Genome and Proteome Analysis of Bacillus cereus TSU1 for Bacterial Function and Biopolymer Production Study
Bacillus cereus tsu1 with poly (3-hydroxybutyrate) (PHB)-producing and cellulose degrading activities was isolated at Tennessee State University. This project contained three parts for genotypic and phenotypic characterization of B. cereus tsu1. In the first part, the whole genome of the bacterium was sequenced using the next generation sequencing (NGS) technique. Draft genome of ‘tsu1’ was assembled into 36 scaffolds. Genes encoding for xylanase and cellulase, and the cluster of genes in the PHB biosynthesis pathway were identified in ‘tsu1’ genome. The five PHB genes (phaA, phaB, phaR, phaC, phaP) were cloned using TOPO cloning system. Successful recombinant gene expression was confirmed using 1D and 2D gel electrophoresis followed by mass spectrometry peptide mapping analysis. The transformed E. coli (over)expressing the recombinant phaC gene were found accumulating PHB after overnight culture. In the second part, biochemical analyses of ‘tsu1’ were conducted for cellulolytic and PHB accumulation activity. Bacterium ‘tsu1’ was cultured in M9 supplemented with 1% carboxymethylcellulose sodium salt medium, both Congo red staining and gel permeation chromatography methods were used to confirm Na-CMC degradation. To determine PHB-producing activity of ‘tsu1’, the bacteria were cultured in rapeseed cake substrate (RCS) and stained with Sudan black. The ‘tsu1’ batch culture in RCS for PHB production results in 558mg of dry cell biomass and 14% PHB content. The physical properties of the ‘tsu1’ PHB extracts were found comparable with standard PHB compound (sigma) using Xplora Raman spectrometer and Nicolet IS10 Fourier transform infrared spectroscopy. In the third part, ‘tsu1’ was cultured in RCS, bacterium was found reaching the maxima of PHB accumulation within 12 hours, and nearly complete depletion of PHB after 48 hours. Proteins were extracted from cell pellet harvested at three time-points 12, 24 and 48 hours, and a quantitative proteomics analysis was conducted using the 10-plex tandem mass tags (TMT) followed by nano-LC-MS/MS analysis. The mass spectrometry data were searched against the ‘tsu1’ annotated protein database. In total, 3,237 proteins were detected and enzymes for PHB biosynthesis (PhaA, PhaJ, PhaC, PhaB, PhaR, PhaP) and intracellular degradation (PhaZ) were identified from 12-24-48hr cultures.
"Genome and Proteome Analysis of Bacillus cereus TSU1 for Bacterial Function and Biopolymer Production Study"
ETD Collection for Tennessee State University.