Two dnaB genes are associated with the origin of replication of pQBR55, an exogenously isolated plasmid from the rhizosphere of sugar beet.
Plasmid pQBR55 ( approximately 149 kb) represents one of five (Groups I-V) genetically distinct transfer proficient, mercury resistance plasmid groups that have been observed in the phytosphere pseudomonad community at a single geographic location in Oxford, UK. A 4.9-kb HindIII fragment was cloned from pQBR55 (a Group III plasmid) that facilitates autonomous replication of a narrow host range cloning vector, pKIL29, in the non-permissive host Pseudomonas putida UWC1. Sequencing revealed that the fragment contains a unique tandem array of dnaB genes (one partial and one complete), a homologue of the traA gene of plasmid RP4 and several potential open reading frames with little homology to any known sequences. The fragment also contains: a short region of direct and indirect repeats, two sequences that resemble the consensus Escherichia coli DnaA box motif, an A+T rich region and evidence of a GC-skew inversion. All features associated with origins of replication. Two specific oligonucleotide primer pairs, one targeted at the tandem dnaB genes and the other at the dnaB-traA arrangement were used for PCR analysis of other plasmids isolated from the same field site. PCR generated amplification products were only amplified from Group III plasmids (defined by RFLP typing) but not from plasmids belonging to Group I, II, IV or V. The nucleotide sequences of the amplified fragments were identical to those of pQBR55, even though the other Group III plasmids had distinct RFLP patterns. The results of a more general PCR screen, using primers to amplify a 389-bp fragment of all described pseudomonad dnaB genes, suggests that closely related dnaB sequences are only associated with Group III plasmids.
Turner
Sarah L
SL
Molecular Microbial Ecology Laboratory, Institute of Virology and Environmental Microbiology, Centre for Ecology and Hydrology-Oxford, Mansfield Road, Oxford OX1 3SR, UK.
Lilley
Andrew K
AK
Bailey
Mark J
MJ