The major objectives of this work were cloning, functional expression and primary biochemical characterization of Yp-NhaP, putative sodium-proton antiporter from the dangerous human pathogen Yersinia pestis. We expressed Yp-NhaP in its functional form in the antiporter-deficient strain of E. coli, TO114. When assayed in inside-out sub-bacterial membrane vesicles, Yp-NhaP acted as an electroneutral cation/proton antiporter, exchanging Ca2+, K+, Na+ and Li+ ions for H+. Competition experiments suggested that in vivo Yp-NhaP operates as Ca2+/H+ and, possibly, Ca2+/Na+ antiporter rather than K+/H+ or Na+/H+ antiporter. Ca2+/H+ and Li+/H+ antiport catalyzed by Yp-NhaP peaked at pH close to 8.0, while K+/H+ and Na+/H+ antiport were smoothly increasing from pH 6.5 to pH 9.0. We also observed inhibition by the excess of substrate in the case of Ca2+/H+ and Li+/H+ antiport mediated by Yp-NhaP. As expected, chromosomal deletion of Yp-nhaP gene did not affect resistance of Y. pestis cells to alkali cations.

The major objectives of this work were cloning, functional expression and primary biochemical characterization of Yp-NhaP, putative sodium-proton antiporter from the dangerous human pathogen Yersinia pestis. We expressed Yp-NhaP in its functional form in the antiporter-deficient strain of E. coli, TO114. When assayed in inside-out sub-bacterial membrane vesicles, Yp-NhaP acted as an electroneutral cation/proton antiporter, exchanging Ca2+, K+, Na+ and Li+ ions for H+. Competition experiments suggested that in vivo Yp-NhaP operates as Ca2+/H+ and, possibly, Ca2+/Na+ antiporter rather than K+/H+ or Na+/H+ antiporter. Ca2+/H+ and Li+/H+ antiport catalyzed by Yp-NhaP peaked at pH close to 8.0, while K+/H+ and Na+/H+ antiport were smoothly increasing from pH 6.5 to pH 9.0. We also observed inhibition by the excess of substrate in the case of Ca2+/H+ and Li+/H+ antiport mediated by Yp-NhaP. As expected, chromosomal deletion of Yp-nhaP gene did not affect resistance of Y. pestis cells to alkali cations.

Degree Not Listed / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Anaplasmataceae family rickettsial bacteria are mostly vector-transmitted pathogens causing important diseases in several vertebrates, including humans, canines, and ruminants. Ehrlichia chaffeensis, a tick-transmitted intraphagosomal rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis (HME). Little is known about how this and other related rickettsial organisms are able to reside and replicate within an acidified phagosome environment. Similarly, it is unclear how the infectious form of the bacterium maintains pH homeostasis in the extracellular milieu where the pH is about 7.35-7.45, before its infection to a naïve host cell. Sodium/cation: proton antiporters are integral membrane proteins reported from a wide range of species. They exchange sodium or other monovalent cations against protons across a plasma membrane in maintaining the cytoplasmic pH of a cell. We recently described a mutation within the Ech_0379 gene of E. chaffeensis that is predicted to encode for a Na+/H+ antiporter protein. The mutation caused the attenuated growth of the organism in vertebrate hosts, resulting in a reduced level of the bacterial presence in the circulation. In this study, we evaluated several antiporter protein genes of E. chaffeensis. Its genome contains 10 coding sequences encoding for polypeptides which may form at least six functional proteins. To define their function, a sodium sensitive Escherichia coli strain having a mutation in two of its three antiporter protein genes (EP432) is used to carry out the functional complementation assay with E. chaffeensis genes from their respective promoters. The EP432 strain has a growth defect during its replication in the presence of NaCl that can be restored with functional complementation. All six E. chaffeensis genes could complement the growth defect of EP432 under acidic pH, while Ech_0379 and Ech_0179 also complemented at basic pH. Ech_0179 complemented at neutral pH as well. The complementation of all genes at neutral and basic pHs, except Ech_0179 and Ech_0379, made EP432 E. coli strain be more sensitive to the presence of 200 mM NaCl. The channeling activity is verified independently by constructing a proteoliposome in vitro with the recombinant protein Ech_0379. The recombinant protein showed antiporter activity at all three pHs in the presence of 100 or 200 mM NaCl when assessed using the recombinant proteoliposome. This research is the first description of antiporter proteins of E. chaffeensis.

