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RepA/C is a replicon specific to the IncA/C incompatibility group of plasmids and was isolated recently from plasmid RA1. The sequence of this autoreplicative region was established; it contains 13 repeats, suggesting that the replicon uses iterons to control its copy number. The sequence contains two ORFs, one potentially coding for a 33-kDa protein (ORF1) and a second potentially coding for a 14-kDa...
Expression of the plasmid pBR322-encoded tetracycline-resistant gene (tet) is known to cause other pleiotropic effects in addition to mediating the efflux of tetracycline from bacterial host cells. We have recently reported that expression of thetetgene in plasmid pKH47, a high-copy-number derivative of pBR322, causes growth inhibition ofEscherichia colicells harboring this plasmid. In this paper...
Few vectors suitable for cloning inActinobacillus actinomycetemcomitans,a periodontal pathogen, have been described. These plasmids were based either on the nativeA. actinomycetemcomitansreplicon, pVT736-1, or derivatives of the IncP and IncQ family of plasmids. Their usefulness as cloning vectors is limited because of instability or size. Therefore, the ability of additional replicons to function...
A shuttle vector composed of pBL1, a 4.46-kb cryptic plasmid from coryneform bacteriumBrevibacterium lactofermentum,andEscherichia colivector pHSG298 was found to inhibit growth and cause cell filamentation inE. coli.After construction of several deletions of pBL1, a 1.23-kbAccI–HindIII fragment was found responsible. DNA sequence analysis showed that this fragment contained a 726-bp-long open reading...
Analysis of the nucleotide sequence ofLeuconostoc oenosplasmid pOg32 (2544 bp) revealed three open reading frames (ORFs) in one of the DNA strands preceded by putative promoters and ribosome binding sites. ORF1 corresponded to a polypeptide similar to the replication proteins (Rep) of pT181 plasmid family. A nucleotide sequence like the double strand origin (DSO) of the same plasmid family was found...
Brevibacterium linensRBL strain cryptic plasmid pRBL1 (8.0 kb) is described. A region involved in pRBL1 autonomous replication inCorynebacterium glutamicumwas identified by insertion and deletion mapping and partially sequenced. This region encodes for a hypothetical 310-amino acid (aa) protein closely related to the replicases of plasmids pXZ10142 (C. glutamicum) and pAL5000 (Mycobacterium fortuitum)...
The complete nucleotide sequence of the insertion sequence IS199has been determined. This novel element was found inStreptococcus mutansV403 where DNA hybridization studies suggested that it was present in multiple copies on the genome. IS199is 1220 bp and appears unique to the mutans streptococci. A limited epidemiological survey revealed this element to occur infrequently in this group (4/50 strains...
TheEnterococcus faecalisconjugative plasmid pPD1 (59 kb) produces an aggregation substance in response to pheromone. ThetraAgene (962 bp) is a member of a gene cluster involved in the regulation of the pPD1 pheromone response. A chimericE. faecalissuicide plasmid containing a 762-bp DNA fragment from the middle portion of thetraAgene was constructed. The pPD1traAwas disrupted by integration of this...
Tolerance ofStaphylococcus lugdunensisto relatively high levels of cadmium is mediated by a 3117-bp plasmid, pLUG10. Sequencing reveals three major open reading frames (ORFs) in a single orientation. One ORF encompasses the origin of replication and its predicted product (RepL; 350 amino acids (aa)) shows 70% homology in its deduced aa sequence with Rep proteins of the pT181 family. A lagging strand...
In this report, a new method for the selection of tetracycline-sensitiveEscherichia colicells from a mixed population is described. This method is simpler and more effective than previous methods, does not utilize toxic reagents, and allows selection in liquid as well as solid media. This method should be of considerable aid in molecular cloning procedures involving inactivation of the tetracycline-resistance...
The replication origin region,ori,of theLactococcus lactissubsp.lactisplasmid pCI305 contains three-and-one-half directly repeated 22-bp sequences and two inverted repeat sequences, IR1 and IR2. These inverted repeat sequences overlap the promoter of therepB gene, which encodes a protein (RepB) essential for plasmid replication. Gel retardation assays, using lactococcal crude cell extracts in which...
Transformation ofEscherichia coliwithDictyostelium discoideumgenomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements...
ThetrfAoperon of broad-host-range IncP plasmids is essential to activate the origin of vegetative replication in diverse species. Thetrboperon encodes most of the apparatus for mating pair formation, the first step in conjugative transfer. Comparison of the nucleotide sequence of the IncPβ plasmid R751 presented here with the equivalent IncPα sequence identifies conserved features of the organization...
Lactobacillus reuteriG4 contains a 7.0-kb plasmid (pTC82) encoding resistance to chloramphenicol (Cm r ). Determination of the nucleotide sequence of the genetic determinant (cat-TC) encoding resistance to Cm on pTC82 revealed an open reading frame for a 238-amino-acid Cm acetyltransferase (CAT) monomer. This structuralcatgene, 714 bp in length, was highly related (ca. 95% nucleotide and ca...
The aggregation-mediated conjugation system ofBacillus thuringiensissubsp.israelensis(Bti), encoded by the 200-kb plasmid pXO16, is highly potent in transferring itself and efficient in mobilizing other nonconjugative plasmids. In the present study we have analyzed the nativeBacillus cereusplasmid pBC16. This plasmid has previously been shown to harbor amobgene (ORFβ) and a locus functioning as anoriT...
The conjugative transposon Tn916moves intercellularly via an excision/insertion mechanism that involves products ofint-Tnandxis-Tn.Tn5-insertion mutations in these genes were found to be complemented in anEnterococcus faecalishost by specific coresident transposons harboring the corresponding wild-type allele. A determinant designatedtraA,partially overlapping and divergently transcribed fromxis-Tn,is...
Rts1 is a large naturally occurring plasmid which has a kanamycin resistance gene and exhibits various temperature-sensitive phenotypes. A smaller derivative of plasmid, pOK, contains the Rts1 replicon and the kanamycin resistance gene of Rts1. This plasmid, pOK, is much more unstable than Rts1 at 42.5°C. A DNA fragment, G 3 , 1590 nucleotides long from Rts1 DNA, stabilized pOK completely...
Two methods for estimation of plasmid loss rates were tested on data obtained from traditional (serial transfer) stability experiments. The first method was based on the assumption that the plasmid does not inhibit the growth of its host, whereas the second method takes differences in the interdivision time of plasmid-free and plasmid-carrying cells into account. In the cases where the loss rate is...
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