Sequence analysis of plasmid pKJ50 from Bifidobacterium longum.
The complete nucleotide sequence of a plasmid, pKJ50, isolated from an intestinal bacterium, Bifidobacterium longum 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 pI 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 pI of 10.97. It also shows a high level of amino acid sequence similarity with some plasmid mobilization proteins. Upstream of ORFIII, a 12 bp stretch resembles an oriT DNA sequence with inverted repeats identical to those found in conjugative plasmids. Hydropathy plot analysis of ORFII, encoding an acidic protein (pI = 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 in vitro 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. In vitro translation showed protein bands of the expected sizes, corresponding to each ORF. A shuttle vector capable of transforming Bifidobacterium animalis MB209 was constructed by cloning pKJ50 and a chloramphenicol resistance gene into pBR322.
Park
Myeong S
MS
Department of Food Science and Technology, Seoul National University, Suwon, 441-744, Korea.
Shin
Dong W
DW
Department of Food Science and Technology, Seoul National University, Suwon, 441-744, Korea.
Lee
Ke H
KH
Department of Food Science and Technology, Seoul National University, Suwon, 441-744, Korea.
Ji
Geun E
GE
Department of Food Science and Nutrition, Seoul National University, Seoul, 151-742, Korea.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001644:Bifidobacterium / Q000235:genetics*; D003001:Cloning, Molecular; D004265:DNA Helicases; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics*; D017433:Protein Structure, Secondary; D011506:Proteins / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011506:Proteins; D012329:RNA, Bacterial; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases