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An intracellular pH gradient in the anammox bacterium Kuenenia stuttgartiensis as evaluated by 31P NMR.

van der Star WR, Dijkema C, de Waard P, Picioreanu C, Strous M, van Loosdrecht MC - Appl. Microbiol. Biotechnol. (2009)

Bottom Line: At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra.These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis.This study provides for the second time--after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens--evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

ABSTRACT
The cytoplasm of anaerobic ammonium oxidizing (anammox) bacteria consists of three compartments separated by membranes. It has been suggested that a proton motive force may be generated over the membrane of the innermost compartment, the "anammoxosome". 31P nuclear magnetic resonance (NMR) spectroscopy was employed to investigate intracellular pH differences in the anammox bacterium Kuenenia stuttgartiensis. With in vivo NMR, spectra were recorded of active, highly concentrated suspensions of K. stuttgartiensis in a wide-bore NMR tube. At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra. These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis. This study provides for the second time--after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens--evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

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Full in vivo 31P spectra of K. stuttgartiensis converting ammonium nitrite. Acquisition time = 4 h (26,800 spectra), line width = 25 Hz. The three pH related peaks are 1.5 ppm (pH 6.3, assigned to the anammoxosome), 2.9 ppm (shoulder, pH 7.4, assigned to the riboplasm), and 3.1 ppm (pH 7.8, the extracellular pH). Four non-pH peaks are visible at −18.6, −10.8, −5.2, −0.8, 4.1, and 4.5 ppm, which can probably be assigned to β-ATP (−18.6), NAD/NADP (−10.8 ppm), γ-ATP (−5.2), phosphoenolpyruvate (PEP, −0.8 ppm). The peaks at 4.1 and 4.5 ppm are probably a phosphate diesther and a phosphate monoesther, respectively
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Fig5: Full in vivo 31P spectra of K. stuttgartiensis converting ammonium nitrite. Acquisition time = 4 h (26,800 spectra), line width = 25 Hz. The three pH related peaks are 1.5 ppm (pH 6.3, assigned to the anammoxosome), 2.9 ppm (shoulder, pH 7.4, assigned to the riboplasm), and 3.1 ppm (pH 7.8, the extracellular pH). Four non-pH peaks are visible at −18.6, −10.8, −5.2, −0.8, 4.1, and 4.5 ppm, which can probably be assigned to β-ATP (−18.6), NAD/NADP (−10.8 ppm), γ-ATP (−5.2), phosphoenolpyruvate (PEP, −0.8 ppm). The peaks at 4.1 and 4.5 ppm are probably a phosphate diesther and a phosphate monoesther, respectively

Mentions: To show that the K. stuttgartiensis cells had been active in the previous incubations, spectra were also recorded during a longer time (4 h, 26,800 spectra) to increase the sensitivity in order to enable the detection of phosphate-containing organic metabolites. Besides the abovementioned peaks of the inorganic phosphate species, these spectra showed peaks at −18.6, −10.8, −5.2, −0.8, 4.1, and 4.5 ppm (Fig. 5). The peaks at −18.6 and −5.2 could be ascribed to γ- and β-ATP, respectively, whereas the peak at −10.8 was NADH/NADPH. The peak at −0.8 is consistent with phosphoenolpyruvate (PEP). The peaks above 4 ppm may indicate the presence of a phosphate esthers (a monoesther at 4.5 ppm and a diesther at 4.1). No signs of poly-phosphate were found.Fig. 5


An intracellular pH gradient in the anammox bacterium Kuenenia stuttgartiensis as evaluated by 31P NMR.

van der Star WR, Dijkema C, de Waard P, Picioreanu C, Strous M, van Loosdrecht MC - Appl. Microbiol. Biotechnol. (2009)

Full in vivo 31P spectra of K. stuttgartiensis converting ammonium nitrite. Acquisition time = 4 h (26,800 spectra), line width = 25 Hz. The three pH related peaks are 1.5 ppm (pH 6.3, assigned to the anammoxosome), 2.9 ppm (shoulder, pH 7.4, assigned to the riboplasm), and 3.1 ppm (pH 7.8, the extracellular pH). Four non-pH peaks are visible at −18.6, −10.8, −5.2, −0.8, 4.1, and 4.5 ppm, which can probably be assigned to β-ATP (−18.6), NAD/NADP (−10.8 ppm), γ-ATP (−5.2), phosphoenolpyruvate (PEP, −0.8 ppm). The peaks at 4.1 and 4.5 ppm are probably a phosphate diesther and a phosphate monoesther, respectively
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2822221&req=5

Fig5: Full in vivo 31P spectra of K. stuttgartiensis converting ammonium nitrite. Acquisition time = 4 h (26,800 spectra), line width = 25 Hz. The three pH related peaks are 1.5 ppm (pH 6.3, assigned to the anammoxosome), 2.9 ppm (shoulder, pH 7.4, assigned to the riboplasm), and 3.1 ppm (pH 7.8, the extracellular pH). Four non-pH peaks are visible at −18.6, −10.8, −5.2, −0.8, 4.1, and 4.5 ppm, which can probably be assigned to β-ATP (−18.6), NAD/NADP (−10.8 ppm), γ-ATP (−5.2), phosphoenolpyruvate (PEP, −0.8 ppm). The peaks at 4.1 and 4.5 ppm are probably a phosphate diesther and a phosphate monoesther, respectively
Mentions: To show that the K. stuttgartiensis cells had been active in the previous incubations, spectra were also recorded during a longer time (4 h, 26,800 spectra) to increase the sensitivity in order to enable the detection of phosphate-containing organic metabolites. Besides the abovementioned peaks of the inorganic phosphate species, these spectra showed peaks at −18.6, −10.8, −5.2, −0.8, 4.1, and 4.5 ppm (Fig. 5). The peaks at −18.6 and −5.2 could be ascribed to γ- and β-ATP, respectively, whereas the peak at −10.8 was NADH/NADPH. The peak at −0.8 is consistent with phosphoenolpyruvate (PEP). The peaks above 4 ppm may indicate the presence of a phosphate esthers (a monoesther at 4.5 ppm and a diesther at 4.1). No signs of poly-phosphate were found.Fig. 5

Bottom Line: At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra.These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis.This study provides for the second time--after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens--evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

ABSTRACT
The cytoplasm of anaerobic ammonium oxidizing (anammox) bacteria consists of three compartments separated by membranes. It has been suggested that a proton motive force may be generated over the membrane of the innermost compartment, the "anammoxosome". 31P nuclear magnetic resonance (NMR) spectroscopy was employed to investigate intracellular pH differences in the anammox bacterium Kuenenia stuttgartiensis. With in vivo NMR, spectra were recorded of active, highly concentrated suspensions of K. stuttgartiensis in a wide-bore NMR tube. At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra. These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis. This study provides for the second time--after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens--evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

Show MeSH