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• W2026465513 abstract "The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity at pH values ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism. The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity at pH values ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism. Since identification of the first aquaporin from red blood cells (1Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1697) Google Scholar), genes encoding aquaporins have been found in both prokaryotic and eukaryotic cells. In animals, abundant major intrinsic protein isoforms are involved in a number of diseases and are known to have a role in the regulation of water homeostasis (2Agre P. Kozono D. FEBS Lett. 2003; 555: 72-78Crossref PubMed Scopus (445) Google Scholar). In plants, aquaporins regulate water permeability and transport in response to external changes in water supply (3Czempinski K. Frachisse J.M. Maurel C. Barbier-Brygoo H. Mueller-Roeber B. Plant J. 2002; 29: 809-820Crossref PubMed Scopus (99) Google Scholar). The first aquaporin to be isolated from plant cells was the tonoplast intrinsic protein, γ-TIP (4Maurel C. Reizer J. Schroeder J.I. Chrispeels M.J. EMBO J. 1993; 12: 2241-2247Crossref PubMed Scopus (423) Google Scholar). This finding established that aquaporins reside not only in the plasma membrane but also in endomembranes, presumably to coordinate water transport inside the cell. In Synechocystis sp. PCC 6803 (henceforth referred to as Synechocystis) a single copy gene encoding an aquaporin homolog, aqpZ, is present in the genome. The functional characteristics of AqpZ and its subcellular localization in Synechocystis have not been determined, although microarray experiments have identified a list of genes induced by hyperosmotic stress in both the wild type (WT) and a ΔaqpZ strain (5Shapiguzov A. Lyukevich A.A. Allakhverdiev S.I. Sergeyenko T.V. Suzuki I. Murata N. Los D.A. Microbiology. 2005; 151: 447-455Crossref PubMed Scopus (54) Google Scholar). Moreover, loss of aquaporins in microorganisms in general does not result in growth defects under a range of environmental conditions (6Tanghe A. Van Dijck P. Thevelein J.M. Trends Microbiol. 2006; 14: 78-85Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Hence, the question as to the physiological role of aquaporins in microbial cells remains open. In microorganisms, the best studied aquaporin is the AqpZ protein from Escherichia coli. The aqpZ null mutant forms smaller colonies and has reduced viability in medium with low osmolarity compared with the parental wild-type cells (7Calamita G. Kempf B. Bonhivers M. Bishai W.R. Bremer E. Agre P. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 3627-3631Crossref PubMed Scopus (77) Google Scholar). However, another study failed to detect any growth defects of an aqpZ disruption mutant under any condition tested (8Soupene E. King N. Lee H. Kustu S. J. Bacteriol. 2002; 184: 4304-4307Crossref PubMed Scopus (29) Google Scholar). Although wild-type E. coli cells have higher water permeability compared with an aqpZ null mutant, it has not been demonstrated that aquaporins are important for proper osmotic adjustment (9Delamarche C. Thomas D. Rolland J.P. Froger A. Gouranton J. Svelto M. Agre P. Calamita G. J. Bacteriol. 1999; 181: 4193-4197Crossref PubMed Google Scholar). Although the physiological relevance of E. coli AqpZ remains unclear, other functions of aquaporins that are related to specific ecological lifestyles or developmental stages have received increased attention (6Tanghe A. Van Dijck P. Thevelein J.M. Trends Microbiol. 