Adaptation of Gut Microbiotas to Transgenic Pigs Secreting β-Glucanase, Xylanase and Phytase

BackgroundThe gut microbiotas play an important role in digestive function and feed efficiency in pigs. However, the effect of exogenous digestive enzymes on the composition and functional contributions of swine intestinal microbes is unclear. The objective of this study was to investigate the change of gut microbiotas in the transgenic pigs secreting microbial digestive enzymes in their salivary glands.MethodsEGFP marker-free transgenic (MF-TG) pigs were generated by deleted the EGFP coding genes in the transgenic pigs we previously generated. Samples of chyme from the ileum, caecum and colon of five MF-TG and five wild-type (WT) sows were collected for investigating the gut microbiomes via metagenomics analyses.ResultsThe levels of probiotics were abundant in the caecum of MF-TG pigs and higher than those of WT pigs. By contrast, the levels of some harmful microorganisms were higher in the caecum of WT pigs than those of MF-TG pigs. In addition, the microorganisms in the colon of MF-TG pigs had high fractional abundance in DNA (cytosine-5)-methyltransferase 1 and serine-type D-Ala-D-Ala carboxypeptidase, whereas the aspartate carbamoyltransferase regulatory subunit and outer membrane protein pathways were enriched in WT pigs. Moreover, the levels of numerous carbohydrases in the caecum of MF-TG pigs were higher than those of WT pigs. ConclusionsThe results indicated that intestinal microbes can change adaptively to the secretion of transgenic enzymes, thereby forming a benign cooperation with their host.

Proteome Cold-Shock Response in the Extremely Acidophilic Archaeon, Cuniculiplasma divulgatum

The archaeon Cuniculiplasma divulgatum is ubiquitous in acidic environments with low-to-moderate temperatures. However, molecular mechanisms underlying its ability to thrive at lower temperatures remain unexplored. Using mass spectrometry (MS)-based proteomics, we analysed the effect of short-term (3 h) exposure to cold. The C. divulgatum genome encodes 2016 protein-coding genes, from which 819 proteins were identified in the cells grown under optimal conditions. In line with the peptidolytic lifestyle of C. divulgatum, its intracellular proteome revealed the abundance of proteases, ABC transporters and cytochrome C oxidase. From 747 quantifiable polypeptides, the levels of 582 proteins showed no change after the cold shock, whereas 104 proteins were upregulated suggesting that they might be contributing to cold adaptation. The highest increase in expression appeared in low-abundance (0.001–0.005 fmol%) proteins for polypeptides’ hydrolysis (metal-dependent hydrolase), oxidation of amino acids (FAD-dependent oxidoreductase), pyrimidine biosynthesis (aspartate carbamoyltransferase regulatory chain proteins), citrate cycle (2-oxoacid ferredoxin oxidoreductase) and ATP production (V type ATP synthase). Importantly, the cold shock induced a substantial increase (6% and 9%) in expression of the most-abundant proteins, thermosome beta subunit and glutamate dehydrogenase. This study has outlined potential mechanisms of environmental fitness of Cuniculiplasma spp. allowing them to colonise acidic settings at low/moderate temperatures.

Allostery and cooperativity in multimeric proteins: bond-to-bond propensities in ATCase

Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of various substrates at distal points from the active sites. A recently developed method, bond-to-bond propensity analysis, has proven capable of predicting allosteric sites in a wide range of proteins using an energy-weighted atomistic graph obtained from the protein structure and given knowledge only of the location of the active site. Bond-to-bond propensity establishes if energy fluctuations at given bonds have significant effects on any other bond in the protein, by considering their propagation through the protein graph. In this work, we use bond-to-bond propensity analysis to study different aspects of ATCase activity using three different protein structures and sources of fluctuations. First, we predict key residues and bonds involved in the transition between inactive (T) and active (R) states of ATCase by analysing allosteric substrate binding as a source of energy perturbations in the protein graph. Our computational results also indicate that the effect of multiple allosteric binding is non linear: a switching effect is observed after a particular number and arrangement of substrates is bound suggesting a form of long range communication between the distantly arranged allosteric sites. Second, cooperativity is explored by considering a bisubstrate analogue as the source of energy fluctuations at the active site, also leading to the identification of highly significant residues to the T↔R transition that enhance cooperativity across active sites. Finally, the inactive (T) structure is shown to exhibit a strong, non linear communication between the allosteric sites and the interface between catalytic subunits, rather than the active site. Bond-to-bond propensity thus offers an alternative route to explain allosteric and cooperative effects in terms of detailed atomistic changes to individual bonds within the protein, rather than through phenomenological, global thermodynamic arguments.

Study of Uridylic Nucleotides Contents and the Investigation Aspartate Carbamoyltransferase in Liver and Small Intestine Mucosa Exposed when Administered to Rats Orotic Acid and Perftoran

Purpose: Study of uridylic nucleotides content and aspartate carbamoyltransferase which was a key enzyme on the pathway for the synthesis of pyrimidine nucleotides in tissues of irradiated animals upon administration of orotic acid and perftorane was conducted. Material and methods: Studies were performed on random-bred albino rats subjected to a single γ-ray exposure at a total dose of 6 Gy. Orotic acid was injected as potassium salt in a dose of 60 mg / kg, perftoran salt in a dose of 1 ml / 100 g water. Results: A decrease in the content of UTP and UDP, as well as an increase in UMP after irradiation, especially on the 7th day, was established. The most pronounced changes in the studied biochemical parameters take place in the mucosa of the small intestine. The administration of orotic acid and perftorane to irradiated animals contributes to a significant correction of both the nucleotide content and the activity of aspartate carbamoyltransferase. Conclusion: The radiation leads to some decrease in the content of UDP and UTP. Changes in the content of nucleotides and activity of aspartate carbamoyltransferase in the mucosa of the small intestine are more pronounced in comparison with liver tissue. The combined administration of orotic acid and perftorane promotes the normalization of the content of nucleotides, and the activity of aspartate carbamoyltransferase in the liver and mucous membrane of the small intestine of irradiated animals.