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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.

Permafrost is an extreme habitat yet it hosts microbial populations that remain active over millennia. Using permafrost collected from a Pleistocene chronosequence (19 ka 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.

The annotation of short-reads metagenomes is an essential process to understand the functional potential of sequenced microbial communities. Annotation techniques based solely on the identification of local matches tend to confound local sequence similarity and overall protein homology and thus don’t mirror the complex multidomain architecture and the shuffling of functional domains in many protein families. Here, we present MetaGeneHunt to identify specific protein domains and to normalize the hit-counts based on the domain length. We used MetaGeneHunt to investigate the potential for carbohydrate processing in the mouse gastrointestinal tract. We sampled, sequenced, and analyzed the microbial communities associated with the bolus in the stomach, intestine, cecum, and colon of five captive mice. Focusing on Glycoside Hydrolases (GHs) we found that, across samples, 58.3% of the 4,726,023 short-read sequences matching with a GH domain-containing protein were located outside the domain of interest. Next, before comparing the samples, the counts of localized hits matching the domains of interest were normalized to account for the corresponding domain length. Microbial communities in the intestine and cecum displayed characteristic GH profiles matching distinct microbial assemblages. Conversely, the stomach and colon were associated with structurally and functionally more diverse and variable microbial communities. Across samples, despite fluctuations, changes in the functional potential for carbohydrate processing correlated with changes in community composition. Overall MetaGeneHunt is a new way to quickly and precisely identify discrete protein domains in sequenced metagenomes processed with MG-RAST. In addition, using the sister program “GeneHunt” to create custom Reference Annotation Table, MetaGeneHunt provides an unprecedented way to (re)investigate the precise distribution of any protein domain in short-reads metagenomes.

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.

The identification of glycoside hydrolases (GHs) for efficient polysaccharide deconstruction is essential for the development of biofuels. Here, we investigate the potential of sequential HMM-profile identification for the rapid and precise identification of the multi-domain architecture of GHs from various datasets. First, as a validation, we successfully reannotated >98% of the biochemically characterized enzymes listed on the CAZy database. Next, we analyzed the 43 million non-redundant sequences from the M5nr data and identified 322,068 unique GHs. Finally, we searched 129 assembled metagenomes retrieved from MG-RAST for environmental GHs and identified 160,790 additional enzymes. Although most identified sequences corresponded to single domain enzymes, many contained several domains, including known accessory domains and some domains never identified in association with GH. Several sequences displayed multiple catalytic domains and few of these potential multi-activity proteins combined potentially synergistic domains. Finally, we produced and confirmed the biochemical activities of a GH5-GH10 cellulase-xylanase and a GH11-CE4 xylanase-esterase. Globally, this “gene to enzyme pipeline” provides a rationale for mining large datasets in order to identify new catalysts combining unique properties for the efficient deconstruction of polysaccharides.

Carnobacterium maltaromaticumis a well-known pathogen of bony fish. More recently,C. maltaromaticumhave been isolated from the brain and inner ear of disorientated and stranded common thresher (Alopias vulpinus) and salmon shark (Lamna ditropis). While thresher shark strandings are recent, salmon sharks have been stranding for decades, suggesting a long-term association betweenC. maltaromaticumand sharks. Interestingly, some strains ofC. maltaromaticumare used by the food industry for their probiotic and antimicrobial activity. Here, we sequenced the genome of 9C. maltaromaticumstrains (SK-isolates) from diseased common thresher and salmon sharks and compared them to otherC. maltaromaticumstrains in order to identify the genomic signatures that differentiate the disease-associated from the innocuousC. maltaromaticumisolates. SK strains formed a monophyletic clade, with a conserved gene repertoire, and shared a high degree of pseudogenization even though isolates were from different shark species, locations, and across years. In addition, these strains displayed few virulence associated genes and unique genomic regions, some resulting from horizontal gene transfer. The association of diseased sharks and SK strains suggests their role as potential pathogens. Although the high degree of pseudogenization suggests a transition to a host-adapted lifestyle, a set of conserved functional genes highlights the need of essential functions required for a host-independent life style. Globally, this work identifies specific genomic signatures ofC. maltaromaticumstrains isolated from infected sharks, provides the framework to elucidate the role of SK strains in the development of the disease in sharks, and further investigate the dissemination of SK strains in populations of wild fish.

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., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

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.

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).

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.

© 2017 International Society for Microbial Ecology. 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.

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 Fil: Berlemont, Renaud. Universite de Liege; Bélgica. University Of California At Irvine; Estados Unidos Fil: Jacquin, Olivier. Universite de Liege; Bélgica Fil: Delsaute, Maud. Universite de Liege; Bélgica Fil: La Salla, Marcello. Universite de Liege; Bélgica Fil: Georis, Jacques. Puratos Group; Bélgica Fil: Verté, Fabienne. Puratos Group; Bélgica Fil: Galleni, Moreno. Universite de Liege; Bélgica Fil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universite de Liege; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

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.

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., United States. Dept. of Energy. Office of Biological and Environmental Research (Award DE-PS02-09ER09-25), United States. Dept. of Energy (Grant DESC0008743)

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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0538-6) contains supplementary material, which is available to authorized users.

© 2015 International Society for Microbial Ecology 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.The ISME Journal advance online publication, 15 May 2015; doi:10.1038/ismej.2015.58.

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.

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.

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.

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 A 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.

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 β-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.