Physical characterization of the replication origin of the cryptic plasmid pCB101 isolated from Clostridium butyricum NCIB 7423.
The complete nucleotide sequence of a 3484-bp Sau3A fragment, previously shown to carry the replication origin of the Clostridium butyricum NCIB 7423 plasmid pCB101 (6.05 kb), has been determined. Of the four open reading frames (ORF A-D) identified within this fragment, two (B and C) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that both polypeptides are required for autonomous replication of the plasmid in Bacillus subtilis. ORF C is immediately preceded by a small ORF (C') that encodes a relatively small polypeptide (50 amino acids) that demonstrates significant homology with RepA of plasmid pLS1. Whereas the ORF C polypeptide (27,100 Da) exhibits no homology to any known protein, that encoded by ORF B (RepB, 43,039 Da) exhibits significant homology with the Rep proteins of the pC194/pUB110 subfamily of single-strand (ss) DNA plasmids, which are widely distributed in gram-positive bacteria. Conserved amino acids include the presumed active site of topoisomerase activity and four cysteine residues in the N-terminus of all Rep proteins compared. The repB gene is preceded by a sequence motif exhibiting substantial homology to the "plus" origins of this family of ss DNA plasmids and was shown to act as a "hot spot" for deletion formation in certain plasmid chimaeras. The compelling suggestion that pCB101 replicates via a rolling circle mechanism was substantiated by the demonstration of ss DNA replication intermediates in B. subtilis cells carrying a pCB101-derived plasmid.
Brehm
J K
JK
Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, United Kingdom.
Pennock
A
A
Bullman
H M
HM
Young
M
M
Oultram
J D
JD
Minton
N P
NP
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism; D001483:Base Sequence; D003013:Clostridium / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000096:biosynthesis; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis; D012093:Replicon / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded