Protein Stable Isotope Probing Research Articles

Energetically exploiting lignocellulose-rich residues in anaerobic digestion technologies: from bioreactors to proteogenomics

The biogas produced through anaerobic digestion (AD) of renewable feedstocks is one of the promising alternatives to replace fossil-derived energy. Even though lignocellulosic biomass is the most abundant biomass on earth, only a small fraction is being used towards resources recovery, leaving a great potential unexploited. In this study, the combination of state-of-art genomic techniques and engineered systems were used to further advance the knowledge on biogas production from lignocellulosic-rich residues and the microbiome involved in the anaerobic digestion hereof. A long-term adapted anaerobic microbiome capable of degrading wheat straw as the sole substrate was investigated using protein stable isotope probing (protein-SIP). The results indicated that a diverse microbial community, primarily composed of Firmicutes and Methanogens, played crucial roles in cellulose degradation and methane production. Notably, Defluviitoga tunisiensis, Syntrophothermus lipocalidus, and Pelobacter carbinolicus were identified as direct metabolizers of cellulose, while Dehalobacterium assimilated labelled carbon through cross-feeding. This study provides direct evidence of primary cellulose degraders and sheds light on their genomic composition. By harnessing the potential of lignocellulosic biomass and understanding the microbial communities involved, we can promote sustainable biogas production, contributing to energy security and environmental preservation.

Proteogenomics identification of TBBPA degraders in anaerobic bioreactor

Tetrabromobisphenol A (TBBPA) is the most used flame retardant worldwide and has become a threat to aquatic ecosystems. Previous research into the degradation of this micropollutant in anaerobic bioreactors has suggested several identities of putative TBBPA degraders. However, the organisms actively degrading TBBPA under in situ conditions have so far not been identified. Protein-stable isotope probing (protein-SIP) has become a cutting-edge technique in microbial ecology for enabling the link between identity and function under in situ conditions. Therefore, it was hypothesized that combining protein-based stable isotope probing with metagenomics could be used to identify and provide genomic insight into the TBBPA-degrading organisms. The identified 13C-labelled peptides were found to belong to organisms affiliated to Phytobacter, Clostridium, Sporolactobacillus, and Klebsilla genera. The functional classification of identified labelled peptides revealed that TBBPA is not only transformed by cometabolic reactions, but also assimilated into the biomass. By application of the proteogenomics with labelled micropollutants (protein-SIP) and metagenome-assembled genomes, it was possible to extend the current perspective of the diversity of TBBPA degraders in wastewater and predict putative TBBPA degradation pathways. The study provides a link to the active TBBPA degraders and which organisms to favor for optimized biodegradation.

Characterisation of cellulose-degrading organisms in an anaerobic digester

The recalcitrant nature of lignocellulosic biomass hinders efficient exploitation of this fraction for energy production. A better understanding of the microorganisms able to convert plant-based feedstocks is needed to improve anaerobic digestion of lignocellulosic biomass. In this study, active thermophilic cellulose-degrading microorganisms were identified from a full-scale anaerobic digester fed with maize by using metagenome-resolved protein stable isotope probing (protein-SIP). 13C-cellulose was converted into 13C-methane with a 13/12C isotope ratio of 0.127 after two days of incubation. Metagenomic analysis revealed 238 different genes coding for carbohydrate-active enzymes (CAZymes), six of which were directly associated with cellulose degradation. The protein-SIP analysis identified twenty heavily labelled peptides deriving from microorganisms actively assimilating labelled carbon from the degradation of 13C-cellulose, highlighting several members of the order Clostridiales. Corynebacterium was identified through CAZyme screening, amplicon analysis, and in the metagenome giving a strong identification of being a cellulose degrader.

