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Metabolic engineering and synthetic biology are predicated on the precise control of gene expression. As synthetic biology expands beyond model organisms, more tools will be required that function robustly in a wide range of bacterial hosts. The pGinger family of plasmids constitutes 43 plasmids that will enable both constitutive and inducible gene expression in a wide range of nonmodel Proteobacteria .
The raw data (metagenomes and metatranscriptomes) for this study are available in the Joint Genomes Institute Genome Portal (https://genome.jgi.doe.gov/portal/ Project ID 503249) with projects designated by year and product type. The MAG genomes analyzed in this paper are available on NCBI, as bioproject PRJNA800073. Plants and microorganisms form beneficial associations. Understanding plant-microbe interactions will inform microbiome management to enhance crop productivity and resilience to stress. Here, we apply a genome-centric approach to identify ecologically important leaf microbiome members on field-grown switchgrass and miscanthus and to quantify their activities for switchgrass over two growing seasons. We integrate metagenome and metatranscriptome sequencing from 192 leaf samples collected over representative time points in crop phenology. We curated 40 medium- and high-quality metagenome-assembled-genomes (MAGs) and focused analysis on seasonal transcript recruitment to them. Classes represented by these focal MAGs (Actinomycetia, Alpha- and Gamma- Proteobacteria, and Bacteroidota) were active and had increases in transcripts for short-chain dehydrogenase, molybdopterin oxidoreductase, and polyketide cyclase in the late season. The majority of MAGs had activated stress-associated pathways, including trehalose metabolism, indole acetic acid degradation, betaine biosynthesis, and reactive oxygen species degradation, suggesting direct engagement with the host environment. We also detected seasonally activated biosynthetic pathways for terpenes (carotenoids and isoprenoids) and for various non-ribosomal peptide pathways that were poorly annotated. Overall, this study overcame laboratory and bioinformatic challenges associated with field-based leaf metatranscriptome analysis to inform both general and likely specialized activities of these phyllosphere populations. These activities collectively support that leaf-associated bacterial populations are seasonally dynamic, responsive to host cues, and interactively engage in feedback with the plant. This analysis represented quality filtering of metagenomes and metatranscriptomes (data-preparation folder), metagenome assemblies (metagenome-assembly folder) and metagenome-assembled genome binning, curation, refinement, annotation (mag-evaluation folder). Abundances of sequencing libraries were calculated based on reads mapped (mapping folder). Additionallly, analysis of our annotated results are also included (analysis folder).
Limited genetic tools for non-model bacteria are one of the limiting factors for genetic studies. This review compiles genetic tools used for three non-model alpha-proteobacteria, such as Zymomonas mobilis, Cereibacter (Rhodobacter) sphaeroides, and Novosphingobium aromaticivorans, which hold significant potential to produce industrially essential bioenergy compounds due to their distinctive metabolic pathways and resilience in extreme environments. Each of these strains has a unique genetic profile that enables them to efficiently carry out key reactions relevant to producing bioenergy compounds, such as converting sugars into bioenergy compounds and breaking down lignotoxins. Genetic tools can further optimize these strains for enhanced bioenergy compound production. This review explores the metabolic advantages of these organisms. It highlights the available array of genetic toolkits that can be shared among them to unlock their full potential for sustainable biofuel production.
Abstract Oilcane is a metabolically engineered sugarcane ( Saccharum spp. hybrid) that hyper-accumulates lipids in its vegetable biomass to provide an advanced feedstock for biodiesel production. The potential impact of hyper-accumulation of lipids in vegetable biomass on microbiomes and the consequences of altered microbiomes on plant growth and lipid accumulation have not been explored so far. Here, we explore differences in the microbiome structure of different oilcane accessions and non-modified sugarcane. 16S SSU rRNA and ITS rRNA amplicon sequencing were performed to compare the characteristics of the microbiome structure from different plant compartments (leaf, stem, root, rhizosphere, and bulk soil) of four greenhouse-grown oilcane accessions and non-modified sugarcane. Significant differences were only observed in the bacterial microbiomes. In leaf and stem microbiomes, more than 90% of the entire microbiome of non-modified sugarcane and oilcane was dominated by similar core taxa. Taxa associated with Proteobacteria led to differences in the non-modified sugarcane and oilcane microbiome structure. While differences were observed between multiple accessions, accession 1566 was notable in that it was consistently observed to differ in its microbial membership than other accessions and had the lowest abundance of taxa associated with plant-growth-promoting bacteria. Accession 1566 is also unique among oilcane accessions in that it has the highest constitutive expression of the WRI 1 transgene. The WRI 1 transcription factor is known to contribute to significant changes in the global gene expression profile, impacting plant fatty acid biosynthesis and photomorphogenesis. This study reveals for the first time that genetically modified oilcanes associate with distinct microbiomes. Our findings suggest potential relationships between core taxa, biomass yield, and TAG in oilcane accessions and support further research on the relationship between plant genotypes and their microbiomes.
