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• W2046473633 abstract "Acidocalcisomes are acidic, calcium storage compartments with a H+ pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H+-pyrophosphatase (H+-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H+-PPase with pyrophosphate (PPi) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane. polyP was also localized to the acidocalcisomes by using 4′,6′-diamino-2-phenylindole staining and identified by using 31P NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PPi and polyP increased when bacteria were grown in the light. Taken together, these results suggest that the H+-PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PPi synthesis in the chromatophore membranes. Acidocalcisomes are acidic, calcium storage compartments with a H+ pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H+-pyrophosphatase (H+-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H+-PPase with pyrophosphate (PPi) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane. polyP was also localized to the acidocalcisomes by using 4′,6′-diamino-2-phenylindole staining and identified by using 31P NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PPi and polyP increased when bacteria were grown in the light. Taken together, these results suggest that the H+-PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PPi synthesis in the chromatophore membranes. Membrane-bound H+-pyrophosphatases (H+-PPases) 1The abbreviations used are: H+-PPase, H+-pyrophosphatase; polyP, polyphosphate; PPi, pyrophosphate; PBS, phosphate-buffered saline; AMDP, aminomethylenediphosphonate; DAPI, 4′,6′-diamino-2-phenylindole; DCCD, dicyclohexylcarbo-diimide.1The abbreviations used are: H+-PPase, H+-pyrophosphatase; polyP, polyphosphate; PPi, pyrophosphate; PBS, phosphate-buffered saline; AMDP, aminomethylenediphosphonate; DAPI, 4′,6′-diamino-2-phenylindole; DCCD, dicyclohexylcarbo-diimide. have been found in various organelles. Mitochondria possess an H+-PPase that may be dimeric (1Lundin M. Deopujari S.W. Lichko L. Pereira da Silva L. Baltscheffsky H. Biochim. Biophys. Acta. 1992; 1098: 217-223Crossref PubMed Scopus (26) Google Scholar) and that is oriented in such a way as to pump protons out into the cytosol (2Pereira da Silva L. Sherman M. Lundin M. Baltscheffsky H. Arch. Biochem. Biophys. 1993; 30: 310-313Crossref Scopus (22) Google Scholar, 3Vianello A. Zancani M. Casolo V. Macri F. Plant Cell Physiol. 1997; 38: 87-90Crossref Scopus (14) Google Scholar). Vacuoles from plants as well as those from charophyte algae and Acetabularia have a monomeric H+-PPase that functions to acidify them (4Rea P.A. Poole R.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 4: 157-180Crossref Scopus (264) Google Scholar, 5Zhen R.-G. Kim E.J. Rea P.A. Adv. Bot. Res. 1997; 25: 297-337Crossref Scopus (65) Google Scholar). Acidocalcisomes of trypanosomatid and Apicomplexan parasites (6Docampo R. Moreno S.N.J. Mol. Biochem. Parasitol. 2001; 114: 151-159Crossref PubMed Scopus (133) Google Scholar), as well as from the green alga Chlamydomonas reinhardtii (7Ruiz F.A. Marchesini N. Seufferheld M. Govindjee Docampo R. J. Biol. Chem. 2001; 276: 46196-46203Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), the slime mold Dictyostelium discoideum (8Marchesini N. Ruiz F.A. Vieira M. Docampo R. J. Biol. Chem. 2002; 277: 8146-8153Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and the bacterium Agrobacterium tumefaciens (9Seufferheld M. Viera M.C.F. Ruiz F.A. Rodrigues C.O. Moreno S.N.J. Docampo R. J. Biol. Chem. 2003; 278: 29971-29978Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) also possess an H+-PPase that is responsible for their acidification. An H+-PPase is also present in the chromatophores (10Baltscheffsky H. Von Stedingk L.-V. Heldt H.-W. Klingenberg M. Science. 1966; 153: 1120-1122Crossref PubMed Scopus (140) Google Scholar, 11Baltscheffsky M. Nature. 