Wood-Ljungdahl pathway utilisation during in situ H2 biomethanation

Biogas production from organic waste is a waste-to-energy technology with the potential to contribute significantly to sustainable energy production. Upgrading of biogas using in situ biomethanation with hydrogen has the potential for surplus electricity storage, and delivery of biogas with a methane content of >90%, allowing for easier integration into the natural gas grid, as well as conversion to other products. Microbial communities in biomethanation reactors undergo changes, however, these changes are largely unexplored. In the present study, metagenome-resolved protein stable isotope probing (Protein-SIP) was applied to laboratory scale batch incubations operating under anaerobic digestion, and (pre-adapted) biomethanation conditions, fed with 13C-labelled bicarbonate, in order to gain insight into the microbial activities during CO2-reduction. The strongest and most microbially diverse isotopic incorporation was observed in the pre-adapted biomethanation incubation. Furthermore, divergent incorporation of 13C-labelled bicarbonate was also observed in the Wood-Ljungdahl pathway, with the anaerobic digester incubations primarily showing labelled proteins in the peripheral pathways leading toward production of energy and biomass. The pre-adapted biomethanation incubations consumed H2 and CO2, but did not convert it to CH4, suggesting the production of acetate in these incubations, which was supported by heavy labelling of key enzymes in the Wood-Ljungdahl pathway. Twelve (ten high quality) metagenome-assembled genomes (MAGs) coding for 13C-incorporated proteins were extracted from the metagenome, eight of which contained one or more of the key genes in the Wood-Ljungdahl pathway, one of which was affiliated to Methanosarcina. Together, the findings in the present study deepen our knowledge surrounding microbial communities in biomethanation systems, and contribute to the development of better strategies for implementation of biogas upgrading and microbial management.

Studying the Temporal Dynamics of the Gut Microbiota Using Metabolic Stable Isotope Labeling and Metaproteomics.

The gut microbiome and its metabolic processes are dynamic systems. Surprisingly, our understanding of gut microbiome dynamics is limited. Here, we report a metaproteomic workflow that involves protein stable isotope probing (protein-SIP) and identification/quantification of partially labeled peptides. We also developed a package, which we call MetaProfiler, that corrects for false identifications and performs phylogenetic and time series analysis for the study of microbiome dynamics. From the stool sample of five mice that were fed with 15N hydrolysate from Ralstonia eutropha, we identified 12 326 nonredundant unlabeled peptides, of which 8256 of their heavy counterparts were quantified. These peptides revealed incorporation profiles over time that were different between and within taxa, as well as between and within clusters of orthologous groups (COGs). Our study helps unravel the complex dynamics of protein synthesis and bacterial dynamics in the mouse microbiome. MetaProfiler and the bioinformatic pipeline are available at https://github.com/northomics/MetaProfiler.git.

Protein stable isotope probing with H218O differentiated cold stress response at permissive temperatures from general growth at optimal conditions in Escherichia coli K12

Tracing isotopically labeled water into proteins allows for the detection of species-specific metabolic activity in complex communities. However, a stress response may alter the newly synthesized proteins. We traced 18-oxygen from heavy water into proteins of Escherichia coli K12 grown from permissive to retardant temperatures. All samples were analyzed using UPLC/Orbitrap Q-Exactive-MS/MS operating in positive electrospray ionization mode. We found that warmer temperatures resulted in significantly (P-value < 0.05) higher incorporation of 18-oxygen as seen by both substrate utilization as relative isotope abundance (RIA) and growth as labeling ratio (LR). However, the absolute number of peptides with incorporation of 18-oxygen showed no significant correlation to temperature, potentially caused by the synthesis of different proteins at low temperatures, namely, proteins related to cold stress response. Our results unveil the species-specific cold stress response of E. coli K12 that could be misinterpreted as general growth; this is why the quantity as RIA and LR but also the quality as absolute number of peptides with incorporation (relative abundance, RA) and their function must be considered to fully understand the activity of microbial communities.

Identification of dominant sulfamethoxazole-degraders in pig farm-impacted soil by DNA and protein stable isotope probing

Increasing introduction of antibiotic residues from humans and animal farming into the environment impacts the functioning of natural ecosystems and significantly contributes to the propagation of antibiotic resistance. Microbial degradation is the major sink of antibiotics in soil but the identification of in situ degrading populations is challenging. Here, we investigated sulfamethoxazole-degrading bacteria in soil microcosms by culture-independent DNA and protein stable isotope probing. 0.5% of the carbon from 13C6-labeled sulfamethoxazole amended to soil microcosms was transformed to 13CO2 demonstrating partial mineralization of the antibiotic. DNA stable isotope probing revealed incorporation of 13C from 13C6-labeled sulfamethoxazole into Actinobacteria and among them into the families Intrasporangiaceae, Nocardioidaceae, and Gaiellaceae and the order Solirubrobacterales. Protein stable isotope probing demonstrated the incorporation of 13C from 13C6-labeled sulfamethoxazole into proteins of bacteria of the families Intrasporangiaceae, Nocardioidaceae and the order Solirubrobacterales, which is consistent with the results of DNA stable isotope probing. The 13C abundance of 60 to 80% in several taxonomically relevant proteins indicated that Intrasporangiaceae directly acquired carbon from 13C6-labeled sulfamethoxazole. The results highlight the crucial role of yet-uncultivated indigenous bacteria for antibiotics degradation, and the potential of cultivation-independent stable isotope based molecular approaches to elucidate the structure of antibiotic-degrading populations in complex microbial communities under natural conditions.

