Cellular Metrology: Scoping for a Value Proposition in Extra- and Intracellular Measurements.

The symptomatic irreproducibility of data in biomedicine and biotechnology prompts the need for higher order measurements of cells in their native and near-native environments. Such measurements may support the adoption of new technologies as well as the development of research programs across different sectors including healthcare and clinic, environmental control and national security. With an increasing demand for reliable cell-based products and services, cellular metrology is poised to help address current and emerging measurement challenges faced by end-users. However, metrological foundations in cell analysis remain sparse and significant advances are necessary to keep pace with the needs of modern medicine and industry. Herein we discuss a role of metrology in cell and cell-related R&D activities to underpin growing international measurement capabilities. Relevant measurands are outlined and the lack of reference methods and materials, particularly those based on functional cell responses in native environments, is highlighted. The status quo and current challenges in cellular measurements are discussed in the light of metrological traceability in cell analysis and applications (e.g., a functional cell count). An emphasis is made on the consistency of measurement results independent of the analytical platform used, high confidence in data quality vs. quantity, scale of measurements and issues of building infrastructure for end-users.

Biochemical and Structural Analyses of Two Cryptic Esterases in Bacteroides intestinalis and their Synergistic Activities with Cognate Xylanases.

Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s-1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98Å resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases.

Bacteroides intestinalis DSM 17393, a member of the human colonic microbiome, upregulates multiple endoxylanases during growth on xylan.

Many human diets contain arabinoxylan, and the ease of genome sequencing coupled with reduced cost have led to unraveling the arsenal of genes utilized by the colonic Bacteroidetes to depolymerize this polysaccharide. The colonic Bacteroidetes with potential to ferment arabinoxylans include Bacteroides intestinalis. In this study, we analyzed the hydrolytic activities of members of a xylan degradation cluster encoded on the genome of Bacteroides intestinalis DSM 17393. Here, it is demonstrated that a cocktail of the xylanolytic enzymes completely hydrolyze arabinoxylans found in human diets. We show that this bacterium and relatives have evolved and secrete a unique bifunctional endoxylanase/arabinofuranosidase in the same polypeptide. The bifunctional enzyme and other secreted enzymes attack the polysaccharides extracellularly to remove the side-chains, exposing the xylan backbone for cleavage to xylo-oligosaccharides and xylose. These end products are transported into the cell where a ß-xylosidase cleaves the oligosaccharides to fermentable sugars. While our experiments focused on B. intestinalis, it is likely that the extracellular enzymes also release nutrients to members of the colonic microbial community that practice cross-feeding. The presence of the genes characterized in this study in other colonic Bacteroidetes suggests a conserved strategy for energy acquisition from arabinoxylan, a component of human diets.

Microbial community diversity in the gut of the South American termite Cornitermes cumulans (Isoptera: Termitidae).

Termites inhabit tropical and subtropical areas where they contribute to structure and composition of soils by efficiently degrading biomass with aid of resident gut microbiota. In this study, culture-independent molecular analysis was performed based on bacterial and archaeal 16S rRNA clone libraries to describe the gut microbial communities within Cornitermes cumulans, a South American litter-feeding termite. Our data reveal extensive bacterial diversity, mainly composed of organisms from the phyla Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and Fibrobacteres. In contrast, a low diversity of archaeal 16S rRNA sequences was found, comprising mainly members of the Crenarchaeota phylum. The diversity of archaeal methanogens was further analyzed by sequencing clones from a library for the mcrA gene, which encodes the enzyme methyl coenzyme reductase, responsible for catalyzing the last step in methane production, methane being an important greenhouse gas. The mcrA sequences were diverse and divided phylogenetically into three clades related to uncultured environmental archaea and methanogens found in different termite species. C. cumulans is a litter-feeding, mound-building termite considered a keystone species in natural ecosystems and also a pest in agriculture. Here, we describe the archaeal and bacterial communities within this termite, revealing for the first time its intriguing microbiota.

Metagenomic analysis of the microbiota from the crop of an invasive snail reveals a rich reservoir of novel genes.

The shortage of petroleum reserves and the increase in CO(2) emissions have raised global concerns and highlighted the importance of adopting sustainable energy sources. Second-generation ethanol made from lignocellulosic materials is considered to be one of the most promising fuels for vehicles. The giant snail Achatina fulica is an agricultural pest whose biotechnological potential has been largely untested. Here, the composition of the microbial population within the crop of this invasive land snail, as well as key genes involved in various biochemical pathways, have been explored for the first time. In a high-throughput approach, 318 Mbp of 454-Titanium shotgun metagenomic sequencing data were obtained. The predominant bacterial phylum found was Proteobacteria, followed by Bacteroidetes and Firmicutes. Viruses, Fungi, and Archaea were present to lesser extents. The functional analysis reveals a variety of microbial genes that could assist the host in the degradation of recalcitrant lignocellulose, detoxification of xenobiotics, and synthesis of essential amino acids and vitamins, contributing to the adaptability and wide-ranging diet of this snail. More than 2,700 genes encoding glycoside hydrolase (GH) domains and carbohydrate-binding modules were detected. When we compared GH profiles, we found an abundance of sequences coding for oligosaccharide-degrading enzymes (36%), very similar to those from wallabies and giant pandas, as well as many novel cellulase and hemicellulase coding sequences, which points to this model as a remarkable potential source of enzymes for the biofuel industry. Furthermore, this work is a major step toward the understanding of the unique genetic profile of the land snail holobiont.

Gut bacterial communities in the giant land snail Achatina fulica and their modification by sugarcane-based diet.

The invasive land snail Achatina fulica is one of the most damaging agricultural pests worldwide representing a potentially serious threat to natural ecosystems and human health. This species is known to carry parasites and harbors a dense and metabolically active microbial community; however, little is known about its diversity and composition. Here, we assessed for the first time the complexity of bacterial communities occurring in the digestive tracts of field-collected snails (FC) by using culture-independent molecular analysis. Crop and intestinal bacteria in FC were then compared to those from groups of snails that were reared in the laboratory (RL) on a sugarcane-based diet. Most of the sequences recovered were novel and related to those reported for herbivorous gut. Changes in the relative abundance of Bacteroidetes and Firmicutes were observed when the snails were fed a high-sugar diet, suggesting that the snail gut microbiota can influence the energy balance equation. Furthermore, this study represents a first step in gaining a better understanding of land snail gut microbiota and shows that this is a complex holobiont system containing diverse, abundant and active microbial communities.