Hyperosmotic Conditions Research Articles

Drinking and Water Handling in the Medaka Intestine: A Possible Role of Claudin-15 in Paracellular Absorption?

When euryhaline fish move between fresh water (FW) and seawater (SW), the intestine undergoes functional changes to handle imbibed SW. In Japanese medaka, the potential transcellular aquaporin-mediated conduits for water are paradoxically downregulated during SW acclimation, suggesting paracellular transport to be of principal importance in hyperosmotic conditions. In mammals, intestinal claudin-15 (CLDN15) forms paracellular channels for small cations and water, which may participate in water transport. Since two cldn15 paralogs, cldn15a and cldn15b, have previously been identified in medaka, we examined the salinity effects on their mRNA expression and immunolocalization in the intestine. In addition, we analyzed the drinking rate and intestinal water handling by adding non-absorbable radiotracers, 51-Cr-EDTA or 99-Tc-DTPA, to the water. The drinking rate was >2-fold higher in SW than FW-acclimated fish, and radiotracer experiments showed anterior accumulation in FW and posterior buildup in SW intestines. Salinity had no effect on expression of cldn15a, while cldn15b was approximately 100-fold higher in FW than SW. Despite differences in transcript dynamics, Cldn15a and Cldn15b proteins were both similarly localized in the apical tight junctions of enterocytes, co-localizing with occludin and with no apparent difference in localization and abundance between FW and SW. The stability of the Cldn15 protein suggests a physiological role in water transport in the medaka intestine.

Cover Image, Volume 100, Issue 5

The cover image is based on the Research Article Betaine addition as a potent ruminal fermentation modulator under hyperthermal and hyperosmotic conditions in vitro by Mubarik Mahmood et al., https://doi.org/10.1002/jsfa.10255. image

SLC26A7 protein is a chloride/bicarbonate exchanger and its abundance is osmolarity- and pH-dependent in renal epithelial cells

Acid-secreting intercalated cells of the collecting duct express the chloride/bicarbonate kidney anion exchanger 1 (kAE1) as well as SLC26A7, two proteins that colocalize in the basolateral membrane. The latter protein has been reported to function either as a chloride/bicarbonate exchanger or a chloride channel. Both kAE1 and SLC26A7 are detected in the renal medulla, an environment hyper-osmotic to plasma. Individuals with mutations in the SLC4A1 gene encoding kAE1 and mice lacking Slc26a7 develop distal renal tubular acidosis (dRTA). Here, we aimed to (i) confirm that SLC26A7 can function as chloride/bicarbonate exchanger in Madin-Darby canine kidney (MDCK) cells, and (ii) examine the behavior of SLC26A7 relative to kAE1 wild type or carrying the dRTA mutation R901X in iso- or hyper-osmotic conditions mimicking the renal medulla. Although we found that SLC26A7 abundance increases in hyper-osmotic growth medium, it is reduced in low pH growth conditions mimicking acidosis when expressed at high levels in MDCK cells. In these cells, SLC26A7 exchange activity was independent from extracellular osmolarity. When SLC26A7 protein was co-expressed with kAE1 WT or the R901X dRTA mutant, the cellular chloride/bicarbonate exchange rate was not additive compared to when proteins are expressed individually, possibly reflecting a decreased overall protein expression. Furthermore, the cellular chloride/bicarbonate exchange rate was osmolarity-independent. Together, these results show that (i) in MDCK cells, SLC26A7 is a chloride/bicarbonate exchanger whose abundance is up-regulated by high osmolarity growth medium and (ii) acidic extracellular pH decreases the abundance of SLC26A7 protein.

Claudins of sea lamprey (Petromyzon marinus) - organ-specific expression and transcriptional responses to water of varying ion content.