Identification of syntrophic acetate-oxidizing bacteria in anaerobic digesters by combined protein-based stable isotope probing and metagenomics.

Inhibition of anaerobic digestion through accumulation of volatile fatty acids occasionally occurs as the result of unbalanced growth between acidogenic bacteria and methanogens. A fast recovery is a prerequisite for establishing an economical production of biogas. However, very little is known about the microorganisms facilitating this recovery. In this study, we investigated the organisms involved by a novel approach of mapping protein-stable isotope probing (protein-SIP) onto a binned metagenome. Under simulation of acetate accumulation conditions, formations of (13)C-labeled CO2 and CH4 were detected immediately following incubation with [U-(13)C]acetate, indicating high turnover rate of acetate. The identified (13)C-labeled peptides were mapped onto a binned metagenome for improved identification of the organisms involved. The results revealed that Methanosarcina and Methanoculleus were actively involved in acetate turnover, as were five subspecies of Clostridia. The acetate-consuming organisms affiliating with Clostridia all contained the FTFHS gene for formyltetrahydrofolate synthetase, a key enzyme for reductive acetogenesis, indicating that these organisms are possible syntrophic acetate-oxidizing (SAO) bacteria that can facilitate acetate consumption via SAO, coupled with hydrogenotrophic methanogenesis (SAO-HM). This study represents the first study applying protein-SIP for analysis of complex biogas samples, a promising method for identifying key microorganisms utilizing specific pathways.

Pulsed (13)C2-Acetate Protein-SIP Unveils Epsilonproteobacteria as Dominant Acetate Utilizers in a Sulfate-Reducing Microbial Community Mineralizing Benzene.

In a benzene-degrading and sulfate-reducing syntrophic consortium, a clostridium affiliated to the genus Pelotomaculum was previously described to ferment benzene while various sulfate-reducing Deltaproteobacteria and a member of the Epsilonproteobacteria were supposed to utilize acetate and hydrogen as key metabolites derived from benzene fermentation. However, the acetate utilization network within this community was not yet unveiled. In this study, we performed a pulsed (13)C2-acetate protein stable isotope probing (protein-SIP) approach continuously spiking low amounts of acetate (10μM per day) in addition to the ongoing mineralization of unlabeled benzene. Metaproteomics revealed high abundances of Clostridiales followed by Syntrophobacterales, Desulfobacterales, Desulfuromonadales, Desulfovibrionales, Archaeoglobales, and Campylobacterales. Pulsed acetate protein-SIP results indicated that members of the Campylobacterales, the Syntrophobacterales, the Archaeoglobales, the Clostridiales, and the Desulfobacterales were linked to acetate utilization in descending abundance. The Campylobacterales revealed the fastest and highest (13)C incorporation. Previous experiments suggested that the activity of the Campylobacterales was not essential for anaerobic benzene degradation in the investigated community. However, these organisms were consistently detected in various hydrocarbon-degrading and sulfate-reducing consortia enriched from the same aquifer. Here, we demonstrate that this member of the Campylobacterales is the dominant acetate utilizer in the benzene-degrading microbial consortium.

Bacteria dominate the short-term assimilation of plant-derived N in soil

In comparison to inorganic N cycling, only little is known regarding the assimilation of organic N in soil. Therefore, we used 16S and 18S rDNA gene profiling and functional metaproteomics to characterize the composition of a soil microbial community assimilating (15)N-labeled plant-derived organic matter (OM).Genomic results showed an increase of the abundance of fungi and Proteobacteria related to the utilization of plant-derived OM within the first days of exposure. Similarly, metaproteomic analysis revealed Proteobacteria as the most abundant phylum followed by Actinobacteria and Ascomycota. Finally, protein stable isotope probing (protein-SIP) demonstrated copiotrophic behavior for Rhizobiales belonging to Proteobacteria, Actinomycetales belonging to Actinobacteria and Chroococcales belonging to Cyanobacteria as these phylotypes immediately incorporated (15)N from the added plant tissue. Conversely, the fungal Saccharomycetales and the bacterial Enterobacteriales, Pseudomonadales, Sphingomonadales and Xanthomonadales displayed slower (15)N-assimilation.We showed that, in contrast to the dominance of fungi in the degradation of complex carbon compounds, mostly bacteria were involved in the short-term assimilation of plant-derived N. The combined use of genomic and proteomic approaches allowed to track the flow of N within the soil microbial community.

Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined deoxyribonucleic acid-stable isotope probing (DNA-SIP) with metagenomics and metaproteomics to characterize an uncultivated marine methylotroph that actively incorporated carbon from (13) C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which the culture-independent techniques of DNA-SIP and protein-SIP have been used to characterize the metabolism of a naturally occurring Methylophaga-like bacterium in the marine environment (i.e. Methylophaga thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment.

Metaproteogenomic analysis of a sulfate-reducing enrichment culture reveals genomic organization of key enzymes in the m-xylene degradation pathway and metabolic activity of proteobacteria

This study aimed to ascertain the functional and phylogenetic relationships within an m-xylene degrading sulfate-reducing enrichment culture, which had been maintained for several years in the laboratory with m-xylene as the sole source of carbon and energy. Previous studies indicated that a phylotype affiliated to the Desulfobacteraceae was the main m-xylene assimilating organism. In the present study, genes and gene products were identified by a metaproteogenomic approach using LC-MS/MS analysis of the microbial community, and 2426 peptides were identified from 576 proteins. In the metagenome of the community, gene clusters encoding enzymes involved in fumarate addition to a methyl moiety of m-xylene (nms, bss), as well as gene clusters coding for enzymes involved in modified beta-oxidation to (3-methyl)benzoyl-CoA (bns), were identified in two separate contigs. Additionally, gene clusters containing homologues to bam genes encoding benzoyl-CoA reductase (Bcr) class II, catalyzing the dearomatization of (3-methyl)benzoyl-CoA, were identified. Time-resolved protein stable isotope probing (protein-SIP) experiments using 13C-labeled m-xylene showed that the respective gene products were highly 13C-labeled. The present data suggested the identification of gene products that were similar to those involved in methylnaphthalene degradation even though the consortium was not capable of growing in the presence of naphthalene, methylnaphthalene or toluene as substrates. Thus, a novel branch of enzymes was found that was probably specific for anaerobic m-xylene degradation.

Automated Data Extraction from In Situ Protein-Stable Isotope Probing Studies

Protein-stable isotope probing (protein-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. While most protein-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism(s), a key application will be in situ studies of microbial communities for short periods of time under natural conditions that result in small degrees of partial labeling. One hurdle restricting large-scale in situ protein-SIP studies is the lack of algorithms and software for automated data processing of the massive data sets resulting from such studies. In response, we developed Stable Isotope Probing Protein Extraction Resources software (SIPPER) and applied it for large-scale extraction and visualization of data from short-term (3 h) protein-SIP experiments performed in situ on phototrophic bacterial mats isolated from Yellowstone National Park. Several metrics incorporated into the software allow it to support exhaustive analysis of the complex composite isotopic envelope observed as a result of low amounts of partial label incorporation. SIPPER also enables the detection of labeled molecular species without the need for any prior identification.

Elucidation of in situ polycyclic aromatic hydrocarbon degradation by functional metaproteomics (protein‐SIP)

Current knowledge of the physiology and phylogeny of polycyclic aromatic hydrocarbon (PAH) degrading bacteria often relies on laboratory enrichments and isolations. In the present study, in situ microcosms consisting of activated carbon pellets (BACTRAP®s) were loaded with either (13) C-naphthalene or (13) C-fluorene and were subsequently exposed in the contaminant source and plume fringe region of a PAH-contaminated aquifer. Metaproteomic analysis and protein-stable isotope probing revealed Burkholderiales, Actinomycetales, and Rhizobiales as the most active microorganisms in the groundwater communities. Proteins identified of the naphthalene degradation pathway showed a relative (13) C isotope abundance of approximately 50 atom% demonstrating that the identified naphthalene-degrading bacteria gained at least 80% of their carbon by PAH degradation. Although the microbial community grown on the fluorene-BACTRAPs showed a structure similar to the naphthalene-BACTRAPs, the identification of fluorene degraders and degradation pathways failed in situ. In complementary laboratory microcosms, a clear enrichment in proteins related to Rhodococcus and possible fluorene degradation enzymes was observed. This result demonstrates the impact of laboratory conditions on microbial community structure and activity of certain species and underlines the need on in situ exploration of microbial community functions. In situ microcosms in combination with protein-stable isotope probing may be a significant tool for in situ identification of metabolic key players as well as degradation pathways.

