Sequence Analysis of Two Cryptic Plasmids from Bifidobacterium longum DJO10A and Construction of a Shuttle Cloning Vector

• DOI: 10.1128/aem.72.1.527-535.2006
• Publication Date: 2006-01-03T20:33:36Z
• Authors: Ju-Hoon Lee; Daniel J. O’Sullivan
• Keywords: Replicon; Bifidobacterium longum; Cloning vector; Shuttle vector; T-DNA Binary system; Autonomously replicating sequence

ABSTRACT Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L pDOJH10S. The complete sequences of both these plasmids have now been determined consist circular DNA molecules 10,073 3,661 bp, G+C contents 62.2% 66.2%, respectively. Plasmid cointegrate plasmid consisting regions exhibiting very high sequence identity to other B. pNAC2 (98%) pKJ50 (96%), together another region. Interestingly, the rolling replication (RCR) pNAC2- pKJ50-like were disrupted during recombination event leading further acquire functional replicon. This consists new fused rep gene an RCR-type ori conserved DnaA box in AT-rich region followed by four contiguous repeated consistent iteron structure inverted repeat. smaller pDOJH10S had no similarity any characterized from bifidobacteria. In addition, it did not contain features RCR, which mechanism proposed for all bifidobacteria date. It exhibit several theta replication-related proteins gram-positive, high-G+C bacteria, closest match Rhodococcus rhodochrous plasmid, suggesting replication. S1 nuclease analysis revealed single-strand intermediates pDOJH10L, but none detected As content similar that (67%) significantly higher than (60.1%), may acquired through horizontal transfer species, as genera are members Actinomycetes intestinal inhabitants. An Escherichia coli-B. shuttle cloning vector was constructed E. coli p15A, lacZ multiple site pUC18, chloramphenicol resistance (CAT) pCI372 transformed successfully into . could be introduced lactic acid bacteria ( Lactococcus Lactobacillus ), showing promiscuous. stably maintained absence antibiotic pressure 92 generations, segregational stability theta-replicating gram-positive bacteria. first does utilize RCR should useful stable introduction heterologous genes dominant inhabitants large intestine.