2. Our hypothesis was that major changes would be observed in the hydrogenases and/or redox-related proteins and the WLP to compensate for the elevated hydrogen feed gas. Our analyses did reveal protein abundance differences between the two conditions largely related to reduction–oxidation (redox) pathways and cofactor biosynthesis, but the changes were more minor than we would have expected. While the Wood–Ljungdahl pathway proteins remained consistent across the conditions, other post-translational regulatory processes, such as lysine-acetylation, were observed and appeared to be more important for fine-tuning this carbon metabolism pathway. Metabolomic analyses showed that the increase in H2:CO ratio drives the organism to higher carbon dioxide utilization resulting in lower carbon storages and accumulated fatty acid metabolite levels. Conclusions: This research delves into the intricate dynamics of carbon fixation in C. autoethanogenum, examining the influence of highly elevated H2:CO ratios on metabolic processes and product outcomes. The study underscores the significance of optimizing gas feed composition for enhanced industrial efficiency, shedding light on potential mechanisms, such as post-translational modifications (PTMs), to fine-tune enzymatic activities and improve desired product yields. [ABSTRACT FROM AUTHOR]

During the first weeks of life, microbial colonization of the gut impacts human immune system maturation and other developmental processes. In premature infants, aberrant colonization has been implicated in the onset of necrotizing enterocolitis (NEC), a life-threatening intestinal disease. To study the premature infant gut colonization process, genome-resolved metagenomics was conducted on 343 fecal samples collected during the first 3 months of life from 35 premature infants housed in a neonatal intensive care unit, 14 of whom developed NEC, and metaproteomic measurements were made on 87 samples. Microbial community composition and proteomic profiles remained relatively stable on the time scale of a week, but the proteome was more variable. Although genetically similar organisms colonized many infants, most infants were colonized by distinct strains with metabolic profiles that could be distinguished using metaproteomics. Microbiome composition correlated with infant, antibiotics administration, and NEC diagnosis. Communities were found to cluster into seven primary types, and community type switched within infants, sometimes multiple times. Interestingly, some communities sampled from the same infant at subsequent time points clustered with those of other infants. In some cases, switches preceded onset of NEC; however, no species or community type could account for NEC across the majority of infants. In addition to a correlation of protein abundances with organism replication rates, we found that organism proteomes correlated with overall community composition. Thus, this genome-resolved proteomics study demonstrated that the contributions of individual organisms to microbiome development depend on microbial community context.IMPORTANCE Humans are colonized by microbes at birth, a process that is important to health and development. However, much remains to be known about the fine-scale microbial dynamics that occur during the colonization period. We conducted a genome-resolved study of microbial community composition, replication rates, and proteomes during the first 3 months of life of both healthy and sick premature infants. Infants were found to be colonized by similar microbes, but each underwent a distinct colonization trajectory. Interestingly, related microbes colonizing different infants were found to have distinct proteomes, indicating that microbiome function is not only driven by which organisms are present, but also largely depends on microbial responses to the unique set of physiological conditions in the infant gut.

BackgroundEstablishment of the human gut microbiota begins at birth. This early-life microbiota development can impact host physiology during infancy and even across an entire life span. However, the functional stability and population structure of the gut microbiota during initial colonization remain poorly understood. Metaproteomics is an emerging technology for the large-scale characterization of metabolic functions in complex microbial communities (gut microbiota).ResultsWe applied a metagenome-informed metaproteomic approach to study the temporal and inter-individual differences of metabolic functions during microbial colonization of preterm human infants' gut. By analyzing 30 individual fecal samples, we identified up to 12,568 protein groups for each of four infants, including both human and microbial proteins. With genome-resolved matched metagenomics, proteins were confidently identified at the species/strain level. The maximum percentage of the proteome detected for the abundant organisms was ~45%. A time-dependent increase in the relative abundance of microbial versus human proteins suggested increasing microbial colonization during the first few weeks of early life. We observed remarkable variations and temporal shifts in the relative protein abundances of each organism in these preterm gut communities. Given the dissimilarity of the communities, only 81 microbial EggNOG orthologous groups and 57 human proteins were observed across all samples. These conserved microbial proteins were involved in carbohydrate, energy, amino acid and nucleotide metabolism while conserved human proteins were related to immune response and mucosal maturation. We identified seven proteome clusters for the communities and showed infant gut proteome profiles were unstable across time and not individual-specific. Applying a gut-specific metabolic module (GMM) analysis, we found that gut communities varied primarily in the contribution of nutrient (carbohydrates, lipids, and amino acids) utilization and short-chain fatty acid production.ConclusionsOverall, this study reports species-specific proteome profiles and metabolic functions of human gut microbiota during early colonization. In particular, our work contributes to reveal microbiota-associated shifts and variations in the metabolism of three major nutrient sources and short-chain fatty acid during colonization of preterm infant gut.

