Accessing the Inaccessible: Molecular Tools for Bifidobacteria

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Bifidobacteria are an important group of the human intestinal microbiota that have been shown to exert a number of beneficial probiotic effects on the health status of their host. Due to these effects, bifidobacteria have attracted strong interest in health care and food industries for probiotic applications and several species are listed as so-called “generally recognized as safe” (GRAS) microorganisms. Moreover, recent studies have pointed out their potential as an alternative or supplementary strategy in tumor therapy or as live vaccines. In order to study the mechanisms by which these organisms exert their beneficial effects and to generate recombinant strains that can be used as drug delivery vectors or live vaccines, appropriate molecular tools are indispensable. This review provides an overview of the currently available methods and tools to generate recombinant strains of bifidobacteria. The currently used protocols for transformation of bifidobacteria, as well as replicons, selection markers, and determinants of expression, will be summarized. We will further discuss promoters, terminators, and localization signals that have been used for successful generation of expression vectors.</jats:p>

Expression of Fluorescent Proteins in Bifidobacteria for Analysis of Host-Microbe Interactions

“…To our knowledge, this represents the widest range of bifidobacterial hosts for any E. coli/Bifidobacterium shuttle vector described so far (2,21,52).…”

“…While some progress has been made in improving transformation efficiencies, directed gene inactivation, and transposon mutagenesis for single strains (19,20), the vast majority of strains of interest remain difficult if not impossible to modify genetically. Moreover, the available vector systems for bifidobacteria have a rather limited host range (i.e., replicons function only in some species of the genus Bifidobacterium but not in others), and expression systems are poorly developed (2,21).…”

Tough nuts to crack

“…In all of the 23 fully sequenced and annotated bifidobacterial genomes 5 restriction-modification (R-M) systems have been identified. was induced in positive clones and integration was verified by PCR as described.…”

“…The genomes of a number of strains of different species have been sequenced and annotated and are publically available. 1,5 However, the detailed analysis of the probiotic effects of bifidobacteria is hampered by the lack of appropriate tools for their genetic modification. While there has been some progress in the development of expression vectors, the currently available protocols do not yield transformation efficiencies above the threshold required to achieve chromosomal integration of non-replicative vectors by homologous recombination.…”

“…While there has been some progress in the development of expression vectors, the currently available protocols do not yield transformation efficiencies above the threshold required to achieve chromosomal integration of non-replicative vectors by homologous recombination. 5 The gold standard to investigate the role of single genes and their products is site-directed mutagenesis and the subsequent phenotypic analysis of the obtained mutants. So far, no system for bifidobacteria has been described that allows for directed mutagenesis in at least a number of different strains and species.…”