Background: Spaceflight impacts astronauts in many ways but little is known on how spaceflight affects the salivary microbiome and the consequences of these changes on astronaut health, such as viral reactivation. In order to understand this, the salivary microbiome was analyzed with 16S rRNA gene amplicon sequencing, and saliva viral titers were analyzed with quantitative polymerase chain reaction (qPCR) with primers specific for Epstein-Barr virus (EBV), herpes simplex virus (HSV), and varicella zoster virus (VZV) from 10 astronauts pre-flight, in-flight, and post-flight. Results: Streptococcus was the most abundant organism in the saliva, making up 8% of the total organisms detected, and their diversity decreased during spaceflight. Other organisms that had statistically significant changes were Proteobacteria and Fusobacteria which increased during flight and Actinobacteria which decreased during flight. At the genus level, Catonella , Megasphera , and Actinobacillus were absent in more than half of saliva samples collected pre-flight but were then detected during flight. In those subjects that already had these genera pre-flight, their relative abundances increased during flight. Correlation analyses between the microbiome and viral titers revealed a positive correlation with Gracilibacteria , Absconditabacteria , and Abiotrophia and a negative correlation between Oribacterium , Veillonella , and Haemophilus . There was also a significant positive correlation between microbiome richness and EBV viral titers. Conclusions: This is the first study to look at how the salivary microbiome changes as a result of spaceflight and the search for bacterial biomarkers for viral reactivation. Further studies examining the role of specific organisms that were shown to be correlative and predictive in viral reactivation, a serious problem in astronauts during spaceflight, could lead to mitigation strategies to help prevent disease during both short and long duration space missions.
Cryptoendolithic communities are microbial ecosystems dwelling inside porous rocks that are able to persist at the edge of the biological potential for life in the ice-free areas of the Antarctic desert. These regions include the McMurdo Dry Valleys, often accounted as the closest terrestrial counterpart of the Martian environment and thought to be devoid of life until the discovery of these cryptic life-forms. Despite their interest as a model for the early colonization by living organisms of terrestrial ecosystems and for adaptation to extreme conditions of stress, little is known about the evolution, diversity, and genetic makeup of bacterial species that reside in these environments. Using the Illumina Novaseq platform, we generated the first metagenomes from rocks collected in Continental Antarctica over a distance of about 350 km along an altitudinal transect from 834 up to 3100 m above sea level (a.s.l.). A total of 497 draft bacterial genome sequences were assembled and clustered into 269 candidate species that lack a representative genome in public databases. Actinobacteria represent the most abundant phylum, followed by Chloroflexi and Proteobacteria. The “Candidatus Jiangella antarctica” has been recorded across all samples, suggesting a high adaptation and specialization of this species to the harshest Antarctic desert environment. The majority of these new species belong to monophyletic bacterial clades that diverged from related taxa in a range from 1.2 billion to 410 Ma and are functionally distinct from known related taxa. Our findings significantly increase the repertoire of genomic data for several taxa and, to date, represent the first example of bacterial genomes recovered from endolithic communities. Their ancient origin seems to not be related to the geological history of the continent, rather they may represent evolutionary remnants of pristine clades that evolved across the Tonian glaciation. These unique genomic resources will underpin future studies on the structure, evolution, and function of these ecosystems at the edge of life.