1967; 216: 241-243Crossref PubMed Scopus (78) Google Scholar). H+-PPases may also be present in the plasma membranes of some plant cells (12Long A.R. Williams L.E. Nelson S.J. Hall J.L. J. Plant Physiol. 1995; 146: 629-638Crossref Scopus (48) Google Scholar, 13Robinson D.G. Haschke H.-P Hinz G. Hoh B. Maeshima M. Marty F. Planta. 1996; 198: 95-103Crossref Scopus (92) Google Scholar), as well as unicellular eukaryotes (14Martinez R. Wang Y. Benaim G. Benchimol M. de Souza W. Scott D.A. Docampo R. Mol. Biochem. Parasitol. 2002; 120: 205-213Crossref PubMed Scopus (27) Google Scholar). The H+-PPase from the phototrophic bacterium Rhodospirillum rubrum was the first H+-PPase discovered (10Baltscheffsky H. Von Stedingk L.-V. Heldt H.-W. Klingenberg M. Science. 1966; 153: 1120-1122Crossref PubMed Scopus (140) Google Scholar, 11Baltscheffsky M. Nature. 1967; 216: 241-243Crossref PubMed Scopus (78) Google Scholar). This enzyme is unique in that it catalyzes not only the hydrolysis of PPi but also the synthesis of PPi in the light (15Baltscheffsky M. Schultz A. Baltscheffsky H. FEBS Lett. 1999; 457: 527-533Crossref PubMed Scopus (108) Google Scholar). Synthesis is driven by the energy derived from the electrochemical H+ gradient generated across the membrane of the chromatophores during illumination (15Baltscheffsky M. Schultz A. Baltscheffsky H. FEBS Lett. 1999; 457: 527-533Crossref PubMed Scopus (108) Google Scholar). Acidocalcisomes have recently been found (9Seufferheld M. Viera M.C.F. Ruiz F.A. Rodrigues C.O. Moreno S.N.J. Docampo R. J. Biol. Chem. 2003; 278: 29971-29978Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) to be morphologically similar to the volutin granules described in bacteria (16Meyer A. Bot. Zeit. 1904; 62: 113-152Google Scholar). Volutin or metachromatic granules were the first subcellular entities to be recognized in bacteria (16Meyer A. Bot. Zeit. 1904; 62: 113-152Google Scholar, 17Kornberg A. J. Bacteriol. 1995; 177: 491-496Crossref PubMed Scopus (462) Google Scholar). Because R. rubrum is known to possess volutin granules that accumulate PPi under illumination (18Salih G.F. Nyrén P. Curr. Res. Photosynth. 1990; 3: 209-212Google Scholar), we investigated whether the H+-PPase was also present in these organelles. In this report, we describe the isolation and biochemical properties of the acidocalcisomes of R. rubrum and show that, as with the acidocalcisomes of A. tumefaciens (9Seufferheld M. Viera M.C.F. Ruiz F.A. Rodrigues C.O. Moreno S.N.J. Docampo R. J. Biol. Chem. 2003; 278: 29971-29978Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), they are surrounded by a membrane, are acidic because of the presence of the H+-PPase in their membrane, are rich in PPi and polyP, and are able to accumulate calcium and other elements. The number of acidocalcisomes as well as the amount of PPi and polyP significantly increase when the bacteria are grown in light. We also demonstrate that the H+-PPase is predominantly located in the acidocalcisomes of R. rubrum. Cell Cultures—R. rubrum cells (strain Esmarch, Molisch ATCC 17031) were obtained from the American Type Culture Collection. The cells were grown in liquid Sistrom succinate medium (19Sistrom W.R. J. Gen. Microbiol. 1960; 22: 778-785Crossref PubMed Scopus (331) Google Scholar) with agitation (160 rpm) in the dark or anaerobically in the light (an intensity of 80 μm photons/m2 × s-1) at 30 °C. The cells were cultured for 4 days and harvested at the stationary phase. Chemicals—Dulbecco's PBS and reagents for marker enzyme assays were purchased from Sigma. Silicon carbide (400 mesh) was bought from Aldrich. Iodixanol (40% solution; OptiPrep; Nycomed) was obtained from Invitrogen. Benzonase® was from Novagen (Wisconsin, MD). Cycloprodigiosin was a gift from Prof. Hajime Hirata (Himeji Institute of Technology, Hyogo, Japan). Polyclonal antibodies raised against a keyhole limpet hemocyanin-conjugated synthetic peptide corresponding to the hydrophilic loop XII (antibody PABHK or 326) of plant V-H+-PPase (20Zhen R.