Cloning, Expression and Phylogenetic Analysis of Two GABA Shunt-related Proteins from Bacillus thuringiensis

Abstract:

The γ-aminobutyric acid (GABA) shunt is a metabolic pathway that bypasses two successive steps of the tricarboxylic acid cycle (TCA cycle) in both prokaryotes and eukaryotes. In this pathway, the enzymes involved in GABA catabolism are GABA transaminase (GABASE), which converts GABA to succinic semialdhyde, and succinic-semialdehyde dehydrogenase (SSADH), which oxidizes succinic semialdhyde to succinate. In this report, we characterized gabT and gabD genes cloned from B.thuringiensis strain G03 isolated in China. Both gabT gene, 1440 bp encoding a protein with 52.6 kD, and gabD gene, 1449 bp encoding a protein with 52.2 kD were expressed in E.coli BL21(DE3) strain, and their products were purified by affinity chromatography. By enzyme assay,GabT protein showed the activity of GABASE, while GabD protein exhibited SSADH activity. The amino acid sequences of two proteins, GABASE and SSADH, showed significant identity with those in the B.cereus group, but their similarity score between G03 and B.subtilis 168 was lower, only 58% and 51%, respectively. In the present study, we provided evidence to identify the genes involved in GABA metabolism, and it will be helpful for further assessment the biological function(s) and regulation mechanism of GABA shunt in B.thuringiensis.