The role of lamprey epithelium tight junctions (TJs) in the regulation of salt and water balance is poorly understood. This study reported on claudin (Cldn) TJ protein transcripts of pre-metamorphic larval and post-metamorphic juvenile sea lamprey (Petromyzon marinus) and the transcriptional response of genes encoding Cldns to changed environmental ion levels. Transcripts encoding Cldn-3b, -4, -5, -10, -14, -18 and -19 were identified, and mRNA expression profiles revealed the organ-specific presence of cldn-5 and -14, broad expression of cldn-3b, -4, -10, -18 and -19 and spatial differences in the mRNA abundance of cldn-4, -3b and -14 along the ammocoete intestine. Expression profiles were qualitatively similar in ammocoetes and juvenile fishes. Transcript abundance of genes encoding Cldns in osmoregulatory organs (gill, kidney, intestine and skin) was subsequently investigated after exposure of ammocoetes to ion-poor water (IPW) and juveniles to hyperosmotic conditions [60% sea water (SW)]. IPW-acclimated ammocoetes increased mRNA abundance of nearly all cldns in the gill. Simultaneously, cldn-10 abundance increased in the skin, whereas cldn-4, -14 and -18 decreased in the kidney. Ammocoete cldn mRNA abundance in the intestine was altered in a region-specific manner. In contrast, cldn transcript abundance was mostly downregulated in osmoregulatory organs of juvenile fish acclimated to SW - cldn-3b, -10 and -19 in the gill; cldn-3b, -4, -10 and -19 in the skin; cldn-3b in the kidney; and cldn-3b and -14 in the intestine. Data support the idea that Cldn TJ proteins play an important role in the osmoregulatory physiology of pre- and post-metamorphic sea lamprey and that Cldn participation can occur across organs, in an organ-specific manner, as well as differ spatially within organs, which contributes to the regulation of salt and water balance in these fishes.

Deletion of Von Hippel-Lindau Interferes with Hyper Osmolality Induced Gene Expression and Induces an Unfavorable Gene Expression Pattern.

Loss of von Hippel–Lindau (VHL) protein function can be found in more than 90% of patients with clear cell renal carcinoma (ccRCC). Mice lacking Vhl function in the kidneys have urine concentration defects due to postulated reduction of the hyperosmotic gradient. Hyperosmolality is a kidney-specific microenvironment and induces a unique gene expression pattern. This gene expression pattern is inversely regulated in patients with ccRCC with consequences for cancer-specific survival. Within this study, we tested the hypothesis if Vhl function influences the hyperosmolality induced changes in gene expression. We made use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to inhibit functional Vhl expression in murine collecting duct cell line. Loss of Vhl function induced morphological changes within the cells similar to epithelial to mesenchymal transition like phenotype. Vhl-deficient cells migrated faster and proliferated slower compared to control cells. Gene expression profiling showed significant changes in gene expression patterns in Vhl-deficient cells compared to control cells. Several genes with unfavorable outcomes showed induced and genes with favorable outcomes for patients with renal cancer reduced gene expression level. Under hyperosmotic condition, the expression of several hyperosmolality induced genes, with favorable prognostic value, was downregulated in cells that do not express functional Vhl. Taken together, this study shows that Vhl interferes with hyperosmotic signaling pathway and hyperosmolality affected pathways might represent new promising targets.

Identification of Histone H3 and H4 Amino Acid Residues Important for the Regulation of Arsenite Stress Signaling in Saccharomyces cerevisiae

Arsenic is an environmental carcinogen that causes many diseases in humans, including cancers and organ failures, affecting millions of people in the world. Arsenic trioxide is a drug used for the treatment of acute promyelocytic leukemia (APL). In the present study, we screened the synthetic histone H3 and H4 library in the presence of arsenite to understand the role of histone residues in arsenic toxicity. We identified residues of histone H3 and H4 crucial for arsenite stress response. The residues H3T3, H3G90, H4K5, H4G13, and H4R95 are required for the activation of Hog1 kinase in response to arsenite exposure. We showed that a reduced level of Hog1 activation increases the intracellular arsenic content in these histone mutants through the Fps1 channel. We have also noticed the reduced expression of ACR3 exporter in the mutants. The growth defect of mutants caused by arsenite exposure was suppressed in hyperosmotic conditions, in a higher concentration of glucose, and upon deletion of the FPS1 gene. The arsenite sensitive histone mutants also showed a lack of H3K4 methylation and reduced H4K16 acetylation. Altogether, we have identified the key residues in histone H3 and H4 proteins important for the regulation of Hog1 signaling, Fps1 activity, and ACR3 expression during arsenite stress.

