Two Trk/Ktr/HKT-type potassium transporters, TrkG and TrkH, perform distinct functions in Escherichia coli K-12.

Escherichia coli K-12 possesses two versions of Trk/Ktr/HKT-type potassium ion (K+) transporters, TrkG and TrkH. The current paradigm is that TrkG and TrkH have largely identical characteristics, and little information is available regarding their functional differences. Here, we show using cation uptake experiments with K+ transporter knockout mutants that TrkG and TrkH have distinct ion transport activities and physiological roles. K+-transport by TrkG required Na+, whereas TrkH-mediated K+ uptake was not affected by Na+. An aspartic acid located five residues away from a critical glycine in the third pore-forming region might be involved in regulation of Na+-dependent activation of TrkG. In addition, we found that TrkG but not TrkH had Na+ uptake activity. Our analysis of K+ transport mutants revealed that TrkH supported cell growth more than TrkG; however, TrkG was able to complement loss of TrkH-mediated K+ uptake in E. coli. Furthermore, we determined that transcription of trkG in E. coli was downregulated but not completely silenced by the xenogeneic silencing factor H-NS (histone-like nucleoid structuring protein or heat-stable nucleoid-structuring protein). Taken together, the transport function of TrkG is clearly distinct from that of TrkH, and TrkG seems to have been accepted by E. coli during evolution as a K+ uptake system that coexists with TrkH.

Two cyanobacterial response regulators with diguanylate cyclase activity, Rre2 and Rre8, participate in biofilm formation.

Phototrophic bacteria face diurnal variations of environmental conditions such as light and osmolarity that affect their carbon metabolism and ability to generate organic compounds. The model cyanobacterium, Synechocystis sp. PCC 6803 forms a biofilm when it encounters extreme conditions like high salt stress, but the molecular mechanisms involved in perception of environmental changes that lead to biofilm formation are unknown. Here, we studied two two-component regulatory systems (TCSs) that contain diguanylate cyclases (DGCs), which produce the second messenger c-di-GMP, as potential components of the biofilm-inducing signaling pathway in Synechocystis. Analysis of single mutants provided evidence for involvement of the response regulators, Rre2 and Rre8 in biofilm formation. A bacterial two-hybrid assay showed that Rre2 and Rre8 each formed a TCS with a specific histidine kinase, Hik12 and Hik14, respectively. The in vitro assay showed that Rre2 had DGC activity regardless of its de/phosphorylation status, whereas Rre8 required phosphorylation for DGC activity. Hik14-Rre8 likely functioned as an inducible sensing system in response to environmental change. Biofilm assays with Synechocystis mutants suggested that pairs of hik12-rre2 and hik14-rre8 responded to high salinity-induced biofilm formation. Inactivation of hik12-rre2 and hik14-rre8 did not affect the performance of the light reactions of photosynthesis. These data suggest that Hik12-Rre2 and Hik14-Rre8 participate in biofilm formation in Synechocystis by regulating c-di-GMP production via the DGC activity of Rre2 and Rre8.

Diverse Physiological Functions of Cation Proton Antiporters across Bacteria and Plant Cells.

Membrane intrinsic transport systems play an important role in maintaining ion and pH homeostasis and forming the proton motive force in the cytoplasm and cell organelles. In most organisms, cation/proton antiporters (CPAs) mediate the exchange of K+, Na+ and Ca2+ for H+ across the membrane in response to a variety of environmental stimuli. The tertiary structure of the ion selective filter and the regulatory domains of Escherichia coli CPAs have been determined and a molecular mechanism of cation exchange has been proposed. Due to symbiogenesis, CPAs localized in mitochondria and chloroplasts of eukaryotic cells resemble prokaryotic CPAs. CPAs primarily contribute to keeping cytoplasmic Na+ concentrations low and controlling pH, which promotes the detoxification of electrophiles and formation of proton motive force across the membrane. CPAs in cyanobacteria and chloroplasts are regulators of photosynthesis and are essential for adaptation to high light or osmotic stress. CPAs in organellar membranes and in the plasma membrane also participate in various intracellular signal transduction pathways. This review discusses recent advances in our understanding of the role of CPAs in cyanobacteria and plant cells.

DAY-LENGTH-DEPENDENT DELAYED-GREENING1, the Arabidopsis Homolog of the Cyanobacterial H+-Extrusion Protein, Is Essential for Chloroplast pH Regulation and Optimization of Non-Photochemical Quenching.

