Cloning, sequencing and molecular characterisation of a cryptic plasmid from a urease-positive thermophilic Campylobacter (UPTC) isolate.
Cloning, sequencing and molecular characterisation of a cryptic plasmid, pUPTC237, from a urease-positive thermophilic Campylobacter (UPTC) isolate obtained from the natural environment in Northern Ireland is reported in this study. Based on the determined DNA sequence, the pUPTC237 DNA was identified as a circular molecule of 3828 bp with a G+C content of 29.5%. As with other plasmid DNAs from Gram-negative bacteria, pUPTC237 contained an A+T-rich region (A+T content: 95%), followed by multiple direct tandem repeat units of 22 bp, characteristic of a replication origin and iteron sequence. A possible open reading frame (ORF)-1 was located upstream of the A+T-rich region and the iteron sequence that encoded a 460 amino acid protein similar to the mobilisation (mob) protein and two putative promoter structure sequences at the -35 and -10 regions and a possible ribosome binding site occurred upstream of the start codon for the ORF-1. Moreover, three possible ORFs (a short ORF-2 encoding 26 amino acids, similar to repA; an ORF-3 encoding 341 amino acids, similar to repB; and an ORF-4 encoding 96 amino acids with unknown function) were also identified. There are also two putative promoter structures for these three ORFs at the -35 and -10 regions upstream of the possible ORF-2. A possible transcription termination region was identified downstream of ORF-4. Northern blot hybridisation analysis suggested that these four ORFs constitute an operon and generate a messenger RNA (mRNA) transcript.
Ito
T
T
Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Sagamihara, Japan.
Sekizuka
T
T
Murayama
O
O
Moore
J E
JE
Millar
B C
BC
Taneike
I
I
Matsuda
M
M
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D044863:Campylobacter lari / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D004751:Enteritis / Q000382:microbiology*; D005516:Food Microbiology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010125:Ostreidae / Q000382:microbiology*; D010957:Plasmids; D017422:Sequence Analysis, DNA; D014510:Urease / Q000378:metabolism
D015342:DNA Probes; D004269:DNA, Bacterial; D014510:Urease