Conjugation and heterologous gene expression in Gluconobacter oxydans ssp. suboxydans

Condon, Ciarán

doi:10.1016/0378-1097(91)90590-7

Cited by 7 publications

(9 citation statements)

References 4 publications

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“…After digestion using Kpn1 and Xba1 and ligation in the corresponding sites of pKOS6b, the resulting vector pKos6bΔ1432 was transferred into competent E. coli (NEB 5-α). Finally, pKos6bΔ1432 was transferred from E. coli NEB 5-α into G. oxydans with the help of E. coli ECG 18 by triparental mating (Condon et al 1991). The markerless deletion of gox1432 was generated by plating G. oxydans pKos6 bΔ1432 on 5-fluorocytosine containing agar plates as described by Kostner et al (2013).…”

Section: Standard Molecular Biology Techniques and Cloningmentioning

confidence: 99%

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans

Zahid¹,

Deppenmeier²

2016

Appl Microbiol Biotechnol

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Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP-dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450 mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP-dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP-dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.

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Section: Standard Molecular Biology Techniques and Cloningmentioning

confidence: 99%

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans

Zahid¹,

Deppenmeier²

2016

Appl Microbiol Biotechnol

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“…Some vector systems in G. oxydans with a broad-hostrange plasmid [8,9] or a cryptic plasmid [10][11][12][13][14] have been reported. The pGE1 vector and its derivative pGE2 were transferred into Gluconobacter by conjugal mating at a frequency of 10 -1 transconjugants per recipient by Masako et al [15].…”

Section: Introductionmentioning

confidence: 99%

Construction of a Novel Shuttle Vector for Use in Gluconobacter oxydans

Zhang

Lin

et al. 2010

Mol Biotechnol

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A shuttle vector pZL1 which can replicate both in Gluconobacter oxydans and Escherichia coli was constructed based on G. oxydans DSM2003 cryptic plasmid pGOX3, a homology of G. oxydans 621H pGOX3, and E. coli cloning vector pUC18. It was found to be stably maintained in G. oxydans during the serial subcultures in the absence of antibiotic pressure for 144 h. With pGOX3 as the reference sample, the relative copy number of pZL1 in G. oxydans is 13 determined by real-time fluorescence quantitative PCR (qPCR). The copy number of pZL1 is much higher than pBBR1MCS5 in E. coli. The vector pZL1 contains six commonly used restriction endonuclease sites, HindIII, SalI, XbaI, BamHI, SmaI, KpnI, and SacI, and is easy to manipulate in molecular biology experiments. The shuttle vector was used to express a reporter protein wasabi successfully in G. oxydans DSM2003 under the control of the tufB promoter.

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“…Transposon Tn951, carrying the lacIZY genes (3), was conjugally transferred from E. coli into G. oxydans. The Tn951-encoded ␤-galactosidase was weakly expressed; however, the transconjugants were unable to grow on lactose as the sole carbon source (2).…”

mentioning

confidence: 99%

Cloning of Escherichia coli lacZ and lacY Genes and Their Expression in Gluconobacter oxydans and Acetobacter liquefaciens

Mostafa

Heller

Geis

2002

Appl Environ Microbiol

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An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G. oxydans and A. liquefaciens. Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both lac genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.

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Conjugation and heterologous gene expression in Gluconobacter oxydans ssp. suboxydans

Cited by 7 publications

References 4 publications

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans

Construction of a Novel Shuttle Vector for Use in Gluconobacter oxydans

Cloning of Escherichia coli lacZ and lacY Genes and Their Expression in Gluconobacter oxydans and Acetobacter liquefaciens

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