Diversity Of Urease Gene And Regulation Of Urease Activity In The Rumen Of Cattle

Urease hydrolyzes urea to ammonia which can be absorbed by microbes in rumen, so urease is thekey enzyme in the urea cycle. However, the high rumen urease activity leads to ammonia quickformulation, which blocks the utilization of urea and induces ammonia toxication. The purposes of thisstudy are to regulate urease activity and slow down the hydrolysis of urea. The combined methodsincluding anaerobic culturing, high-throughput sequencing, protein expression or expression, andimmunology are used in this study. We isolated partial urease producing bacteria from rumen, comparedthe urease gene diversity from rumen wall and content, expressed urease protein which was used toanalyze immunological homology to rumen urease protein, regulated rumen urease activity immunizedwith urease in vivo or vitro.To isolate urease producing bacteria from rumen content and wall, we use the anaerobic culturesrich in urea to enrich the bacteria which is detected by qPCR. After dilution plating, the single colonieswere sequenced by urease and bacterial16S rDNA primers. Results showed that we isolated2ureaseproducing bacteria from rumen wall, UB1belongs to Bacillus licheniformis, UB2belongs to Proteusmirabilis, and isolated3urease producing bacteria from rumen content, UB3belongs to Ruminococcusalbus, UB4belongs to Succinivibrio dextrinosolvens, UB5belongs to Treponema bryantii. These fivebacteria had different cell modalities, and UB3had the highest urease activity. The urease sequencesfrom UB1, UB4and UB5just had80%-89%identities to the known urease sequences, which revealedthat these urease genes are new sequences.To compare the urease gene diversity in rumen wall and content, we sequenced PCR productsgenerated from urease gene degenerate primers using Roche454sequencing. The QIIME software wasused to do quality controlling, OTU, diversity and clustering analysis. Results showed that urease geneα diversity was similar, but β diversity was different compared rumen wall with content with the cutoffvalue of0.03. Urease gene sequences in rumen wall and content were clustered separately, whichimplied rumen wall contained unique and new urease gene and urease producing populations. TheOTU2and OTU4were most obvious rich population in rumen wall and content, respectively. The qPCRrevealed that the proportion of urease gene in rumen wall was higher than that in rumen wallsignificantly.To express bacterial urease protein by genetic engineering, we cloned urease gene fromHelicobacter pylori to pET vector, transformed into E.coli BL21(DE3). The urease protein was inducedby IPTG and purified by Ni-NTA. The anti-urease polyclonal antibody was prepared by immunizingrabbits. The results showed that about5mg urease protein was expressed and purified. The titer ofanti-urease polyclonal antibody was about1:12800. This supplied the antigen for the following animalimmunization.To detect the immunological homology between Helicobacter pylori urease and rumen urease, we collected rumen bacterial cells from dairy cows. After ultrasonication and precipitation, the fluid wasapplied to a HiTrap Capto Q ion exchange column. Urease was then verified by modified Fishbeinstaining and LC-MS/MS. The anti-urease polyclonal antibody prepared above was used as primaryantibody to do western-blotting analysis. The results revealed that higher activity urease was isolatedand purified from cell free rumen fluid (151.53U/mg) and microbial intracellular fluid (541.90U/mg).After straining, one brownish red strap which containing urease from microbial intracellular fluid wasobserved. The purified rumen urease was confirmed by LC-MS/MS. Western-blotting showed that therewas immunological homology between Helicobacter pylori urease and rumen urease.To verify the effect of anti-urease polyclonal antibody on the inhibition of urea hydrolysis in vitro,we added0,0.5and1.0ml anti-urease polyclonal antibody serum into the bottle and incubation forsome time, then detected fermentation, urea and ammonia concentration, checked bacterial populationchanges by DGGE. Results showed that the antibody reduced the speed of urea hydrolysis (70%) andammonia formation (18%), evaluated the concentration of total VFA, acetate, propionate and valerate.DGGE profile revealed that antibody changed the quantity, location and OD of bands, and reduced thebacterial diversity. The bacterial population treated with antibody just had51%identity to the control.The ratio of urease-producing bacteria to total bacteria decreased when treated with antibody, whichmaybe the reason of low urease activity in fermention.To inhibited rumen urease activity by immunology, we selected8dairy cows immunized withurease antigen and Freund adjuvant followed by3boosters. Results revealed that the serum and salivaIgG and IgA antibody titers to specific urease were evaluated by immunization with urease. The IgGand IgA titers reached the highest level after the4th and3rd immunization. After the forth booster,vaccine group had a significant reduction (17%) in urease activity compared to control. There was noobvious effect of immunization on rumen pH, VFA, ruminal dynamic degradation of dry matter andcrude protein. After urea infusion into the rumen, ammonia concentration in immunization groupdescent36%compared to control at the first hour, and there was no significant different after4h. Thisindicated that the inhibition just happened at the initial stage.