Quantification of Water Flux in Large Unilamellar Vesicles

Water homeostasis is fundamental to all forms of life and plays a major role in human health and disease, plant growth or bacterial survival. It is balanced through a complex interplay of osmotically active substance across lipid membranes and narrow membrane channels. The endeavors to understand the determinants of water permeation and the effect of the lipid or polymer membrane on channel function, the development of specific water flow inhibitors, the design of artificial water channels and aquaporins for the use in industrial water filtration applications rely on accurate ways to quantify water permeabilities (Pf). A commonly used method is to reconstitute membrane channels into large unilamellar vesicles (LUVs) and to subject these vesicles to an osmotic gradient in a stopped-flow device. Fast recordings of either scattered light intensity or fluorescence self-quenching signals are taken as a readout for vesicle volume change, which in turn can be recalculated to accurate Pf values (Horner at al., Science Advances 1, e1400083, 2015; Hannesschläger et al., Sci Rep 8, 8516, 2018). By means of computational and experimental data, we discuss the pros and cons of using scattering versus self-quenching experiments or subjecting vesicles to hypo- or hyperosmotic conditions. In addition, we explicate the influence of the LUVs size distribution and remaining detergent after protein reconstitution on Pf values. We point out that results such as that the single channel water permeability (pf) depends on the membrane matrix or on the direction of the applied osmotic gradient may be direct results of the measurement and analysis procedure.

Betaine addition as a potent ruminal fermentation modulator under hyperthermal and hyperosmotic conditions in vitro.

BACKGROUNDClimatic and dietary shifts predispose ruminal microbes to hyperthermal and hyperosmotic stress, leading to poor fermentation and subsequently adverse effects on ruminant productivity. Betaine may function as substrate, osmolyte, antioxidant, and methyl donor for microbes. However, its effect depends on the extent of microbial catabolism. This study revealed the ruminal disappearance kinetics of betaine and its dose effect on ruminal fermentation during thermal and osmotic stress using a rumen simulation technique.RESULTSThree different betaine doses were used: 0, 50, and 286 mg L−1; each was assigned to two incubation temperatures (39.5 and 42 °C) and two osmotic conditions (295 and 420 mOsmol kg−1). Betaine disappeared rapidly within the first 6 h of incubation; however, the rate was lower during hyperosmotic stress (P < 0.05), the stress condition that also suppressed the overall fermentation and degradation of organic nutrients and decreased the bacterial diversity (P < 0.001). During hyperosmotic stress, betaine shifted the fermentation pathway to more propionate (P < 0.05). Betaine counteracted the negative effect of hyperthermal stress on total short‐chain fatty acid concentration (P < 0.05) without affecting the composition. Both stress conditions shifted the bacterial composition, but the effect of betaine was minimal.CONCLUSIONDespite its rapid ruminal disappearance, betaine modulated microbial fermentation in different ways depending on stress conditions, indicating the plasticity of the betaine effect in response to various kinds of physicochemical stress. Although betaine did not affect the abundance of ruminal microbiota, the enhanced fermentation suggests an improved microbial metabolic activity under stress conditions. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Is the protective effect of egg yolk against osmotic and cryogenic damage on dog spermatozoa dose-dependent?

