Green fluorescent protein labeling of ylyA gene in Bacillus subtilis

Abstract:

[Objective] In this study we labeled the ylyA gene of Bacillus subtilis with green fluorescent protein (GFP) to investigate the subcellular localization of YlyA protein. [Methods] Chromosomal DNA was prepared from different strains of Bacillus subtilis, ylyA amplified by PCR and the products sequenced. Full-length ylyA was then amplified and inserted into the GFP plasmid vector pSG1729, to give pNG426 containing a gfpmut1-ylyA fusion. Finally, Bacillus subtilis 168 was transformed with pNG426, resulting in insertion of the gfpmut1-ylyA fusion into the chromosome at the amyE locus. Double crossover integrants (subsequently named BS363) were identified by their inability to hydrolyse starch and verified by colony-PCR. Following induction of gfpmut1-ylyA expression with 0.5% xylose, localization of the fusion protein was determined by epifluorescence microscopy. [Results] The correct sequence and translation start site of ylyA was identified from sequence analysis of the several amplified PCR products permitting construction of a gfpmut1-ylyA fusion. Microscopic observation of strain BS363 showed that the GFP labeled YlyA was distributed around the cell periphery, closely juxtaposed with the cytoplasmic membrane. [Conclusion] GFP labeled YlyA produced by BS363 cultured on the nutrient agar solid medium distributed around the cell periphery.This suggests it may play a role in membrane biology.