Replacement of a Phosphoenolpyruvate-dependent Phosphotransferase by a Nicotinamide Adenine Dinucleotide-linked Dehydrogenase for the Utilization of Mannitol

Tanaka, Shuji; Lerner, Stephen A.; Lin, E. C. C.

doi:10.1128/jb.93.2.642-648.1967

Cited by 387 publications

(116 citation statements)

References 26 publications

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“…Bacterial strains and growth of cells E. coli strain DK8 [44] lacking the atp operon was transformed with plasmid pBWU13 [45] carrying the atp operon except atpI. Cells were grown on minimal medium [46] supplemented with thiamine (2 mg´mL 21 ), thymine, asparagine, isoleucine and valine (50 mg´mL 21 each) together with 75 mm glycerol as carbon source, harvested at late logarithmic phase and stored at 280 8C.…”

Section: Methodsmentioning

confidence: 99%

Secondary structure composition of reconstituted subunit b of the Escherichia coli ATP synthase

2000

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Subunit b of the Escherichia coli ATP synthase was isolated by preparative gel electrophoresis, acetone precipitated and after ion-pair extraction redissolved in a buffer either containing n-dodecyl-b-d-maltoside or sodium cholate. The secondary structure of isolated subunit b was shown to be the same as within the F O complex, but was strongly dependent on the detergent used for replacement of the phospholipid environment. This was shown by an identical tryptic digestion pattern, which was strongly influenced by the detergent used for solubilization. An influence of the detergent n-dodecyl-b-d-maltoside on the secondary structure of the hydrophilic part of subunit b was also shown for the soluble part of the polypeptide comprising residues Val25 to Leu156 (b sol ) using CD spectroscopy. In order to determine the secondary structure of subunit b in its native conformation, isolated subunit b was reconstituted into E. coli lipid vesicles and analyzed with CD spectroscopy. The resulting spectrum revealed a secondary structure composition of 80% a helix together with 14% b turn conformation. These results suggest that subunit b is not a rigid rod-like a helix simply linking F 1 to F O , but rather provides an inherent flexibility for the storage of elastic energy within the second stalk generated by rotational movements within the F 1 F O complex.

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Section: Methodsmentioning

confidence: 99%

Secondary structure composition of reconstituted subunit b of the Escherichia coli ATP synthase

2000

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“…These were carried out according to published methods (Ito et al, 1981 ;Gebert et al, 1988) with some modifications. An overnight culture of E. coli in mineral salts medium (Tanaka et al, 1967) was freshly inoculated (100 pl) into 4 ml mineral salts medium containing thiamin, glycerol and the methionine-assay medium (Difco), and was grown under shaking at 37°C to an A,,, of about 0.25. The culture was diluted with medium to an A,,, of 0.1 in a total volume of 2 ml in large Eppendorf tubes, and incubated at 30°C.…”

Section: Methodsmentioning

confidence: 99%

Expression of the Zymomonas Mobilis Gfo Gene for NADP‐Containing Glucose: Fructose Oxidoreductase (GFOR) in Escherichia Coli— Formation of Enzymatically Active Pregfor but Lack of Processing Into a Stable Periplasmic Protein

Wiegert

Sahm

Sprenger

1997

European Journal of Biochemistry

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G1ucose:fructose oxidoreductase (GFOR) of the gram-negative bacterium Zyrnornonas rnobilis is a periplasmic enzyme with tightly bound cofactor NADP. The preprotein carries an unusually long Nterminal signal peptide of 52 amino acid residues. Expression of the afo gene in cells of Escherichia coli K12, under the control of a tuc promoter, led to immunologically detectable proteins in western blots, and to the formation of an enzymatically active precursor form (preGFOR), located in the cytosol. Processing of preGFOR to the mature form was not observed in E. coli. Replacement of the authentic GFOR signal peptide by the shorter signal peptides of PhoA or OmpA from E. coli led to processing of the respective GFOR precursor proteins. However, the processed proteins were unstable and rapidly degraded in the periplasm unless an E. coli mutant was used that carried a triple lesion for periplasmic and outermembrane proteases. When fusion-protein export was inhibited by sodium azide or carboxylcyanide rnchlorophenylhydrazone, the cytoplasmic precursor forms of the respective preGFOR were not degraded. A major protease-resistant GFOR peptide from the OmpA-GFOR fusion was found within spheroplasts of E. coli to which NADP had been added externally. The formation of this peptide did not occur in the presence of NAD. It is concluded that NADP is required for GFOR to fold into its native conformation and that its absence from the E. coli periplasm is responsible for failure to form a stable periplasmic protein. The results strongly suggest that, in Z. rnobilis, additional protein factors are required for the transport of NADP across the plasma membrane andor incorporation of NADP into the GFOR apoenzyme.

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“…Louis, MO) and 250 mg/L neomycin sulfate (Sigma ChemicalCo.). For shakeflaskexperiments, RB79l(pKN) was cultured at 20 "C in Tanaka medium (Tanaka et al, 1967), pH 7.2, supplemented with 2.0 g/L glucose and 2.0 g/L casein amino acids, acid hydrolysate (Sigma Chemical Co.). Cells were induced by adding lo4 M IPTG (Sigma Chemical Co. or Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) to the medium.…”

Section: Methodsmentioning

confidence: 99%

Escherichia coli Host Cell Modifications in Continuous Culture Affecting Heterologous Protein Overproduction: A Population Dynamics Study

Togna

Shuler

et al. 1992

Biotechnology Progress

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There are many published studies of plasmid segregational instability in Escherichia coli in the literature. However, the formation of plasmid-free segregants can be controlled by the addition of selective chemical agents like antibiotics. This solution has become commonplace in both the laboratory and industry. On the other hand, host cell modifications, which result in low production of plasmid-encoded protein and lead to loss of culture productivity, have not been adequately addressed. Continuous culture of an inducible (ptac) Escherichia coli vector containing strain, RB791(pKN), was characterized by strong dynamic changes in the cell population and product (beta-lactamase) expression. Long-term cultivation resulted in the loss of high-level production of beta-lactamase. Loss of productivity was not due to the formation of plasmid-free cells or structural modifications to the plasmid; instead, continuous operation resulted in a culture dominated by irreversibly altered, low-producing cells. Two distinct classes of lac- mutants which inhibited induction were identified (Y- and I(s)).

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Replacement of a Phosphoenolpyruvate-dependent Phosphotransferase by a Nicotinamide Adenine Dinucleotide-linked Dehydrogenase for the Utilization of Mannitol

Cited by 387 publications

References 26 publications

Secondary structure composition of reconstituted subunit b of the Escherichia coli ATP synthase

Secondary structure composition of reconstituted subunit b of the Escherichia coli ATP synthase

Expression of the Zymomonas Mobilis Gfo Gene for NADP‐Containing Glucose: Fructose Oxidoreductase (GFOR) in Escherichia Coli— Formation of Enzymatically Active Pregfor but Lack of Processing Into a Stable Periplasmic Protein

Escherichia coli Host Cell Modifications in Continuous Culture Affecting Heterologous Protein Overproduction: A Population Dynamics Study

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