Genome Structure of Bacillus cereus tsu1 and Genes Involved in Cellulose Degradation and Poly-3-Hydroxybutyrate Synthesis

Figure 3

Characterization of genes in the poly-3-hydroxybutyrate (PHB) biosynthesis pathways in Bacillus cereus tsu1. (a) Genes in the PHB biosynthesis pathways: phaR, phaB, and phaC on the same operon, phaJ, phaP, and phaQ on reverse direction, and phaA located on a separate locus. (b) Sudan Black staining of B. cereus tsu1 showing accumulation of PHB granules in the bacterial cells. (c) The PHB gene fragments amplified from the genomic DNA of Bacillus cereus tsu1 using polymerase chain reaction (PCR) and separated on a 0.7% agarose gel. (d) Two-dimensional gel electrophoresis of recombinant proteins of PhaA, PhaB, PhaC, PhaR, and PhaP. The PCR amplified gene fragments showing identical sequence matches with the PHB synthesis genes were cloned into TOPO pET101 vector (Invitrogen, CA); recombinant proteins were expressed in DE3 cells. Recombinant proteins were separated on 2D gels. Protein spot with matching molecular size and isoelectric point (pI) of each predicted protein was picked from the gel. Protein identity was confirmed using the peptide fingerprinting (70–100% coverage with identical predicated peptides) using mass spectrometry analysis of tryptic digests of these proteins. (e) Sudan Black staining of the recombinant E. coli (over)expressing phaC gene showing intracellular PHB granules; E. coli transformed with the empty vector had no PHB accumulation. The arrow refers to the black spots inside of the bacterial cell, which is the PHB accumulation stained with Sudan Black.

(a) Loci of genes in PHB biosynthesis pathways

(b) Accumulation of PHB granules (indicated by arrows) in Bacillus cereus tsu1

(c) PCR cloning of PHB genes

(d) Recombinant PHB proteins (indicated by arrows) on 2D gels and confirmation using peptide mapping

(e) E. coli with phaC genes displayed PHB producing activity