The complete nucleotide sequence of a plasmid, pKJ50, isolated from an intestinal bacterium, KJ, has been determined. The plasmid was analysed and found to be 4960 bp in size with a G+C content of 61·7 mol%. Computer analysis of sequence data revealed three major ORFs encoding putative proteins of 31·5 (ORFI), 24·5 (ORFII) and 38·6 kDa (ORFIII). ORFI encodes a protein with a pl of 10·18 and shows relatively high amino acid sequence similarity (more than 60%) with several plasmid replication proteins from Gram-positive and -negative bacteria. Southern blot analysis showed that pKJ50 accumulates an ssDNA intermediate, suggesting that it replicates by a rolling-circle mechanism. Upstream of ORFI, three sets of repeated sequences resembling iteron structures of related plasmids were identified. ORFIII encodes a protein with a pl of 10·97. It also shows a high level of amino acid sequences similarity with some plasmid mobilization proteins. Upstream of ORFIII, a 12 bp stretch resembles an DNA sequence with inverted repeats identical to those found in conjugative plasmids. Hydropathy plot analysis of ORFII, encoding an acidic protein (pl = 4·95), suggests it is a transmembrane protein. Several interesting palindromic sequences, repeat sequences and hairpin-loop structures around ORFI, which might confer regulatory effects on the replication of the plasmid, were also noted. Reverse transcriptase PCR (RT-PCR) and translation confirmed the expression of ORFI and ORFII. RT-PCR produced amplified DNA fragments of the expected sizes, corresponding to ORFI and ORFII. However, no RT-PCR product corresponding to ORFIII was obtained. translation showed protein bands of the expected sizes, corresponding to each ORF. A shuttle vector capable of transforming MB209 was constructed by cloning pKJ50 and a chloramphenicol resistance gene into pBR322.


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