-G. Kim E.J. Rea P.A. J. Biol. Chem. 1997; 272: 22340-22348Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) were kindly provided by Prof. Philip Rea (University of Pennsylvania, Philadelphia, PA). Aminomethylenediphosphonate (AMDP) was synthesized by Michael Martin (Department of Chemistry, University of Illinois at Urbana-Champaign). Monoclonal antibody against a keyhole limpet hemocyanin-conjugated synthetic peptide corresponding to the hydrophilic loop XII of Trypanosoma cruzi H+-PPase (21Luo S. Vieira M. Graves J. Zhong L. Moreno S.N.J. EMBO J. 2001; 20: 55-64Crossref PubMed Scopus (75) Google Scholar) was prepared at the University of Illinois Biotechnology Center. Molecular weight markers and Coomassie Blue protein assay reagent were from Bio-Rad. EnzChek phosphate assay kit and LysoSensor blue DND-167 (9,10-bis (N-morpholinomethyl) anthracene) were from Molecular Probes (Eugene, OR). Prof. Arthur Kornberg (Stanford University School of Medicine, Stanford, CA), kindly provided Escherichia coli strain CA38 pTrcPPX1. Prof. Mary Lynne Perille Collins (University of Wisconsin, Milwaukee, WI) provided a polyclonal antibody raised against crude membranes of phototrophic R. rubrum cells. All other reagents were of analytical grade. Isolation of Acidocalcisomes—Bacteria were collected by centrifugation at 3,900 × g, and the pellet was resuspended in lysis buffer (125 mm sucrose, 50 mm KCl, 4 mm MgCl2, 0.5 mm EDTA, 20 mm K-Hepes, 5 mm dithiothreitol, 0.1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 μm pepstatin, 10 μm leupeptin, 10 μmtrans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane, and 10 μmN-tosyl-l-lysine chloromethyl ketone, pH 7.2) containing 2 mg/ml lysozyme. Benzonase® (1 μl/ml) was added, and the bacteria then passed through a French press (SLM-Aminco, Spectometric Instruments) twice at 1,000 p.s.i. The lysate was incubated on ice under agitation for 1 h with an equal volume of silica/silicon carbide (1:1) to remove DNA and RNA fragments. The lysate was then centrifuged at 1,000 × g for 5 min and washed two times under the same conditions. The supernatant fractions were combined and centrifuged for 10 min at 14,500 × g. The pellet was resuspended in 2 ml of lysis buffer with the aid of a 22-gauge needle. The suspension was diluted 1:1 in OptiPrep (60% iodixanol) and applied as the 30% concentration step of a discontinuous gradient of Optiprep, with 4-ml steps of 24, 28, 30, 35, and 40% iodixanol, diluted in lysis buffer. The gradient was centrifuged at 50,000 × g in a Beckman SW 28 rotor for 60 min. The acidocalcisome fraction pelleted at the bottom of the tube and was resuspended in lysis buffer. Gradient fractions and markers were assayed as previously described (23Scott D.A. Docampo R. J. Biol. Chem. 2000; 275: 24215-24221Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Analytical Methods and Immunoblotting—Bacteria were washed once with Dulbecco's PBS, and then PPi and long chain and short chain polyPs were extracted as described previously (24Ruiz F.A. Rodrigues C.O. Docampo R. J. Biol. Chem. 2001; 276: 26114-26121Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Pyrophosphatase activity was assayed by measuring released phosphate using the EnzChek phosphate assay (23Scott D.A. Docampo R. J. Biol. Chem. 2000; 275: 24215-24221Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 25Scott D.A. de Souza W. Benchimol M. Zhong L. Lu H.-G. Moreno S.N.J. Docampo R. J. Biol. Chem. 1998; 273: 22151-22158Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The apparent Km for PPi was calculated by using a nonlinear regression program (Sigma Plot 1.0, Jandel Scientific) using the Hill equation. Protein determination was carried out by using the Coomassie Blue protein assay reagent from Bio-Rad. The proteins were separated by SDS-PAGE using 10% gels and then blotted onto nitrocellulose using a Bio-Rad Transblot apparatus. Subsequent processing steps were performed in Dulbecco's PBS containing 0.1% Tween 20. The blots were blocked for 1 h in 5% nonfat dry milk, washed three times, and then incubated with polyclonal antibody 326 against Arabidopsis H+-PPase (20Zhen R.-G. Kim E.J. Rea P.A. J. Biol. Chem. 1997; 272: 22340-22348Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) (1:1,000) for 1 h at room temperature. The blots were then washed three times, incubated for 1 h with horseradish peroxidase-labeled anti-rabbit IgG (1:20,000), washed three times, and processed for chemiluminiscence detection as per the manufacturer's instructions (Amersham Biosciences). Molecular weights were calculated using prestained molecular weight markers. Immunofluorescence Microscopy—For subcellular localization of H+-PPase, bacteria were washed with Dulbecco's PBS and fixed in 4% freshly prepared formaldehyde for 10 min at room temperature and 50 min at 4 °C, attached to poly l-lysine treated glass slides, and permeabilized with 0.2% Nonidet P-40 in PBS for 10 min. The samples were blocked for 1 h with PBS containing 3% bovine serum albumin, 1% cold fish gelatin, and 50 mm NH4Cl and were first incubated for 1 h at room temperature with the polyclonal antibody against the Arabidopsis thaliana H+-PPase (20Zhen R.-G. Kim E.J. Rea P.A. J. Biol. Chem. 1997; 272: 22340-22348Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) or monoclonal antibody against T. cruzi H+-PPase (21Luo S. Vieira M. Graves J. Zhong L. Moreno S.N.J. EMBO J. 2001; 20: 55-64Crossref PubMed Scopus (75) Google Scholar), diluted 1: 50 (polyclonal) or 1:100 (monoclonal) in 1% cold fish gelatin. Bacteria were subsequently incubated for 60 min at room temperature with fluorescein-conjugated secondary antibody diluted 1:200 in PBS plus 1% cold fish gelatin. Coverslips were mounted in glass slides with Vectashield® media and sealed. The images were collected with an Olympus laser scanning confocal microscope or an Olympus BX-60 fluorescence microscope. For polyP localization, bacteria were washed twice with Dulbecco's PBS and resuspended in the same buffer and fixed for 30 min with 4% formaldehyde. 45 μl of this suspension was incubated at room temperature with 10 μg/ml DAPI. After 10 min, the samples were mounted on a slide and observed using the fluorescence microscope (9Seufferheld M. Viera M.C.F. Ruiz F.A. Rodrigues C.O. Moreno S.N.J. Docampo R. J. Biol. Chem. 2003; 278: 29971-29978Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). For localization of LysoSensor blue DND-167, bacteria were centrifuged and resuspended in prewarmed (30 °C) Sistrom medium containing 1 μm LysoSensor. Bacteria were incubated for 1 h at 30 °C, centrifuged, and resuspended in fresh prewarmed Sistrom medium. Bacteria were mounted on a slide and observed with the fluorescence microscope using UV excitation. For cycloprodigiosin detection, bacteria were centrifuged, resuspended in Dulbecco's PBS containing 100 nm cycloprodigiosin, and incubated for 30 min. Bacteria were mounted on a slide and observed with the fluorescence microscope using a red emission filter. Bacteria resuspended in Dulbecco's PBS or Sistrom medium, but without cycloprodigiosin or LysoSensor, respectively, were used as controls. For co-localization studies of chromatophore proteins and H+-PPase, polyclonal antibodies against crude membranes of phototropic R. rubrum cells were adsorbed with R. rubrum grown aerobically in the dark, to remove nonspecific antibodies (22Crook S.M. Treml S.B. Collins M.L.P. J. Bacteriol. 1986; 167: 89-95Crossref PubMed Google Scholar). Cells grown aerobically were fixed with 4% formaldehyde for 1 h followed by permeabilization with 0.3% Triton X-100 in PBS for 10 min and then washed twice with PBS. The cells were then incubated with the antibodies against crude membranes for 45 min at 37 °C and centrifuged, and the antibody that was not adsorbed was collected from the supernatant. For co-localization studies, the cells were incubated 1 h with antibodies diluted 1:20 and with a mouse monoclonal antibody against T. cruzi H+-PPase, diluted 1:50. The cells were subsequently incubated for 60 min at room temperature with rabbit fluorescein-conjugated