Complete Genome Analysis And Safety Exploration Of Christensenella Minuta

Obesity,a chronic metabolic disease which closely related to diabetes,cardiovascular disease,fatty liver and other metabolic syndrome,has become a global public health problem due to its significant morbidity and high-risk consequences.Therefore,how to prevent and treat obesity has become research hotspots.Christensenella minuta（C.minuta）is a strictly anaerobic and gram-negative enteric bacterium.Studies have shown that C.minuta is closely related to weight loss,thus it has potential application for the treatment of obesity.However,C.minuta is difficult to culture in vitro and its genetic characteristics remain elusive,which restricts its in-depth study.In this paper,the genome of C.minuta was sequenced by the second and third generation sequencing technology and the genetic information related to safety was analyzed.The putative hemolysin gene product activity and lipopolysaccharide（LPS）toxicity of C.minuta were further investigated.These results preliminary clarified the genetic information and pathogenic characteristics of C.minuta.The growth medium of C.minuta was optimized for its difficulty in culture,and a huge amount of C.minuta could be efficiently obtained by culture in vitro.These not only lay a foundation for microbiology research of C.minuta,but also provide a train of thought for the development and application of C.minuta as probiotics in the future.The detailed research contents are as follows:The complete genome of C.minuta was sequenced using the combination of second and third generation sequencing technology.It comprises one circular chromosome of 2,969,292bp with an average G+C content of 51.44%.There are 3053 predicted protein-coding genes,67 tandem repeats,36 small satellite sequences,9 microsatellites sequences,49 tRNA,6rRNA and 1 sRNA in C.minuta genome.The safety of C.minuta was analyzed at genome level.The results showed that C.minuta genome didn’t have gene island and prophage which were releated to pathogenicity and drug resistances of pathogenic bacteria.Moreover,the C.minuta genome has 12 predicted drug resistance genes with low risk of transfer,10 genes associated with harmful metabolites which may not be expressed or its products with low activity,one putative hemolysin gene and some LPS synthetic genes.The hypothetical hemolysin protein was prepared by Escherichia coli（E.coli）expression system.Then,the hemolytic activity of the hypothetical hemolysin protein and C.minuta was detected respectively.Results showed that they did not cause a clearing zone on blood agar plates,indicating that the putative hemolysin gene expression product had no hemolytic activity,and C.minuta had no hemolysis.The structure of C.minuta LPS was compared with pathogenic E.coli LPS by SDS-PAGE and sliver staining.The results showed that C.minuta LPS displayed three bands at 18 kDa,20-28 kDa and 33 kDa,which was obviously different from the structure of E.coli LPS that exhibited a characteristic ladder-like pattern of bands.Subsequently,the results of cell experiments showed that C.minuta LPS could not activate RAW 264.7 macrophages efficiently compared to pathogenic E.coli LPS,in which C.minuta LPS induced low level of cell proliferation,phagocytosis,NF-κB activation and inflammatory mediator（TNF-α,IL-6,IL-1β,NO and reactive oxygen species）production.Thus,the toxicity of C.minuta LPS is weak even lost.Based on the peptone-yeast extract-glucose（PYG）medium,the optimal medium for the growth of C.minuta was obtained by the combination of single factor test and response surface method.It contanined 5.41 g/L fructose,63.21 g/L beef extract,1.90 g/L L-cysteine hydrochloride,0.032 g/L MgSO₄,0.16 g/L K₂HPO₄ and 1.6 g/L NaHCO₃.The viable count of C.minuta cultured in this medium could reach 8.15×10¹⁰ cfu/mL,which was 48.22-fold higher than that in PYG medium.