Sequence analysis of a cryptic plasmid pCJ419 from Campylobacter jejuni and construction of an Escherichia coli-Campylobacter shuttle vector.
A small cryptic plasmid, pCJ419, was identified in a human clinical isolate of Campylobacter jejuni, cloned and sequenced. pCJ419 is a circular molecule of 4013 bp with a G+C content of 27.1%. The products of four open reading frames (ORFs) share significant sequence similarity with putative proteins from known C. jejuni and Campylobacter coli plasmids. ORF-1 encodes a putative mobilisation protein (Mob). ORF-2 and ORF-3 encode proteins that have high identity to putative RepA and RepB proteins, respectively, of known C. jejuni and C. coli plasmids. ORF-4 encodes a protein that has high identity to a hypothetical protein of unknown function, Cjp32, previously described in a pVir plasmid of C. jejuni. Tandem repeating 22-bp sequences typical of a plasmid replication origin (ori) were identified upstream of the DNA sequences encoding putative replication initiation proteins. An Escherichia coli-Campylobacter shuttle cloning vector, pGU0202, was constructed using plasmid pMW2 that harbours a Campylobacter-derived kanamycin resistance gene [aph(3')-III]. The sequences encoding pCJ419 mob, RepA and RepB proteins were inserted upstream of aph(3')-III resulting in a stable construct of 6174 bp that was used to transform both E. coli and Campylobacter.
Alfredson
David A
DA
Microbial Glycobiology, Institute for Glycomics, Griffith University, Gold Coast, Southport, Qld. 4215, Australia. david_alfredson@health.qld.gov.au
Korolik
Victoria
V
D001483:Base Sequence; D015139:Blotting, Southern; D016123:Campylobacter jejuni / Q000235:genetics*; D002874:Chromosome Mapping; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
D017931:DNA Primers