Technological Advances in Bifidobacterial Molecular Genetics: Application to Functional Genomics and Medical Treatments

Abstract

Bifidobacteria are well known as beneficial intestinal bacteria that exert health-promoting effects in humans. In addition to physiological and immunological investigations, molecular genetic technologies have been developed and have recently started to be applied to clarify the molecular bases of host-Bifidobacterium interactions. These technologies include transformation technologies and Escherichia coli-Bifidobacterium shuttle vectors that enable heterologous gene expression. In this context, a plasmid artificial modification method that protects the introduced plasmid from the restriction system in host bifidobacteria has recently been developed to increase transformation efficiency. On the other hand, targeted gene inactivation systems, which are vital for functional genomics, seemed far from being practically applicable in bifidobacteria. However, remarkable progress in this technology has recently been achieved, enabling functional genomics in bifidobacteria. Integrated use of these molecular genetic technologies with omics-based analyses will surely boost characterization of the molecular basis underlying beneficial effects of bifidobacteria. Applications of recombinant bifidobacteria to medical treatments have also progressed.

Bifidobacteria and Their Role as Members of the Human Gut Microbiota

“…The first successful mutation created in a bifidobacterial strain was in the gene apuB using this single-crossover plasmid insertion approach whereby a combination of plasmids facilitated the conditional replication of the non-replicative plasmid pORI19 (Table 3 ; O'Connell Motherway et al, 2008 ). Although, it successfully led to the inactivation of the apuB gene, this method was quite time consuming and laborious (Fukiya et al, 2012 ).…”

“…(A) represents the single-crossover plasmid insertion, (B) illustrates the double-crossover gene disruption and finally (C) illustrates the double-crossover markerless gene deletion approach. (Figure was adapted from Fukiya et al, 2012 ).…”

“…Double-crossover gene disruption was first demonstrated in B. longum NCC2705 (Table 3 ; Fukuda et al, 2011 ). This method makes use of a non-replicating plasmid and a double-cross over event in order to create a gene deletion (Fukiya et al, 2012 ). The non-replicative vector harbors two homologous regions of the target gene between which an antibiotic resistance gene is inserted (Figure 2B ).…”

“…The double cross-over approaches are more time consuming due to multiple transformations and extensive screening for positive mutants, although the improved double crossover gene deletion system aims to address this (Table 3 ; Fukiya et al, 2012 ). For single cross-over mutants it is theoretically possible for additional cross-over events to occur between the homologous regions left in the target gene thus resulting in the excision of the integrated plasmid (Fukiya et al, 2012 ).…”

“…The double cross-over approaches are more time consuming due to multiple transformations and extensive screening for positive mutants, although the improved double crossover gene deletion system aims to address this (Table 3 ; Fukiya et al, 2012 ). For single cross-over mutants it is theoretically possible for additional cross-over events to occur between the homologous regions left in the target gene thus resulting in the excision of the integrated plasmid (Fukiya et al, 2012 ). It is also worth noting that the marker-less method is a superior approach as it does not leave an antibiotic resistance gene, the presence of which may cause polar effects affecting genes down-stream of the mutated gene (Table 3 ; Fukiya et al, 2012 ).…”