Conditionally Replicative and Conjugative Plasmids CarryinglacZα for Cloning, Mutagenesis, and Allele Replacement in Bacteria

Metcalf, William W.; Jiang, Weihong; Daniels, Larry; Kim, Soo Ki; Haldimann, Andreas; Wanner, Barry L.

doi:10.1006/plas.1996.0001

Cited by 405 publications

(311 citation statements)

References 1 publication

Supporting

Mentioning

311

Contrasting

Order By: Relevance

“…A 545-bp EcoRI-BamHI fragment containing 323 bp of 5Ј upstream noncoding sequence, along with the first 332 bp of the traI coding region and a 1,499-bp XbaI-EcoRI fragment containing 219 bp from the 3Ј end of traI and the entire trbB gene, were amplified using Pfu DNA polymerase (Stratagene), ligated together, and cloned between the BamHI and SpeI sites of the suicide plasmid pWM91 (23). This vector also contains a sacB gene, which can be used for selecting for loss of the plasmid.…”

Section: Methodsmentioning

confidence: 99%

Induction and Loss of Ti Plasmid Conjugative Competence in Response to the Acyl-Homoserine Lactone Quorum-Sensing Signal

Khan

Farrand

2008

J Bacteriol

View full text Add to dashboard Cite

Conjugative transfer of the Ti plasmids ofConjugative transfer of the Ti plasmids of Agrobacterium tumefaciens is a tightly regulated process involving two signaling systems arranged in a hierarchy. Expression of the three Ti plasmid transfer operons, traAFB, traCDG, and traI-trb, requires first a small molecule signal, the conjugative opine, which is produced by the crown gall tumors induced on susceptible plants by this phytopathogen (7,8). In addition to acting as a signal, the conjugative opine also can be catabolized by the bacteria by using functions coded for by the Ti plasmid. The conjugative opines regulate transfer by controlling the expression of a Ti plasmid operon of which TraR, the direct regulator of the three transfer operons, is a member. For example, transfer of pTiC58 is induced by the sugar phosphodiester opines agrocinopine A and agrocinopine B. In this plasmid, traR is a member of the five-gene arc operon, the expression of which is regulated by the opine-responsive repressor AccR (1). In the absence of the conjugative opines, the expression of arc is repressed. When the opines are present, as when the bacteria are colonizing an agrocinopine-producing crown gall tumor, repression by AccR is relieved, and the arc operon, including traR, is expressed.TraR, a member of the LuxR family of quorum-sensing transcription factors, activates expression of the transfer operons in response to the second signal, an acyl-homoserine lactone (acyl-HSL) (27, 34). The cognate quormone N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL) is produced by the bacteria themselves via the acyl-HSL synthase TraI, also encoded by the Ti plasmid (16,24). This signal transits out of and back into the cells in a stochastic fashion, and its concentration in a diffusion-limited environment increases with in-

show abstract

Section: Methodsmentioning

confidence: 99%

Induction and Loss of Ti Plasmid Conjugative Competence in Response to the Acyl-Homoserine Lactone Quorum-Sensing Signal

Khan

Farrand

2008

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…E. coli strains DH5␣ pir, SM10 pir (18), and BW20767 (19) and M. smegmatis strain mc 2 155 (20) were maintained by standard methods. Plasmids pPR23 (21) and pBMML2S were maintained in E. coli.…”

Section: Methodsmentioning

confidence: 99%

“…This was digested with XbaI, and an XbaI-SpeI fragment from pEMKan was cloned to create pMME. A fragment of DNA containing oriR6K␥ was added to the transposon by PCR, amplifying a fragment of plasmid pWM41 (19) with primers 5Ј-AGATCT-CAAACTGGAACAACACTCAACCC-3Ј and 5Ј-TTAATT-AACCCCGAAAAGTGCCACCTGACG-3Ј and cloning the product into the SmaI site of pMME to create pMyr6K. This was digested with XbaI and SpeI, and the fragment containing the transposon and transposase was cloned into XbaI-digested pPR23 to make pMycoMar.…”

Section: Methodsmentioning

confidence: 99%

In vivo transposition of mariner -based elements in enteric bacteria and mycobacteria

Rubín

Akerley

Novik

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

340

287

View full text Add to dashboard Cite

ABSTRACTmariner family transposons are widespread among eukaryotic organisms. These transposons are apparently horizontally transmitted among diverse eukaryotes and can also transpose in vitro in the absence of added cofactors. Here we show that transposons derived from the mariner element Himar1 can efficiently transpose in bacteria in vivo. We have developed simple transposition systems by using minitransposons, made up of short inverted repeats f lanking antibiotic resistance markers. These elements can efficiently transpose after expression of transposase from an appropriate bacterial promoter. We found that transposition of mariner-based elements in Escherichia coli produces diverse insertion mutations in either a targeted plasmid or a chromosomal gene. With Himar1-derived transposons we were able to isolate phage-resistant mutants of both E. coli and Mycobacterium smegmatis. mariner-based transposons will provide valuable tools for mutagenesis and genetic manipulation of bacteria that currently lack well developed genetic systems.

show abstract

“…They are isogenic, except that CP78 is relA + and CP79 is relA2. The argR mutants (DargR : : cat), DR78 and DR79 (derived from CP78 and CP79 respectively) were obtained by homologous recombination using the pWM91 suicide vector (Metcalf et al, 1996) containing the entire argR gene disrupted with a cat cassette. Successful recombination resulted in chloramphenicolresistant cells.…”

Section: Methodsmentioning

confidence: 99%

Increased transcription rates correlate with increased reversion rates in leuB and argH Escherichia coli auxotrophs

et al. 2004

View full text Add to dashboard Cite

Escherichia coli auxotrophs of leuB and argH were examined to determine if higher rates of transcription in derepressed genes were correlated with increased reversion rates. Rates of leuB and argH mRNA synthesis were determined using half-lives and concentrations, during exponential growth and at several time points during 30 min of amino acid starvation. Changes in mRNA concentration were primarily due to increased mRNA synthesis and not to increased stability. Four strains of E. coli amino acid auxotrophs, isogenic except for relA and argR, were examined. In both the leuB and argH genes, rates of transcription and mutation were compared. In general, strains able to activate transcription with guanosine tetraphosphate (ppGpp) had higher rates of mRNA synthesis and mutation than those lacking ppGpp (relA2 mutants). argR knockout strains were constructed in relA + and relA mutant strains, and rates of both argH reversion and mRNA synthesis were significantly higher in the argR knockouts than in the regulated strains. A statistically significant linear correlation between increased rates of transcription and mutation was found for data from both genes. In general, changes in mRNA half-lives were less than threefold, whereas changes in rates of mRNA synthesis were often two orders of magnitude. The results suggest that specific starvation conditions target the biosynthetic genes for derepression and increased rates of transcription and mutation.

show abstract

Conditionally Replicative and Conjugative Plasmids CarryinglacZα for Cloning, Mutagenesis, and Allele Replacement in Bacteria

Cited by 405 publications

References 1 publication

Induction and Loss of Ti Plasmid Conjugative Competence in Response to the Acyl-Homoserine Lactone Quorum-Sensing Signal

Induction and Loss of Ti Plasmid Conjugative Competence in Response to the Acyl-Homoserine Lactone Quorum-Sensing Signal

In vivo transposition of mariner -based elements in enteric bacteria and mycobacteria

Increased transcription rates correlate with increased reversion rates in leuB and argH Escherichia coli auxotrophs

Contact Info

Product

Resources

About