Plants convert solar energy into chemical energy through photosynthesis, which supports almost all life activities on earth. Because the intensity and quality of sunlight can change dramatically throughout the day, various regulatory mechanisms help plants adjust their photosynthetic output accordingly, including the regulation of light energy accumulation to prevent the generation of damaging reactive oxygen species. Non-photochemical quenching (NPQ) is a regulatory mechanism that dissipates excess light energy, but how it is regulated is not fully elucidated. In this study, we report a new NPQ-regulatory protein named Day-Length-dependent Delayed-Greening1 (DLDG1). The Arabidopsis DLDG1 associates with the chloroplast envelope membrane, and the dldg1 mutant had a large NPQ value compared with wild type. The mutant also had a pale-green phenotype in developing leaves but only under continuous light; this phenotype was not observed when dldg1 was cultured in the dark for ≥8 h/d. DLDG1 is a homolog of the plasma membrane-localizing cyanobacterial proton-extrusion-protein A that is required for light-induced H+ extrusion and also shows similarity in its amino-acid sequence to that of Ycf10 encoded in the plastid genome. Arabidopsis DLDG1 enhances the growth-retardation phenotype of the Escherichia coli K+/H+ antiporter mutant, and the everted membrane vesicles of the E. coli expressing DLDG1 show the K+/H+ antiport activity. Our findings suggest that DLDG1 functionally interacts with Ycf10 to control H+ homeostasis in chloroplasts, which is important for the light-acclimation response, by optimizing the extent of NPQ.

A long natural-antisense RNA is accumulated in the conidia of Aspergillus oryzae.

Analysis of expressed sequence tag libraries from various culture conditions revealed the existence of conidia-specific transcripts assembled to putative conidiation-specific reductase gene (csrA) in Aspergillus oryzae. However, the all transcripts were transcribed with opposite direction to the gene csrA. The sequence analysis of the transcript revealed that the RNA overlapped mRNA of csrA with 3'-end, and did not code protein longer than 60 amino acid residues. We designated the transcript Conidia Specific Long Natural-antisense RNA (CSLNR). The real-time PCR analysis demonstrated that the CSLNR is conidia-specific transcript, which cannot be transcribed in the absence of brlA, and the amount of CSLNR was much more than that of the transcript from csrA in conidia. Furthermore, the csrA deletion, also lacking coding region of CSLNR in A. oryzae reduced the number of conidia. Overexpression of CsrA demonstrated the inhibition of growth and conidiation, while CSLNR did not affect conidiation.

Instantaneous extracellular solution exchange for concurrent evaluation of membrane permeability of single cells.

The osmotic stress imposed on microorganisms by hypotonic conditions is perceived to regulate water and solute flux via cell membranes, which are crucial for survival. Some cells that fail to perceive osmotic stress die because this results in the rupture of the cell membrane. The flux through the membrane is characterized by the membrane permeability, which is measured using a stopped-flow apparatus in response to a millisecond-order osmolarity change. However, the obtained data are an ensemble average of each cell response. Additionally, the measurement of permeability, considering cellular viability, contributes to a more accurate evaluation of osmoadaptation. Here, we present a novel on-chip instantaneous extracellular solution exchange method using an air-liquid interface. The presented method provides a concurrent evaluation at the single-cell level in response to a millisecond-order osmotic shock, considering cellular viability by solution exchange. This method utilizes a liquid bridge with a locally formed droplet on the surface of a micropillar fabricated inside a microchannel. We evaluated a solution exchange time of 3.6 ms and applied this method to Synechocystis PCC 6803 under two different osmolarity conditions. The live/dead ratio of 1 M to 0.5 M osmotic down shock condition was 78.8/21.2% while that of 1 M to 0.25 M osmotic down shock condition was 40.0/60.0%. We evaluated the water permeability of two groups: cells that were still live before and after osmotic shock (hereafter named cell type 1), and cells that were live before but were dead 10 minutes after osmotic shock (hereafter named cell type 2). The results indicated that the water permeability of cell type 2 was higher than that of cell type 1. The results obtained using the presented methods confirmed that the effect of osmotic stress can be accurately evaluated using single-cell analysis.

Integration of Microfluidic Chip and Probe with a Dual Pump System for Measurement of Single Cells Transient Response.