2006; 14: 78-85Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 10Hill A.E. Shachar-Hill B. Shachar-Hill Y. J. Membr. Biol. 2004; 197: 1-32Crossref PubMed Scopus (187) Google Scholar). Some aquaporin isoforms mediate permeation of glycerol, H2O2, CO2, silicon, or boron in addition to water (11Yasui M. Hazama A. Kwon T.H. Nielsen S. Guggino W.B. Agre P. Nature. 1999; 402: 184-187Crossref PubMed Scopus (408) Google Scholar, 12Maurel C. FEBS Lett. 2007; 581: 2227-2236Crossref PubMed Scopus (269) Google Scholar). The range of specificities of aquaporins implies that they are involved in processes as diverse as nutrient acquisition, control of development, and growth and defense responses against environmental stress. Cyanobacteria are prokaryotic microorganisms that perform oxygenic photosynthesis and are adapted to a regular cycle of light and dark periods, in which they are different from non-photosynthetic microorganisms. In most species of cyanobacteria, glycogen accumulated during the day serves as the predominant metabolic fuel at night. Glucose derived from glycogen or supplied exogenously is catabolized via the oxidative pentose phosphate pathway, glycolysis, and the tricarboxylic acid (TCA) cycle, leading to the production of ATP and carbon skeletons. A glucose-tolerant strain of the cyanobacterium Synechocystis has been isolated previously (13Williams J.G.K. Methods Enzymol. 1988; 167: 776-778Google Scholar). These cells grow photoautotrophically under light conditions but are also capable of photomixotrophic growth or light-activated heterotrophic growth in glucose-supplemented media (14Anderson S.L. McIntosh L. J. Bacteriol. 1991; 173: 2761-2767Crossref PubMed Google Scholar). In the present study, we determined the membrane localization and investigated the physiological role of aquaporin AqpZ in Synechocystis. The addition of glucose to ΔaqpZ cells triggered structural aberrations and morphological abnormalities. Moreover, ΔaqpZ cells growing on medium containing glucose accumulated more glycogen, and their glucose catabolysis was down-regulated. These data suggest that AqpZ plays a crucial role in the regulation of glucose metabolism under photomixotrophic conditions. To our knowledge, this is the first evidence of a physiological role of AqpZ in addition to its role in the osmotic stress response. The aqpZ coding region of Synechocystis slr2057 was amplified from genomic DNA by PCR using gene-specific primers (sense, 5′-CAGTAGATCTATGAAAAAGTACATTGCTG-3′; antisense, 5′-CAGTGCTAGCTCACTCTGCTTCGGGTTCG-3′). The resulting PCR product was cloned into the BglII and NheI sites of pXβG-ev1 (1Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1697) Google Scholar). To create Myc-tagged AqpZ, another set of primers (sense, 5′-CATGGAATTCCATGAAAAAGTACATTGCTG-3′; antisense, 5′-CAGTGCTAGCTCACTCTGCTTCGGGTTCG-3′) was used to amplify the coding region of aqpZ from genomic DNA by PCR, and the resulting PCR product was cloned into the EcoRI and NheI sites of pXβG-ev1, placing it in frame with the N-terminal Myc tag contained in the vector. The correct frame was verified by sequencing. Myc-Y69 (AQP-3) from C. elegans and the human aquaporin hAQP1 were used as controls (1Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1697) Google Scholar). Capped cRNAs were synthesized in vitro from XbaI-linearized pXβG-ev1 plasmids using the mMESSAGE mMACHINE T3 kit (Ambion, Austin, TX). Defolliculated Xenopus laevis oocytes were injected with 5 or 10 ng of cRNA or diethyl pyrocarbonate-treated water (1Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1697) Google Scholar, 15Uozumi N. Gassmann W. Cao Y. Schroeder J.I. J. Biol. Chem. 1995; 270: 24276-24281Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Injected oocytes were incubated for 2–3 days at 18 °C in 200 mosm modified Barth's solution (10 mm Tris-HCl (pH 7.6), 88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.3 mm Ca(NO3)2, 0.4 mm CaCl2, 0.8 mm MgSO4). An oocyte swelling assay was used to determine the osmotic water permeability, Pf (1Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1697) Google Scholar). Oocytes were transferred to modified Barth's solution diluted to 70 mosm with distilled water, and the time course of volume increase was monitored at room temperature by video microscopy with an on-line computer (16Ma T. Frigeri A. Skach W. Verkman A.S. Biochem. Biophys. Res. Commun. 