Complete Genome Sequence And Nitrogen Utilization In Ureolytic Succinovibrio Dextrinosolvens Z6 Isolated From Rumen Of Dairy Cows

Succinivibrio dextrinosolvens?SD?is a gram-negative anaerobe bacterium which mainly found in large number when ruminants fed with high grain diet.It actively ferments dextrin and maltose in the rumen which makes it the most important fermenter of the hydrolytic products of starch and some strains of SD are the dominant urease producer.However,the description of novel SD strains given by different researchers earlier was mainly based upon partial characterization and there is limited information about the role it plays in nitrogen metabolism.For our SD-Z6 characterization,we have applied the integration of whole genome sequence,MALDI-TOF MS,phenotypic and chemotaxonomic analysis.To analyze the role SD in nitrogen metabolism the strain grown in different nitrogen sources was analyzed by using transcriptome analysis methods.Succinivibrio dextrinosolvens-Z6 was isolated from rumen fluid by using four different anaerobic bacterial isolation media.The urease and bacterial 16S rRNA gene were amplified by appropriate primers and sequenced.The results were used to ascertain the identity of SD-Z6 using BLASTN search at NCBI.Bacterial identification was also made by MALDI-TOF protein analysis.Whole genome sequencing of isolated SD-Z6 was performed using Oxford Nanopore Technology?ONT?.Genome assembly,annotation and comparison were done using recommended software and databases.Cell morphology and gram-staining were examined by using scanning electron microscope and Hucker's modification.The growth was checked at different temperatures,pH and salt concentration under anaerobic conditions;at 39^oC.SD-Z6 was grown using ammonia,urea,or Amicase to the level of 9.4 m M of NH₃-N as the sole nitrogen source.The concentration of ammonia,amino acid and urea in the supernatant were analyzed by using modified colorimetric method,spectrophotometric ninhydrin procedure and urea assay kit,respectively.Enzyme activity related nitrogen utilization were also analyzed using enzyme activity test kit.Gene expression profiles for cell grown on different nitrogen sources were evaluated using RNA-seq by Illumina Hiseq sequencing platform.The whole genome sequence of SD-Z6 revealed that genome size of 3.47 Mbp with 38.9%of G+C content.A total of 2,993 coding sequences accounts 98%,21 rRNA genes and 69tRNAs were identified.The genes for regulating carbohydrate 10.6%?182?and amino acid9%?155?transport and metabolism were the most abundant.ANI?Average nucleotide identity?showed SD-Z6 was most closely related to SD-22B?99.96%?,SD-ACV-10?86.73%?,SD-H5?83.87%?and SD-DSM3072?83.14%?.The whole genome alignment of SD-Z6 with SD-22B showed than more than 0.34 Mb nucleotide difference.Growth of SD-Z6 occurred at a temperature 36 to 42^oC with optimum 39.7^oC,the range of pH 6 to 8,the optimum pH was 6.9 and with 0 to 1%?w/v?NaCl with the maximum growth at 1%.The maximum growth(OD₆₀₀ 0.825±0.12)and MCP?microbial crude protein??178.2?g/ml?was observed in cell gown in amino acid.The maximum concentration of ammonia?3.96±1.2?was observed in urea containing media and 1.06mM remains after 24hr incubation which around 26.7%of the amount produced.Enzyme activities varied according to nitrogen source and growth phase.Activities of urease and GS?Glutamine synthase??P<0.01?and GDH?Glutamate dehydrogenase??P<0.05?were significantly different in both nitrogen and growth phase whereas,GOGAT?Glutamate synthase??P<0.01?was significantly different only at different growth phase.In total,1246 differentially expressed genes?DEGs?were identified in all nitrogen sources.Among differentially expressed genes 33 were related to nitrogen metabolism.The genes?ilvE,CysK,PurF and FabG?related to amino acid degradation and biosynthesis were differentially expressed in cell grown on amino acid.Urea transporter?urtE&urtD?and urease structural genes?ureA?which are urea hydrolysis related genes were differentially expressed in cell grown in media containing urea.Their expression directly correlated with type of nitrogen sources utilized and the intensity of enzyme activity.In conclusion,this study revealed that the genome comparison of S.dextrinosolvens strains revealed a significant genetic diversity between them in genus Succinovibio.Furthermore,ammonia,amino acid and urea can all used as nitrogen sources for Succinovibrio dextrinosolvens,and enzyme activities and expression of genes involved in nitrogen metabolism of Succinovibrio dextrinosolvens strain Z6 are regulated by the type of nitrogen source.