Genome Structure ofBacillus cereustsu1 and Genes Involved in Cellulose Degradation and Poly-3-Hydroxybutyrate Synthesis

Abstract

In previous work, we reported on the isolation and genome sequence analysis ofBacillus cereusstrain tsu1 NCBI accession number JPYN00000000. The 36 scaffolds in the assembled tsu1 genome were all aligned withB. cereusB4264 genome with variations. Genes encoding for xylanase and cellulase and the cluster of genes in the poly-3-hydroxybutyrate (PHB) biosynthesis pathway were identified in tsu1 genome. The PHB accumulation inB. cereustsu1 was initially identified using Sudan Black staining and then confirmed using high-performance liquid chromatography. Physical properties of these PHB extracts, when analyzed with Raman spectra and Fourier transform infrared spectroscopy, were found to be comparable to the standard compound. The five PHB genes in tsu1(phaA,phaB,phaR,phaC,andphaP)were cloned and expressed with TOPO cloning, and the recombinant proteins were validated using peptide mapping of in-gel trypsin digestion followed by mass spectrometry analysis. The recombinantE. coliBL21 (DE3) (over)expressingphaCwas found to accumulate PHB particles. The cellulolytic activity of tsu1 was detected using carboxymethylcellulose (CMC) plate Congo red assay and the shift towards low-molecular size forms of CMC revealed by gel permeation chromatography in CMC liquid culture and the identification of a cellulase in the secreted proteome.