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops.

Remediation of industrial wastewater is important for preventing environmental contamination and enabling water reuse. Biological treatment for one industrial contaminant, thiocyanate (SCN-), relies upon microbial hydrolysis, but this process is sensitive to high loadings. To examine the activity and stability of a microbial community over increasing SCN- loadings, we established and operated a continuous-flow bioreactor fed increasing loadings of SCN-. A second reactor was fed ammonium sulfate to mimic breakdown products of SCN-. Biomass was sampled from both reactors for metagenomics and metaproteomics, yielding a set of genomes for 144 bacteria and one rotifer that constituted the abundant community in both reactors. We analyzed the metabolic potential and temporal dynamics of these organisms across the increasing loadings. In the SCN- reactor, Thiobacillus strains capable of SCN- degradation were highly abundant, whereas the ammonium sulfate reactor contained nitrifiers and heterotrophs capable of nitrate reduction. Key organisms in the SCN- reactor expressed proteins involved in SCN- degradation, sulfur oxidation, carbon fixation, and nitrogen removal. Lower performance at higher loadings was linked to changes in microbial community composition. This work provides an example of how meta-omics can increase our understanding of industrial wastewater treatment and inform iterative process design and development.

Already a proven mechanism for drought resilience, crassulacean acid metabolism (CAM) is a specialized type of photosynthesis that maximizes water-use efficiency by means of an inverse (compared to C3 and C4 photosynthesis) day/night pattern of stomatal closure/opening to shift CO2 uptake to the night, when evapotranspiration rates are low. A systems-level understanding of temporal molecular and metabolic controls is needed to define the cellular behaviour underpinning CAM. Here, we report high-resolution temporal behaviours of transcript, protein and metabolite abundances across a CAM diel cycle and, where applicable, compare the observations to the well-established C3 model plant Arabidopsis. A mechanistic finding that emerged is that CAM operates with a diel redox poise that is shifted relative to that in Arabidopsis. Moreover, we identify widespread rescheduled expression of genes associated with signal transduction mechanisms that regulate stomatal opening/closing. Controlled production and degradation of transcripts and proteins represents a timing mechanism by which to regulate cellular function, yet knowledge of how this molecular timekeeping regulates CAM is unknown. Here, we provide new insights into complex post-transcriptional and -translational hierarchies that govern CAM in Agave. These data sets provide a resource to inform efforts to engineer more efficient CAM traits into economically valuable C3 crops.

Nitrogen, sulfur and carbon fluxes in the terrestrial subsurface are determined by the intersecting activities of microbial community members, yet the organisms responsible are largely unknown. Metagenomic methods can identify organisms and functions, but genome recovery is often precluded by data complexity. To address this limitation, we developed subsampling assembly methods to re-construct high-quality draft genomes from complex samples. We applied these methods to evaluate the interlinked roles of the most abundant organisms in biogeochemical cycling in the aquifer sediment. Community proteomics confirmed these activities. The eight most abundant organisms belong to novel lineages, and two represent phyla with no previously sequenced genome. Four organisms are predicted to fix carbon via the Calvin-Benson-Bassham, Wood-Ljungdahl or 3-hydroxyproprionate/4-hydroxybutarate pathways. The profiled organisms are involved in the network of denitrification, dissimilatory nitrate reduction to ammonia, ammonia oxidation and sulfate reduction/oxidation, and require substrates supplied by other community members. An ammonium-oxidizing Thaumarchaeote is the most abundant community member, despite low ammonium concentrations in the groundwater. This organism likely benefits from two other relatively abundant organisms capable of producing ammonium from nitrate, which is abundant in the groundwater. Overall, dominant members of the microbial community are interconnected through exchange of geochemical resources.

