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Expression of Alcaligenes eutrophus flavohemoprotein and engineered Vitreoscilla hemoglobin-reductase fusion protein for improved hypoxic growth of Escherichia coli.

ABSTRACT: Expression of the vhb gene encoding hemoglobin from Vitreoscilla sp. (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb. However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) reductase activities. To examine whether the carboxy-terminal redox-active site of flavohemoproteins can be used to improve the positive effects of VHb in microaerobic Escherichia coli cells, we fused sequences encoding NAD(P)H, FAD, or NAD(P)H-FAD reductase activities of A. eutrophus in frame after the vhb gene. Similarly, the gene for FHP was modified, and expression cassettes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed. Biochemically active heme proteins were produced from all of these constructions in Escherichia coli, as indicated by their ability to scavenge carbon monoxide. The presence of FHP or of VHb-FAD-NAD reductase increased the final cell density of transformed wild-type E. coli cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb. Approximately the same final optical densities were achieved with the E. coli strains expressing FHPg and VHb. The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-expressing control under the same cultivation conditions. The expression of VHb-NAD or FHPg-NAD fusion proteins reduced the final cell densities approximately 20% relative to the VHb-expressing control. The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant beta-lactamase relative to the VHb-expressing control.

SUBMITTER: Frey AD

PROVIDER: S-EPMC91791 | biostudies-literature | 2000 Jan

REPOSITORIES: biostudies-literature

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Expression of Alcaligenes eutrophus flavohemoprotein and engineered Vitreoscilla hemoglobin-reductase fusion protein for improved hypoxic growth of Escherichia coli.

Frey A D AD Bailey J E JE Kallio P T PT

Applied and environmental microbiology 20000101 1

Expression of the vhb gene encoding hemoglobin from Vitreoscilla sp. (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb. However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin aden ...[more]

PMID: 10618209

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Project description:Hypoxia, a naturally occurring phenomenon, results in tremendous losses in the growth of plants and in their productivity. On the physiological level oxygen deprivation results in distinct metabolic rearrangements, is accompanied by dehydration, and oxidative and nitrosative stresses. Six to seven core hypoxia-responsive genes, which are always induced under oxygen deprivation, have been shown to include hemoglobin 1 (Hb1) across a wide range of plant species. However, the function of this induced Hb1 is not completely understood .The importance of elevated Hb expression has been attributed mainly to its nitric oxide (NO) scavenging properties, and hence, to the elimination of this free-radical and signaling species. NO is known to accumulate under hypoxia and its level is in reciprocal relationship with the Hb expression levels. However, evidence has accumulated on Hbs as essential for normal plant development and they take part in the regulation of e.g. flowering, germination and lateral and adventitious root emergence. The physiological role of induced hemoglobin (Hb) expression and its role in NO scavenging were assessed by introducing bacterial (Vitreoscilla stercoraria) hemoglobin (VHb) to the cytoplasm and mitochondria of Arabidopsis thaliana. Heterologous expression was chosen to avoid interference with developmental functions of endogenous Hbs. Sixteen day old transformants were subjected to hypoxia or to NO donor treatment (DETA, 500 µM) for 2h and 24h and global changes in gene expression were assessed using a microarray approach. Both hypoxic and NO-treatment experiments were aimed also to reveal novel functions for Hb under abiotic stress conditions, exemplified by oxygen deprivation and NO/oxidative stresses and during normal development of Arabidopsis transformants on the background of normally expressed endogenous Hbs Hypoxia, a naturally occurring phenomenon, results in tremendous losses in the growth of plants and in their productivity. On the physiological level oxygen deprivation results in distinct metabolic rearrangements, is accompanied by dehydration, and oxidative and nitrosative stresses. Six to seven core hypoxia-responsive genes, which are always induced under oxygen deprivation, have been shown to include hemoglobin 1 (Hb1) across a wide range of plant species. However, the function of this induced Hb1 is not completely understood .The importance of elevated Hb expression has been attributed mainly to its nitric oxide (NO) scavenging properties, and hence, to the elimination of this free-radical and signaling species. NO is known to accumulate under hypoxia and its level is in reciprocal relationship with the Hb expression levels. However, evidence has accumulated on Hbs as essential for normal plant development and they take part in the regulation of e.g. flowering, germination and lateral and adventitious root emergence. The physiological role of induced hemoglobin (Hb) expression and its role in NO scavenging were assessed by introducing bacterial (Vitreoscilla stercoraria) hemoglobin (VHb) to the cytoplasm and mitochondria of Arabidopsis thaliana. Heterologous expression was chosen to avoid interference with developmental functions of endogenous Hbs. Sixteen day old transformants were subjected to hypoxia or to NO donor treatment (DETA, 500 µM) for 2h and 24h and global changes in gene expression were assessed using a microarray approach. Both hypoxic and NO-treatment experiments were aimed also to reveal novel functions for Hb under abiotic stress conditions, exemplified by oxygen deprivation and NO/oxidative stresses and during normal development of Arabidopsis transformants on the background of normally expressed endogenous Hbs

Project description:The linked resistance to nickel and cobalt of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) is encoded by the cnr operon, which is localized on the megaplasmid pMOL28. The regulatory genes cnrYXH have been cloned, overexpressed, and purified in Escherichia coli. CnrY fractionated as a 10.7-kDa protein in in vitro translation assays. CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in E. coli. His-CnrX was found to possess a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the presence of E. coli core RNA polymerase enzyme, could retard at least two different promoter DNA targets, cnrYp and cnrHp, localized within the cnrYXH locus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL M91650 description entry). To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of cnrYXH and the luxCDABE, luciferase reporter genes. Nickel and cobalt specifically induced the cnrYXH-luxCDABE fusion at optimal concentrations of 0.3 mM Ni(2+) and 2.0 mM Co(2+) in a noncomplexing medium for metals. The two promoter regions P(Y) (upstream cnrY) and P(H) (upstream cnrH) were probed and characterized using this vector and were found to control the nickel-inducible regulatory response of the cnr operon. The cnrHp promoter was responsible for full transcription of the cnrCBA structural resistance genes, while the cnrYp promoter was necessary to obtain metal-inducible transcription from the cnrHp promoter. The zinc resistance phenotype (ZinB) of a spontaneous cnr mutant strain, AE963, was investigated and could be attributed to an insertion of IS1087, a member of the IS2 family of insertion elements, within the cnrY gene.

Dataset Information

Expression of Alcaligenes eutrophus flavohemoprotein and engineered Vitreoscilla hemoglobin-reductase fusion protein for improved hypoxic growth of Escherichia coli.

Publications

Expression of Alcaligenes eutrophus flavohemoprotein and engineered Vitreoscilla hemoglobin-reductase fusion protein for improved hypoxic growth of Escherichia coli.

Similar Datasets

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