No description available.

Background: In Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction. Here, we studied the expression patterns and regulation of CNGC19 and CNGC20, which constitute one of the five CNGC subfamilies. Results: GUS, GFP and luciferase reporter assays were used to study the expression of CNGC19 and CNGC20 genes from Arabidopsis thaliana in response to developmental cues and salt stress. CNGC19 and CNGC20 were differentially expressed in roots and shoots. The CNGC19 gene was predominantly active in roots already at early growth stages. Major expression was observed in the phloem. CNGC20 showed highest promoter activity in mesophyll cells surrounding the veins. Its expression increased during development and was maximal in mature and senescent leaves. Both genes were upregulated in the shoot in response to elevated NaCl but not mannitol concentrations. While in the root, CNGC19 did not respond to changes in the salt concentration, in the shoot it was strongly upregulated in the observed time frame (6-72 hours). Salt-induction of CNGC20 was also observed in the shoot, starting already one hour after stress treatment. It occurred with similar kinetics, irrespective of whether NaCl was applied to roots of intact plants or to the petiole of detached leaves. No differences in K and Na contents of the shoots were measured in homozygous T-DNA insertion lines for CNGC19 and CNGC20, respectively, which developed a growth phenotype in the presence of up to 75 mM NaCl similar to that of the wild type. Conclusion: Together, the results strongly suggest that both channels are involved in the salinity response of different cell types in the shoot. Upon salinity both genes are upregulated within hours. CNGC19 and CNGC20 could assist the plant to cope with toxic effects caused by salt stress, probably by contributing to a re-allocation of sodium within the plant.

Large, membrane-bound vacuoles of plant cells are suitable organelles for the compartmentation of ions. These vacuoles contain Na+/H+ antiporters for movement of Na+ within the organelle in exchange for H+. They provide an efficient mechanism to prevent the occurance of detrimental outcomes of Na+ accumulation in the cytosol. Identification of AtNHX1 gene that confers resistance to salinity by expressing a Na+/H+ antiport pump facilitates the understanding of the salt stress tolerance mechanisms of plants. The aim of the present study was to isolate and clone the Arabidopsis thaliana AtNHX1 coding sequence for transformation of Nicotiana tabacum plants via Agrobacterium tumefaciens mediated gene transfer. For this purpose, total RNA was isolated from Arabidopsis thaliana plants and cDNA synthesis was performed. AtNHX1 (1614bp) was amplified by using cDNA of Arabidopsis via specific primers. The amplified PCR product was verified by sequencing. AtNHX1 coding sequence was cloned into the plant transformation vector pCVB1 and 10 independent putative transgenic tobacco plants were obtained via Agrobacterium tumefaciens mediated gene transfer sysytem. Transfer of selected 8 putative transgenic plants to soil provided the regeneration of T1 seeds. Germination of the seeds under different salt treatments (0, 50, 100, 150, 200, 250 mM NaCl) was observed for evaluating the salt tolerance of transformed plants. The 82% and 60% of the transgenic T1 seeds were germinated on 150 mM NaCl and 200 mM NaCl containing media, respectively. In contrast the germination percentage of wild type tobacco seeds under 150 mM NaCl and 200 mM NaCl concentrations were 39% and 21%, respectively. The germination rate of the transgenic T1 seeds were significantly higher (p=0,001) when compared to the control seeds especially under high salt stress conditions (150 and 200 mM NaCl). Taken all together, our results demonstrated that the germination efficiencies and growth of the plants transformed with AtNHX1 were higher than the wild type tobacco plants under high salt concentrations.

