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To better understand mechanisms of carbon (C) and nitrogen (N) dynamics under anthropogenic N enrichment, we examined frequencies of C- and N-targeting genes in litter fungi. In particular, we tested the hypothesis that N enrichment selects for C-targeting genes but against N-targeting genes, if fungi preferentially invest resources in acquisition of growth-limiting nutrients. We conducted a fully-factorial litter and microbial transplant in a N fertilization experiment in Southern California grassland. The transplant design enabled us to contrast direct effects of N fertilization in the environment, indirect effects of N-induced shifts in the microbial community, and indirect effects of N-induced changes in plant litter chemistry. For each treatment, we assessed frequencies of select well-annotated fungal functional genes: cellulose-targeting AA9 genes (for C acquisition) versus ammonium transporter genes and amino acid permease genes (for N acquisition). We found that our hypothesis was upheld only with regard to shifts in the microbial community. Specifically, when grown in the same environment and litter, fungi from the N-fertilized plots displayed greater frequencies of cellulose-targeting AA9 genes from basidiomycetes, but smaller frequencies of ammonium transporter genes and amino acid permease genes, when compared to fungi from the control plots. In contrast, N fertilization in the plot environment was associated with higher frequencies of amino acid permease genes and ammonium transporter genes. Likewise, plant litter from the N-fertilized plots selected for higher frequencies of ammonium transporter genes. Altogether, we found fairly inconsistent effects of N enrichment on fungal functional genes related to C and N acquisition. Even if the genetic capacity of the fungal community to acquire C versus N changes owing to shifts in the microbial community, direct effects of N fertilization and indirect effects of litter chemistry may offset the response.

CEA has manufactured a series of Fukushima Daiichi fuel debris simulants, either with depleted uranium oxide or with hafnium oxide as a surrogate of UO2. In ex-vessel compositions resulting from an interaction between corium and concrete, the oxidic phase density becomes lighter than that of the metallic phase, which segregates at the bottom. Three of these metallic phases have been mechanically cut at CEA Cadarache with handsaw and with core boring tool in FUJISAN facility. It appeared that two of these metallic blocks were extremely hard to cut (one from a fabrication with uranium oxide, the other from a simulant block) while the last one was more easily cut. The similarities and differences in metallographic analyses (SEM-EDS and XRD) of these three metal blocks will be presented and discussed. This experience provides useful learnings in view of the cutting and retrieval of fuel debris from Fukushima Daiichi. [ABSTRACT FROM AUTHOR]

Bacteria and fungi drive the decomposition of dead plant biomass (litter), an important step in the terrestrial carbon cycle. Here we investigate the sensitivity of litter microbial communities to simulated global change (drought and nitrogen addition) in a California annual grassland. Using 16S and 28S rDNA amplicon pyrosequencing, we quantify the response of the bacterial and fungal communities to the treatments and compare these results to background, temporal (seasonal and interannual) variability of the communities. We found that the drought and nitrogen treatments both had significant effects on microbial community composition, explaining 2-6% of total compositional variation. However, microbial composition was even more strongly influenced by seasonal and annual variation (explaining 14-39%). The response of microbial composition to drought varied by season, while the effect of the nitrogen addition treatment was constant through time. These compositional responses were similar in magnitude to those seen in microbial enzyme activities and the surrounding plant community, but did not correspond to a consistent effect on leaf litter decomposition rate. Overall, these patterns indicate that, in this ecosystem, temporal variability in the composition of leaf litter microorganisms largely surpasses that expected in a short-term global change experiment. Thus, as for plant communities, future microbial communities will likely be determined by the interplay between rapid, local background variability and slower, global changes.