Annually, half of all plant-derived carbon is added to soil where it is microbially respired to CO2. However, understanding of the microbiology of this process is limited because most culture-independent methods cannot link metabolic processes to the organisms present, and this link to causative agents is necessary to predict the results of perturbations on the system. We collected soil samples at two sub-root depths (10-20 cm and 30-40 cm) before and after a rainfall-driven nutrient perturbation event in a Northern California grassland that experiences a Mediterranean climate. From ten samples, we reconstructed 198 metagenome-assembled genomes that represent all major phylotypes. We also quantified 6,835 proteins and 175 metabolites and showed that after the rain event the concentrations of many sugars and amino acids approach zero at the base of the soil profile. Unexpectedly, the genomes of novel members of the Gemmatimonadetes and Candidate Phylum Rokubacteria phyla encode pathways for methylotrophy. We infer that these abundant organisms contribute substantially to carbon turnover in the soil, given that methylotrophy proteins were among the most abundant proteins in the proteome. Previously undescribed Bathyarchaeota and Thermoplasmatales archaea are abundant in deeper soil horizons and are inferred to contribute appreciably to aromatic amino acid degradation. Many of the other bacteria appear to breakdown other components of plant biomass, as evidenced by the prevalence of various sugar and amino acid transporters and corresponding hydrolyzing machinery in the proteome. Overall, our work provides organism-resolved insight into the spatial distribution of bacteria and archaea whose activities combine to degrade plant-derived organics, limiting the transport of methanol, amino acids and sugars into underlying weathered rock. The new insights into the soil carbon cycle during an intense period of carbon turnover, including biogeochemical roles to previously little known soil microbes, were made possible via the combination of metagenomics, proteomics, and metabolomics.

The ecosystem roles of fungi have been extensively studied by targeting one organism and/or biological process at a time, but the full metabolic potential of fungi has rarely been captured in an environmental context. We hypothesized that fungal genome sequences could be assembled directly from the environment using metagenomics and that transcriptomics and proteomics could simultaneously reveal metabolic differentiation across habitats. We reconstructed the near-complete 27 Mbp genome of a filamentous fungus, Acidomyces richmondensis, and evaluated transcript and protein expression in floating and streamer biofilms from an acid mine drainage (AMD) system. A. richmondensis transcripts involved in denitrification and in the degradation of complex carbon sources (including cellulose) were up-regulated in floating biofilms, whereas central carbon metabolism and stress-related transcripts were significantly up-regulated in streamer biofilms. These findings suggest that the biofilm niches are distinguished by distinct carbon and nitrogen resource utilization, oxygen availability, and environmental challenges. An isolated A. richmondensis strain from this environment was used to validate the metagenomics-derived genome and confirm nitrous oxide production at pH 1. Overall, our analyses defined mechanisms of fungal adaptation and identified a functional shift related to different roles in carbon and nitrogen turnover for the same species of fungi growing in closely located but distinct biofilm niches.

Microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. To this end, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data. More specifically, the function of the microbial community initially involved biomass growth, protein production, and lipid metabolism, and then switched to more complex metabolic functions, such as carbohydrate metabolism, once the community stabilized and matured. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Likewise, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. This study provides the first snapshot of coordinated human and microbial protein expression in a preterm infant's gut during early development.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO2 uptake, facilitates increased water-use efficiency (WUE), and enables CAM plants to inhabit water-limited environments such as semi-arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave and Opuntia, for biomass production on semi-arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management.

