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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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The experimental setup for in vivo 31P NMR employing a high purity, well-mixed anammox culture consisting of fully suspended K. stuttgartiensis cells
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Fig2: The experimental setup for in vivo 31P NMR employing a high purity, well-mixed anammox culture consisting of fully suspended K. stuttgartiensis cells

Mentions: For the experiment, the 20 mm NMR tube was employed as a miniature fed-batch reactor (Fig. 2). Ten to fifteen milliliters of concentrated biomass was added to the NMR tube. Mixing and anoxic conditions were maintained by sparging continuously with N2. Ammonium nitrite (200 mM) was added as the substrate via a syringe pump at 1 mL/h for 1 to 20 h. The first spectra were recorded without substrate supply or N2 flushing. Subsequently, spectra were recorded for 1–2 h while the tube was flushed with N2 without substrate supply. Finally, spectra were recorded during 1–4 h with substrate supply and flushing with N2. For experiments at different external pH values, the concentrated cell solution was buffered with 200–400 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer at pH 6.5, 6.9, 7.4, and 7.8. After 1 h of anoxic conditions without substrate supply, 200 mM ammonium nitrite solution was added for 2 h.Fig. 2


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)

The experimental setup for in vivo 31P NMR employing a high purity, well-mixed anammox culture consisting of fully suspended K. stuttgartiensis cells
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2822221&req=5

Fig2: The experimental setup for in vivo 31P NMR employing a high purity, well-mixed anammox culture consisting of fully suspended K. stuttgartiensis cells
Mentions: For the experiment, the 20 mm NMR tube was employed as a miniature fed-batch reactor (Fig. 2). Ten to fifteen milliliters of concentrated biomass was added to the NMR tube. Mixing and anoxic conditions were maintained by sparging continuously with N2. Ammonium nitrite (200 mM) was added as the substrate via a syringe pump at 1 mL/h for 1 to 20 h. The first spectra were recorded without substrate supply or N2 flushing. Subsequently, spectra were recorded for 1–2 h while the tube was flushed with N2 without substrate supply. Finally, spectra were recorded during 1–4 h with substrate supply and flushing with N2. For experiments at different external pH values, the concentrated cell solution was buffered with 200–400 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer at pH 6.5, 6.9, 7.4, and 7.8. After 1 h of anoxic conditions without substrate supply, 200 mM ammonium nitrite solution was added for 2 h.Fig. 2

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