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.

Analysis of structure, function, and activity of a benzene-degrading microbial community

We identified phylotypes performing distinct functions related to benzene degradation in complex microbial biofilms from an aerated treatment pond containing coconut textile. RNA- and protein-stable isotope probing (SIP) and compound-specific stable isotope analysis were applied to delineate bacteria and predominant pathways involved in the degradation of benzene. In laboratory microcosms, benzene was degraded at rates of ≥ 11 μM per day and per gram coconut textile under oxic conditions. Carbon isotope fractionation with isotopic enrichment factors (ε) of -0.6 to -1‰ and no significant hydrogen isotope fractionation indicated a dihydroxylation reaction for the initial ring attack. The incubation with [(13)C₆]-benzene led to (13)CO₂ formation accompanied by (13)C-labeling of RNA and proteins of the active biomass. Phylogenetic analysis of the (13)C-labeled RNA revealed that phylotypes related to Zoogloea, Ferribacterium, Aquabacterium, and Hydrogenophaga within the Betaproteobacteria predominantly assimilated carbon from benzene. Although the phylogenetic classification of identified (13)C-labeled proteins was biased by the incomplete metagenome information of public databases, it matched with RNA-SIP results at genus level. The detection of (13)C-labeled proteins related to toluene dioxygenase and catechol 2,3-dioxygenase suggests benzene degradation by a dihydroxylation pathway with subsequent meta-cleavage of formed catechol.

Protein‐based stable isotope probing (protein‐SIP) in functional metaproteomics

The community phenotype as the sum of molecular functions of organisms living in consortia strongly depends on interactions within these communities. Therefore, the analyses of the most significant molecules in terms of the phenotype, the proteins, have to be performed on samples without disrupting the meta-species environment. Due to the increasing genomic information, proteins provide insights into a potential molecular function and the phylogenetic structure of the community. Unfortunately, the lists of identified proteins are often based first on the technical capacity of the used methods or instruments, and second on the interpretation of them by the assignment of molecular functions to proteins in databases. Especially in non-model organisms the functions of many proteins are often not known and an increasing number of studies indicate a significant amount of uncertainty. To decrease the dependency on assumptions and to enable functional insights by metaproteome approaches, the metabolic labeling from an isotopically labeled substrate can be used. Since the metabolites deriving from the substrate are very rarely species-specific, the incorporation of the stable isotope into proteins can be used as a surrogate marker for metabolic activity. The degree of incorporation can be determined accurately on the peptide level by mass spectrometry; additionally, the peptide sequence provides information on the metabolic active species. Thereby, protein-stable isotope probing (protein-SIP) adds functional information to metaproteome approaches. The classical metaproteome approaches will be reviewed with an emphasis on their attempts towards functional interpretation. The gain from functional insights into metaproteomics by using metabolic labeling of stable isotopes of carbon, nitrogen, and sulfur is reviewed with a focus on the techniques of measurement, calculation of incorporation and data processing.

Assimilation of benzene carbon through multiple trophic levels traced by different stable isotope probing methodologies

The flow of benzene carbon along a food chain consisting of bacteria and eukaryotes, including larvae (Diptera: Chironomidae), was evaluated by total lipid fatty acids (TLFAs)-, amino acid- and protein-stable isotope probing (SIP). A coconut-fibre textile, colonized by a benzene-degrading biofilm, was sampled in a system established for the remediation of benzene, toluene, ethylbenzene and xylenes (BTEX)-polluted groundwater and incubated with (12)C- and [(13)C(6)]-benzene (>99 at.%) in a batch-scale experiment for 2-8 days. After 8 days, Chironomus sp. larvae were added to study carbon flow to higher trophic levels. Gas chromatography-combustion-isotope ratio monitoring mass spectrometry of TLFA showed increased isotope ratios in the (13)C-benzene-incubated biofilm. A higher (13)C-enrichment was observed in TLFAs, indicative of Gram-negative bacteria than for Gram-positive. Fatty acid indicators of eukaryotes showed significant (13)C-incorporation, but to a lower extent than bacterial indicators. Fatty acids extracted from larvae feeding on (13)C-biofilm reached an isotopic ratio of 1.55 at.%, illustrating that the larvae feed, to some extent, on labelled biomass. No (13)C-incorporation was detectable in larval proteins after their separation by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis and analysis by nano-liquid-chromatography-mass spectrometry. The flow of benzene-derived carbon could be traced in a food web consisting of bacteria and eukaryotes.