Sequence Analysis of Plasmid pKJ50 From Bifidobacterium Longum

Microbiology. 1999 Mar;145 ( Pt 3):585-592. doi: 10.1099/13500872-145-3-585.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins*
  • Base Sequence
  • Bifidobacterium / genetics*
  • Cloning, Molecular
  • DNA Helicases*
  • DNA Replication
  • DNA, Single-Stranded / isolation & purification
  • DNA-Binding Proteins / genetics
  • Genes, Bacterial
  • Genetic Vectors
  • Molecular Sequence Data
  • Open Reading Frames
  • Plasmids / genetics*
  • Protein Structure, Secondary
  • Proteins / genetics
  • RNA, Bacterial / genetics
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Trans-Activators / genetics

Substances

  • Bacterial Proteins
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Proteins
  • RNA, Bacterial
  • Trans-Activators
  • replication initiator protein
  • DNA Helicases

Associated data

  • GENBANK/U76614