1993; 197: 654-659Crossref PubMed Scopus (48) Google Scholar, 17Chakrabarti N. Roux B. Pomès R. J. Mol. Biol. 2004; 343: 493-510Crossref PubMed Scopus (102) Google Scholar). The relative volume (V/V0) was calculated essentially as described by Preston et al. (1Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1697) Google Scholar). The relative volume (V/V0) was calculated from the area at the initial time (A0) and after a time interval (At): V/V0 = (At/A0) 32. The coefficient of osmotic water permeability (Pf) was determined from the initial slope of the time course (d(V/V0)/dt), initial oocyte volume (V0 = 9 × 10−4 cm3), initial oocyte surface area (S = 0.045 cm2), and the molar volume of water (Vw = 18 cm3/mol): Pf = (V0 × d(V/V0)/dt)/(S × Vw × (osMin − osMout)). Oocytes were incubated in fixing solution (80 mm Pipes, pH 6.8, 5 mm EGTA, 1 mm MgCl2, 3.7% formaldehyde, 0.2% Triton X-100) at room temperature for 4 h, transferred to methanol at −20 °C for 24 h, equilibrated in PBS (3.2 mm Na2HPO4, 0.5 mm KH2PO4, 1.3 mm KCl, 135 mm NaCl, pH 7.4) at room temperature for 2 h, incubated in PBS with 100 mm NaBH4 at room temperature for 24 h, and bisected with a razor blade (18Liu K. Kozono D. Kato Y. Agre P. Hazama A. Yasui M. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2192-2197Crossref PubMed Scopus (71) Google Scholar). Fixed oocytes were blocked in PBS containing 2% BSA for 1 h at room temperature and then incubated at 4 °C with the anti-AqpZ antibody (see below) for 24 h followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG in PBS containing 2% BSA for 24 h. Samples were mounted in Fluoromount-G (Southern Biotechnology Associates) and visualized with a PerkinElmer UltraView LCI confocal laser-scanning microscope. For each sample, 10 oocytes were homogenized together by pipetting up and down in hypotonic lysis buffer (7.5 mm sodium phosphate, 1 mm EDTA, pH 7.5) containing a protease inhibitor mixture (Sigma-Aldrich) (19Meetam M. Keren N. Ohad I. Pakrasi H.B. Plant Physiol. 1999; 121: 1267-1272Crossref PubMed Scopus (33) Google Scholar). The oocyte yolk was removed by discarding the pellet after a centrifugation at 735 × g and 4 °C for 10 min. The supernatant was then centrifuged at 200,000 × g, 4 °C, for 1 h. The pellet containing the oocyte membrane fraction was solubilized in buffer (50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 50 mm EDTA-2Na, 10% (w/v) glycerol, and 2% SDS). Total protein content was determined by the bicinchoninic acid (BCA) assay method (Pierce). Equal amounts of protein were separated by SDS-PAGE on a 12% gel. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, probed with either anti-AqpZ antibody (see below) or anti-Myc antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences). The enhanced chemiluminescence detection system (Amersham Biosciences) was used to visualize the immunoreactive proteins by exposure to x-ray films. Thylakoid and plasma membrane fractions were prepared from Synechocystis cells as described previously (20Norling B. Zak E. Andersson B. Pakrasi H. FEBS Lett. 1998; 436: 189-192Crossref PubMed Scopus (107) Google Scholar, 21Tsunekawa K. Shijuku T. Hayashimoto M. Kojima Y. Onai K. Morishita M. Ishiura M. Kuroda T. Nakamura T. Kobayashi H. Sato M. Toyooka K. Matsuoka K. Omata T. Uozumi N. J. Biol. Chem. 2009; 284: 16513-16521Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). An anti-AqpZ antibody was raised against two synthetic peptides with the sequences NH2-GSNPLATNGFGDHS-COOH and NH2-VLEDLGRPEPEAE-COOH (Operon, Tokyo, Japan). Polyclonal antibodies raised against the plasma membrane nitrate transporter NrtA (22Omata T. Plant Cell Physiol. 