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PMID: 10322022 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Replication mechanism and sequence analysis of the replicon of pAW63, a conjugative plasmid from Bacillus thuringiensis.

Journal of bacteriology ·Vol. 181 ·No. 10 ·1999-05-00 ·Pages 3193-200

Wilcks A, Smidt L, Okstad OA, Kolsto AB, Mahillon J, Andrup L

Abstract

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.

MeSH Terms
Bacillus thuringiensis/drug effects,enzymology,genetics Bacterial Proteins/genetics Base Sequence Cloning, Molecular Conjugation, Genetic DNA Polymerase I/metabolism DNA Replication/drug effects,genetics DNA, Single-Stranded/analysis,genetics Gram-Positive Bacteria/genetics Molecular Sequence Data Molecular Weight Open Reading Frames/genetics Plasmids/genetics,metabolism Replication Origin/genetics Replicon/genetics Restriction Mapping Rifampin/pharmacology Sequence Analysis, DNA Sequence Deletion Sequence Homology
Chemicals
Bacterial Proteins DNA, Single-Stranded DNA Polymerase I Rifampin
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Wilcks A
National Institute of Occupational Health, Copenhagen, Denmark.
Smidt L
Okstad O A
Kolsto A B
Mahillon J
Andrup L
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.181.10.3193-3200.1999
Published
1999-05-00
Pages
3193-200
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC93776
Subset
IM
Databases
GENBANK
AJ011655
External Links
PubMed source DOI: 10.1128/JB.181.10.3193-3200.1999 PMC Full Text View journal details
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