Abstract Background Phytoplankton communities significantly contribute to global biogeochemical cycles of elements and underpin marine food webs. Although their uncultured genomic diversity has been estimated by planetary-scale metagenome sequencing and subsequent reconstruction of metagenome-assembled genomes (MAGs), this approach has yet to be applied for complex phytoplankton microbiomes from polar and non-polar oceans consisting of microbial eukaryotes and their associated prokaryotes. Results Here, we have assembled MAGs from chlorophyll a maximum layers in the surface of the Arctic and Atlantic Oceans enriched for species associations (microbiomes) with a focus on pico- and nanophytoplankton and their associated heterotrophic prokaryotes. From 679 Gbp and estimated 50 million genes in total, we recovered 143 MAGs of medium to high quality. Although there was a strict demarcation between Arctic and Atlantic MAGs, adjacent sampling stations in each ocean had 51–88% MAGs in common with most species associations between Prasinophytes and Proteobacteria . Phylogenetic placement revealed eukaryotic MAGs to be more diverse in the Arctic whereas prokaryotic MAGs were more diverse in the Atlantic Ocean. Approximately 70% of protein families were shared between Arctic and Atlantic MAGs for both prokaryotes and eukaryotes. However, eukaryotic MAGs had more protein families unique to the Arctic whereas prokaryotic MAGs had more families unique to the Atlantic. Conclusion Our study provides a genomic context to complex phytoplankton microbiomes to reveal that their community structure was likely driven by significant differences in environmental conditions between the polar Arctic and warm surface waters of the tropical and subtropical Atlantic Ocean.
Abstract Background The role of bacterial symbionts that populate octocorals (Cnidaria, Octocorallia) is still poorly understood. To shed light on their metabolic capacities, we examined 66 high-quality metagenome-assembled genomes (MAGs) spanning 30 prokaryotic species, retrieved from microbial metagenomes of three octocoral species and seawater. Results Symbionts of healthy octocorals were affiliated with the taxa Endozoicomonadaceae , Candidatus Thioglobaceae , Metamycoplasmataceae , unclassified Pseudomonadales , Rhodobacteraceae , unclassified Alphaproteobacteria and Ca. Rhabdochlamydiaceae . Phylogenomics inference revealed that the Endozoicomonadaceae symbionts uncovered here represent two species of a novel genus unique to temperate octocorals, here denoted Ca. Gorgonimonas eunicellae and Ca. Gorgonimonas leptogorgiae . Their genomes revealed metabolic capacities to thrive under suboxic conditions and high gene copy numbers of serine-threonine protein kinases, type 3-secretion system, type-4 pili, and ankyrin-repeat proteins, suggesting excellent capabilities to colonize, aggregate, and persist inside their host. Contrarily, MAGs obtained from seawater frequently lacked symbiosis-related genes. All Endozoicomonadaceae symbionts harbored endo-chitinase and chitin-binging protein-encoding genes, indicating that they can hydrolyze the most abundant polysaccharide in the oceans. Other symbionts, including Metamycoplasmataceae and Ca. Thioglobaceae , may assimilate the smaller chitin oligosaccharides resulting from chitin breakdown and engage in chitin deacetylation, respectively, suggesting possibilities for substrate cross-feeding and a role for the coral microbiome in overall chitin turnover. We also observed sharp differences in secondary metabolite production potential between symbiotic lineages. Specific Proteobacteria taxa may specialize in chemical defense and guard other symbionts, including Endozoicomonadaceae , which lack such capacity. Conclusion This is the first study to recover MAGs from dominant symbionts of octocorals, including those of so-far unculturable Endozoicomonadaceae , Ca. Thioglobaceae and Metamycoplasmataceae symbionts. We identify a thus-far unanticipated, global role for Endozoicomonadaceae symbionts of corals in the processing of chitin, the most abundant natural polysaccharide in the oceans and major component of the natural zoo- and phytoplankton feed of octocorals. We conclude that niche partitioning, metabolic specialization, and adaptation to low oxygen conditions among prokaryotic symbionts likely contribute to the plasticity and adaptability of the octocoral holobiont in changing marine environments. These findings bear implications not only for our understanding of symbiotic relationships in the marine realm but also for the functioning of benthic ecosystems at large.
Abstract Background Biofilms in sulfide-rich springs present intricate microbial communities that play pivotal roles in biogeochemical cycling. We studied chemoautotrophically based biofilms that host diverse CPR bacteria and grow in sulfide-rich springs to investigate microbial controls on biogeochemical cycling. Results Sulfide springs biofilms were investigated using bulk geochemical analysis, genome-resolved metagenomics, and scanning transmission X-ray microscopy (STXM) at room temperature and 87 K. Chemolithotrophic sulfur-oxidizing bacteria, including Thiothrix and Beggiatoa , dominate the biofilms, which also contain CPR Gracilibacteria, Absconditabacteria, Saccharibacteria, Peregrinibacteria, Berkelbacteria, Microgenomates, and Parcubacteria. STXM imaging revealed ultra-small cells near the surfaces of filamentous bacteria that may be CPR bacterial episymbionts. STXM and NEXAFS spectroscopy at carbon K and sulfur L 2,3 edges show that filamentous bacteria contain protein-encapsulated spherical elemental sulfur granules, indicating that they are sulfur oxidizers, likely Thiothrix . Berkelbacteria and Moranbacteria in the same biofilm sample are predicted to have a novel electron bifurcating group 3b [NiFe]-hydrogenase, putatively a sulfhydrogenase, potentially linked to sulfur metabolism via redox cofactors. This complex could potentially contribute to symbioses, for example, with sulfur-oxidizing bacteria such as Thiothrix that is based on cryptic sulfur cycling. One Doudnabacteria genome encodes adjacent sulfur dioxygenase and rhodanese genes that may convert thiosulfate to sulfite. We find similar conserved genomic architecture associated with CPR bacteria from other sulfur-rich subsurface ecosystems. Conclusions Our combined metagenomic, geochemical, spectromicroscopic, and structural bioinformatics analyses of biofilms growing in sulfide-rich springs revealed consortia that contain CPR bacteria and sulfur-oxidizing Proteobacteria, including Thiothrix , and bacteria from a new family within Beggiatoales. We infer roles for CPR bacteria in sulfur and hydrogen cycling.
