Molecular characterization of Bifidobacterium longum biovar longum NAL8 plasmids and construction of a novel replicon screening system.
In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids.
Guglielmetti
Simone
S
Department of Food Science and Microbiology, Industrial Microbiology Section, University of Milan, Via Celoria 2, 20133 Milan, Italy. simone.guglielmetti@unimi.it
Karp
Matti
M
Mora
Diego
D
Tamagnini
Isabella
I
Parini
Carlo
C
D001644:Bifidobacterium / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000737:chemistry*; D010802:Phylogeny; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D012093:Replicon