| Bacillus coagulans is a spore-forming gram-positive soil bacterium. This specieswas first isolated from spoiled canned milk by Hammer in1915. Recently, it has beenreported that the strains have many good fermentation features such as growing athigh temperature50~55°C and high carbon-efficiency. In addition, it can fermentvarious biomass-derived sugars to platform bio-chemicals, such as lactic acid.Moreover, high fermentation temperature of B. coagulans strains enablesnon-sterilized batch and fed-batch fermentations for L-lactic acid production. Besidesthe production of lactic acid, B. coagulans has also been found to be a source of manyother commercial valuable products, such as thermostable enzymes, and coagulin, anantimicrobial peptide. More recently, this species has been regarded as a novel safetyprobiotic. These studies suggest that B. coagulans strains can readily achieve theGRAS (generally regarded as safe) status required for large-scale commercial use.Compared with other probiotic strains such as those belonging to the Lactobacillusspecies, B. coagulans strains are able to survive in the extreme environments, such assevere heat or acidity.Although B. coagulans is a good producer of platform biochemicals, littleinformation is known about this species. As the development of high-throughputsequence technology, it is much easier to obtain the genome sequence of variousstrains of this species. In this study, we used different methods (Solexa from Illuminaand ABI3730from AB) to obtain the complete genome sequence of B. coagulans2-6and the draft genome sequences of the strains XZL4, XZL9, H-1and DSM1. We haveaccordingly submitted all of these sequences to the open access database (GenBank).Based on the genome sequences, we compared the B. coagulans strains withinspecies and with different species. We analyzed the evolutionary process of B.coagulans, carbohydrate metabolism systems, microbial immune system, cell wallsynthesis and material transport systems, which help B. coagulans to own the goodfermentation characteristics. By evolutionary analysis, we found that it is likely that the strains of B. coagulans and Geobacillus came from the same ancestor. In thexylose metabolism, we comprehensively analyzed the xylose metabolism anddiscovered a new gene of xylose isomerase. In the cell wall synthesis pathway, thepresence of a new fusion gene improves efficiency of cell wall teichoic acid synthesis,which may enhance the ability of the bacteria to withstand high temperatures. TheCRISPR-Cas and restriction-modification systems, which were found in the B.coagulans strains, provide a resistance to phage and other forigen DNA.According to theory of molecular evolution, during the organisms’ adaption toenvironment, some key genes with positive selection pressure such asresistance-related genes, and virulence genes, provide competitive advantage fororganisms. In order to explore the genes related to the thermophilie features in thegenomes of B. coagulans strains, we have established a pipeline to calculate thepositive selection pressure, named PSP. The PSP web server has been developed torapidly identify orthologous coding genes under positive selection across up to thirtyprokaryotic genomes. PSP excludes the effect of gene recombination and incorporatesfunctional investigation at the metabolic pathway level. Remarkably, PSP is able tofacilitate efficient exploration of the identified orthologous genes by assignments andenrichments of KO, GO or COG terms. In the analysis of genus Bacillus, the geneswith evidence of significnat positive selection were enriched among the amino acidsynthesis pathways, implying that these genes might play important roles during theenvironmental adaptation in B. coagulans strains.With strand-specific RNA-Seq technology, we studied the transcriptionalprofiling of B. coagulans strains under different temperatures. According to theexpression analysis, a total of170significantly differentially expressed genes wereobtained. About64genes were down-regulated and106up-regulated during growingin different temperatures. Based on the sequence coverage, we built the completetranscript structures, and found20genes belonging to devise operons under differenttemperatures. It is also very interesting to find that among the down-regulated genes,the genes related to purine synthesis have been strongly suppressed, which wereconfirmed by RT-qPCR. In addition, the genes involved in the carbohydrate metabolism were enriched and strongly up regulated, which maybe the main reasonfor the B. coagulans strain to stably produce L-lactic acid under high temperature.Finally, we used a label-free proteomic technology to determine the change ofprtoein content under different temperatures. Totally275significantly differentialexpressed proteins were obtained using iBAQ as quantitative measure, of which35proteins matched the transcript profiling, such as the genes related to purinemetabolism, and chaperones. And, by the enrichment analysis, results of two methodshave a similar regulation pattern.In this work, we analyzed the genomes and evolutionary process of industrial B.coagulans strains systemically, and compared the genome content with other Bacillusstrains in-depth. By means of the transcriptomic and proteomic analyses of the strainscultured at different temperatures, we explored the unique transcript structure of B.coagulans2-6and suggested that amino acid synthesis pathway, especially the genesof purine synthesis, play important roles in heat resistant. |
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