Egg yolk (EY) is conventionally used to reduce sperm cryodamage, however, there has not be evaluation of whether there is a dose-dependent effect with inclusion of EY in semen extender. To enhance the knowledge about the protective effect of EY during cryopreservation of dog semen, a specific study was designed to evaluate the dose-dependent protection of the EY against osmotic and cryogenic damage of dog sperm. In the first experiment, sperm stored in an extender that contained graded EY concentrations (0 %, 5 %, 10 %, and 20 %) were diluted with hypo- or hyper-osmotic solutions (final osmolality of 75, 150, 300, 500, 1000 mOsm/kg). Results from sperm kinetic, membrane integrity (MI), mitochondrial activity, and normal morphology evaluations indicated osmotic stress has especially marked effects on the kinetic capacity of spermatozoa, however, there were no direct effects on mitochondrial activity. In both hypo- and hyper-osmotic conditions, EY had a protective effect regardless of concentration. In the second experiment, semen samples were diluted in extenders at increasing EY concentrations (0 %, 5 %, 10 %, and 20 %) and cryopreserved. Effects on sperm kinetics, membrane and acrosome integrity and mitochondrial membrane potential indicated there was improved sperm viability after thawing when the EY concentration was 5 % and 10 %, and lesser viability when it was 20 %. These results indicate, for the first time, that EY reduces osmotic and cryogenic damage when used at 5 % or 10 % concentrations, and that these concentrations can be used to protect dog spermatozoa more effectively than the conventionally used concentration (20 %).

Decreased placental apoptosis and autophagy in pregnancies complicated by gestational diabetes with large-for-gestational age fetuses

IntroductionDysregulation of placental apoptosis and autophagy are observed in pregnancy complications including preeclampsia and fetal growth restriction. However, studies of their changes in the placentas of women with gestational diabetes mellitus (GDM) show inconsistent results. We aimed to compare the changes in apoptosis, autophagy, and Bcl-2 family proteins in the placentas from women with normal pregnancies and those with GDM, with or without large-for-gestational age (LGA) infants and to investigate the effect of hyperglycemia on the changes in apoptosis, autophagy, and Bcl-2 family proteins in primary cytotrophoblastic cells. MethodsVillous tissues were obtained from normal pregnant women and those with GDM, with or without LGA infants. Primary cytotrophoblast cells were isolated from normal term placentas and cultured under standard, hyperglycemic, or hyperosmotic conditions. ResultsCompared to placentas from normal pregnant women, those from GDM women with LGA infants were heavier, had lower beclin-1 and DRAM levels, less M30 and cleaved PARP immunoreactivity, and increased Ki-67 immunoreactivity. These changes were associated with increased Bcl-xL and decreased Bak levels. Increased glucose concentration led to lower ATG5, beclin-1, LC3B-II, p62, and DRAM levels, lower annexin V and M30-positive cell percentages, and less cleaved PARP changes compared with standard culture conditions. Hyperglycemia caused higher Bcl-xL levels and lower Bak and Bad levels than did standard culture conditions. DiscussionThere were differential changes in apoptosis and autophagy between placentas from normal pregnant women and those from GDM women with LGA infants. Bcl-2 family proteins are likely involved in the regulation of these changes.

Hypertonic external medium represses cellular respiration and promotes Warburg/Crabtree effect.

Hyperosmotic conditions are associated to several pathological states. In this article, we evaluate the consequence of hyperosmotic medium on cellular energy metabolism. We demonstrate that exposure of cells to hyperosmotic conditions immediately reduces the mitochondrial oxidative phosphorylation rate. This causes an increase in glycolysis, which represses further respiration. This is known as the Warburg or Crabtree effect. In addition to aerobic glycolysis, we observed two other cellular responses that would help to preserve cellular ATP level and viability: A reduction in the cellular ATP turnover rate and a partial mitochondrial uncoupling which is expected to enhance ATP production by Krebs cycle. The latter is likely to constitute another metabolic adaptation to compensate for deficient oxidative phosphorylation that, importantly, is not dependent on glucose.