In most terrestrial environments, our knowledge of the elemental composition and stoichiometry of microorganisms stems from indirect whole community analyses. In contrast, we have little direct knowledge of the elemental composition of specific microorganisms and the variation between and within Fungi and Bacteria. To address this issue, we isolated and identified the elemental content of 87 strains of Fungi and Bacteria isolated from grassland leaf litter. The isolated strains were affiliated with a broad range of diversity including Ascomycota and Basidiomycota for Fungi, and Proteobacteria, Bacteroidetes, and Actinobacteria for Bacteria. The C:P and C:N but not N:P ratios were significantly higher in Fungi than in Bacteria. Extensive strain variation in elemental composition was partly linked to phylogeny and growth rate. Across all strains, the geometric mean C:N:P was 88:15:1. This overall ratio was significantly higher than reported for other leaf litter and terrestrial whole communities but closer to the canonical Redfield ratio characterizing marine microorganisms. This result warrants further investigation into the discrepancy between whole community and isolated strain elemental ratios. © 2014 Elsevier Ltd.

Many microorganisms contain cellulases that are important for plant cell wall degradation and overall soil ecosystem functioning. At present, we have extensive biochemical knowledge of cellulases but little is known about the phylogenetic distribution of these enzymes. To address this, we analyzed the distribution of 21,985 genes encoding proteins related to cellulose utilization in 5,123 sequenced bacterial genomes. First, we identified the distribution of glycoside hydrolases involved in cellulose utilization and synthesis at different taxonomic levels, from the phylum to the strain. Cellulose degradation/utilization capabilities appeared in nearly all major groups and resulted in strains displaying various enzyme gene combinations. Potential cellulose degraders, having both cellulases and β-glucosidases, constituted 24% of all genomes whereas potential opportunistic strains, having β-glucosidases only, accounted for 56%. Finally, 20% of the bacteria have no relevant enzymes and do not rely on cellulose utilization. The latter group was primarily connected to specific bacterial lifestyles like autotrophy and parasitism. Cellulose degraders, as well as opportunists, have multiple enzymes with similar functions. However, the potential degraders systematically harbor about twice more β-glucosidases than their potential opportunistic relatives. Although scattered, the distribution of functional types, in bacterial lineages, is not random but mostly follows a Brownian motion evolution model. Degraders form clusters of relatives at the species level, whereas opportunists are clustered at the genus level. This information can form a mechanistic basis for the linking of changes in microbial community composition to soil ecosystem processes.

Glycoside hydrolases (GHs) are carbohydrate-active enzymes essential for many environmental (e.g., carbon cycling) and biotechnological (e.g., biofuels) processes. The complete processing of carbohydrates by bacteria requires many enzymes acting synergistically. Here, I investigated the clustered or scattered distribution of 406,337 GH-genes and their association with transporter genes identified in 15,640 completely sequenced bacterial genomes. Different bacterial lineages displayed conserved levels of clustered or scattered GH-genes, but overall, the GH-genes clustering was generally higher than in randomized genomes. In lineages with highly clustered GH-genes (e.g., Bacteroides, Paenibacillus ), clustered genes shared the same orientation. These codirectional gene clusters potentially facilitate the genes' co-expression by allowing transcriptional read-through and, at least in some cases, forming operons. In several taxa, the GH-genes clustered with distinct types of transporter genes. The type of transporter genes and the distribution of the so-called GH:TR-genes clusters were conserved in selected lineages. Globally, the phylogenetically conserved clustering of the GH-genes with transporter genes highlights the central function of carbohydrate processing across bacterial lineages. In addition, in bacteria with the most identified GH-genes, the genomic adaptations for carbohydrate processing also mirrored the broad environmental origin of the sequenced strains (e.g., soil and mammal gut) suggesting that a combination of evolutionary history and the environment selects for the specific supragenic organization of the GH-genes supporting the carbohydrate processing in bacterial genomes., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Berlemont.)

