A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium.

Stachel, Scott E.; An, Gynheung; Flores, Carlos; Nester, Eugene W.

doi:10.1002/j.1460-2075.1985.tb03715.x

Cited by 412 publications

(306 citation statements)

References 52 publications

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“…The library was constructed using pVK102 and 8399 chromosomal DNA partially digested with HindIII as described previously (Genti-Raimondi et al, 1991). The Tn3-HoHo1 transposon (Stachel et al, 1985) was used as described previously (Tolmasky et al, 1988) to generate insertional derivatives of recombinant cosmids harbouring A. baumannii 8399 DNA. Recombinant cosmids and Tn3-HoHo1 insertional derivatives were introduced into E. coli DH5α by electroporation.…”

Section: Methodsmentioning

confidence: 99%

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron This paper is dedicated to the memory of Dr M. A. Vides, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Argentina, who was a great mentor and colleague. The GenBank accession number for the sequence reported in this paper is AF130307.

et al. 2001

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The adhC1 gene from Acinetobacter baumannii 8399, which encodes a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), was identified and cloned after mapping the insertion site of Tn3-HoHo1 in a recombinant cosmid isolated from a gene library. Sequence analysis showed that this gene encodes a protein exhibiting significant similarity to alcohol dehydrogenases in bacterial, yeast, plant and animal cells. The expression of the adhC1 gene was confirmed by the detection of GSH-FDH enzyme activity in A. baumannii and Escherichia coli cells that expressed the cloned gene. However, the construction and analysis of an A. baumannii 8399 adhC1 ::Tn3-HoHo1 isogenic derivative revealed the presence of adhC2, a second copy of the gene encoding GSH-FDH activity. Enzyme assays and immunoblot analysis showed that adhC2 encodes a 465 kDa protein that is produced in similar amounts under iron-rich and iron-limited conditions. In contrast, the expression of adhC1, which encodes a 45 kDa protein with GSH-FDH activity, is induced under iron limitation and repressed when the cells are cultured in the presence of free inorganic iron. The differential expression of adhC1 is controlled at the transcriptional level and mediated through the Fur ironrepressor protein, which has potential binding sites within the promoter region of this adhC copy. The expression of both adhC copies is significantly enhanced by the presence of sub-inhibitory concentrations of formaldehyde in the culture media. Examination of different A. baumannii isolates indicates that they can be divided into two groups based on the type of GSH-FDH they produce. One group contains only the constitutively expressed 465 kDa protein, whilst the other produces this GSH-FDH type in addition to the ironregulated isoenzyme. Further analysis showed that the presence and expression of the two adhC genes does not confer resistance to exogenous formaldehyde, nor does it enable it to utilize methylated compounds as a sole carbon source when cultured under iron-rich as well as iron-deficient conditions.

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Section: Methodsmentioning

confidence: 99%

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron This paper is dedicated to the memory of Dr M. A. Vides, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Argentina, who was a great mentor and colleague. The GenBank accession number for the sequence reported in this paper is AF130307.

et al. 2001

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“…p-Galactosidase activity was determined essentially as described by Miller (1972) with the modifications of Stachel et al (1985).…”

Section: Methodsmentioning

confidence: 99%

Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358

et al. 1998

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Transposon Tn5 genomic mutants of plant-growth-promoting Pseudomonas putida strain WCS358 have been isolated which no longer utilize ferufic and coumaric acids as sole sources of carbon and energy. Genetic studies confirmed previous biochemical data showing that ferulic acid is degraded via vanillic acid, and coumaric acid via hydroxybenzoic acid. The genes involved in these enzymic steps were cloned and characterized. Two proteins designated Fca (26-5 kDa) and Vdh (50.3 kDa) were identified as responsible for the conversion of ferulic acid to vanillic acid; the proteins are encoded by the fca and vdh genes which are organized in an operon structure in the chromosome. The Vdh protein is 69% identical a t the amino acid level to the Vdh protein recently identified in Pseudomonas sp. strain HR199 and converts vanillin to vanillic acid. Homology studies revealed that the Vdh proteins exhibited significant identity to aldehyde dehydrogenases from different organisms whereas Fca belonged to the enoyl-CoA hydratase family of proteins. Two proteins, designated VanA (39.9 kDa) and VanB (343 kDa), encoded by two genes, vanA and van& are organized in an operon in the chromosome. They were found to be responsible for the demethylation of vanillic acid to protocatechuic acid. The VanA proteins showed no homology to any other known protein, while VanB belonged to the ferredoxin family of proteins. This two-component enzyme system demethylated another phenolic monomer, veratric acid, thus indicating broad specificity. Studies of the regulation of the vanAB operon demonstrated that the genes were induced by the substrate, vanillic acid; however, the strongest induction was observed when cells were grown in the presence of the product of the reaction, protocatechuic acid.

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“…Transcriptional/translational IacZ fusions were made in the 1.7-and 2.3-kb HindIII fragments of pR32 (see Fig. 1) using Tn3-HoHol as described by Stachel et al (32). The location and orientation of inserts were determined by restriction mapping.…”

Section: Methodsmentioning

confidence: 99%

The Bradyrhizobium japonicum nolA gene and its involvement in the genotype-specific nodulation of soybeans.

Sadowsky

Cregan

Göttfert

et al. 1991

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

139

115

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Several soybean genotypes have been identified which specifically exclude modulation by members of Bradyrhizobiumjaponicum serocluster 123. We have identified and sequenced a DNA region from B. japonicum strain USDA 110 which is involved in genotype-specifc modulation of soybeans. This 2.3-kilobase region, cloned in pMJS12, allows B. japonicum serocluster 123 isolates to form nodules on plants of serogroup 123-restricting genotypes. The nodules, however, were ineffective for symbiotic nitrogen fixation. The nodulation-complementing region is located approximately 590 base pairs transcriptionally downstream from nodD2. The 5' end of pMJS12 contains a putative open reading frame (ORF) of 710 base pairs, termed nowl. Transposon Tn3-HoHo mutations only within the ORF abolished modulation complementation. The N terminus of the predicted noL4 gene product has strong similarity with the N terminus of MerR, the regulator of mercury resistance genes. Translational IacZ fusion experiments indicated that nolA was moderately induced by soybean seed extract and the isoflavone genistein. Restriction fragments that hybridize to pMJS12 were detected in genomic DNAs from both nodulation-restricted and -unrestricted strains.

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A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium.

Cited by 412 publications

References 52 publications

Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358

The Bradyrhizobium japonicum nolA gene and its involvement in the genotype-specific nodulation of soybeans.

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