Summary auto-generated
Researchers isolated and completely sequenced pKJ50, a 4960 bp plasmid from Bifidobacterium longum KJ, an intestinal bacterium with beneficial health properties. The plasmid contains three genes: ORFI encodes a replication protein (31.5 kDa) showing 60% amino acid similarity to related plasmids; ORFII encodes a predicted transmembrane protein (24.5 kDa); and ORFIII encodes a mobilization protein (38.6 kDa). Southern blot analysis confirmed pKJ50 replicates via rolling-circle mechanism, producing single-stranded DNA intermediates. The replication protein region contains AT-rich sequences and iteron-like repeats typical of plasmid origins of replication. ORFIII possesses DNA sequences resembling oriT (origin of transfer) regions found in conjugative plasmids, suggesting potential for conjugative gene transfer. Gene expression analysis using RT-PCR confirmed ORFI and ORFII transcription, while ORFIII showed no RT-PCR product despite in vitro translation of all three ORFs. The researchers successfully constructed shuttle vectors capable of transforming both E. coli and Bifidobacterium animalis, demonstrating practical application for genetic modification of beneficial Bifidobacterium strains.
Key findings
- pKJ50 is a 4960 bp plasmid with three open reading frames encoding replication, transmembrane, and mobilization proteins
- The plasmid replicates via rolling-circle mechanism, evidenced by accumulation of single-stranded DNA intermediates detected by S1 nuclease treatment
- ORFIII contains DNA sequences resembling oriT and inverted repeats characteristic of conjugative transfer regions, suggesting potential for bacterial conjugation in Bifidobacterium
- E. coli-Bifidobacterium shuttle vectors were successfully constructed and transformed into B. animalis MB209, enabling genetic modification capabilities
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Abstract
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.