Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis.
The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAM beta 1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAM beta 1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 X 10(-2) to 3.0-4.0 X 10(-3) plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAM beta 1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.
Swinfield
T J
TJ
Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Salisbury, Wiltshire, England.
Jannière
L
L
Ehrlich
S D
SD
Minton
N P
NP
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D013293:Enterococcus faecalis / Q000235:genetics*; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial