Genome Analysis Of A Hemolytic Bacillus Cereus And Screen Its Hemolysis Genes

Bacillus cereus is a Gram-positive and spore-forming bacterium which can be found in foods, rice dishes, eggs, spices, meat, dairy products and even the drugs. Severe food poisoning accidents have been reported to be caused by the B. cereus with vomiting and diarrhea in many countries, including the USA, Canada and the Netherlands.In this study, a hemolytic bacterium strain isolated from a farmed turbot, and now Scophthalmus maximus(turbot) became one of the dominant species in mariculture fishes in China with an annual production more than 60000 tons, during the rapid development of turbot aquaculture, fish diseases emerged and became serious problems, many pathogenic bacteria were reported to cause hemolysis of the host, the isolates could be a consistent pathogenic germs, so we have a following study on the hemolytic bacteria:1. Based on morphological and phylogenetic analyses, the isolate was identified as Bacillus cereus and was named as WH2015 according to the isolation place and time.2. Blood agar hemolytic assay confirmed that the WH2015 strain could lyse the sheep red blood cells, electronic scanning electron microscopy(sem) analysis confirmed B. cereus WH2015 have a certain ability to damage the blood cells.3. We conducted genome sequencing and annotation of WH2015 strain. The results showed that B. cereus WH2015 has a genome size around 5.8 Mb, with an average G+C content of 35%, 6 568 annotated protein-coding genes and 98 tRNA sequence. Comparing with other sequenced B. cereus strains, the WH2015 genome possesses almost all of the core hemolytic genes, a total of 23 virulence factor genes, including 1 HBL operon gene, a hlyIII gene, strongly suggest that its pathogenesis mechanisms are similar to other reported B. cereus isolates.4. To test and verify the function of hemolytic genes, this paper also successfully construct prokaryotic expression plasmid pET-28a-hblC, pET-28a-hblD, pET-28a-hblA, pET-28a-hlyIII, and using protein immunoblot analysis shows that the recombinant proteins have been expressed, this laid the foundation for the next step of function research.Based on the isolation and identification of Bacillus cereus WH2015 from farmed turbot surface, we also sequenced its genome and conducted the analysis and identification of pathogenic genes and virulence factors, which facilitates our understanding of the farmed turbot disease caused by B. cereus, and provides valuable information in developing new method for disease prevention and therapy in farmed turbot.