1887

Abstract

Bifidobacteria are widely used as probiotics and have attracted increasing research interest worldwide. However, molecular techniques are still very scarce mainly due to the low efficiencies and strain-specific electroporation protocols that have been developed. Bacterial conjugation enables the transfer of genetic material among a relatively wide range of organisms and with virtually no size limitation. A conjugation protocol was developed based on the RP4 conjugative machinery in the strain WM3064(pBB109). Using this machinery, the newly constructed transmissible shuttle vector, pDOJHR-WD2, was successfully and consistently transferred into several strains representing four species at efficiencies which correlated with the to bifidobacteria ratios. Higher ratios were found to significantly improve transfer frequency per recipient, with almost 100 % transfer frequency occurring when the ratio was 10 : 1. The incompatible resident plasmid, pDOJH10S, in DJO10A was able to coexist, albeit at lower copy numbers, with the incoming vector pDOJHR-WD2 even though they possess the same . In some cases the copy number of this resident plasmid was too low to observe via gel electrophoresis, but it could be detected by Southern hybridization. Plasmid curing resulted in a strain, DJO10A-W3, that had lost both plasmids and this showed a one-log increase in conjugation efficiency due to the lack of plasmid incompatibility. In conclusion, this novel conjugative gene transfer protocol can be used for the introduction of genetic material (without size restriction) into species and is particularly useful for strains that are recalcitrant to electroporation.

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2013-02-01
2024-03-29
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