Identification and characterization of the Bacillus thuringiensis phaZ gene, encoding new intracellular...

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Poly-3-hydroxybutyrate (PHB) is a biopolyester used for energy storage in bacteria.
• Intracellular PHB depolymerases are crucial for PHB mobilization and metabolism.
• Previous identification of PHB-related genes often led to misannotations.

Purpose of the Study:

• To identify and characterize a novel intracellular PHB depolymerase in Bacillus thuringiensis subsp. israelensis.
• To determine the enzymatic activity and substrate specificity of the identified protein.
• To elucidate the in vivo function of the novel depolymerase in PHB metabolism.

Main Methods:

• Gene identification and annotation in Bacillus thuringiensis genome.
• Protein purification (His-tagged PhaZ and PcaD).
• Enzymatic assays using native and artificial PHB granules, and other substrates.
• Construction and analysis of a phaZ gene knockout mutant.
• Enzyme activity assays using cell sonication supernatants.

Main Results:

• A novel gene, phaZ, encoding an intracellular PHB depolymerase was identified and characterized.
• Purified His-tagged PhaZ efficiently degraded amorphous PHB granules but not semicrystalline PHB.
• The phaZ mutant exhibited higher PHB accumulation than the wild type, confirming in vivo function.
• PhaZ possesses a lipase box-like motif and requires a specific serine residue for activity.

Conclusions:

• Bacillus thuringiensis harbors a new type of intracellular PHB depolymerase, designated PhaZ.
• PhaZ plays a significant role in the in vivo degradation of intracellular PHB granules.
• This discovery expands the known repertoire of bacterial PHB depolymerases and their mechanisms.