1995; 36: 207-213Crossref PubMed Scopus (84) Google Scholar) or against the thylakoid membrane proteins NdhD3 and NdhF3 (23Ohkawa H. Price G.D. Badger M.R. Ogawa T. J. Bacteriol. 2000; 182: 2591-2596Crossref PubMed Scopus (91) Google Scholar, 24Zhang P. Battchikova N. Jansen T. Appel J. Ogawa T. Aro E.M. Plant Cell. 2004; 16: 3326-3340Crossref PubMed Scopus (189) Google Scholar) were used to identify the Synechocystis plasma membrane, or thylakoid membrane fractions, respectively. Proteins were separated by SDS-PAGE on a 12.5 or 15% gel and then transferred to PVDF membranes. Membranes were incubated for 1 h with the primary antibody (1:2000 in blocking buffer), followed by incubation for 30 min with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham Biosciences; 1:1000)) and subsequently developed by ECL (Amersham Biosciences). Synechocystis cells grown to an OD730 of about 1.0 in BG11 medium were fixed with 50 mm phosphatase buffer containing 5% glutaraldehyde and 2% osmium tetroxide (pH 7.2). The samples were dehydrated through a graded acetone series and embedded in Spurr's resin and polymerized. Ultrathin sections were first labeled with AqpZ antiserum (1:20) in Tris-buffered saline and then with 12-nm colloidal gold particles coupled to goat anti-rabbit IgG. IgG was purified from the serum using the MelonTM Gel IgG spin purification kit (Pierce). The sections were stained with uranyl acetate followed by lead citrate solution and examined with a transmission electron microscope (H-7500, Hitachi). The GT (glucose tolerance) strain of Synechocystis sp. PCC 6803 and its derivatives were grown at 30 °C in BG11 medium (25Stanier R.Y. Kunisawa R. Mandel M. Cohen-Bazire G. Bacteriol. Rev. 1971; 35: 171-205Crossref PubMed Google Scholar) buffered with 20 mm TES 2The abbreviations used are: TES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidBicineN,N-bis(2-hydroxyethyl)glycineCHES2-(cyclohexylamino)ethanesulfonic acid. -KOH (pH 8.0) under aerobic conditions. Solid medium consisted of BG11 buffered at pH 8.0, with 1.5% agar, and 0.3% sodium thiosulfate. To test the effects of pH on growth of Synechocystis cells, a range of BG11 media were prepared by replacing TES buffer with MES buffer for pH 6.5, TES buffer for pH 7.0–8.0, Bicine buffer for pH 8.5, and CHES buffer for pH 9.0–10.0. Continuous illumination was provided by white fluorescent lamps (50 μmol of photons m−2 s−1). For growth in the dark, culture vessels were wrapped with aluminum foil. For light-activated heterotrophic growth, BG11 plates supplemented with 5 mm glucose were incubated in the dark with a daily 15-min pulse of white light. Unless otherwise stated, cells were grown in volumes of 30 ml in flasks. In all experiments, cells grown in fresh BG11 media as precultures were transferred to BG11 (photoautotrophic growth condition) or BG11 containing 5 mm glucose (photomixotrophic growth condition) at an OD730 = 0.05. 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid N,N-bis(2-hydroxyethyl)glycine 2-(cyclohexylamino)ethanesulfonic acid. In this study, several mutant strains (ΔglcP (sll0771), Δsll1961, and ΔaqpZ/ΔglcP) were generated by homologous recombination using plasmids described elsewhere (21Tsunekawa K. Shijuku T. Hayashimoto M. Kojima Y. Onai K. Morishita M. Ishiura M. Kuroda T. Nakamura T. Kobayashi H. Sato M. Toyooka K. Matsuoka K. Omata T. Uozumi N. J. Biol. Chem. 2009; 284: 16513-16521Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The plasmids for disruption of the glcP or sll1961 gene contained an insertion of a kanamycin (Kmr) resistance cassette in the middle of the respective coding sequence. For reintegration of the Synechocystis aqpZ (slr2057) gene into the genome of the ΔaqpZ cells, the full-length coding sequence of aqpZ was amplified by PCR using an NdeI site-containing forward primer 5′-CAGTCATATGATGAAAAAGTACATTGCTG-3′ and SalI site-containing reverse primer 5′-CAGTGTCGACTCACTCTGCTTCGGGTTCG-3′. The NdeI-SalI DNA fragment encoding AqpZ was inserted into the corresponding sites in p68TS4OxCm and integrated by homologous recombination into targeting site 4 of the chromosomal DNA of Synechocystis ΔaqpZ cells (21Tsunekawa K. Shijuku T. Hayashimoto M. Kojima Y. Onai K. Morishita M. Ishiura M. Kuroda T. Nakamura T. Kobayashi H. Sato M. Toyooka K. Matsuoka K. Omata T. Uozumi N. J. Biol. Chem. 2009; 284: 16513-16521Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Cultures were grown to a cell density of OD730 = 1.0 and then transferred to fresh BG11 medium either with or without 5 mm glucose. The cells were lysed mechanically by vortexing with glass beads, and the proteins were separated by SDS-PAGE (12.5 or 15% gels) and then transferred to PVDF membranes. The membranes were probed with anti-AqpZ antibody and anti-OpcA antibody (26Sundaram S. Karakaya H. Scanlan D.J. Mann N.H. Microbiology. 1998; 144: 1549-1556Crossref PubMed Scopus (27) Google Scholar, 27Hagen K.D. Meeks J.C. J. Biol. Chem. 2001; 276: 11477-11486Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The membranes were incubated for 1 h with the primary antibody (1:2000 in blocking buffer) and then incubated for 30 min with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000)) and subsequently subjected to chemiluminescence detection. Cells were collected at mid-exponential phase by centrifugation at 5,000 × g for 5 min and then resuspended in BG11 supplemented with 5 mm glucose at an OD730 of 0.05. Glucose uptake was determined by measuring the concentration of glucose in the medium enzymatically using the d-glucose kit (Roche Applied Science, R-Biopharm), following the manufacturer's instructions. Aliquots (200 μl) of the cultures were harvested every 12 h and centrifuged at 1,500 rpm for 2 min, followed by the determination of glucose concentration in the supernatant. Optical microscopy was performed with a microscope (Axioskop FL, Carl Zeiss, Gottingen) equipped with a high definition image capture camera (HC-1000, Fujix, Tokyo) as described previously (21Tsunekawa K. Shijuku T. Hayashimoto M. Kojima Y. Onai K. Morishita M. Ishiura M. Kuroda T. Nakamura T. Kobayashi H. Sato M. Toyooka K. Matsuoka K. Omata T. Uozumi N. J. Biol. Chem. 2009; 284: 16513-16521Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 28Toyooka K. Moriyasu Y. Goto Y. Takeuchi M. Fukuda H. Matsuoka K. Autophagy. 2006; 2: 96-106Crossref PubMed Scopus (91) Google Scholar). The cell size was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD) and beads of a defined diameter (3.005 ± 0.027 μm) as size standards. Cells grown under either photoautotrophic or photomixotrophic conditions were harvested. Cells were fixed in 4% paraformaldehyde and 2% glutaraldehyde in 100 mm cacodylate buffer (pH 7.2) and then postfixed in 1% osmium tetroxide in 50 mm cacodylate buffer. Samples were dehydrated through a graded methanol series, then embedded in Epon812 resin (28Toyooka K. Moriyasu Y. Goto Y. Takeuchi M. Fukuda H. Matsuoka K. Autophagy. 2006; 2: 96-106Crossref PubMed Scopus (91) Google Scholar). The ultrathin sections were stained with uranyl acetate followed by lead citrate solution and examined with a 1010 transmission electron microscope (JEOL) at 80 kV (28Toyooka K. Moriyasu Y. Goto Y. Takeuchi M. Fukuda H. Matsuoka K. Autophagy. 