The oxidoreductase activity of Rnf balances redox cofactors during fermentation of glucose to propionate in Prevotella.

Propionate is a microbial metabolite formed in the gastrointestinal tract, and it affects host physiology as a source of energy and signaling molecule. Despite the importance of propionate, the biochemical pathways responsible for its formation are not clear in all microbes. For the succinate pathway used during fermentation, a key enzyme appears to be missing-one that oxidizes ferredoxin and reduces NAD. Here we show that Rnf [ferredoxin-NAD+ oxidoreductase (Na+-transporting)] is this key enzyme in two abundant bacteria of the rumen (Prevotella brevis and Prevotella ruminicola). We found these bacteria form propionate, succinate, and acetate with the classic succinate pathway. Without ferredoxin:NAD+ oxidoreductase, redox cofactors would be unbalanced; it would produce almost equal excess amounts of reduced ferredoxin and oxidized NAD. By combining growth experiments, genomics, proteomics, and enzyme assays, we point to the possibility that these bacteria solve this problem by oxidizing ferredoxin and reducing NAD with Rnf [ferredoxin-NAD+ oxidoreductase (Na+-transporting)]. Genomic and phenotypic data suggest many bacteria may use Rnf similarly. This work shows the ferredoxin:NAD+ oxidoreductase activity of Rnf is important to propionate formation in Prevotella species and other bacteria from the environment, and it provides fundamental knowledge for manipulating fermentative propionate production.

Dietary supplementation of botanical blends enhanced performance and disease resistance of weaned pigs experimentally infected with enterotoxigenic Escherichia coli F18.

Botanicals exhibit promising impacts on intestinal health, immune-regulation, and growth promotion in weaned pigs. However, these benefits may vary depending on major active components in the final feed additive products. Therefore, this study aimed to investigate two types of botanical blends (BB) that were comprised of 0.3% capsicum oleoresin and 12% garlic extracts from different sources on performance, diarrhea, and health of weaned piglets experimentally infected with a pathogenic Escherichia coli F18. Sixty weanling pigs (7.17 ± 0.97 kg body weight (BW)) blocked by weight and gender were assigned to one of five dietary treatments: negative control (NC), positive control (PC), or dietary supplementation with 100 mg/kg of BB1, 50 mg/kg or 100 mg/kg of BB2. This study lasted 28 d with 7 d before and 21 d after the first E. coli inoculation (day 0). All pigs, except negative control, were orally inoculated with 1010 cfu E. coli F18/3-mL dose for 3 consecutive days. Blood samples were collected periodically to analyze systemic immunity. Intestinal tissues and mucosa were collected on days 5 and 21 PI for analyzing histology and gene expression. All data, except for frequency of diarrhea, were analyzed by ANOVA using the PROC MIXED of SAS. The Chi-square test was used for analyzing frequency of diarrhea. Escherichia coli infection reduced (P < 0.05) growth rate and feed intake and increased (P < 0.05) frequency of diarrhea of weaned pigs throughout the experiment. Supplementation of 100 mg/kg BB1 or BB2 alleviated (P < 0.05) frequency of diarrhea of E. coli challenged pigs during the entire experiment. Escherichia coli infection also enhanced (P < 0.05) serum TNF-α and haptoglobin concentrations on day 4 post-inoculation (PI) but reduced (P < 0.05) duodenal villi height and area on day 5 PI, while pigs supplemented with 100 mg/kg BB1 or BB2 had lower (P < 0.05) serum TNF-α than pigs in PC on day 4 PI. Pigs fed with 100 mg/kg BB2 had higher (P < 0.05) jejunal villi height than pigs in PC on day 5 PI. Pigs fed with 100 mg/kg BB2 had reduced (P < 0.05) gene expression of IL1B, PTGS2, and TNFA in ileal mucosa than pigs in PC on day 21 PI. In conclusion, dietary supplementation of botanical blends at 100 mg/kg could enhance disease resistance of weaned pigs infected with E. coli F18 by enhancing intestinal morphology and regulating local and systemic immunity of pigs.

This experiment aimed to investigate two botanical blends consisting of 0.3% capsicum oleoresin and 12% garlic extracts on performance, diarrhea, and health of weaned piglets experimentally infected with a pathogenic Escherichia coli F18. The two botanical blends have the same formulation, except that different garlic oils were used. A total of 60 weaned pigs were randomly allotted to one of five experimental treatments: 1) a complex control diet without E. coli F18 challenge; 2) control diet with E. coli F18 challenge; 3) supplementing 100 mg/kg of botanical blend type 1 to pigs challenged with E. coli F18; 4) and 5) supplementing 50 or 100 mg/kg of botanical blend type 2 to pigs challenged with E. coli F18. The experiment lasted 28 d with 7 d adaptation and 21 d after the first F18 E. coli inoculation. Results of this experiment demonstrate that supplementation of 100 mg/kg of botanical blend enhanced disease resistance and tended to improve growth of weaned pigs, regardless of garlic oil variety. An improved intestinal morphology and reduced systemic inflammation was also observed in pigs supplemented with 100 mg/kg of botanical blends. In conclusion, supplementation of 100 mg/kg of botanical blends could reduce diarrhea of E. coli infected pigs and modify local or systemic immunity of pigs.

A New Pathway for Forming Acetate and Synthesizing ATP during Fermentation in Bacteria.

Many bacteria and other organisms carry out fermentations forming acetate. These fermentations have broad importance for foods, agriculture, and industry. They also are important for bacteria themselves because they often generate ATP. Here, we found a biochemical pathway for forming acetate and synthesizing ATP that was unknown in fermentative bacteria. We found that the bacterium Cutibacterium granulosum formed acetate during fermentation of glucose. It did not use phosphotransacetylase or acetate kinase, enzymes found in nearly all acetate-forming bacteria. Instead, it used a pathway involving two different enzymes. The first enzyme, succinyl coenzyme A (succinyl-CoA):acetate CoA-transferase (SCACT), forms acetate from acetyl-CoA. The second enzyme, succinyl-CoA synthetase (SCS), synthesizes ATP. We identified the genes encoding these enzymes, and they were homologs of SCACT and SCS genes found in other bacteria. The pathway resembles one described in eukaryotes, but it uses bacterial, not eukaryotic, gene homologs. To find other instances of the pathway, we analyzed sequences of all biochemically characterized homologs of SCACT and SCS (103 enzymes from 64 publications). Homologs with similar enzymatic activity had similar sequences, enabling a large-scale search for them in genomes. We searched nearly 600 genomes of bacteria known to form acetate, and we found that 6% encoded homologs with SCACT and SCS activity. This included >30 species belonging to 5 different phyla, showing that a diverse range of bacteria encode the SCACT/SCS pathway. This work suggests the SCACT/SCS pathway is important for acetate formation in many branches of the tree of life. IMPORTANCE Pathways for forming acetate during fermentation have been studied for over 80 years. In that time, several pathways in a range of organisms, from bacteria to animals, have been described. However, one pathway (involving succinyl-CoA:acetate CoA-transferase and succinyl-CoA synthetase) has not been reported in prokaryotes. Here, we discovered enzymes for this pathway in the fermentative bacterium Cutibacterium granulosum. We also found >30 other fermentative bacteria that encode this pathway, demonstrating that it could be common. This pathway represents a new way for bacteria to form acetate from acetyl-CoA and synthesize ATP via substrate-level phosphorylation. It could be a target for controlling yield of acetate during fermentation, with relevance for foods, agriculture, and industry.