Culture-independent characterization of microbial communities associated with popular plant model systems have increased our understanding of the plant microbiome. However, the integration of other model systems, such as duckweed, could facilitate our understanding of plant microbiota assembly and evolution. Duckweeds are floating aquatic plants with many characteristics, including small size and reduced plant architecture, that suggest their use as a facile model system for plant microbiome studies. Here, we investigated the structure and assembly of the duckweed bacterial microbiome. First, a culture-independent survey of the duckweed bacterial microbiome from different locations in New Jersey revealed similar phylogenetic profiles. These studies showed that Proteobacteria is a dominant phylum in the duckweed bacterial microbiome. To observe the assembly dynamics of the duckweed bacterial community, we inoculated quasi-gnotobiotic duckweed with wastewater effluent from a municipal wastewater treatment plant. Our results revealed that duckweed strongly shapes its bacterial microbiome and forms distinct associations with bacterial community members from the initial inoculum. Additionally, these inoculation studies showed the bacterial communities of different duckweed species were similar in taxa composition and abundance. Analysis across the different duckweed bacterial communities collected in this study identified a set of “core” bacterial taxa consistently present on duckweed irrespective of the locale and context. Furthermore, comparison of the duckweed bacterial community to that of rice and Arabidopsis revealed a conserved taxonomic structure between the duckweed microbiome and the terrestrial leaf microbiome. Our results suggest that duckweeds utilize similar bacterial community assembly principles as those found in terrestrial plants and indicate a highly conserved structuring effect of leaf tissue on the plant microbiome.
Lignin is nature’s largest source of phenolic compounds. Its recalcitrance to enzymatic conversion is still a limiting step to increase the value of lignin. Although bacteria are able to degrade lignin in nature, most studies have focused on lignin degradation by fungi. To understand which bacteria are able to use lignin as the sole carbon source, natural selection over time was used to obtain enriched microbial consortia over a 12-week period. The source of microorganisms to establish these microbial consortia were commercial and backyard compost soils. Cultivation occurred at two different temperatures, 30°C and 37°C, in defined culture media containing either Kraft lignin or alkaline-extracted lignin as carbon source. iTag DNA sequencing of bacterial 16S rDNA gene was performed for each of the consortia at six timepoints (passages). The initial bacterial richness and diversity of backyard compost soil consortia was greater than that of commercial soil consortia, and both parameters decreased after the enrichment protocol, corroborating that selection was occurring. Bacterial consortia composition tended to stabilize from the fourth passage on. After the enrichment protocol, Firmicutes phylum bacteria were predominant when lignin extracted by alkaline method was used as a carbon source, whereas Proteobacteria were predominant when Kraft lignin was used. Bray-Curtis dissimilarity calculations at genus level, visualized using NMDS plots, showed that the type of lignin used as a carbon source contributed more to differentiate the bacterial consortia than the variable temperature. The main known bacterial genera selected to use lignin as a carbon source were Altererythrobacter , Aminobacter , Bacillus , Burkholderia , Lysinibacillus , Microvirga , Mycobacterium , Ochrobactrum , Paenibacillus , Pseudomonas , Pseudoxanthomonas , Rhizobiales and Sphingobium . These selected bacterial genera can be of particular interest for studying lignin degradation and utilization, as well as for lignin-related biotechnology applications.
