Na؉ /H ؉ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na ؉ /H؉ antiporters, nhaS1-5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na ؉ /H ؉ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na ؉ concentrations. NhaS3 had no K ؉ /H ؉ exchange activity itself but enhanced K ؉ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO 2 to high CO 2 conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO 2 consumption caused by the ceasing of O 2 -evolving photosynthesis. This is the first report of a Na

Elucidation of the structure-function relationship of a small number of prokaryotic ion channels characterized so far greatly contributed to our knowledge on basic mechanisms of ion conduction. We identified a new potassium channel (SynK) in the genome of the cyanobacterium Synechocystis sp. PCC6803, a photosynthetic model organism. SynK, when expressed in a K+-uptake-system deficient E.coli strain, was able to recover growth of these organisms. The protein functions as a potassium selective ion channel when expressed in Chinese Hamster Ovary cells. The location of SynK in cyanobacteria in both thylakoid and plasmamembranes was revealed by immunogold electron microscopy and Western blotting of isolated membrane fractions. SynK seems to be conserved during evolution, giving rise to a TPK (two-pore K+ channel) family member which is shown here to be located in the thylakoid membrane of Arabidopsis. Our work characterizes a novel cyanobacterial potassium channel and indicates the molecular nature of the first higher plant thylakoid cation channel, opening the way to functional studies.

f Photoautotrophic bacteria have developed mechanisms to maintain K ؉ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K ؉ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K ؉ (Kdp). The contributions of each of these K ؉ transport systems to cellular K ؉ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K ؉ uptake, albeit with lower rates than were conferred by Ktr. An onchip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K ؉ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K ؉ depletion. Correspondingly, Kdp-mediated K ؉ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K ؉ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K Living cells have developed specific responses to hyperosmotic shock. Upon exposure to this stress, cells initially lose water and their volume shrinks. In all living cells, K ϩ is the major intracellular cation used for the maintenance of turgor pressure, cytosolic osmolarity, protein structuring, and membrane potential (1-3). In contrast to animals, Na ϩ /K ϩ ATP pumps are generally missing in bacteria and plants. Hence, these cells possess K ϩ uptake transporters to supply K ϩ to the cells. Particularly after hyperosmotic stress, cells quickly take up K ϩ from the medium to increase the intracellular osmolarity, which prevents water efflux from the cell. Data from genetic and biochemical experiments indicate that the activity and the expression of these transporters respond to hyperosmotic stress. In the later phase of acclimation to hyperosmotic stress, cells also induce the synthesis of osmoprotective molecules, such as glutamate, trehalose, proline, and glucosylglycerol (4, 5). Despite an increasing amount of data on cellular osmoregulation involving ion flux across the membrane, direct evidence for the involvement of specific transporters in the cellular response to osmotic up-shock is lacking for photoautotrophic organisms.The cyanobacterium Synechocystis sp. strain PCC 6803 (hereinafter referred to as Synechocystis) is a frequently used unicellular photosynthetic prokaryote that can survive under a wide range of environmental conditions (6). Unlike Escherichia coli, Synechocystis possesses an internal thylakoid membrane system, which...

The moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 contains a plasma membrane aquaporin, AqpZ. We previously reported that AqpZ plays a role in glucose metabolism under photomixotrophic growth conditions, suggesting involvement of AqpZ in cytosolic osmolarity homeostasis. To further elucidate the physiological role of AqpZ, we have studied its gene expression profile and its function in Synechocystis. The expression level of aqpZ was regulated by the circadian clock. AqpZ activity was insensitive to mercury in Xenopus oocytes and in Synechocystis, indicating that the AqpZ can be categorized as a mercury-insensitive aquaporin. Stopped-flow light-scattering spectrophotometry showed that addition of sorbitol and NaCl led to a slower decrease in cell volume of the Synechocystis ⌬aqpZ strain than the wild type. The ⌬aqpZ cells were more tolerant to hyperosmotic shock by sorbitol than the wild type. Consistent with this, recovery of oxygen evolution after a hyperosmotic shock by sorbitol was faster in the ⌬aqpZ strain than in the wild type. In contrast, NaCl stress had only a small effect on oxygen evolution. The amount of AqpZ protein remained unchanged by the addition of sorbitol but decreased after addition of NaCl. This decrease is likely to be a mechanism to alleviate the effects of high salinity on the cells. Our results indicate that Synechocystis AqpZ functions as a water transport system that responds to daily oscillations of intracellular osmolarity.

Synechocystis sp strain PCC 6803 contains one gene encoding a putative large conductance mechanosensitive channel homolog [named SyMscL (slr0875)]. However, it is unclear whether SyMscL contributes to the adaptation to hypoosmotic stress in Synechocystis. Here we report the in vivo characteristics of SyMscL. SyMscL was mainly expressed in the plasma membrane of Synechocystis. Cell volume monitoring using stopped-flow spectrophotometry showed that ΔsymscL cells swelled more rapidly than wild-type cells under hypoosmotic stress conditions. Expression of symscL was under circadian control, and its peak corresponded to the beginning of subjective night. These results indicate that SyMscL functioned as one component of the osmotic homeostatic regulatory system of the cell coordinating the response of Synechocystis to daily metabolic osmotic fluctuations and environmental changes.