Effects Of Selenomonas Ruminantium And Its Gene Deletion Engineering Bacteria On The Fermentation Of Ruminal Microorganism

Selenomonas ruminantium is one of dominant bacteria in the rumen, and of theimportant microorganism in participating rumen fermentation. Transposon taggingmethod was applied to construct the engineered strain Selenomonas ruminantium K6.The eight mutants were screened from the numerous transconjugants with selectiveculture medium containing kanamycin and fluoroacetic acid. The 16S ribosomalRNA gene and Tn5 transposon gene of the strains TnK6 were amplified bypolymerase chain reaction (PCR) and sequenced. The sequence obtained was 98%identical to that of Selenomonas ruminantium, Tn5 transposon gene inGenBank.PCR of 16S ribosomal RNA and Tn5 analyses revealed that the mutantshad the transposon inserted in chromosomal DNA of Selenomonas ruminantium K6.It was proven that the eight mutants (TnK6) were pta gene deletants by measuringthe specific activity of acetate kinase(AK) and phosphotransacetylase (PTA).In vitro fermentation of ruminal fluid, primary substrates (lactic acid andpyruvate) were cultured with K6 and TnK6 for 48h, respectively. Culture fluid wassampled for analysis of pH and VFA. The results were as follows: the lactate in theculture fluid was utilized by the strains of K6 and TnK6. Compared to strains K6,theacetate concentrations and the ratio of acetate to propionate were obviously reducedin the culture fluid with the strains TnK6, even though the concentration ofpropionate was slightly reduced. This demonstrate that the transposition engineeringbacteriums belong to defect of acetic acid-producing strain.