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Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication.

ABSTRACT: A 5-kbp region of pAD1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. The complete DNA sequence of this approximately 3-kbp region and the precise positions of several transposon inserts were determined, and the phenotypic effects of the transposon inserts were correlated with the physical locations of individual determinants. The following three genes, apparently involved in plasmid maintenance, were identified; repA, which encodes a protein required for replication; repB, which encodes a protein involved in copy control; and repC, which may be involved in stable inheritance. In addition, two clusters of repeats composed of a consensus sequence, TAGTARRR, were identified, one located between the divergently transcribed repA and repB genes and another located downstream of repC. The region between repA and repB contained 25 repeats divided into two subregions of 13 and 12 repeats separated by 78 bp. The region located downstream of repC contained only three repeats but may be essential for plasmid replication, since deletion of this determinant resulted in loss of ability to replicate in Enterococcus faecalis. We hypothesize that the repeat units represent protein-binding sites required for assembly of the replisome and control of plasmid copy number. Another region of unrelated repeat units that may also be involved in replication is located within the repA gene. Possible mechanisms of action of these determinants are discussed.

SUBMITTER: Weaver KE

PROVIDER: S-EPMC204257 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication.

Weaver K E KE Clewell D B DB An F F

Journal of bacteriology 19930401 7

A 5-kbp region of pAD1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. The complete DNA sequence of this approximately 3-kbp reg ...[more]

PMID: 8384618

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Project description:The conjugative pheromone-responsive plasmid pAD1 (59.6 kb) of Enterococcus faecalis encodes a UV resistance determinant (uvr) in addition to the hemolysin-bacteriocin determinant. pAD1 enhances the UV resistance of wild-type E. faecalis FA2-2 and E. faecalis UV202, which is a UV-sensitive derivative of E. faecalis JH2-2. A 2.972-kb fragment cloned from between 27.7 and 30.6 kb of the pAD1 map conferred UV resistance function on UV202. Sequence analysis showed that the cloned fragment contained three open reading frames designated uvrA, uvrB, and uvrC. The uvrA gene is located on the pAD1 map between 28.1 and 29.4 kb. uvrB is located between 30.1 and 30.3 kb, and uvrC is located between 30.4 and 30.6 kb on the pAD1 map. The uvrA, uvrB, and uvrC genes encode sequences of 442, 60, and 74 amino acids, respectively. The deduced amino acid sequence of the uvrA-encoded protein showed 20% homology of the identical residues with the E. coli UmuC protein. Tn917 insertion mutagenesis and deletion mutant analysis of the cloned fragment showed that uvrA conferred UV resistance. A palindromic sequence, 5'-GAACNGTTC-3', which is identical to the consensus sequence found within the putative promoter region of the Bacillus subtilis DNA damage-inducible genes, was located within the promoter region of uvrA. Two uvrA transcripts of different lengths (i.e., 1.54 and 2.14 kb) which terminate at different points downstream of uvrA were detected in UV202 carrying the deletion mutant containing uvrA. The longer transcript, 2.14 kb, was not detected in UV202 carrying the deletion mutant containing both uvrA and uvrB, which suggests that uvrB encodes a terminator for the uvrA transcript. The uvrA transcript was not detected in any significant quantity in UV202 carrying the cloned fragment containing uvrA, uvrB, and uvrC; on the other hand, the 1.54-kb uvrA transcript was detected in the strain exposed to mitomycin C, which suggests that the UvrC protein functions as a regulator of uvrA.

Dataset Information

Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication.

Publications

Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication.

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