Juvenile common thresher sharks ( Alopias vulpinus ) have been recently stranding along the California coastline. Using Illumina sequencing of the bacterial 16S rRNA gene along with necropsy, cytological, bacteriological, and histological techniques, we screened microbial communities and described lesions characterizing affected sharks with the purpose of identifying potential pathogen sources and pathologic processes. Histopathological assessment of moribund sharks revealed severe meningoencephalitis, as previously described in stranded salmon sharks ( Lamna ditropis ), along with inflammation of the inner ear and subcutaneous tissues surrounding the endolymphatic ducts. Furthermore, inflamed areas were characterized by the prevalence of Carnobacterium maltaromaticum , suggesting this bacterium as a potential pathogen that gains access to the inner ear through the endolymphatic ducts, with subsequent spread into the brain. The absence or low abundance of this bacterium in the spiral valve in both healthy and infected sharks suggests that Carnobacterium is not a commensal member of their digestive communities and the spiral valve is unlikely to be the source of the pathogen. Furthermore, phylogenetic analysis suggests that C. maltaromaticum strains isolated from diseased sharks have minimal genetic variation and differ from other strains originating from food or diseased teleosts. While a C. maltaromaticum- like organism has previously been associated with meningoencephalitis in salmon shark strandings, this is the first study to report common thresher shark strandings associated with C. maltaromaticum , involving the endolymphatic ducts as portals of entry to the brain.

Most plants rely on specialized root-associated microbes to obtain essential nitrogen (N), yet not much is known about the evolutionary history of the rhizosphere-plant interaction. We conducted a common garden experiment to investigate the plant root-rhizosphere microbiome association using chloridoid grasses sampled from around the world and grown from seed in a greenhouse. We sought to test whether plants that are more closely related phylogenetically have more similar root bacterial microbiomes than plants that are more distantly related. Using metagenome sequencing, we found that there is a conserved core and a variable rhizosphere bacterial microbiome across the chloridoid grasses. Additionally, phylogenetic distance among the host plant species was correlated with bacterial community composition, suggesting the plant hosts prefer specific bacterial lineages. The functional potential for N utilization across microbiomes fluctuated extensively and mirrored variation in the microbial community composition across host plants. Variation in the bacterial potential for N fixation was strongly affected by the host plants' phylogeny, whereas variation in N recycling, nitrification, and denitrification was unaffected. This study highlights the evolutionary linkage between the N fixation traits of the microbial community and the plant host and suggests that not all functional traits are equally important for plant-microbe associations.

Permafrost is an extreme habitat yet it hosts microbial populations that remain active over millennia. Using permafrost collected from a Pleistocene chronosequence (19 to 33 ka), we hypothesized that the functional genetic potential of microbial communities in permafrost would reflect microbial strategies to metabolize permafrost soluble organic matter (OM) in situ over geologic time. We also hypothesized that changes in the metagenome across the chronosequence would correlate with shifts in carbon chemistry, permafrost age, and paleoclimate at the time of permafrost formation. We combined high-resolution characterization of water-soluble OM by Fourier-transform ion-cyclotron-resonance mass spectrometry (FT-ICR MS), quantification of organic anions in permafrost water extracts, and metagenomic sequencing to better understand the relationships between the molecular-level composition of potentially bioavailable OM, the microbial community, and permafrost age. Both age and paleoclimate had marked effects on both the molecular composition of dissolved OM and the microbial community. The relative abundance of genes associated with hydrogenotrophic methanogenesis, carbohydrate active enzyme families, nominal oxidation state of carbon (NOSC), and number of identifiable molecular formulae significantly decreased with increasing age. In contrast, genes associated with fermentation of short chain fatty acids (SCFAs), the concentration of SCFAs and ammonium all significantly increased with age. We present a conceptual model of microbial metabolism in permafrost based on fermentation of OM and the buildup of organic acids that helps to explain the unique chemistry of ancient permafrost soils. These findings imply long-term in situ microbial turnover of ancient permafrost OM and that this pooled biolabile OM could prime ancient permafrost soils for a larger and more rapid microbial response to thaw compared to younger permafrost soils., (Copyright © 2020 Leewis, Berlemont, Podgorski, Srinivas, Zito, Spencer, McFarland, Douglas, Conaway, Waldrop and Mackelprang.)

The discovery of new glycoside hydrolases that can be utilized in the chemoenzymatic synthesis of carbohydrates has emerged as a promising approach for various biotechnological processes. In this study, recombinant Ps_Cel5A from Pseudomonas stutzeri A1501, a novel member of the GH5_5 subfamily, was expressed, purified and crystallized. Preliminary experiments confirmed the ability of Ps_Cel5A to catalyze transglycosylation with cellotriose as a substrate. The crystal structure revealed several structural determinants in and around the positive subsites, providing a molecular basis for a better understanding of the mechanisms that promote and favour synthesis rather than hydrolysis. In the positive subsites, two nonconserved positively charged residues (Arg178 and Lys216) were found to interact with cellobiose. This adaptation has also been reported for transglycosylating β-mannanases of the GH5_7 subfamily.

Although water is a critical resource for organisms, microbially-mediated processes such as decomposition and nitrogen (N) transformations can endure within ecosystems even when water is scarce. To identify underlying mechanisms, we examined the genetic potential for fungi to contribute to specific aspects of carbon (C) and N cycling in a drought manipulation in Southern California grassland. In particular, we measured the frequency of fungal functional genes encoding enzymes that break down cellulose and chitin, and take up ammonium and amino acids, in decomposing litter. Furthermore, we used "microbial cages" to reciprocally transplant litter and microbes between control and drought plots. This approach allowed us to distinguish direct effects of drought in the plot environment versus indirect effects via shifts in the microbial community or changes in litter chemistry. For every fungal functional gene we examined, the frequency of that gene within the microbial community increased significantly in drought plots compared to control plots. In contrast, when plot environment was held constant, frequencies of these fungal functional genes did not differ significantly between control-derived microbes versus drought-derived microbes, or between control-derived litter versus drought-derived litter. It appears that drought directly selects for fungi with the genetic capacity to acquire these specific C- and N-containing compounds. This genetic trait may allow fungi to take advantage of ephemeral water supplies. Altogether, proliferation of fungi with the genetic capacity for C and N acquisition may contribute to the maintenance of biogeochemical cycling under drought., Competing Interests: The authors have declared that no competing interests exist.

Here, we report the draft genome sequences of 9 strains of Carnobacterium maltaromaticum (SK_LD1 to SK_LD3 and SK_AV1 to SK_AV6), a member of the Carnobacteriaceae family (phylum Firmicutes ). These strains were isolated from the brain and the inner ear of three diseased thresher sharks and two diseased salmon sharks. The genome assembly resulted in an average of 3,306,205.9 ± 29,143.9 bp and 3,085 ± 32.67 coding DNA sequences (CDS)., (Copyright © 2018 Martinez-Steele et al.)

The enzymatic deconstruction of structural polysaccharides, which relies on the production of specific glycoside hydrolases (GHs), is an essential process across environments. Over the past few decades, researchers studying the diversity and evolution of these enzymes have isolated and biochemically characterized thousands of these proteins. The carbohydrate-active enzymes database (CAZy) lists these proteins and provides some metadata. Here, the sequences and metadata of characterized sequences derived from GH families associated with the deconstruction of cellulose, xylan, and chitin were collected and discussed. First, although few polyspecific enzymes are identified, characterized GH families are mostly monospecific. Next, the taxonomic distribution of characterized GH mirrors the distribution of identified sequences in sequenced genomes. This provides a rationale for connecting the identification of GH sequences to specific reactions or lineages. Finally, we tested the annotation of the characterized GHs using HMM scan and the protein families database (Pfam). The vast majority of GHs targeting cellulose, xylan, and chitin can be identified using this publicly accessible approach.

Fungi are important polysaccharide degraders in the environment and for biotechnology. Here, the increasing number of sequenced fungal genomes allowed for systematic identification of genes and proteins involved in polysaccharide degradation in 218 fungi. Globally, 9,003 sequences for glycoside hydrolases and lytic polysaccharide mono-oxygenases targeting cellulose, xylan, and chitin, were identified. Although abundant in most lineages, the distribution of these enzymes is variable even between organisms from the same genus. However, most fungi are generalists possessing several enzymes for polysaccharide deconstruction. Most identified enzymes were small proteins with simple domain organization or eventually consisted of one catalytic domain associated with a non-catalytic accessory domain. Thus unlike bacteria, fungi's ability to degrade polysaccharides relies on apparent redundancy in functional traits and the high frequency of lytic polysaccharide mono-oxygenases, as well as other physiological adaptation such as hyphal growth. Globally, this study provides a comprehensive framework to further identify enzymes for polysaccharide deconstruction in fungal genomes and will help identify new strains and enzymes with potential for biotechnological application.

Terrestrial ecosystem models assume that microbial communities respond instantaneously, or are immediately resilient, to environmental change. Here we tested this assumption by quantifying the resilience of a leaf litter community to changes in precipitation or nitrogen availability. By manipulating composition within a global change experiment, we decoupled the legacies of abiotic parameters versus that of the microbial community itself. After one rainy season, more variation in fungal composition could be explained by the original microbial inoculum than the litterbag environment (18% versus 5.5% of total variation). This compositional legacy persisted for 3 years, when 6% of the variability in fungal composition was still explained by the microbial origin. In contrast, bacterial composition was generally more resilient than fungal composition. Microbial functioning (measured as decomposition rate) was not immediately resilient to the global change manipulations; decomposition depended on both the contemporary environment and rainfall the year prior. Finally, using metagenomic sequencing, we showed that changes in precipitation, but not nitrogen availability, altered the potential for bacterial carbohydrate degradation, suggesting why the functional consequences of the two experiments may have differed. Predictions of how terrestrial ecosystem processes respond to environmental change may thus be improved by considering the legacies of microbial communities.

Across many environments microbial glycoside hydrolases support the enzymatic processing of carbohydrates, a critical function in many ecosystems. Little is known about how the microbial composition of a community and the potential for carbohydrate processing relate to each other. Here, using 1,934 metagenomic datasets, we linked changes in community composition to variation of potential for carbohydrate processing across environments. We were able to show that each ecosystem-type displays a specific potential for carbohydrate utilization. Most of this potential was associated with just 77 bacterial genera. The GH content in bacterial genera is best described by their taxonomic affiliation. Across metagenomes, fluctuations of the microbial community structure and GH potential for carbohydrate utilization were correlated. Our analysis reveals that both deterministic and stochastic processes contribute to the assembly of complex microbial communities., Competing Interests: The authors have declared that no competing interests exist.

Assigning ecological roles to bacterial taxa remains imperative to understanding how microbial communities will respond to changing environmental conditions. Here we analyze the genus Curtobacterium , as it was found to be the most abundant taxon in a leaf litter community in southern California. Traditional characterization of this taxon predominantly associates it as the causal pathogen in the agricultural crops of dry beans. Therefore, we sought to investigate whether the abundance of this genus was because of its role as a plant pathogen or another ecological role. By collating >24,000 16S rRNA sequences with 120 genomes across the Microbacteriaceae family, we show that Curtobacterium has a global distribution with a predominant presence in soil ecosystems. Moreover, this genus harbors a high diversity of genomic potential for the degradation of carbohydrates, specifically with regards to structural polysaccharides. We conclude that Curtobacterium may be responsible for the degradation of organic matter within litter communities.

Background: Glycoside hydrolases (GH) targeting cellulose, xylan, and chitin are common in the bacterial genomes that have been sequenced. Little is known, however, about the architecture of multi-domain and multi-activity glycoside hydrolases. In these enzymes, combined catalytic domains act synergistically and thus display overall improved catalytic efficiency, making these proteins of high interest for the biofuel technology industry., Results: Here, we identify the domain organization in 40,946 proteins targeting cellulose, xylan, and chitin derived from 11,953 sequenced bacterial genomes. These bacteria are known to be capable, or to have the potential, to degrade polysaccharides, or are newly identified potential degraders (e.g., Actinospica, Hamadaea, Cystobacter, and Microbispora). Most of the proteins we identified contain a single catalytic domain that is frequently associated with an accessory non-catalytic domain. Regarding multi-domain proteins, we found that many bacterial strains have unique GH protein architectures and that the overall protein organization is not conserved across most genera. We identified 217 multi-activity proteins with at least two GH domains for cellulose, xylan, and chitin. Of these proteins, 211 have GH domains targeting similar or associated substrates (i.e., cellulose and xylan), whereas only six proteins target both cellulose and chitin. Fifty-two percent of multi-activity GHs are hetero-GHs. Finally, GH6, -10, -44 and -48 domains were mostly C-terminal; GH9, -11, -12, and -18 were mostly N-terminal; and GH5 domains were either N- or C-terminal., Conclusion: We identified 40,946 multi-domain/multi-activity proteins targeting cellulase, chitinase, and xylanase in bacterial genomes and proposed new candidate lineages and protein architectures for carbohydrate processing that may play a role in biofuel production.

The high diversity of microbial communities hampers predictions about their responses to global change. Here we investigate the potential for using a phylogenetic, trait-based framework to capture the response of bacteria and fungi to global change manipulations. Replicated grassland plots were subjected to 3+ years of drought and nitrogen fertilization. The responses of leaf litter bacteria and fungi to these simulated changes were significantly phylogenetically conserved. Proportional changes in abundance were highly correlated among related organisms, such that relatives with approximately 5% ribosomal DNA genetic distance showed similar responses to the treatments. A microbe's change in relative abundance was significantly correlated between the treatments, suggesting a compromise between numerical abundance in undisturbed environments and resistance to change in general, independent of disturbance type. Lineages in which at least 90% of the microbes shared the same response were circumscribed at a modest phylogenetic depth (τD 0.014-0.021), but significantly larger than randomized simulations predict. In several clades, phylogenetic depth of trait consensus was higher. Fungal response to drought was more conserved than was response to nitrogen fertilization, whereas bacteria responded equally to both treatments. Finally, we show that a bacterium's response to the manipulations is correlated with its potential functional traits (measured here as the number of glycoside hydrolase genes encoding the capacity to degrade different types of carbohydrates). Together, these results suggest that a phylogenetic, trait-based framework may be useful for predicting shifts in microbial composition and functioning in the face of global change.

Because microorganisms have different abilities to utilize nitrogen (N) through various assimilatory and dissimilatory pathways, microbial composition and diversity likely influence N cycling in an ecosystem. Terrestrial plant litter decomposition is often limited by N availability; however, little is known about the microorganisms involved in litter N cycling. In this study, we used metagenomics to characterize the potential N utilization of microbial communities in grassland plant litter. The frequencies of sequences associated with eight N cycling pathways differed by several orders of magnitude. Within a pathway, the distributions of these sequences among bacterial orders differed greatly. Many orders within the Actinobacteria and Proteobacteria appeared to be N cycling generalists, carrying genes from most (five or six) of the pathways. In contrast, orders from the Bacteroidetes were more specialized and carried genes for fewer (two or three) pathways. We also investigated how the abundance and composition of microbial N cycling genes differed over time and in response to two global change manipulations (drought and N addition). For many pathways, the abundance and composition of N cycling taxa differed over time, apparently reflecting precipitation patterns. In contrast to temporal variability, simulated global change had minor effects on N cycling potential. Overall, this study provides a blueprint for the genetic potential of N cycle processes in plant litter and a baseline for comparisons to other ecosystems., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Glycoside hydrolases are important enzymes that support bacterial growth by enabling the degradation of polysaccharides (e.g., starch, cellulose, xylan, and chitin) in the environment. Presently, little is known about the overall phylogenetic distribution of the genomic potential to degrade these polysaccharides in bacteria. However, knowing the phylogenetic breadth of these traits may help us predict the overall polysaccharide processing in environmental microbial communities. In order to address this, we identified and analyzed the distribution of 392,166 enzyme genes derived from 53 glycoside hydrolase families in 8,133 sequenced bacterial genomes. Enzymes for oligosaccharides and starch/glycogen were observed in most taxonomic groups, whereas glycoside hydrolases for structural polymers (i.e., cellulose, xylan, and chitin) were observed in clusters of relatives at taxonomic levels ranging from species to genus as determined by consenTRAIT. The potential for starch and glycogen processing, as well as oligosaccharide processing, was observed in 85% of the strains, whereas 65% possessed enzymes to degrade some structural polysaccharides (i.e., cellulose, xylan, or chitin). Potential degraders targeting one, two, and three structural polysaccharides accounted for 22.6, 32.9, and 9.3% of genomes analyzed, respectively. Finally, potential degraders targeting multiple structural polysaccharides displayed increased potential for oligosaccharide deconstruction. This study provides a framework for linking the potential for polymer deconstruction with phylogeny in complex microbial assemblages.