The microbial and molecular characterization of the ectorhizosphere is an important step towards developing a more complete understanding of how the cultivation of biofuel crops can be undertaken in nutrient poor environments. The ectorhizosphere of Setaria is of particular interest because the plant component of this plant-microbe system is an important agricultural grain crop and a model for biofuel grasses. Importantly, Setaria lends itself to high throughput molecular studies. As such, we have identified important intra- and interspecific microbial and molecular differences in the ectorhizospheres of three geographically distant Setaria italica accessions and their wild ancestor S . viridis . All were grown in a nutrient-poor soil with and without nutrient addition. To assess the contrasting impact of nutrient deficiency observed for two S . italica accessions, we quantitatively evaluated differences in soil organic matter, microbial community, and metabolite profiles. Together, these measurements suggest that rhizosphere priming differs with Setaria accession, which comes from alterations in microbial community abundances, specifically Actinobacteria and Proteobacteria populations. When globally comparing the metabolomic response of Setaria to nutrient addition, plants produced distinctly different metabolic profiles in the leaves and roots. With nutrient addition, increases of nitrogen containing metabolites were significantly higher in plant leaves and roots along with significant increases in tyrosine derived alkaloids, serotonin, and synephrine. Glycerol was also found to be significantly increased in the leaves as well as the ectorhizosphere. These differences provide insight into how C 4 grasses adapt to changing nutrient availability in soils or with contrasting fertilization schemas. Gained knowledge could then be utilized in plant enhancement and bioengineering efforts to produce plants with superior traits when grown in nutrient poor soils.
The soil microbiome is central to the cycling of carbon and other nutrients and to the promotion of plant growth. Despite its importance, analysis of the soil microbiome is difficult due to its sheer complexity, with thousands of interacting species. Here, we reduced this complexity by developing model soil microbial consortia that are simpler and more amenable to experimental analysis but still represent important microbial functions of the native soil ecosystem. Samples were collected from an arid grassland soil and microbial communities (consisting mainly of bacterial species) were enriched on agar plates containing chitin as the main carbon source. Chitin was chosen because it is an abundant carbon and nitrogen polymer in soil that often requires the coordinated action of several microorganisms for complete metabolic degradation. Several soil consortia were derived that had tractable richness (30-50 OTUs) with diverse phyla representative of the native soil, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Verrucomicrobia. The resulting consortia can be stored as glycerol or lyophilized stocks at -80° C and revived while retaining community composition, greatly increasing their use as tools for the research community at large. One of the consortia that was particularly stable was chosen as a model soil consortium (MSC-1) for further analysis. MSC-1 species interactions were studied using both pairwise co-cultivation in liquid media and during growth in soil under several perturbations. Co-abundance analyses highlighted interspecies interactions and helped to define keystone species, including Mycobacterium, Rhodococcus, and Rhizobiales taxa. These experiments demonstrate the success of an approach based on naturally enriching a community of interacting species that can be stored, revived, and shared. The knowledge gained from querying these communities and their interactions will enable better understanding of the soil microbiome and the roles these interactions play in this environment.
The economy of biorefineries is influenced not only by biofuel production from carbohydrates but also by the production of valuable compounds from largely underutilized industrial residues. Currently, the demand for many chemicals that could be made in a biorefinery, such as succinic acid (SA), medium-chain fatty acids (MCFAs), and lactic acid (LA), is fulfilled using petroleum, palm oil, or pure carbohydrates as raw materials, respectively. Thin stillage (TS), the residual liquid material following distillation of ethanol, is an underutilized coproduct from the starch biofuel industry. This carbon-rich material has the potential for chemical upgrading by microorganisms. Here, we explored the formation of different fermentation products by microbial communities grown on TS using different bioreactor conditions. At the baseline operational condition (6-day retention time, pH 5.5, 35°C), we observed a mixture of MCFAs as the principal fermentation products. Operation of a bioreactor with a 1-day retention time induced an increase in SA production, and a temperature increase to 55°C resulted in the accumulation of lactic and propionic acids. In addition, a reactor operated with a 1-day retention time at 55°C conditions resulted in LA accumulation as the main fermentation product. The prominent members of the microbial community in each reactor were assessed by 16S rRNA gene amplicon sequencing and phylogenetic analysis. Under all operating conditions, members of the Lactobacillaceae family within Firmicutes and the Acetobacteraceae family within Proteobacteria were ubiquitous. Members of the Prevotellaceae family within Bacteroidetes and Lachnospiraceae family within the Clostridiales order of Firmicutes were mostly abundant at 35°C and not abundant in the microbial communities of the TS reactors incubated at 55°C. The ability to adjust bioreactor operating conditions to select for microbial communities with different fermentation product profiles offers new strategies to explore and compare potentially valuable fermentation products from TS and allows industries the flexibility to adapt and switch chemical production based on market prices and demands.
