Recombination between compatible plasmids containing homologous segments requires the Bacillus subtilis recE gene product

Keggins, K M; Duvall, Elizabeth J.; Lovett, Paul S.

doi:10.1128/jb.134.2.514-520.1978

Cited by 27 publications

(6 citation statements)

References 19 publications

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“…The molecular weight of the vector plasmid is approximately 3 x 10' (11). (16,17). Incubation was at 370C; liquid cultures were grown with rotary shaking.…”

Section: Methodsmentioning

confidence: 99%

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Insertional Inactivation of trpC in Cloned Bacillus trp Segments: Evidence for a Polar Effect on trpF

et al. 1979

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Plasmid pUB110 was previously used as a vector to clone fragments of deoxyribonucleic acid that complement the trpC2 mutation in Bacillus subtilis from endonuclease EcoRI digested B. licheniformis, B. pumilus, and B. subtilis cellular deoxyribonucleic acid. Each of several such trp plasmids was subsequently shown to contain a segment of the trp gene cluster on the basis of genetic complementing activity. In the present study, analysis of the Trp enzyme levels in B. subtilis harboring the constructed trp plasmids confirms the genetic constitution of the plasmids. Thus, plasmids that complement mutations in specific trp genes specify the corresponding enzyme activities. The levels of the plasmid-specified Trp enzymes in B. subtilis were generally above the repressed level of the chromosomally specified Trp enzymes and equal to or below the derepressed levels of the chromosomally specified.Trp enzymes. Certain cloned trp segments contain a single HindIlI-sensitive site. Insertion of HindIll-generated deoxyribonucleic acid fragments into these trp plasmids resulted in inactivation of trpC complementing activity, loss of the trpC-specified enzyme activity, and a 10-fold reduction in the specific activity of the plasmid-specified trpF product. The HindIII insertions had no detectable effect on the level of the trpD product, nor did the insertions detectably alter plasmid-specified complementing activity other than to abolish trpC complementation. Removal of the HindIII insertions was accompanied by recovery of trpC complementing activity and restoration of the trpCand trpF-determined enzymes to the levels specified by the parent plasmids.

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“…The molecular weight of the vector plasmid is approximately 3 x 10' (11). (16,17). Incubation was at 370C; liquid cultures were grown with rotary shaking.…”

Section: Methodsmentioning

confidence: 99%

“…We directed our efforts toward cloning segments of Bacillus chromosome DNA specifying the tryptophan biosynthetic enzymes (16,17). The order of trp genes in B. subtilis is trpE-D-C-F-B-A (1, 2, 5).…”

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Insertional Inactivation of trpC in Cloned Bacillus trp Segments: Evidence for a Polar Effect on trpF

et al. 1979

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“…This XbaI-deleted plasmid was joined to the XbaI site of pE194 in both of the two possible orientations, and the fusion plasmids were transformed into strain BGSC 1A422 (recE4) harboring pPL708AA. The recE4 mutation has been shown previously to block recombination between homologous plasmids in the same cell (12). These attempts at complementation failed to permit cat-86 induction with either chloramphenicol or amicetin (data not shown).…”

Section: Resultsmentioning

confidence: 80%

Chloramphenicol-induced translation of cat-86 mRNA requires two cis-acting regulatory regions

Ambulos¹,

Mongkolsuk²,

Kaufman³

et al. 1985

J Bacteriol

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Sequences essential to the chloramphenicol-inducible expression of cat-86, a chloramphenicol acetyltransferase gene, reside in a 144-base pair (bp) regulatory region that intervenes between the cat-86 coding sequence and its promoter. A key regulatory element within the 144-bp segment consists of a pair of inverted-repeat sequences that immediately precede the cat-86 coding region and span the ribosome-binding site for the gene. Because of the location of the inverted repeats, cat-86 transcripts are predicted to sequester the ribosome-binding site in a stable RNA stem-loop structure which should block translation of cat-86 mRNA. Chloramphenicol induction of gene expression is believed to result from ribosome-mediated destabilization of the RNA stem-loop structure, which frees the cat-86 ribosome-binding site, thereby allowing translation. In this study we demonstrated that deletion of 85 bp from the 5' end of the 144-bp regulatory region abolishes inducible expression of cat-86, although the gene is transcribed. This deletion leaves intact both the inverted repeats and the cat-86 coding sequence, and the deletion mutation is not complementable. Therefore, inducible regulation of cat-86 requires the inverted repeats plus an upstream, cis-acting regulatory region. The cis-acting region is believed to control translation of cat-86 mRNA by its essential participation in chloramphenicol-induced opening of the RNA stem-loop. cat-86 deleted for the 85-bp regulatory region and therefore virtually unexpressed was used to select for mutations that restore expression to the gene. An analysis of one mutant plasmid showed that the cat-86 gene is constitutively expressed and that this results from a duplication of the DNA sequence that spans the ribosome-binding site. The duplication provides cat-86 with two ribosome-binding sites. One of these sites is predicted to be sequestered in an RNA stem-loop, and the other is not involved in RNA secondary structure.

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“…Recombination between plasmids can result in the formation of cointegrate molecules in a cell. Cointegrate formation has been seen in Escherichia coli (12,39), Bacillus subtilis (18), Salmonella typhimurium (14,24), and Agrobacterium species (15,16,37). Cointegrate formation has also been observed between plasmids and bacteriophages (17,29).…”

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Cointegrate formation between homologous plasmids in Escherichia coli

Peterson¹,

Hashimoto²,

Rownd³

1982

J Bacteriol

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Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1. The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate. Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome. When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60%. Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative. Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation. When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids. When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1. These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.

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Recombination between compatible plasmids containing homologous segments requires the Bacillus subtilis recE gene product

Cited by 27 publications

References 19 publications

Insertional Inactivation of trpC in Cloned Bacillus trp Segments: Evidence for a Polar Effect on trpF

Insertional Inactivation of trpC in Cloned Bacillus trp Segments: Evidence for a Polar Effect on trpF

Chloramphenicol-induced translation of cat-86 mRNA requires two cis-acting regulatory regions

Cointegrate formation between homologous plasmids in Escherichia coli

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