Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase

Objective Neisseria meningitidis is a Gram-negative bacterium that causes meningitis. N. meningitidis serogroup W (NmW) capsule polymerase synthesizes capsular polysaccharide of this serogroup. This enzyme could be a tool for meningococcal glycoconjugate vaccine development. Our long-term goal is to control activity of the NmW capsule polymerase for production of defined carbohydrates for vaccines. The enzyme lacks a simple, high-throughput activity assay. Here, we describe the use of high-throughput bioluminescence assays (CMP-Glo and UDP-Glo by Promega) to investigate NmW capsule polymerase activity. These assays detect free nucleotides produced during transfer of sugar from UDP-Galactose and CMP-Sialic Acid to an acceptor. Kinetic studies using NmW hydrolyzed polysaccharide (PS) acceptor are described as well as preliminary work with a sialic acid trimer (DP3) acceptor. Results In CMP-Glo kinetic studies, with constant donor (80 µM) and varied NmW hydrolyzed polysaccharide (0–2000 µg/mL), a Km of 629.2 ± 101.4 µg/mL and a Vmax of 0.8965 ± 0.05823 µM/min was obtained. Using UDP-Glo, Km and Vmax values of 13.84 ± 9.675 µM and 0.6205 ± 0.1331 µM/min were obtained with varied CMP-NeuNAc (0–80 µM) and constant acceptor (400 µg/mL) and UDP-Gal (80 µM). This is the first report of using bioluminescence assays for NmW kinetics.

Umami synergy as the scientific principle behind taste-pairing champagne and oysters

Food and flavour pairing are commonly used as an empirically based phenomenology by chefs and food innovators for creating delicious dishes. However, there is little if any science behind the pairing systems used, and it appears that pairing is determined by food culture and tradition rather than by chemical food composition. In contrast, the pairing implied by the synergy in the umami taste, elicited by free glutamate and free nucleotides, is scientifically founded on an allosteric action at the umami receptor, rendering eggs-bacon and cheese-ham delicious companions. Based on measurement of umami compounds in champagnes and oysters we suggest that a reason why champagne and oysters are considered good companions may be the presence of free glutamate in champagne, and free glutamate and 5′-nucleotides in oysters. By calculations of the effective umami potential we reveal which combinations of oysters and champagnes lead to the strongest umami taste. We also show that glutamate levels and total amount of free amino acids are higher in aged champagnes with long yeast contact, and that the European oyster (Ostrea edulis) has higher free glutamate and nucleotide content than the Pacific oyster (Crassostrea gigas) and is thus a better candidate to elicit synergistic umami taste.

Emergence of ribozyme and tRNA-like structures from mineral-rich muddy pools on prebiotic earth

The RNA world hypothesis, although a viable one regarding the origin of life on earth, has so far failed to provide a compelling explanation for the synthesis of RNA molecules with catalytic functions, from free nucleotides via abiotic processes. To tackle this long-standing problem, we develop a realistic model for the onset of the RNA world, using experimentally determined rates for polymerization reactions. We start with minimal assumptions about the initial state that only requires the presence of short oligomers or just free nucleotides and consider the effects of environmental cycling by dividing a day into a dry, semi-wet and wet phases that are distinguished by the nature of reactions they support. Long polymers, with maximum lengths sometimes exceeding 100 nucleotides, spontaneously emerge due to a combination of non-enzymatic, non-templated polymer extension and template-directed primer extension processes. The former helps in increasing the lengths of RNA strands, whereas the later helps in producing complementary copies of the strands. Strands also undergo hydrolysis in a structure-dependent manner that favour breaking of bonds connecting unpaired nucleotides. We identify the most favourable conditions needed for the emergence of ribozyme and tRNA-like structures and double stranded RNA molecules, classify all RNA strands on the basis of their secondary structures and determine their abundance in the population. Our results indicate that under suitable environmental conditions, non-enzymatic processes would have been sufficient to lead to the emergence of a variety of ribozyme-like molecules with complex secondary structures and potential catalytic functions.

Non-spherical coacervate shapes in an enzyme driven active system

Coacervates are polymer-rich droplets that form through liquid-liquid phase separation in polymer solutions. Liquid-liquid phase separation and coacervation have recently been shown to play an important role in the organization of biological systems. Such systems are highly dynamic and under continuous influence of enzymatic and chemical processes. However, it is still unclear how enzymatic and chemical reactions affect the coacervation process. Here, we present and characterize a system of enzymatically active coacervates containing spermine, RNA, free nucleotides, and the template independent RNA (de)polymerase PNPase. We find that these RNA coacervates display transient non-spherical shapes, and we systematically study how PNPase concentration, UDP concentration and temperature affect coacervate morphology. Furthermore, we show that PNPase localizes predominantly into the coacervate phase and that its depolymerization activity in high-phosphate buffer causes coacervate degradation. Our observations of non-spherical coacervate shapes may have broader implications for the relationship between (bio-)chemical activity and coacervate biology.

The dental calculus metabolome in modern and historic samples

IntroductionDental calculus is a mineralized microbial dental plaque biofilm that forms throughout life by precipitation of salivary calcium salts. Successive cycles of dental plaque growth and calcification make it an unusually well-preserved, long-term record of host-microbial interaction in the archaeological record. Recent studies have confirmed the survival of authentic ancient DNA and proteins within historic and prehistoric dental calculus, making it a promising substrate for investigating oral microbiome evolution via direct measurement and comparison of modern and ancient specimens.ObjectiveWe present the first comprehensive characterization of the human dental calculus metabolome using a multi-platform approach.MethodsUltra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) quantified 285 metabolites in modern and historic (200 years old) dental calculus, including metabolites of drug and dietary origin. A subset of historic samples was additionally analyzed by high-resolution gas chromatography-MS (GC-MS) and UPLC- MS/MS for further characterization of polar metabolites and lipids, respectively. Metabolite profiles of modern and historic calculus were compared to identify patterns of persistence and loss.ResultsDipeptides, free amino acids, free nucleotides, and carbohydrates substantially decrease in abundance and ubiquity in archaeological samples, with some exceptions. Lipids generally persist, and saturated and mono-unsaturated medium and long chain fatty acids appear to be well-preserved, while metabolic derivatives related to oxidation and chemical degradation are found at higher levels in archaeological dental calculus than fresh samples.ConclusionsThe results of this study indicate that certain metabolite classes have higher potential for recovery over long time scales and may serve as appropriate targets for oral microbiome evolutionary studies.