secondary antibody diluted 1:100 and with mouse rodamine-conjugated secondary antibody diluted 1:200 in PBS plus 1% cold fish gelatin. Control preparations were incubated with preimmune serum or without the primary antibody. Three-dimensional Confocal Immunofluorescence Microscopy Reconstruction Analysis of H+-PPase Staining in R. rubrum—Volumetric renderings through a representative bacterial cell were compiled using the average projection ray tracing algorithm (26Drebin, R. Carpenter, L., and Hanrahan, P. (1988) Proceedings of the 15th Annual Conference on Computer Graphics and Interactive Techniques, pp. 65-74, ACM Press, New YorkGoogle Scholar, 27Meissner, M. Huang, J. Bartz, D., Mueller, K., and Crawfis, R. (2000) Proceedings of the 2000 IEEE Symposium on Volume Visualization, pp. 81-91, ACM Press, New YorkGoogle Scholar) in the Olympus FluoView software suite. Further image processing was conducted using the ImageJ software tools and Application Programming Interface originally developed by Wayne Rasband at the National Institutes of Health (rsb.info.nih.gov/ij/). The voxel signal intensities were normalized (28Russ J.C. The Image Processing Handbook. 2nd Ed. CRC Press Inc., Boca Raton, FL1995: 151-210Google Scholar) to enable easier discrimination of relative pixel intensities, and a median filter (28Russ J.C. The Image Processing Handbook. 2nd Ed. CRC Press Inc., Boca Raton, FL1995: 151-210Google Scholar, 29van Kempen G.M.P. van Vliet L.J. Verveer P.J. Cogswell C.J. Conchello J.-A. Wilsonm T. 3-D Microscopy: Image Aquisition and Processing IV. SPIE International Society for Optical Engineering, Bellingham, WA1997: 114-124Google Scholar) was applied to correct image artifacts caused by shot noise (30Russ J.C. The Image Processing Handbook. 2nd Ed. CRC Press Inc., Boca Raton, FL1995: 1-76Google Scholar). A Gaussian filter (28Russ J.C. The Image Processing Handbook. 2nd Ed. CRC Press Inc., Boca Raton, FL1995: 151-210Google Scholar, 29van Kempen G.M.P. van Vliet L.J. Verveer P.J. Cogswell C.J. Conchello J.-A. Wilsonm T. 3-D Microscopy: Image Aquisition and Processing IV. SPIE International Society for Optical Engineering, Bellingham, WA1997: 114-124Google Scholar) was applied to further counteract image artifacts caused by statistical noise (30Russ J.C. The Image Processing Handbook. 2nd Ed. CRC Press Inc., Boca Raton, FL1995: 1-76Google Scholar), and a colorized lookup table (31Pawley J.B. Stevens J.K. Mill L.R. Trogadis J.E. Three Dimensional Confocal Microscopy: Investigation of Biological Specimens. Academic Press, London1994: 47-93Google Scholar) was applied to the 8-bit gray scale image to convey the spectral detection range under which the data was acquired. Average projections through the xyz plane and the zyx plane were selected to depict the relative signal intensities through the volumetric dataset. Size bars and arrows were applied as overlays to the xyz and zyx images using tools in Adobe Photoshop. Electron Microscopy and X-ray Microanalysis—For routine electron microscopy, bacteria were washed with Dulbecco's PBS and fixed for 1 h with 2.5% grade II glutaraldehyde, 4% freshly prepared formaldehyde, 0.03% CaCl2, and 0.03% picric acid in 0.1 m cacodylate buffer, pH 7.2. Bacteria were post-fixed with OsO4 for 45 min and then for 15 min with potassium ferricyanide, washed, and treated with 2% uranyl acetate for 30 min. Subsequently, the samples were dehydrated by successive incubations of 6 min with increasing concentrations of ethanol (10, 25, 50, 75, 95, and 100%) at room temperature. Epoxy embedding was carried out by resuspending the sample once in 1:1 ethanol/acetonitrile, twice in 100% acetonitrile, then 30 min in 1:1 Epoxy/acetonitrile, 1.5 h in 3:1 Epoxy/acetonitrile, and 4 h in 100% Epoxy. The embedded samples were polymerized for 20 h at 85 °C. Epoxy blocks were ultrathin-sectioned, the sections were deposited on 300-mesh copper grids, and the grids were stained with uranyl acetate for 30 min and triple lead stain (lead citrate, lead