Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria.

@article{Reeve1999ConstructsFI,
  title={Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria.},
  author={Wayne Gerald Reeve and Ravi P. Tiwari and P S Worsley and Michael J. Dilworth and Andrew R. Glenn and John Gregory Howieson},
  journal={Microbiology},
  year={1999},
  volume={145 ( Pt 6)},
  pages={
          1307-16
        }
}
Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia… Expand
A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria.
TLDR
A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon), which have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. Expand
Construction of improved vectors and cassettes containing gusA and antibiotic resistance genes for studies of transcriptional activity and bacterial localization.
TLDR
Broad-host-range, conjugative vectors with a constitutively expressed gusA gene combined with different antibiotic resistance genes have been constructed, designed for tracking Gram-negative bacterial strains without the risk of random mutagenesis. Expand
A histidine kinase sensor protein gene is necessary for induction of low pH tolerance in Sinorhizobium sp. strain BL3
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It was shown that actX was induced at low pH and the method of gene identification used in this study for isolation of actX may be applied for the isolation of other genes involved in tolerance to adverse environmental factors. Expand
Counter-transcribed RNAs of Rhizobium leguminosarum repABC plasmids exert incompatibility effects only when highly expressed.
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For the repABC operons in this study, the intergenic region between repB and repC was the strongest incompatibility factor, and this appears to require a high level of transcription of the ctRNA. Expand
Probing for pH-Regulated Genes in Sinorhizobium medicae Using Transcriptional Analysis
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Findings implicate cytochrome synthesis, potassium ion cycling, lipid biosynthesis and transport processes as key components of pH response in S. medicae. Expand
The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium
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It is shown that Salicylic acid can directly shut down the expression of the Agrobacterium virA/G two-component regulatory system and up-regulated the attKLM operon, which functions in degrading the bacterial quormone N-acylhomoserine lactone. Expand
Genetic Characterization of a Novel Rhizobial Plasmid Conjugation System in Rhizobium leguminosarum bv. viciae Strain VF39SM
TLDR
Experimental evidence suggested the presence of two putative origins of transfer within the gene cluster, and a regulatory gene, trbR, was identified in the region between traA and traG and was mutated and shown to function as a repressor of both trb gene expression and plasmid transfer. Expand
Defining the requirements for the conjugative transfer of Rhizobium leguminosarum plasmid pRleVF39b.
Rhizobium leguminosarum strain VF39 contains a plasmid, pRleVF39b, which encodes a distinctive type of conjugation system (rhizobial type IVa) that is relatively widespread among rhizobial genomes.Expand
Isolation and characterization of Pseudobutyrivibrio ruminis gene promoters
TLDR
A mutation within the -35 element of one promoter revealed that promoter strength, and the choice of transcription start site were both sensitive to single nucleotide, however, no correlation was found between the composition and context of elements within P. ruminis promoters, and promoter strength. Expand
Characterization of genes involved in erythritol catabolism in Rhizobium leguminosarum bv. viciae.
A genetic locus encoding erythritol uptake and catabolism genes was identified in Rhizobium leguminosarum bv. viciae, and shown to be plasmid encoded in a wide range of R. leguminosarum strains. AExpand
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A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction ofExpand
New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions.
TLDR
A set of antibiotic-resistance and promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions was constructed and have been successfully used for mutagenisation and studies of gene transcription in Rhizobium meliloti. Expand
Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria
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The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations, and that do not carry antibiotic resistance markers characteristic of most available cloning vectors. Expand
β-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria
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A series of transposons are described which contain the gusA gene, encoding β-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive, which constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants. Expand
Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419
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Four Tn5-induced mutants of Rhizobium meliloti WSM419 were unable to grow or maintain intracellular pH at an external pH of 5.6 and restriction analysis indicated that all four cloned mutations are unique and that the two strains (TG1-6 and TG1-11) carry Tn 5 insertions which are within 4.4 kilobases of each other on a single EcoRI fragment. Expand
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Several new vectors for the construction of operon and protein fusions to the Escherichia coli lacZ gene are described, improved in that they have very low levels of background lac gene expression, which makes possible the easy detection and accurate quantitation of very weak transcriptional and translational signals. Expand
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TLDR
Four new Escherichia coli cloning vectors are described, pUC6S, p UC21, pUK21 and pOK12, which contain a polylinker or multiple cloning site (MCS) with the recognition sequences for 28 restriction enzymes allowing blue/white screening for inserts. Expand
Acid tolerance in Rhizobium meliloti strain WSM419 involves a two-component sensor-regulator system.
TLDR
Data implicate a two-component sensor-regulator protein pair in acid tolerance in R. meliloti and suggest their involvement in pH sensing and/or response by these bacteria. Expand
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TLDR
Novel features revealed by these studies include nod expression in the meristem, regulated in planta expression of control genes nodD1 and nodD3, disappearance of nod expression late in organogenesis, and properties of syrM. Expand
Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria.
TLDR
A series of derivatives of the omega interposon that can be used for in vitro insertional mutagenesis are constructed and insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli. Expand
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