Bulk and spatially resolved extracellular metabolomics of free-living nitrogen fixation

Soil microorganisms drive ecosystem function, but challenges of scale between microbe and ecosystem hinder our ability to accurately quantify and predictively model the soil microbe-ecosystem function relationship. Quantifying this relationship necessitates studies that systematically characterize multi-omics of soil microorganisms and their activity across sampling scales from spatially resolved to bulk measures, and structural complexity, from liquid pure culture to in situ. To address this need, we cultured two diazotrophic bacteria in liquid and solid media, with and without nitrogen (N) to quantify differences in extracellular metabolites associated with nitrogen fixation under increasing environmental structural complexity. We also quantified extracellular metabolites across sampling scales including bulk sampling via GC-MS analysis and spatially resolved analysis via MALDI mass spectrometry imaging. We found extracellular production of inorganic and organic N during free-living nitrogen fixation activity, highlighting a key mechanism of terrestrial N contributions from this process. Additionally, our results emphasize the need to consider the structural complexity of the environment and spatial scale when quantifying microbial activity. We found differences in metabolite profiles between culture conditions, supporting previous work indicating environmental structure influences microbial function, and across scales, underscoring the need to quantify microbial scale conditions to accurately interpret microbial function.

Rabbit microbiota across the whole body revealed by 16S rRNA gene amplicon sequencing

Background Rabbit can produce meat, fur and leather, and serves as an important biomedical animal model. Understanding the microbial community of rabbits helps to raise rabbits healthily and better support their application as animal models. Results In this study, we selected 4 healthy Belgium gray rabbits to collect the microbial samples from 12 body sites, including skin, lung, uterus, mouth, stomach, duodenum, ileum, jejunum, colon, cecum, cecal appendix and rectum. The microbiota across rabbit whole body was investigated via 16S rRNA gene amplicon sequencing. After quality control, 46 samples were retained, and 3,148 qualified ASVs were obtained, representing 23 phyla and 264 genera. Based on the weighted UniFrac distances, these samples were divided into the large intestine (Lin), stomach and small intestine (SSin), uterus (Uter), and skin, mouth and lung (SML) groups. The diversity of Lin microbiota was the highest, followed by those of the SSin, Uter and SML groups. In the whole body, Firmicutes (62.37%), Proteobacteria (13.44%) and Bacteroidota (11.84%) were the most predominant phyla. The relative abundance of Firmicutes in the intestinal tract was significantly higher than that in the non-intestinal site, while Proteobacteria was significantly higher in the non-intestinal site. Among the 264 genera, 35 were the core microbiota distributed in all body sites. Sixty-one genera were specific in the SML group, while 13, 8 and 1 were specifically found in the Lin, SSin and Uter groups, respectively. The Lin group had the most difference with other groups, there were average 72 differential genera between the Lin and other groups. The functional prediction analysis showed that microbial function within each group was similar, but there was a big difference between the intestinal tracts and the non-intestinal group. Notably, the function of microorganism in uterus and mouth were the most different from those in the gastrointestinal sites; rabbit’s coprophagy of consuming soft feces possibly resulted in little differences of microbial function between stomach and large intestinal sites. Conclusion Our findings improve the knowledge about rabbit microbial communities throughout whole body and give insights into the relationship of microbial communities among different body sites in health rabbits.

Alfalfa Hay Supplementary Intake is Advancing Effect To Rumen Fermentation and Microbial Function of Gansu Alpine Fine Wool Sheep During Cold Season

Bodyweight loss and rumen microbial dysfunction of grazing sheep was a challenge for the sheep production industry during cold season, which were considered to correlated with under-roughage-feeding. The objective of our research was to assess the role of Alfalfa hay supplementary intake in roughage in ewes’ rumen fermentation and microbial function. 120 ewes were allocated randomly into 4 groups, and were fed with different level of alfalfa hay, respectively. Individual ewes’ bodyweight, blood biochemical indexes and rumen microbial characteristics were analyzed after the end of feeding trials. The results showed that alfalfa hay supplementary could significantly enhance sheep body weight, nitrogen components (Total-N, Soluble protein-N and Ammonia-N), blood biochemical indices (LDH, BUN and CHO) and ruminal volatile fatty acids (P < 0.05). Meantime, alfalfa hay supplementary increased the richness and diversity of ruminal fluid microbiota, and decreased ruminal fluid microbiota beta-diversity. The ruminal fluid microbiota of alfalfa hay supplementary feeding showed low immune pathway and high carbohydrate metabolism pathway. Overall, the study suggested that there was an increasing tendency of alfalfa-hay-supplementary group in 30% Yellow Maize Silage + 70% Alfalfa Hay roughage in body weight, ruminal fermentation and microbial function, which improved GS performance through developing hay supplementary system during cold season.

