Development of a reproducible conjugative gene transfer system for bifidobacteria and its use for investigating the stress response of bifidobacterium longum DJO10A in situ in a yogurt fermentation

University of Minnesota Ph.D. dissertation. March, 2013. Major: Food Science. Advisor: Daniel J. O’Sullivan. 1 computer file (PDF) viii, 258 pages.Bifidobacteria are considered a beneficial inhabitant of the human gastrointestinal tract and are widely used as a probiotic in the food industry. However, our understanding of how these bacteria interact with the host and with the stresses encountered during food processing is limited. One of the main research limitations is the lack of molecular tools for these bacteria, particularly gene transfer techniques that currently depend on strain-specific electroporation protocols. Therefore, it was necessary to develop an efficient and reproducible gene transfer system for bifidobacteria. This was achieved by employing a conjugative-base DNA transfer system able to transfer a mobilizable vector from E. coli to bifidobacteria. The developed system was dependent on the donor (E. coli) to recipient (bifidobacteria) ratio, such that higher efficiencies were observed with higher donor ratios. In addition, the system was successful in all the bifidobacteria strains tested. This tool coupled with a genome-wide transcriptional analysis, RT-qPCR and the identification of ncRNAs, allowed the identification of molecular players during a yogurt fermentation with B. longum DJO10A. These molecular players included dnaK that appeared to play an important role at all times during growth, groEL that seemed particularly important for heat stress, and ibpA, a small heat-shock protein, together with the novel bifidobacteria-specific gene, bspA, that were added to the chaperone network as the acid accumulated in the fermentation. In addition, three ncRNAs, termed Bl20, Bl30, and Bl39, were shown to affect the stress response. The constitutive expression of Bl20 reduced the mRNA levels from all the tested stress genes, while the expression of dnaK was specifically targeted by Bl39 and ibpA expression was considerably reduced by the constitutive expression of Bl30. In conclusion, B. longum DJO10A responds to stress conditions during the yogurt fermentation by coordinating the expression of stress genes that are influenced by the expression of ncRNAs. Furthermore, this research revealed for the first time in bifidobacteria the involvement of ibpA in acid stress, the expression in response to stress of the novel gene bspA, the presence and expression of ncRNAs and a reproducible, efficient and strain-independent means of mobilizing DNA into this bacterial group