TY - JOUR
T1 - Developing an efficient and reproducible conjugation-based gene transfer system for Bifidobacteria
AU - Dominguez, Wilfredo
AU - O'Sullivan, Daniel J
PY - 2013/2
Y1 - 2013/2
N2 - 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 Escherichia coli strain WM3064(pBB109). Using this machinery, the newly constructed transmissible E. coli-Bifidobacterium shuttle vector, pDOJHR-WD2, was successfully and consistently transferred into several strains representing four Bifidobacterium species at efficiencies which correlated with the E. coli to bifidobacteria ratios. Higher ratios were found to significantly improve transfer frequency per recipient, with almost 100% transfer frequency occurring when the ratio was 105:1. The incompatible resident plasmid, pDOJH10S, in Bifidobacterium longum DJO10A was able to coexist, albeit at lower copy numbers, with the incoming vector pDOJHR-WD2 even though they possess the same ori. 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 Bifdobacterium species and is particularly useful for strains that are recalcitrant to electroporation.
AB - 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 Escherichia coli strain WM3064(pBB109). Using this machinery, the newly constructed transmissible E. coli-Bifidobacterium shuttle vector, pDOJHR-WD2, was successfully and consistently transferred into several strains representing four Bifidobacterium species at efficiencies which correlated with the E. coli to bifidobacteria ratios. Higher ratios were found to significantly improve transfer frequency per recipient, with almost 100% transfer frequency occurring when the ratio was 105:1. The incompatible resident plasmid, pDOJH10S, in Bifidobacterium longum DJO10A was able to coexist, albeit at lower copy numbers, with the incoming vector pDOJHR-WD2 even though they possess the same ori. 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 Bifdobacterium species and is particularly useful for strains that are recalcitrant to electroporation.
UR - http://www.scopus.com/inward/record.url?scp=84873657190&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84873657190&partnerID=8YFLogxK
U2 - 10.1099/mic.0.061408-0
DO - 10.1099/mic.0.061408-0
M3 - Article
C2 - 23197173
AN - SCOPUS:84873657190
SN - 1350-0872
VL - 159
SP - 328
EP - 338
JO - Microbiology (United Kingdom)
JF - Microbiology (United Kingdom)
IS - 2
ER -