Gut microbiome is affected by gut region but robust to host physiological changes in captive active-season ground squirrels

Background Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) are obligate hibernators and are only active 4–5 months annually. During this period, squirrels rapidly acquire fat for use during hibernation. We investigated how the gut microbiome changed over the active season in the mucosa and lumen of two gut sections: the cecum and ileum. We sequenced the 16S rRNA gene to assess diversity and composition of the squirrel gut microbiome and used differential abundance and network analyses to identify relationships among gut sections. Results Microbial composition significantly differed between the cecum and ileum, and within the ileum between the mucosa and lumen. Cecum mucosa and lumen samples did not differ in alpha diversity and composition, and clustered by individual squirrel. Ileum mucosa and lumen samples differed in community composition, which can likely be attributed to the transient nature of food-associated bacteria in the lumen. We did not detect a shift in microbiome diversity and overall composition over the duration of the active season, indicating that the squirrel microbiome may be relatively robust to changes in physiology. Conclusions Overall, we found that the 13-lined ground squirrel microbiome is shaped by microenvironment during the active season. Our results provide baseline data for new avenues of research, such as investigating potential differences in microbial function among these physiologically unique gut environments.

Elucidation of an anaerobic pathway for metabolism of l-carnitine–derived γ-butyrobetaine to trimethylamine in human gut bacteria

Trimethylamine (TMA) is an important gut microbial metabolite strongly associated with human disease. There are prominent gaps in our understanding of how TMA is produced from the essential dietary nutrient l-carnitine, particularly in the anoxic environment of the human gut where oxygen-dependent l-carnitine–metabolizing enzymes are likely inactive. Here, we elucidate the chemical and genetic basis for anaerobic TMA generation from the l-carnitine–derived metabolite γ-butyrobetaine (γbb) by the human gut bacterium Emergencia timonensis. We identify a set of genes up-regulated by γbb and demonstrate that the enzymes encoded by the induced γbb utilization (bbu) gene cluster convert γbb to TMA. The key TMA-generating step is catalyzed by a previously unknown type of TMA-lyase enzyme that utilizes a putative flavin cofactor to catalyze a redox-neutral transformation. We identify additional cultured and uncultured host-associated bacteria that possess the bbu gene cluster, providing insights into the distribution of anaerobic γbb metabolism. Lastly, we present genetic, transcriptional, and metabolomic evidence that confirms the relevance of this metabolic pathway in the human gut microbiota. These analyses indicate that the anaerobic pathway is a more substantial contributor to TMA generation from l-carnitine in the human gut than the previously proposed aerobic pathway. The discovery and characterization of the bbu pathway provides the critical missing link in anaerobic metabolism of l-carnitine to TMA, enabling investigation into the connection between this microbial function and human disease.

The Development of the Gut Microbiota and Short-Chain Fatty Acids of Layer Chickens in Different Growth Periods

A long-term observation of changes of the gut microbiota and its metabolites would be beneficial to improving the production performance of chickens. Given this, 1-day-old chickens were chosen in this study, with the aim of observing the development of the gut microbiota and gut microbial function using 16S rRNA gene sequencing and metabolites short-chain fatty acids (SCFAs) from 8 to 50 weeks. The results showed that the relative abundances of Firmicutes and genus Alistipes were higher and fiber-degradation bacteria were less at 8 weeks compared with 20 and 50 weeks (P &lt; 0.05). Consistently, gut microbial function was enriched in ATP-binding cassette transporters, the energy metabolism pathway, and amino acid metabolism pathway at 8 weeks. In contrast, the abundance of Bacteroidetes and some SCFA-producing bacteria and fiber-degradation bacteria significantly increased at 20 and 50 weeks compared with 8 weeks (P &lt; 0.05), and the two-component system, glycoside hydrolase and carbohydrate metabolism pathway, was significantly increased with age. The concentration of SCFAs in the cecum at 20 weeks was higher than at 8 weeks (P &lt; 0.01), because the level of fiber and the number of dominant fiber-degradation bacteria and SCFA-producing bacteria were more those at 20 weeks. Notably, although operational taxonomic units (OTUs) and the gut microbial α-diversity including Chao1 and abundance-based coverage estimator (ACE) were higher at 50 than 20 weeks (P &lt; 0.01), the concentration of SCFAs at 50 weeks was lower than at 20 weeks (P &lt; 0.01), suggesting that an overly high level of microbial diversity may not be beneficial to the production of SCFAs.