The pathogen Vibrio cholerae uses cations as a primary currency of virulence and environmental persistence, using gradients of those cations to move, acquire nutrients, and control virulence gene expression. An understanding of the overlapping roles of bioenergetics and chemotaxis in the virulence and environmental survival of V. cholerae issues from a large body of prior work, but the interplay of each component is not yet clearly understood. To this end, the activity of the antiporters Vc-NhaP1, Vc-NhaA, and Vc-NhaB was assayed, as was the sodium transporting respiratory pump NQR, and environmental stimuli were paired with potential motilitylinked sensors. The Vc-NhaP1 antiporter was found to be a K⁺(Na⁺)/H⁺ antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-logarithmic phase, after 4-6 hours of culturing. Using a pH-sensitive GFP protein, cytosolic pH was shown to be dependent on K⁺ in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless E. coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K⁺ or Na⁺ ions for protons within a broad pH range (7.25 to 9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K⁺(Na⁺)/H⁺ antiporter of the CPA- 1 family that is required for bacterial pH homeostasis and growth in an acidic environment. Further, a model system comprised of a V. cholerae strain lacking both the nqr operon and the ORFs of Vc-nhaA or Vc-nhaB was generated and tested with and without lactate. These strains, along with the single mutants of nqr, Vc-nhaA, and Vc-nhaB, were assessed for aerobic growth as a function of media pH and cation concentration (Na⁺, Li⁺, or K⁺). Loss of Vc-NhaA and, to a lesser extent, Vc-NhaB, was better observed when NQR was absent but lactate was added to facilitate replenishment of the quinone pool. Loss of Vc-NhaA in this background inhibited growth most at basic pH under increasing Na⁺ and Li⁺ conditions, and loss of Vc- NhaB in this background inhibited was most severe in acidic conditions in the presence of 0-100 mM Na⁺ or Li⁺. We also observed the growth inhibition of Vc- NhaA in the absence of NQR and in the presence of lactate and 100-450 mM Li⁺, which has not been previously reported. These growth defects were restored upon expression of the cognate antiporter gene on an inducible expression vector. Lastly, potential chemotaxis stimuli were correlated with cognate methyl-accepting chemotaxis protein (MCP) receptors. The homology of MCP sensory domains among Vibrionaceae demonstrated a subset were unique to V. cholerae. Of these unique MCPs, transposon insertion in VC0098 significantly reduced chemotaxis swarm diameter towards Na⁺ and K⁺. Additionally, the MCP VCA0663 was shown, by transposon mutagenesis and complementation, to direct chemotaxis towards N-acetylglucosamine. Additional observations are described concerning the chemotaxis defects incurred by transposon mutagenesis of MCPs in vitro towards mucin, bile, or L-serine. MCP strains were also tested in vivo for 4 and 24 hours in the infant mouse model of infection. None of the observed chemotaxis defects showed complete loss of chemotaxis by transposon mutagenesis, in line with the hypothesis that the large number of MCPs encoded by V. cholerae result in redundant chemotaxis sensory functions. These findings add to the understanding of how bioenergetics and chemotaxis interact within V. cholerae, a foundation from which the bacterium can be understood and, eventually, controlled. / Graduation date: 2012

Chapter 1: “Role of cyclic electron transport around photosystem I in the regulation of proton motive force” is based on the following paper. Caijuan Wang, Hiroshi Yamamoto, Toshiharu Shikanai, Role of cyclic electron transport around photosystem I in regulating proton motive force, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1847, Issue 9, September 2015, Pages 931-938, ISSN 0005-2728, http://dx.doi.org/10.1016/j.bbabio.2014.11.013.(http://www.sciencedirect.com/science/article/pii/S0005272814006586) / 京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19535号 / 理博第4195号 / 新制||理||1602(附属図書館) / 32571 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 教授 西村 いくこ, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

3 Abstract Yeast Saccharomyces cerevisiae belongs to important models for alkali-metal-cation homeostasis research. As other cells, certain intracellular content of K+ is necessary for S. cerevisiae, but Na+ or other alkali metal cations (Li+ , Rb+ ) are toxic for yeast cells. Uniporters Trk1 and Trk2 are responsible for K+ accumulation, while efflux of Na+ , Li+ , Rb+ and K+ is ensured by Ena ATPases, Na+ (K+ )/H+ antiporter Nha1 and K+ specific channel Tok1. Several regulators of K+ (Na+ ) transporters are already known, but reciprocal regulation between transporters and overall picture of the maintenance of alkali-metal-cation homeostasis is still unclear. In this work, K+ circulation (simultaneous uptake and export of K+ ) was shown to be important in alkali-metal-cation homeostasis maintenance. K+ circulation is maintained using reciprocal regulation and interactions between K+ exporters and importers. Though obtained results showed that the alkali-metal-cation homeostasis and associated physiological parameters (e.g. membrane potential, cell size, salt sensitivity) are strain specific, Nha1p was verified to be important for cell survival in ever-changing natural environment. Furthermore, two novel positive regulators of Nha1p activity were found, 14-3-3 proteins and Cka1 kinase. 14-3-3 proteins...

