Renaud Berlemont

Phylogenetic Distribution of Potential Cellulases in Bacteria

ABSTRACT 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.

Genomic Potential for Polysaccharide Deconstruction in Bacteria

ABSTRACT 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.

Microbial response to simulated global change is phylogenetically conserved and linked with functional potential.

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.

Temporal variation overshadows the response of leaf litter microbial communities to simulated global change

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.

Cellulolytic potential under environmental changes in microbial communities from grassland litter

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.

Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples

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 °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 Extended-Spectrum TEM-Type beta-Lactamase, TEM-138, from Salmonella enterica Serovar Infantis

ABSTRACT 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 blaTEM-138 gene was located on a 50-kb transferable plasmid. Expression studies with Escherichia coli revealed efficient ceftazidimase and cefotaximase activities for TEM-138.

Microbial legacies alter decomposition in response to simulated global change

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.

Natural diversity of cellulases, xylanases, and chitinases in bacteria

BackgroundGlycoside 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.ResultsHere, 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.ConclusionWe 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.

Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome

An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the α/β 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/β hydrolases.