Table_1_Isolation and pan-genome analysis of Enterobacter hormaechei Z129, a ureolytic bacterium, from the rumen of dairy cow.XLSX

IntroductionUrea is an important non-protein nitrogen source for ruminants. In the rumen, ureolytic bacteria play critical roles in urea-nitrogen metabolism, however, a few ureolytic strains have been isolated and genomically sequenced. The purpose of this study was to isolate a novel ureolytic bacterial strain from cattle rumen and characterize its genome and function.MethodsThe ureolytic bacterium was isolated using an anaerobic medium with urea and phenol red as a screening indicator from the rumen fluid of dairy cattle. The genome of isolates was sequenced, assembled, annotated, and comparatively analyzed. The pan-genome analysis was performed using IPGA and the biochemical activity was also analyzed by test kits.ResultsA gram-positive ureolytic strain was isolated. Its genome had a length of 4.52 Mbp and predicted genes of 4223. The 16S rRNA gene and genome GTDB-Tk taxonomic annotation showed that it was a novel strain of Enterobacter hormaechei, and it was named E. hormaechei Z129. The pan-genome analysis showed that Z129 had the highest identity to E. hormaechei ATCC 49162 with a genome average nucleotide identity of 98.69% and possessed 238 unique genes. Strain Z129 was the first E. hormaechei strain isolated from the rumen as we know. The functional annotation of the Z129 genome showed genes related to urea metabolism, including urea transport (urtA-urtE), nickel ion transport (ureJ, tonB, nixA, exbB, exbD, and rcnA), urease activation (ureA-ureG) and ammonia assimilation (gdhA, glnA, glnB, glnE, glnL, glsA, gltB, and gltD) were present. Genes involved in carbohydrate metabolism were also present, including starch hydrolysis (amyE), cellulose hydrolysis (celB and bglX), xylose transport (xylF-xylH) and glycolysis (pgi, pgk, fbaA, eno, pfkA, gap, pyk, gpmL). Biochemical activity analysis showed that Z129 was positive for alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-glucosidase, β-glucosidase, and pyrrolidone arylaminase, and had the ability to use D-ribose, L-arabinose, and D-lactose. Urea-nitrogen hydrolysis rate of Z129 reached 55.37% at 48 h of incubation.DiscussionTherefore, the isolated novel ureolytic strain E. hormaechei Z129 had diverse nitrogen and carbon metabolisms, and is a preferred model to study the urea hydrolysis mechanism in the rumen.</p

Data_Sheet_1_Contribution of Ruminal Bacteriome to the Individual Variation of Nitrogen Utilization Efficiency of Dairy Cows.docx

High nitrogen utilization efficiency (NUE) is important for increasing milk protein production and decreasing the feed nitrogen cost and nitrogen emission to the environment. Currently, there is a limited whole picture of the relationship between ruminal bacteriome and the NUE of dairy cows, even though some information has been revealed about the bacteriome and milk or milk protein production of dairy cows. The purpose of this study was to compare the rumen bacterial community in dairy cows with different nitrogen utilization efficiency under the same diet. The natural abundance of 15N between the animal proteins and diet (Δ15N) was used as a simple, non-invasive, and accurate biomarker for NUE in ruminants to mark the individual variation. Dairy cows with high NUE (HE_HP, n = 7), medium NUE (ME_MP, n = 7), and low NUE (LE_LP, n = 7) were selected from 284 Holstein dairy cows with the same diet. Measurement of the rumen fermentation indices showed that the proportion of propionate was higher in HE_HP cows and ME_MP cows than in LE_LP cows (P 15N (P 15N (P < 0.05). In conclusion, the study showed that the diversity and relative abundances of rumen bacteria differed among different NUE cows, indicating that rumen bacteriome contributes to nitrogen metabolism in dairy cows.</p

Additional file 11 of Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle

Additional file 11: Supplementary Fig. 6. Urea hydrolysis rates (A) and urea-utilization rates of each ureolytic isolate per unit of culture optical density (OD, n = 3)

Additional file 8 of Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle

Additional file 8: Supplementary Fig. 3. Circular diagrams of the core and unique gene clusters of the ureolytic isolates of individual species

Additional file 6 of Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle

Additional file 6: Supplementary Fig. 1. Optimization of isolation methodology of functional gene-guided enrichment plus in situ microsphere cultivation for ureolytic bacteria. A. A plot of ureC-positive wells vs. dilution; B. Distribution of agarose microsphere sizes (diameter); C. Percentage of agarose microspheres with embedded bacteria at different dilutions; D. Percentage of agarose microspheres with embedded single bacteria at different dilutions; E. Percentage of agarose microspheres with PCR-detected embedded bacteria with increasing incubation time; F. Percentage of agarose microsphere with PCR-detected single bacteria with increasing incubation time

Additional file 9 of Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle

Additional file 9: Supplementary Fig. 4. Pan-genome profiles of ureolytic isolates. Pan-genome (blue) and core-genome (red) sizes were predicted based on all the strains of individual species

Additional file 3 of Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle

Additional file 3: Supplementary Table 3. The major GH families identified in each isolate

Additional file 2 of Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle

Additional file 2: Supplementary Table 2. All ruminal ureolytic bacteria that have been reported in the literature

Additional file 7 of Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle

Additional file 7: Supplementary Fig. 2. Microbial metabolites inside and outside of the dialysis bags. A. The identified metabolites inside the dialysis bags; B. A heatmap of the metabolite profiles at each incubation time both inside and outside of the dialysis bags; C. Correlation of metabolite profiles between insides and outside the dialysis bags

Additional file 1 of Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle

Additional file 1: Supplementary Table 1. Summary of genome features of the ureolytic bacterial isolates