Project description:In this study, we have applied the top-down approach to reduce the genome of B. subtilis in order to obtain minimal strains with robust growth on complex medium at 37°C. For this purpose, we have evaluated the function of each gene of the B. subtilis genome and identified essential, important and dispensable genomic regions. Using an efficient markerless and scarless deletion method and a system allowing induction of genetic competence in the complete cell population, we have constructed two genome-reduced strains lacking about 36% of dispensable genetic information. Multi-omics analyses with the genome-reduced strains revealed substantial changes in the transcriptome, the proteome and in the metabolome. The massive reorganization of metabolism in the two genome-reduced strains can be explained by the underlying genotypes that were determined by genome re-sequencing. Moreover, the transcriptome and proteome analyses uncovered novel dispensable genomic regions that can be removed to further streamline the B. subtilis genome. In conclusion, both minimal strains show interesting metabolic features and they serve as excellent starting points to generate an ultimate reduced-genome B. subtilis cell containing only genes required for robust growth on complex medium.
Project description:Helicobacter pylori, which is known as pathogens of various gastric diseases, have many types of genome sequence variants. That is part of the reason why pathogenesis and infection mechanisms of the H. pylori-driven gastric diseases have not been well clarified yet. Here we performed a large-scale proteome analysis to profile the heterogeneity of the proteome expression of 7 H. pylori strains by using LC/MS/MS-based proteomics approach combined with a customized database consisting of non-redundant tryptic peptide sequences derived from full genome sequences of 52 H. pylori strains. The non-redundant peptide database enabled us to identify more peptides in the database search of MS/MS data, compared with a simply merged protein database. Using the approach we performed proteome analysis of genome-unknown strains of H. pylori in as large-scale as genome-known ones. Clustering of the H. pylori strains using the proteome profiling slightly differed from the genome profiling and more clearly divided the strains into two groups based on the isolated area. Furthermore, we also identified phosphorylated proteins and sites of the H. pylori strains and obtained phosphorylation motif located in the N-terminus, which are commonly observed in bacteria.
