2001 Volume 14 Issue 2 Pages 75-85
Bifidobacteria are Gram-positive, anaerobic, GC-rich bacteria constituting a major part of the normal microflora in the large intestine of human and animals. These intestinal organisms have been believed to have health-promoting properties for their host, involving enhancement of immune response, inhibitory effect for carcinogenesis, protection of the host against virus infection and possible production of antibacterial substances. Some Bifidobacterium species are widely used for the preparation of fermented milk products in the world. Here, we report reproducible and stable transformation of B. longum by plasmid vectors, and transcription mode of a gene encoding histone-like protein of B. longum. Highefficiency transformation of B. longum by electroporation was achieved with novel shuttle vectors pBLES 100 and pMASK 23, which were constructed by cloning B. longum plasmid and a gene encoding spectinomycin AAD (9) from Enterococcus faecalis into Escherichia coli vector pBR 322. Transformation efficiency of 2×104/μg DNA could be obtained under optimal condition of 10.0kV/cm, 200 Ω and 25 mF, using cells harvested at late log phase of growth. Bifidobacterium longum 105-A transformed with these shuttle vectors showed normal cell growth rate, and the plasmids were maintained stably in the transformants without rearrangement of the molecules. Histone-like protein HU is a structural component of bacterial nucleoids, which has been well conserved during evolution of prokaryotes, and is expressed highly and constitutively. We cloned hup gene (gene encoding HU protein) from B. longum ATCC 15707, and found that the ORF is identical to HU family protein HB1, a DNA binding protein of B. longum reported previously. We identified promoter, transcription start point, ribosome binding region and transcription termination site in this gene. The hup gene is expressed constitutively through cell cycle in ATCC 15707, and is transcribed efficiently when it is integrated in pBLES 100 and transferred into B. longum 105-A.