In many ecosystems, global changes are likely to profoundly affect microorganisms. In Southern California, changes in precipitation and nitrogen deposition may influence the composition and functional potential of microbial communities and their resulting ability to degrade plant material. To test whether such environmental changes impact the distribution of functional groups involved in leaf litter degradation, we determined how the genomic diversity of microbial communities in a semi-arid grassland ecosystem changed under reduced precipitation or increased N deposition. We monitored communities seasonally over a period of 2 years to place environmental change responses into the context of natural variation. Fungal and bacterial communities displayed strong seasonal patterns, Fungi being mostly detected during the dry season whereas Bacteria were common during wet periods. Most putative cellulose degraders were associated with 33 bacterial genera and predicted to constitute 18% of the microbial community. Precipitation reduction reduced bacterial abundance and cellulolytic potential whereas nitrogen addition did not affect the cellulolytic potential of the microbial community. Finally, we detected a strong correlation between the frequencies of genera of putative cellulose degraders and cellulase genes. Thus, microbial taxonomic composition was predictive of cellulolytic potential. This work provides a framework for how environmental changes affect microorganisms responsible for plant litter deconstruction.

RBcel1 is an endoglucanase belonging to glycoside hydrolase family 5 subfamily 5 (GH5_5) that was recently identified from a soil metagenome library from the Antarctic. Unlike its closest structural homologue (Cel5A from Thermoascus aurantiacus), this enzyme was reported to be able to catalyze transglycosylation reactions and has putatively been implicated in the bacterial cellulose-synthesis process. Here, the structure of RBcel1 at 1.4 Å resolution, solved by molecular replacement, is reported. The structure and putative substrate-binding site are described and compared with those of other GH5_5 subfamily members.

Many microorganisms contain cellulases that are important for plant cell wall degradation and overall soil ecosystem functioning. At present, we have extensive biochemical knowledge of cellulases but little is known about the phylogenetic distribution of these enzymes. To address this, we analyzed the distribution of 21,985 genes encoding proteins related to cellulose utilization in 5,123 sequenced bacterial genomes. First, we identified the distribution of glycoside hydrolases involved in cellulose utilization and synthesis at different taxonomic levels, from the phylum to the strain. Cellulose degradation/utilization capabilities appeared in nearly all major groups and resulted in strains displaying various enzyme gene combinations. Potential cellulose degraders, having both cellulases and β-glucosidases, constituted 24% of all genomes whereas potential opportunistic strains, having β-glucosidases only, accounted for 56%. Finally, 20% of the bacteria have no relevant enzymes and do not rely on cellulose utilization. The latter group was primarily connected to specific bacterial lifestyles like autotrophy and parasitism. Cellulose degraders, as well as opportunists, have multiple enzymes with similar functions. However, the potential degraders systematically harbor about twice more β-glucosidases than their potential opportunistic relatives. Although scattered, the distribution of functional types, in bacterial lineages, is not random but mostly follows a Brownian motion evolution model. Degraders form clusters of relatives at the species level, whereas opportunists are clustered at the genus level. This information can form a mechanistic basis for the linking of changes in microbial community composition to soil ecosystem processes.

An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6-9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm) at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation). In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/b hydrolases.

in order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 α/β hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.

In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.

A novel natural TEM beta-lactamase with extended-spectrum activity, TEM-138, was identified in a ceftazidime-resistant clinical isolate of Salmonella enterica serovar Infantis. Compared to TEM-1, TEM-138 contains the following mutations: E104K, N175I, and G238S. The bla(TEM-138) gene was located on a 50-kb transferable plasmid. Expression studies with Escherichia coli revealed efficient ceftazidimase and cefotaximase activities for TEM-138.