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Plasmids for nicotine-dependent and -independent gene expression in Arthrobacter nicotinovorans and other arthrobacter species.

ABSTRACT: The first inducible Arthrobacter overexpression system, based on the promoter/operator and the repressor of the 6-D-hydroxynicotine oxidase gene of Arthrobacter nicotinovorans, is described here. Nicotine-dependent overproduction and affinity purification of recombinant proteins are presented. The system will allow the production of complex enzymes and genetic complementation studies in Arthrobacter species.

SUBMITTER: Sandu C

PROVIDER: S-EPMC1317448 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Plasmids for nicotine-dependent and -independent gene expression in Arthrobacter nicotinovorans and other arthrobacter species.

Sandu Cristinel C Chiribau Calin-Bogdan CB Sachelaru Paula P Brandsch Roderich R

Applied and environmental microbiology 20051201 12

The first inducible Arthrobacter overexpression system, based on the promoter/operator and the repressor of the 6-D-hydroxynicotine oxidase gene of Arthrobacter nicotinovorans, is described here. Nicotine-dependent overproduction and affinity purification of recombinant proteins are presented. The system will allow the production of complex enzymes and genetic complementation studies in Arthrobacter species. ...[more]

PMID: 16332890

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Project description:In cells of an Arthrobacter oxidans riboflavin-dependent mutant the specific activity of the DL-nicotine-inducible nicregulon enzymes nicotine dehydrogenase (NDH, EC 1.5.99.4), 6-hydroxy-L-nicotine oxidase (6-HLNO, EC 1.5.3.5) and 6-hydroxy-D-nicotine oxidase (6-HDNO, EC 1.5.3.6) was shown to be dependent on the supply of the vitamin in the growth medium. Experiments designed to identify at which level riboflavin directs the biosynthesis of these flavoenzymes revealed that the steady-state levels of enzyme protein analysed on Western blots correlated directly with riboflavin supply from the minimal concentration of 0.5 microns-riboflavin required for growth up to 8 microns-riboflavin. Mutant cells grown at the higher riboflavin concentration showed on dot-blots increased levels of RNA which hybridized to 32P-labelled probes derived from the nic-regulon genes. When cells grown at 2 microns-riboflavin were shifted to 8 microns-riboflavin, 6-HDNO expression increased as indicated by elevated enzyme and RNA levels. When the rates of synthesis of the 6-HDNO and 6-HLNO polypeptides after DL-nicotine induction was analysed in cells grown at 0.5 microns and 8 microns-riboflavin, only cells grown at the higher riboflavin concentration showed on Western blots an accumulation of the polypeptides. No 6-HDNO or 6-HLNO polypeptide was identified in cell extracts from cells grown on 0.5 microns-riboflavin. Pulse-chase experiments with [35S]methionine showed that 6-HDNO- and 6-HLNO synthesis was prevented in cells grown at the low riboflavin concentration. The absence of detectable enzyme levels seemed not to be caused by proteolytic breakdown. Incubation in vitro of apo-6HDNO with low- or high-riboflavin-grown-cell extracts showed no increased proteolytic activity in 0.5 microns-riboflavin-grown cells. From these results it is concluded that riboflavin supply co-regulates the expression of the nicregulon genes at the level of transcription and/or mRNA turnover.

Dataset Information

Plasmids for nicotine-dependent and -independent gene expression in Arthrobacter nicotinovorans and other arthrobacter species.

Publications

Plasmids for nicotine-dependent and -independent gene expression in Arthrobacter nicotinovorans and other arthrobacter species.

Similar Datasets

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