The integration of liquid exchange and microfluidic chips plays a critical role in the biomedical and biophysical fields as it enables the control of the extracellular environment and allows for the simultaneous stimulation and detection of single cells. In this study, we present a novel approach for measuring the transient response of single cells using a system integrated with a microfluidic chip and a probe with a dual pump. The system was composed of a probe with a dual pump system, a microfluidic chip, optical tweezers, an external manipulator, an external piezo actuator, etc. Particularly, we incorporated the probe with the dual pump to allow for high-speed liquid change, and the localized flow control enabled a low disturbance contact force detection of single cells on the chip. Using this system, we measured the transient response of the cell swelling against the osmotic shock with a very fine time resolution. To demonstrate the concept, we first designed the double-barreled pipette, which was assembled with two piezo pumps to achieve a probe with the dual pump system, allowing for simultaneous liquid injection and suction. The microfluidic chip with on-chip probes was fabricated, and the integrated force sensor was calibrated. Second, we characterized the performance of the probe with the dual pump system, and the effect of the analysis position and area of the liquid exchange time was investigated. In addition, we optimized the applied injection voltage to achieve a complete concentration change, and the average liquid exchange time was achieved at approximately 3.33 ms. Finally, we demonstrated that the force sensor was only subjected to minor disturbances during the liquid exchange. This system was utilized to measure the deformation and the reactive force of Synechocystis sp. strain PCC 6803 in osmotic shock, with an average response time of approximately 16.33 ms. This system reveals the transient response of compressed single cells under millisecond osmotic shock which has the potential to characterize the accurate physiological function of ion channels.

The HKT1 Na+ transporter protects plant fertility by decreasing Na+ content in stamen filaments.

Salinity stress can greatly reduce seed production because plants are especially sensitive to salt during their reproductive stage. Here, we show that the sodium ion transporter AtHKT1;1 is specifically expressed around the phloem and xylem of the stamen in Arabidopsis thaliana to prevent a marked decrease in seed production caused by salt stress. The stamens of AtHKT1;1 mutant under salt stress overaccumulate Na+, limiting their elongation and resulting in male sterility. Specifically restricting AtHKT1;1 expression to the phloem leads to a 1.5-fold increase in the seed yield upon sodium ion stress. Expanding phloem expression of AtHKT1;1 throughout the entire plant is a promising strategy for increasing plant productivity under salinity stress.

Hik36-Hik43 and Rre6 act as a two-component regulatory system to control cell aggregation in Synechocystis sp. PCC6803.

In response to environmental stress the model cyanobacterium, Synechocystis sp. PCC6803 can switch from a planktonic state to autoaggregation and biofilm formation. The precise mechanism of this transition remains unknown. Here we investigated the role of a candidate two-component regulatory system (TCS) in controlling morphological changes, as a way to understand the intermediate molecular steps that are part of the signaling pathway. A bacterial two-hybrid assay showed that the response regulator Rre6 formed a TCS together with a split histidine kinase consisting of Hik36 and Hik43. Individual disruption mutants displayed autoaggregation in a static culture. In contrast, unlike in the wild type, high salinity did not induce biofilm formation in Δhik36, Δhik43 and Δrre6. The expression levels of exopolysaccharide (EPS) production genes were higher in Δhik36 and Δhik43, compared with the wild type, but lower in Δrre6, suggesting that the TCS regulated EPS production in Synechocystis. Rre6 interacted physically with the motor protein PilT2, that is a component of the type IV pilus system. This interaction was enhanced in a phosphomimic version of Rre6. Taken together, Hik36-Hik43-Rre6 function as an upstream component of the pili-related signal transduction cascade and control the prevention of cell adhesion and biofilm formation.

Evidence for potassium transport activity of Arabidopsis KEA1-KEA6.

Arabidopsis thaliana contains the putative K+ efflux transporters KEA1-KEA6, similar to KefB and KefC of Escherichia coli. KEA1-KEA3 are involved in the regulation of photosynthetic electron transport and chloroplast development. KEA4-KEA6 mediate pH regulation of the endomembrane network during salinity stress. However, the ion transport activities of KEA1-KEA6 have not been directly characterized. In this study, we used an E. coli expression system to examine KEA activity. KEA1-KEA3 and KEA5 showed bi-directional K+ transport activity, whereas KEA4 and KEA6 functioned as a K+ uptake system. The thylakoid membrane-localized Na+/H+ antiporter NhaS3 from the model cyanobacterium Synechocystis is the closest homolog of KEA3. Changing the putative Na+/H+ selective site of KEA3 (Gln-Asp) to that of NhaS3 (Asp-Asp) did not alter the ion selectivity without loss of K+ transport activity. The first residue in the conserved motif was not a determinant for K+ or Na+ selectivity. Deletion of the possible nucleotide-binding KTN domain from KEA3 lowered K+ transport activity, indicating that the KTN domain was important for this function. The KEA3-G422R mutation discovered in the Arabidopsis dpgr mutant increased K+ transport activity, consistent with the mutant phenotype. These results indicate that Arabidopsis KEA1-KEA6 act as K+ transport systems, and support the interpretation that KEA3 promotes dissipation of ΔpH in the thylakoid membrane.