nitrate, and lead acetate) for 1 min. For immunocytochemistry, bacteria were washed with Dulbecco's PBS and fixed for 1 h at 4 °C in a solution containing 0.5% grade I glutaraldehyde, 4% freshly prepared formaldehyde, 1% picric acid, in 0.1 m cacodylate buffer, pH 7.2. Fixed bacteria were washed with Dulbecco's PBS and dehydrated by successive incubations of 6 min each with increasing concentrations of ethanol (10, 25, 50, 75, 95, 100, and 100%) at -20 °C. The samples were embedded in Unicryl at 4 °C by incubation with 1:1 ethanol/Unicryl for 1 h and 100% Unicryl for 1, 16, and 8 h. The embedded samples were polymerized under UV irradiation at -20 °C for 48 h. Thin sections were collected on 300-mesh nickel grids and blocked for 30 min with PBS containing 0.1% Tween 20 and 0.5% cold fish gelatin (PBS-TW-FG). The grids were incubated for 3 h with a mouse monoclonal antibody against T. cruzi H+-PPase (21Luo S. Vieira M. Graves J. Zhong L. Moreno S.N.J. EMBO J. 2001; 20: 55-64Crossref PubMed Scopus (75) Google Scholar) diluted 1:10 in PBS-TW-FG. After washing in PBS-TW-FG, the grids were incubated for 1 h with a 5-nm gold conjugate goat anti-mouse antibody. Subsequently, the grids were washed with PBS and then in distilled water, stained with uranyl acetate and lead citrate. Routine and immunocytochemistry samples were observed with a Hitachi H 600 electron microscope. For imaging whole bacteria, the preparations were washed in 0.25 m sucrose, and a 5-μl sample was placed on a Formvar-coated 200-mesh copper grid, allowed to adhere for 10 min at room temperature, blotted dry, and observed directly with a Hitachi 600 transmission electron microscope operating at 100 kV (9Seufferheld M. Viera M.C.F. Ruiz F.A. Rodrigues C.O. Moreno S.N.J. Docampo R. J. Biol. Chem. 2003; 278: 29971-29978Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Energy-dispersive x-ray analysis was done at the Electron Microscopy Center of Southern Illinois University (Carbondale, IL). The spectra shown are the ones that yielded the most counts in 100 s (of 10 spectra obtained from granules of different bacteria in each preparation), but all other spectra taken from acidocalcisomes of the same preparation were qualitatively similar. Specimen grids were examined in a Hitachi H-7100FA transmission electron microscope at an accelerating voltage of 50 kV. Fine probe sizes were adjusted to cover the electron-dense vacuoles (or a similar area of the background), and x-rays were collected for 100 s by utilizing a thin window (Norvar) detector. Analysis was performed by using a Noran Voyager III analyzer with a standardless analysis identification program. Perchloric Acid Extracts—For NMR, bacteria were washed twice with buffer A (116 mm NaCl, 5.4 mm KCl, 0.8 mm MgSO4,50mm Hepes, pH 7.2) and then extracted with ice-cold 0.5 m HClO4 (2 ml/g wet weight cells). After 30 min of incubation on ice, the extracts were centrifuged at 3,000 × g for 5 min. The supernatants were neutralized by the addition of 0.72 m KOH, 0.6 M KHCO3 (32Moreno B. Urbina J.A. Oldfield E. Bailey B.N. Rodrigues C.O. Docampo R. J. Biol. Chem. 2000; 275: 28356-28362Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Precipitated KClO4 was removed by centrifugation at 12,000 × g for 10 min., the supernatant was separated, and EDTA was added to a final concentration of 100 μm prior to adjusting to pH 8. All of the extracts contained 10% D2O (v/v) to provide a field frequency lock. NMR Spectroscopy—Phosphorus NMR spectra were acquired at 303.6 MHz using a Varian INOVA NMR spectrometer equipped with a 17.6 Tesla Oxford Instruments magnet. For perchloric acid extracts, 16,384 transients were collected at room temperature using 25-μs (90°) pulse excitation, 20-kHz spectral width, 32,768 data points, and a 5-s recycle time. Inverse-gated proton decoupling was used to remove nuclear Overhauser effect and J-coupling effects. The chemical shifts of all 31P spectra were referenced to 0 ppm using an 85% phosphoric acid external reference (33Van