Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR

Huang, Guozhong; Zhang, L.; Birch, Robert G.

doi:10.1046/j.1365-2672.2000.00781.x

Cited by 32 publications

(26 citation statements)

References 12 publications

(22 reference statements)

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“…For this analysis, the total DNA of the mutant was isolated (32) and digested with PstI. The resulting DNA fragments were circularized with T4 DNA ligase under conditions promoting intramolecular circularization (26). The regions flanking the inserted mini-Tn5 were amplified with two primers which hybridized specifically to both repetitive ends (the I and O ends) of the mini-Tn5 transposon ( Table 2).…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

Manilla-Pérez

Lange

Hetzler

et al. 2010

Appl Environ Microbiol

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Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

Manilla-Pérez

Lange

Hetzler

et al. 2010

Appl Environ Microbiol

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“…To locate the transposon Tn5 insertion site in the TH2 mutant, the strategy described by Huang et al (27), based on inverse PCR, was carried out. As a modification, primer BR (27) was used in combination with primer BRf1Bam (Table 2).…”

Section: Vol 188 2006mentioning

confidence: 99%

Knockout and Overexpression of Pyrroloquinoline Quinone Biosynthetic Genes in Gluconobacter oxydans 621H

Hölscher

Görisch

2006

J Bacteriol

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In Gluconobacter oxydans, pyrroloquinoline quinone (PQQ) serves as the cofactor for various membranebound dehydrogenases that oxidize sugars and alcohols in the periplasm. Proteins for the biosynthesis of PQQ are encoded by the pqqABCDE gene cluster. Our reverse transcription-PCR and promoter analysis data indicated that the pqqA promoter represents the only promoter within the pqqABCDE cluster of G. oxydans 621H. PQQ overproduction in G. oxydans was achieved by transformation with the plasmid-carried pqqA gene or the complete pqqABCDE cluster. A G. oxydans mutant unable to produce PQQ was obtained by site-directed disruption of the pqqA gene. In contrast to the wild-type strain, the pqqA mutant did not grow with D-mannitol, D-glucose, or glycerol as the sole energy source, showing that in G. oxydans 621H, PQQ is essential for growth with these substrates. Growth of the pqqA mutant, however, was found with D-gluconate as the energy source. The growth behavior of the pqqA mutant correlated with the presence or absence of the respective PQQdependent membrane-bound dehydrogenase activities, demonstrating the vital role of these enzymes in G. oxydans metabolism. A different PQQ-deficient mutant was generated by Tn5 transposon mutagenesis. This mutant showed a defect in a gene with high homology to the Escherichia coli tldD gene, which encodes a peptidase. Our results indicate that the tldD gene in G. oxydans 621H is involved in PQQ biosynthesis, possibly with a similar function to that of the pqqF genes found in other PQQ-synthesizing bacteria.The acetic acid bacterium Gluconobacter oxydans is characterized by its ability to incompletely oxidize various sugars and alcohols by using membrane-associated dehydrogenases that contain pyrroloquinoline quinone (PQQ) as a cofactor (33). Examples are the quinoprotein glucose dehydrogenase (3) and the quinoprotein glycerol dehydrogenase, which also oxidizes D-gluconate, D-mannitol, D-sorbitol, and other polyols (34). The oxidation reactions take place in the periplasmic space and are coupled to the respiratory chain (19,33). Several oxidation reactions carried out by G. oxydans quinoproteins are of industrial importance, e.g., the conversion of D-gluconate to 5-D-ketogluconate, a precursor for the production of L-tartaric acid, and the formation of L-sorbose, an intermediate in the synthesis of vitamin C (reviewed in reference 9). PQQ has invoked considerable attention due to its positive physiological effects in mammals (47). A possible role of PQQ as a vitamin in mammals has been suggested but is controversially debated (13,28,40).Genes involved in PQQ synthesis have been characterized for several bacteria, including Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens AM1, and Pseudomonas sp. (reviewed in reference 19). In Klebsiella pneumoniae, the PQQ biosynthetic genes are clustered in the pqqABCDEF operon (35). In Pseudomonas aeruginosa, the pqqABCDE operon is separated from the pqqF operon (18,48). Methylobacterium extorquens AM1 contains a ...

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“…The location of the transposon insertion was identified either by direct genomic sequencing (1) or by inverse PCR (8). Since the Synechocystis genome has been sequenced (11), the identification of the genes disrupted by the transposon mutagenesis is facile.…”

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Four Novel Genes Required for Optimal Photoautotrophic Growth of the Cyanobacterium Synechocystis sp. Strain PCC 6803 Identified by In Vitro Transposon Mutagenesis

et al. 2004

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Four novel Synechocystis sp. strain PCC 6803 genes (sll1495, sll0804, slr1306, and slr1125) which encode hypothetical proteins were determined by transposon mutagenesis to be required for optimal photoautotrophic growth. Mutations were also recovered in ccmK4, a carboxysome coat protein homologue, and me, the decarboxylating NADP ؉ -dependent malic enzyme. This is the first report that these known genes are required for optimal photoautotrophy.Photosynthesis is one of the most important biological processes and occurs in a very diverse set of organisms ranging from prokaryotes to eukaryotes. Recently, much effort has been directed towards understanding the structure and function of proteins involved in photosynthesis (photosystem I, photosystem II, cytochrome b 6 /f complex, Calvin-Benson cycle enzymes, etc.). While much progress has been made in the understanding of the functional organization of these proteins, relatively little is known concerning the organization of other protein components which must be involved in the regulation, assembly, and turnover of the proteins involved in photosynthesis. Cyanobacteria are photoautotrophic gram-negative eubacteria capable of performing oxygenic photosynthesis in a manner quite similar to that in eukaryotic algae and higher plants. Synechocystis sp. strain PCC 6803 is a naturally competent unicellular cyanobacterium and has proved to be one of the best model organisms for studying the mechanism and regulation of oxygenic photosynthesis (15). We are interested in identifying the genes required for oxygenic photosynthesis. In this study, we used a hyperactive Tn5-based in vitro transposition system to introduce random insertional mutations into Synechocystis and have identified a number of mutants which are incapable of undergoing optimal photoautotrophic growth. Here we describe the production, identification, and characterization of a number of these mutants. The structure and possible function of the affected genes in these mutants will also be discussed.A glucose-tolerant strain of Synechocystis sp., PCC 6803 (15), was used as a parental control and as the DNA recipient strain in the present study. Cells of both the control strain and the derivative photosynthetic mutants were maintained under photoheterotrophic growth conditions at 30°C with a light intensity of 20 mol of photons m Ϫ2 s Ϫ1 (fluorescent light) in liquid BG-11 growth medium (ATCC medium 616) supplemented with 10 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid]-KOH (pH 8.2), 5 mM glucose, and 10 M DCMU [N-(3,4-dichlorophenyl)-NЈ-dimethylurea]. Liquid cultures were bubbled continuously with air. For autotrophic cell culture, the glucose and DCMU were omitted. For cultures grown on plates, the BG-11 medium was supplemented with 1.5% agar and 0.3% sodium thiosulfate. When appropriate, kanamycin was included in the media at a final concentration of 10 g/ml.

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Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR

Cited by 32 publications

References 12 publications

Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

Knockout and Overexpression of Pyrroloquinoline Quinone Biosynthetic Genes in Gluconobacter oxydans 621H

Four Novel Genes Required for Optimal Photoautotrophic Growth of the Cyanobacterium Synechocystis sp. Strain PCC 6803 Identified by In Vitro Transposon Mutagenesis

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