Os receptores medeiam a maioria das respostas fisiológicas em resposta a diversidade de estímulos. A ativação da sinalização mediada pelo receptor de angiotensina II tipo 1 é o principal responsável pelos efeitos do hormônio angiotensina II (Ang II) nos tecidos alvo. No rim concentrações fisiológicas de Ang II aumentam a atividade no túbulo proximal da isoforma 3 do trocador de Na+/H+ (NHE3). Este efeito é crucial para a manutenção do volume extracelular e pressão arterial. Evidências recentes mostraram que a ativação seletiva da sinalização enviesada da beta-arrestina/ receptor AT1 induz diurese e natriurese independentemente da sinalização via proteína G. Neste estudo testamos a hipótese de que a sinalização enviesada do receptor AT1/ beta-arrestina inibe a atividade do NHE3 no túbulo proximal, bem como investigar os possíveis mecanismos moleculares que medeio este efeito. Para tal, nós determinamos os efeitos do composto TRV120023, que se liga ao receptor AT1, bloqueando o acoplamento da proteína G e estimulando a sinalização da beta-arrestina, na função do NHE3 in vivo e in vitro. A atividade do NHE3 foi medida quer em túbulo proximal nativo, por meio de microperfusão estacionária, bem como em uma linha celular de túbulo proximal de gamba (OKP), por meio de recuperação de pH intracelular dependente de Na+. Os nossos resultados mostram que o TRV120023 na concentração de 10-7 M inibe marcadamente a atividade do NHE3 em túbulo proximal quer in vivo quer in vitro, sendo que este efeito é completamente abolido nas células silenciadas para a beta-arrestina 1 e 2 através de RNA de interferência. Adicionalmente, a estimulação do NHE3 pela Ang II é completamente suprimida pelo TRV120023 quer in vivo quer in vitro. A inibição do NHE3 pelo TRV120023 foi associada com a diminuição do NHE3 expresso na superfície da membrana plasmática em células OKP e com a redistribuição entre o corpo e a base das microvilosidades em túbulo proximal de rato. A diminuição do NHE3 na superfície da membrana plasmática em células OKP estava associado com um aumento na internalização do NHE via endocitose mediada por clatrina. A inibição do NHE3 mediada pela beta-arrestina não envolve a sinalização do receptor AT2, cAMP/ PKA, Akt e ERK1/2. Estes achados indicam que a sinalização enviesada do receptor AT1/beta-arretina inibe a atividade do NHE3 em túbulo proximal, pelo menos em parte, devido a alterações na localização subcelular do NHE3 / Cell surface receptors mediate most of our physiological responses to an array of stimulus. The triggering of the angiotensin II type I (AT1) receptor signaling is the major control point in the regulation of the ultimate effects of the peptide hormone angiotensin II (Ang II) on its target tissue. In the kidney physiological concentrations of Ang II upregulate the activity of proximal tubule Na+/H+ exchanger isoform 3 (NHE3). This effect is crucial for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the betaarrestin-biased AT1 receptor signalingpathway induces diuresis and natriuresis independent of G-protein mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule as well as investigate the underlying molecular mechanisms mediating this effect. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G protein coupling, and stimulates beta-arrestin signaling, on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na+-dependent intracellular pH recovery. Our results showed that 10-7 MTRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro, and the effect was completely abolished in OKP cells silenced for beta-arrestin 1 and 2 by small interference RNA. Additionally, stimulation of NHE3 by Ang II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. The decreased surface NHE3 in OKP cells was associated with an increase in NHE3 internalization via clathrin mediated endocytic. Beta-arrestin mediated NHE3 inhibition did not involve AT2 receptor, cAMP/ PKA, Akt and ERK1/2 signaling. These findings indicate that biased signaling of the AT1 receptor/beta-arrestin pathway inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization