AUTHOR=Ortiz Charneco Guillermo , Kelleher Philip , Buivydas Andrius , Streekstra Hugo , van Themaat Emiel Ver Loren , de Waal Paul P. , Mahony Jennifer , van Sinderen Douwe 

TITLE=Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.680920

DOI=10.3389/fmicb.2021.680920

ISSN=1664-302X

ABSTRACT=<p>Plasmid pNP40, which was first identified nearly 40 years ago in <italic>Lactococcus lactis</italic> subsp. <italic>lactis</italic> biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the <italic>tra</italic>R gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery.</p>