[Green fluorescent protein labeling of ylyA gene in Bacillus subtilis]

In this study we labeled the ylyA gene of Bacillus subtilis with green fluorescent protein (GFP) to investigate the subcellular localization of YlyA protein.

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.

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.

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.