Colon cancer cells cultured under hyperosmotic conditions as in vitro model to investigate dehydration effects on cancer drug susceptibility

In most clinical studies older people are underrepresented compared to the demographic reality. However, risk for some severe diseases like cancer typically increase with age. Most insight into cancer treatment comes from mixed-age patient cohorts, leading to a lack of detailed understanding of cancer drug effects in the elderly population. There is growing evidence that cancer drug effects can be influenced by dehydration conditions often found in older people. Colon cancer remains the second leading cause of death by cancer in Europe. Inter- and intra-heterogeneity of tumors contribute to why some individuals do not respond to specific cancer therapies or may often suffer a relapse. Our study applies an in vitro drug test system for simulating treatment with cytostatics of colorectal cancer in elderly patients with dehydration condition. Two well-known colon cancer cell lines, Caco-2 and RKO, harboring defined cancer-related mutations, were step-wisely adapted from routine culture medium to a severe hyperosmotic condition (397 mOmol/kg) by adding sodium chloride to the medium. We investigated the effects of these cell culture conditions, which should mimic cellular dehydration in elderly people, on the growth characteristics of the cells. Therefore, cell proliferation was investigated by measuring population doubling times. Furthermore, we investigated how the metabolic activity of the cells was influenced by treatment with different concentrations of cyclophosphamide (CPA) under normal and hyperosmotic conditions. We found that Caco-2 and RKO cell lines have an identical cell doubling time of 23 hours in normosmotic medium. However, hyperosmotic medium lifted the doubling time of Caco-2 cells to 31 hours while that of RKO cells did not change. Despite reduced cell proliferation rates, hyperosmotic medium sensitized Caco-2 cells to treatment with 10 mM CPA for 48 hours as measured by metabolic activity assays on ATP levels. The two investigated colon cancer cells lines reacted differently to hyperosmotic conditions. Only the growth of Caco-2 cells was reduced by increased osmolality. Despite this reduced growth their sensitivity to an alkylating cytostatic agent was even slightly increased. We are now in line to examine these effects in more detail and with more tumor cell lines.

Genome-Wide Identification and Characterization of Cucumber BPC Transcription Factors and Their Responses to Abiotic Stresses and Exogenous Phytohormones.

BASIC PENTACYSTEINE (BPC) is a small transcription factor family that functions in diverse growth and development processes in plants. However, the roles of BPCs in plants, especially cucumber (Cucumis sativus L.), in response to abiotic stress and exogenous phytohormones are still unclear. Here, we identified four BPC genes in the cucumber genome, and classified them into two groups according to phylogenetic analysis. We also investigated the gene structures and detected five conserved motifs in these CsBPCs. Tissue expression pattern analysis revealed that the four CsBPCs were expressed ubiquitously in both vegetative and reproductive organs. Additionally, the transcriptional levels of the four CsBPCs were induced by various abiotic stress and hormone treatments. Overexpression of CsBPC2 in tobacco (Nicotiana tabacum) inhibited seed germination under saline, polyethylene glycol, and abscisic acid (ABA) conditions. The results suggest that the CsBPC genes may play crucial roles in cucumber growth and development, as well as responses to abiotic stresses and plant hormones. CsBPC2 overexpression in tobacco negatively affected seed germination under hyperosmotic conditions. Additionally, CsBPC2 functioned in ABA-inhibited seed germination and hypersensitivity to ABA-mediated responses. Our results provide fundamental information for further research on the biological functions of BPCs in development and abiotic stress responses in cucumber and other plant species.

Hyperosmotic stress in Chlamydomonas induces metabolomic changes in biosynthesis of complex lipids

ABSTRACT To study the early stress response of the green microalga Chlamydomonas reinhardtii CC-503, we exposed cells to hyperosmotic conditions for a period of 5 hours, sampling at eight time points. We performed metabolomic analysis of primary metabolism using gas chromatography/time-of-flight mass spectrometry (GC-TOF MS), and lipidomic analysis by liquid chromatography/quadrupole time-of-flight mass spectrometry (CSH-QTOF MS). Under hyperosmotic stress, many mono- and disaccharides increased along with the concentrations of sugar alcohols similar to osmoprotectants in higher plants. The levels of 13 proteinogenic amino acids increased, suggesting increased biosynthesis over the course of 5 hours, which is clearly a slower response than carbohydrate metabolism. TCA cycle compounds decreased in a tightly co-regulated manner under hyperosmotic stress as a cataplerotic reaction to provide carbon backbones for amino acid biosynthesis. Of note, levels of free palmitoyl-compounds (palmitic acid and monopalmitin) decreased over the stress time course in an opposite pattern to the increase in stearoyl compounds. Triglycerides that increased in concentration under osmotic stress were on average three carbons longer and had four more double bonds compared with triglycerides that remained unchanged. Along with the difference in C18-fatty acids compared with C16-fatty acids, these changes may indicate a specific activation of elongases and desaturases or remodelling from membrane acyl-lipids towards triacylglcyerides.