2006; 2: 96-106Crossref PubMed Scopus (91) Google Scholar). Cell size distribution was measured using a particle size distribution analyzer (CDA-1000X, Sysmex). Samples (100 μl) were taken from the cultures at the indicated intervals and diluted 100–500 times with modified physiological saline (0.45% sodium chloride, 144 mosm) prior to performing the measurements. Analysis of the intracellular glycogen content was performed as described elsewhere (29Osanai T. Kanesaki Y. Nakano T. Takahashi H. Asayama M. Shirai M. Kanehisa M. Suzuki I. Murata N. Tanaka K. J. Biol. Chem. 2005; 280: 30653-30659Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The glucose produced by acid hydrolysis was quantified using a colorimetric assay (30Forchhammer K. Tandeau de Marsac N. J. Bacteriol. 1995; 177: 2033-2040Crossref PubMed Google Scholar). Deletion of chromosomal genes in E. coli was performed as described previously (31Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar). E. coli MA02 (glpF::km) and E. coli MA03 (aqpZ::cm/glpF::km) were generated from the E. coli K-12 W3110 strain using the following primer sets: for aqpZ (JW0859) gene disruption, primers ΔEcAqpZ-PS1 (5′-CTATAAAACGACCATATTTTTCACAGGGTCAATTTTTAATTGTGGTGGATGTGTAGGCTGGAGCTGCTTC-3′) and ΔEcAqpZ-PS2 (5′-AACAACATCTTAAAAAAAGGCCTGACATTACGCCAGGCCTTCTGCGTTAACATATGAATATCCTCCTTAGT-3′); for glpF (JW3898) gene disruption, primers ΔEcGlpF-PS1 (5′-GTCCGTGACTTTCACGCATACAACAAACATTAACTCTTCAGGATCCGATTGTGTAGGCTGGAGCTGCTTC-3′) and ΔEcGlpF-PS2 (5′-GCGCAACGATATATTTTTTTTCAGTCATGTTTAATTGTCCCGTAGTCATACATATGAATATCCTCCTTAGT-3′). We removed the kanamycin and chloramphenicol resistance genes from E. coli MA02 and E. coli MA03 to generate strains MA05 (glpF−) and MA06 (aqpZ−/glpF−) using the FLP-mediated recombination method (31Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar). Bacterial strains were routinely plated on LB or M9 agar medium containing 50 mg/liter ampicillin. For overnight liquid cultures, bacterial strains were grown aerobically at 30 °C in M9 modified minimal medium with 0.2% casamino acids, 100 mg/liter ampicillin, and 10 mm maltose. Growth was monitored by measuring the A600 of the cultures in M9 modified medium supplemented with 2 mm glycerol (32Froger A. Rolland J.P. Bron P. Lagrée V. Le Cahérec F. Deschamps S. Hubert J.F. Pellerin I. Thomas D. Delamarche C. Microbiology. 2001; 147: 1129-1135Crossref PubMed Scopus (41) Google Scholar). Cultures were harvested by centrifugation at 15,000 × g at 4 °C for 2 min at the times indicated and washed twice with 1 ml of ice-cold distilled water to remove dead cells, and then cell pellets were frozen in liquid nitrogen. Each sample was extracted, derivatized, and analyzed by gas chromatography-time-of-flight mass spectrometry (GC/TOF-MS) as described (33Jonsson P. Gullberg J. Nordström A. Kusano M. Kowalczyk M. Sjöström M. Moritz T. Anal. Chem. 2004; 76: 1738-1745Crossref PubMed Scopus (297) Google Scholar, 34Jonsson P. Johansson E.S. Wuolikainen A. Lindberg J. Schuppe-Koistinen I. Kusano M. Sjöström M. Trygg J. Moritz T. Antti H. J. Proteome Res. 2006; 5: 1407-1414Crossref PubMed Scopus (96) Google Scholar, 35Kusano M. Fukushima A. Kobayashi M. Hayashi N. Jonsson P. Moritz T. Ebana K. Saito K. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007; 855: 71-79Crossref PubMed Scopus (144) Google Scholar, 36Kusano M. Fukushima A. Arita M. Jonsson P. Moritz T. Kobayashi M. Hayashi N. Tohge T. Saito K. BMC Syst. Biol. 2007; 1: 53Crossref PubMed Scopus (99) Google Scholar). We used lyophilized samples for analysis. Each sample was prepared using 2 mg, dry weight, of cells for GC/TOF-MS analysis. Finally, 22 μg of fresh weight of the derivatized extract was injected into the GC/TOF-MS. Nonprocessed data were exported to the netCDF format using ChormaTOF software (version 3.22, Leco) for further data analysis. The raw data were processed using a