The establishment of early life microbiota in the human infant gut is highly variable and plays a crucial role in host nutrient availability/uptake and maturation of immunity. Although high-performance mass spectrometry (MS)-based metaproteomics is a powerful method for the functional characterization of complex microbial communities, the acquisition of comprehensive metaproteomic information in human fecal samples is inhibited by the presence of abundant human proteins. To alleviate this restriction, we have designed a novel metaproteomic strategy based on double filtering (DF) the raw samples, a method that fractionates microbial from human cells to enhance microbial protein identification and characterization in complex fecal samples from healthy premature infants. This method dramatically improved the overall depth of infant gut proteome measurement, with an increase in the number of identified low-abundance proteins and a greater than 2-fold improvement in microbial protein identification and quantification. This enhancement of proteome measurement depth enabled a more extensive microbiome comparison between infants by not only increasing the confidence of identified microbial functional categories but also revealing previously undetected categories.

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.

Microbial communities that underpin global biogeochemical cycles will likely be influenced by elevated temperature associated with environmental change. Here, we test an approach to measure how elevated temperature impacts the physiology of individual microbial groups in a community context, using a model microbial-based ecosystem. The study is the first application of tandem mass tag (TMT)-based proteomics to a microbial community. We accurately, precisely and reproducibly quantified thousands of proteins in biofilms growing at 40, 43 and 46 °C. Elevated temperature led to upregulation of proteins involved in amino-acid metabolism at the level of individual organisms and the entire community. Proteins from related organisms differed in their relative abundance and functional responses to temperature. Elevated temperature repressed carbon fixation proteins from two Leptospirillum genotypes, whereas carbon fixation proteins were significantly upregulated at higher temperature by a third member of this genus. Leptospirillum group III bacteria may have been subject to viral stress at elevated temperature, which could lead to greater carbon turnover in the microbial food web through the release of viral lysate. Overall, these findings highlight the utility of proteomics-enabled community-based physiology studies, and provide a methodological framework for possible extension to additional mixed culture and environmental sample analyses.

Detailed characterization of post-translational modifications (PTMs) of proteins in microbial communities remains a significant challenge. Here we directly identify and quantify a broad range of PTMs (hydroxylation, methylation, citrullination, acetylation, phosphorylation, methylthiolation, S-nitrosylation and nitration) in a natural microbial community from an acid mine drainage site. Approximately 29% of the identified proteins of the dominant Leptospirillum group II bacteria are modified, and 43% of modified proteins carry multiple PTM types. Most PTM events, except S-nitrosylations, have low fractional occupancy. Notably, PTM events are detected on Cas proteins involved in antiviral defense, an aspect of Cas biochemistry not considered previously. Further, Cas PTM profiles from Leptospirillum group II differ in early versus mature biofilms. PTM patterns are divergent on orthologues of two closely related, but ecologically differentiated, Leptospirillum group II bacteria. Our results highlight the prevalence and dynamics of PTMs of proteins, with potential significance for ecological adaptation and microbial evolution.

Sandy coastal sediments are global hotspots for microbial mineralization of organic matter and denitrification. These sediments are characterized by advective porewater flow, tidal cycling and an active and complex microbial community. Metagenomic sequencing of microbial communities sampled from such sediments showed that potential sulfur oxidizing Gammaproteobacteria and members of the enigmatic BD1-5/SN-2 candidate phylum were abundant in situ (>10% and ~2% respectively). By mimicking the dynamic oxic/anoxic environmental conditions of the sediment in a laboratory chemostat, a simplified microbial community was selected from the more complex inoculum. Metagenomics, proteomics and fluorescence in situ hybridization showed that this simplified community contained both a potential sulfur oxidizing Gammaproteobacteria (at 24 ± 2% abundance) and a member of the BD1-5/SN-2 candidate phylum (at 7 ± 6% abundance). Despite the abundant supply of organic substrates to the chemostat, proteomic analysis suggested that the selected gammaproteobacterium grew partially autotrophically and performed hydrogen/formate oxidation. The enrichment of a member of the BD1-5/SN-2 candidate phylum enabled, for the first time, direct microscopic observation by fluorescent in situ hybridization and the experimental validation of the previously predicted translation of the stop codon UGA into glycine.