Recovery and cultivation of diverse environmentally-relevant microorganisms from the terrestrial subsurface remain a challenge despite recent advances in modern molecular technology. Here, we applied complex carbon (C) sources, i.e., sediment dissolved organic matter (DOM) and bacterial cell lysate, to enrich groundwater microbial communities for 30 days. As comparisons, we also included enrichments amended with simple C sources including glucose, acetate, benzoate, oleic acid, cellulose, and mixed vitamins. Our results demonstrate that complex C is far more effective in enriching diverse and distinct microorganisms from groundwater than simple C. Simple C enrichments yield significantly lower biodiversity, and are dominated by few phyla (e.g., Proteobacteria and Bacteroidetes ), while microcosms enriched with complex C demonstrate significantly higher biodiversity including phyla that are poorly represented in published culture collections (e.g., Verrucomicrobia , Planctomycetes , and Armatimonadetes ). Subsequent isolation from complex C enrichments yielded 228 bacterial isolates representing five phyla, 17 orders, and 56 distinct species, including candidate novel, rarely cultivated, and undescribed organisms. Results from this study will substantially advance cultivation and isolation strategies for recovering diverse and novel subsurface microorganisms. Obtaining axenic representatives of “once-unculturable” microorganisms will enhance our understanding of microbial physiology and function in different biogeochemical niches of terrestrial subsurface ecosystems.
The conversion of mercury (Hg) to monomethylmercury (MMHg) is a critical area of concern in global Hg cycling. Periphyton biofilms may harbor significant amounts of MMHg but little is known about the Hg-methylating potential of the periphyton microbiome. Therefore, we used high-throughput amplicon sequencing of the 16S rRNA gene, ITS2 region, and Hg methylation gene pair (hgcAB) to characterize the archaea/bacteria, fungi, and Hg-methylating microorganisms in periphyton communities grown in a contaminated watershed in East Tennes(see United States). Furthermore, we examined how nutrient amendments (nitrate and/or phosphate) altered periphyton community structure and function. We found that bacterial/archaeal richness in experimental conditions decreased in summer and increased in autumn relative to control treatments, while fungal diversity generally increased in summer and decreased in autumn relative to control treatments. Interestingly, the Hg-methylating communities were dominated by Proteobacteria followed by Candidatus Atribacteria across both seasons. Surprisingly, Hg methylation potential correlated with numerous bacterial families that do not contain hgcAB, suggesting that the overall microbiome structure of periphyton communities influences rates of Hg transformation within these microbial mats. To further explore these complex community interactions, we performed a microbial network analysis and found that the nitrate-amended treatment resulted in the highest number of hub taxa that also corresponded with enhanced Hg methylation potential. This work provides insight into community interactions within the periphyton microbiome that may contribute to Hg cycling and will inform future research that will focus on establishing mixed microbial consortia to uncover mechanisms driving shifts in Hg cycling within periphyton habitats.
Rapid microbial growth in the early phase of plant litter decomposition is viewed as an important component of soil organic matter (SOM) formation. However, the microbial taxa and chemical substrates that correlate with carbon storage are not well resolved. The complexity of microbial communities and diverse substrate chemistries that occur in natural soils make it difficult to identify links between community membership and decomposition processes in the soil environment. To identify potential relationships between microbes, soil organic matter, and their impact on carbon storage, we used sand microcosms to control for external environmental factors such as changes in temperature and moisture as well as the variability in available carbon that exist in soil cores. Using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) on microcosm samples from early phase litter decomposition, we found that protein- and tannin-like compounds exhibited the strongest correlation to dissolved organic carbon (DOC) concentration. Proteins correlated positively with DOC concentration, while tannins correlated negatively with DOC. Through random forest, neural network, and indicator species analyses, we identified 42 bacterial and 9 fungal taxa associated with DOC concentration. The majority of bacterial taxa (26 out of 42 taxa) belonged to the phylum Proteobacteria while all fungal taxa belonged to the phylum Ascomycota. Additionally, we identified significant connections between microorganisms and protein-like compounds and found that most taxa (12/14) correlated negatively with proteins indicating that microbial consumption of proteins is likely a significant driver of DOC concentration. This research links DOC concentration with microbial production and/or decomposition of specific metabolites to improve our understanding of microbial metabolism and carbon persistence.
Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil ® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the Is PETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as Is PETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.