custom script as described by Jonsson et al. (33Jonsson P. Gullberg J. Nordström A. Kusano M. Kowalczyk M. Sjöström M. Moritz T. Anal. Chem. 2004; 76: 1738-1745Crossref PubMed Scopus (297) Google Scholar, 34Jonsson P. Johansson E.S. Wuolikainen A. Lindberg J. Schuppe-Koistinen I. Kusano M. Sjöström M. Trygg J. Moritz T. Antti H. J. Proteome Res. 2006; 5: 1407-1414Crossref PubMed Scopus (96) Google Scholar) to perform base-line correction, alignment, and peak deconvolution. Metabolites were identified by comparing their mass spectrum and retention time index with those generated for reference compounds analyzed on our instrumentation as well as those in the MS and retention time index libraries in the Golm Metabolome Database (37Schauer N. Steinhauser D. Strelkov S. Schomburg D. Allison G. Moritz T. Lundgren K. Roessner-Tunali U. Forbes M.G. Willmitzer L. Fernie A.R. Kopka J. FEBS Lett. 2005; 579: 1332-1337Crossref PubMed Scopus (521) Google Scholar, 38Kopka J. Schauer N. Krueger S. Birkemeyer C. Usadel B. Bergmüller E. Dörmann P. Weckwerth W. Gibon Y. Stitt M. Willmitzer L. Fernie A.R. Steinhauser D. Bioinformatics. 2005; 21: 1635-1638Crossref PubMed Scopus (1091) Google Scholar). Analytical bias in metabolite measurements was controlled using cross-contribution-compensating multiple standard normalization (39Redestig H. Fukushima A. Stenlund H. Moritz T. Arita M. Saito K. Kusano M. Anal. Chem. 2009; 81: 7974-7980Crossref PubMed Scopus (131) Google Scholar) with 16 internal standard peaks. Metabolite peaks with more than 30% missing values were removed. Analysis of variance for examining correlation with glycogen levels and cell diameter was fitted for each metabolite. The formula for examining correlation with duration of culture, glycogen content, and cell diameter is shown in Fig. 8A. Model parameters were calculated using LIMMA (40Smyth G.K. Stat. Appl. Genet. Mol. Biol. 2004; 3 (Article3)Crossref PubMed Scopus (9250) Google Scholar) for the statistical environment R project (see the R Project site on the World Wide Web). p values were false discovery rate-adjusted to correct for multiple testing. The model used for studying the effect of genotype, g, glycogen level, d, and cell diameter, p, on the estimated level, m, of metabolite i in sample j was as follows, mi,j=βi+gi,j+di,j+pi,j+g:di,j+g:pi,j+∈i,jEq. 1 where g:d and g:p are the interactions between genotype and glycogen level and between genotype and cell diameter, respectively, β is the intercept, and ∈ is the error. We furthermore considered the following, mi,j=βi+ai,j+ai,j2+a:gi,j+a2:gi,j+∈i,jEq. 2 to examine interaction between the same metabolite levels and the duration of culture a. Cells grown under photoautotrophic or photomixotrophic conditions for 5 days were harvested by centrifugation at 5,000 × g at room temperature and resuspended in 20 mm TES buffer, pH 8.0, at an OD730 = 1.0. Cell suspensions were illuminated with white light at 1000 μmol of photon m−2 s−1, and oxygen evolution was measured using a Clark-type electrode at 25 °C. For the determination of whole chain photosynthetic electron transport activities, cell suspensions were supplemented with 2 mm sodium bicarbonate. PS-II-mediated electron transport activities were measured in the presence of 1 mm 1,4-benzoquinone and 0.8 mm K3Fe(CN)6 i" @default.
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• W2026465513 date "2011-07-01" @default.
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• W2026465513 title "Plasma Membrane Aquaporin AqpZ Protein Is Essential for Glucose Metabolism during Photomixotrophic Growth of Synechocystis sp. PCC 6803" @default.
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• W2026465513 doi "https://doi.org/10.1074/jbc.m111.236380" @default.
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