Wazer J.R. Ditchfield R. Burt C.T. Phosphorus NMR in Biology. CRC Press, Boca Raton, FL1987: 1-23Google Scholar). The specific assignments of individual resonances were initially based on published chemical shifts and 31P-31P scalar couplings (33Van Wazer J.R. Ditchfield R. Burt C.T. Phosphorus NMR in Biology. CRC Press, Boca Raton, FL1987: 1-23Google Scholar). NMR spectra were processed using the VNMR 6.1B software package (Varian Inc., Palo Alto, CA) running on a Sun Ultra5 (Sun Microsystems, Santa Clara, CA) work station, and included base-line correction, zero filling, and a 3-Hz exponential line broadening prior to Fourier transformation. Acidocalcisomes are recognizable by their strong electron density when observed by electron microscopy in unfixed and unstained whole cell mounts (6Docampo R. Moreno S.N.J. Mol. Biochem. Parasitol. 2001; 114: 151-159Crossref PubMed Scopus (133) Google Scholar). Using this technique, R. rubrum grown in the dark showed both large and small granules, in different locations of the cells (Fig. 1A, arrows). The large granules have a diameter of ∼205 ± 24 nm. X-ray microanalyses were performed on these granules, and a representative spectrum (Fig. 1B) shows that the counts for phosphorus were ∼3-fold greater than the counts for magnesium, which were approximately the same as the counts for potassium. The counts for oxygen and phosphorus were approximately the same. The medium used to grow R. rubrum in the previous experiments was calcium-deficient, which might explain the lack of detectable levels of calcium in the x-ray microanalyses of the acidocalcisomes (Fig. 1B). To test this idea, we therefore next cultivated bacteria in the presence of 100 mm CaCl2 for4h before preparing them for x-ray microanalysis. Fig. 1C shows that there is a dramatic increase in the counts for calcium as well as a decrease in the counts for magnesium and potassium in the acidocalcisomes of these cells. The presence of these elements was not detected in spectra taken from the background (Fig. 1D) and demonstrates the ability of acidocalcisomes to accumulate calcium. Peaks for copper and in part for carbon arise from the grid. Examination of cells in thin sections showed round vacuoles of ∼200 nm in cells grown both in the light (Fig. 2A, arrowhead) and in the dark (Fig. 2B, arrowhead), clearly different from the chromatophore membranes detected in cells grown in the light (Fig. 2A). As is characteristic of the morphology of acidocalcisomes (6Docampo R. Moreno S.N.J. Mol. Biochem. Parasitol. 2001; 114: 151-159Crossref PubMed Scopus (133) Google Scholar), an electron-dense ring was observed surrounding the apparently empty vacuoles (Fig. 2A, arrowhead). Each intracellular vacuole appeared to be surrounded by a membrane (Fig. 2B, arrowhead). R. rubrum acidocalcisomes (Fig. 2C) showed a sponge-like appearance that is also typical of acidocalcisomes in different organisms (6Docampo R. Moreno S.N.J. Mol. Biochem. Parasitol. 2001; 114: 151-159Crossref PubMed Scopus (133) Google Scholar, 7Ruiz F.A. Marchesini N. Seufferheld M. Govindjee Docampo R. J. Biol. Chem. 2001; 276: 46196-46203Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8Marchesini N. Ruiz F.A. Vieira M. Docampo R. J. Biol. Chem. 2002; 277: 8146-8153Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 9Seufferheld M. Viera M.C.F. Ruiz F.A. Rodrigues C.O. Moreno S.N.J. Docampo R. J. Biol. Chem. 2003; 278: 29971-29978Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). We purified the acidocalcisomes following a purification procedure used for the isolation of acidocalcisomes from A. tumefaciens (9Seufferheld M. Viera M.C.F. Ruiz F.A. Rodrigues C" @default.
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• W2046473633 date "2004-12-01" @default.
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• W2046473633 title "The H+-pyrophosphatase of Rhodospirillum rubrum Is Predominantly Located in Polyphosphate-rich Acidocalcisomes" @default.
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