Enhancing oxidative stress resistance in Bifidobacterium thermophilum using a novel overexpression vector and transformation protocol

Bifidobactenum thermophilwn is encountered in the GI-tract of pigs and infants. Here we provide a transformation protocol for B. thermophilum and a novel expression vector for this species. The protocol resulted in transformation rates of 1 x 10(3) transformed cells per mu g DNA. Transformation was shown to be dependent on the presence of fructo-oligosaccharides during growth, polyethylene glycol in the electroporation buffer, and on methylation of the vector. The Escherichia coli - B. thermophilum shuttle vector pLFB1012 for heterologous gene expression was constructed harbouring the glyceraldehyde 3-phosphate dehydrogenase promoter from Bifidobacterium longum (P-gap). Activity of the beta-glucoronidase gene gust under control of P-gap could be detected at a 20-fold higher rate compared to the wild type, showing activity of the promoter in B. thermophilum. Thereafter, the B. longurn gene bl_1404, previously proposed to be involved in oxidative stress resistance, was cloned under control of the P-gap. The wild type cell numbers of B. thermophilwn RBL 67 decreased at least 9 log after a 20-mM H2O2 treatment for 60 min whereas the mutant strain expressing bl_1404 showed an increased survival of 2 logs compared to the wild type strain. To our knowledge this is the first report on transformation of B. thermophilum. Further, it is shown that pLFB1002 is suitable for engineering B. thermophilum and that bl_1404 from B. longum is involved in peroxide resistance in bifidobacteria.