Premium
Genes involved in conjugative DNA processing of plasmid R6K
Author(s) -
Núñez Belén,
Avila Pilar,
De La Cruz Fernando
Publication year - 1997
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1997.4111778.x
Subject(s) - biology , bacterial conjugation , plasmid , genetics , gene , transposable element , origin of replication , transposon mutagenesis , dna , mutagenesis , insertional mutagenesis , repressor , pilus , microbiology and biotechnology , mutation , mutant , escherichia coli , gene expression
The conjugative transfer region of the IncX plasmid R6K (TRA X ) was analysed by transposon mutagenesis and DNA sequencing. Tn 5 tac1 insertional mutations localized TRA X to a 14.8 kb segment containing the α origin of transfer ( oriT α), genes involved in conjugative DNA‐processing (Dtr X ) and genes involved in pilus synthesis and assembly (Mpf X ). A second functional oriT , oriT β, was located at a distance of 5.3 kb from oriT α and was outside TRA X . Mpf X occupied a segment of 10 kb, as judged by the location of insertions conferring resistance to infection by the X pilus‐specific phage X‐2. At both sides of Mpf X there were insertions that were Tra − but X‐2 sensitive, suggesting that the mutations were in Dtr X . This region was sequenced and three genes were identified: taxA , taxB , and taxC . The overall organization was oriT α– taxA – taxC –Mpf X – taxB . taxC coded for a oriT ‐relaxase that belongs to the VirD2 family. taxA coded for a protein of 181 amino acids that showed similarity to TraY of F‐like plasmids and to the Arc‐repressor superfamily. TaxB showed similarity to TraG‐like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA‐transport apparatus. TaxA and TaxC are required for oriT nicking in vivo . The nicking reaction was mistakenly assumed by Flashner et al . (1996) to represent a feature of the vegetative replication origins. However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K. Conversely, protein π, which is absolutely required for replication of R6K, was not required for conjugative transfer. In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation. Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein π for conjugation.