Pituitary adenylate cyclase activating polypeptide acts against neovascularization in retinal pigment epithelial cells.

The purpose of this study was to determine whether pituitary adenylate cyclase activating polypeptide (PACAP) could influence the neovascularization processes in hyperosmotic and oxidative stress in retinal pigment epithelial cells. Hyperosmotic conditions and oxidative stress were induced by 200mM sucrose and 250µM hydrogen peroxide (H2 O2 ), respectively. Morphology and elasticity of adult retinal pigment epithelial (ARPE-19) cells were measured by atomic force microscopy, while the investigation of junctional molecules, such as occludin and ZO-1, was carried out using immunofluorescence. For cell viability measurement, the MTT test was used. The effect of PACAP on the key angiogenic factors, such as vascular endothelial growth factor, angiogenin, and endothelin-1, was measured by an angiogenesis array and flow cytometry. Hyperosmotic stress-induced reorganization of the cytoskeleton and impairment of the junctions decreased cell viability and upregulated several angiogenic factors. In oxidative stress, we found that opening of the junctions decreased viability and upregulated the expression of angiogenic factors. PACAP was shown to be protective in both conditions. Retinal pigment epithelium cells play an important role in several diseases, such as diabetic retinopathy and macular edema. Therefore, protecting retinal pigment epithelial (RPE) cells with PACAP could be a novel and potential treatment in these diseases.

Labile Calcium-Permeable AMPA Receptors Constitute New Glutamate Synapses Formed in Hypothalamic Neuroendocrine Cells during Salt Loading.

Magnocellular neuroendocrine cells (MNCs) of the hypothalamus play a critical role in the regulation of fluid and electrolyte homeostasis. They undergo a dramatic structural and functional plasticity under sustained hyperosmotic conditions, including an increase in afferent glutamatergic synaptic innervation. We tested for a postulated increase in glutamate AMPA receptor expression and signaling in magnocellular neurons of the male rat hypothalamic supraoptic nucleus (SON) induced by chronic salt loading. While without effect on GluA1-4 subunit mRNA, salt loading with 2% saline for 5-7 d resulted in a selective increase in AMPA receptor GluA1 protein expression in the SON, with no change in GluA2-4 protein expression, suggesting an increase in the ratio of GluA1 to GluA2 subunits. Salt loading induced a corresponding increase in EPSCs in both oxytocin (OT) and vasopressin (VP) neurons, with properties characteristic of calcium-permeable AMPA receptor-mediated currents. Unexpectedly, the emergent AMPA synaptic currents were silenced by blocking protein synthesis and mammalian target of rapamycin (mTOR) activity in the slices, suggesting that the new glutamate synapses induced by salt loading require continuous dendritic protein synthesis for maintenance. These findings indicate that chronic salt loading leads to the induction of highly labile glutamate synapses in OT and VP neurons that are comprised of calcium-permeable homomeric GluA1 AMPA receptors. The glutamate-induced calcium influx via calcium-permeable AMPA receptors would be expected to play a key role in the induction and/or maintenance of activity-dependent synaptic plasticity that occurs in the magnocellular neurons during chronic osmotic stimulation.

Glucose Metabolism in Cardiac Hypertrophy and Heart Failure.

Glucose Metabolism in Cardiac Hypertrophy and Heart Failure.

Methylated metabolite of arsenite blocks glycerol production in yeast by inhibition of glycerol-3-phosphate dehydrogenase.

The yeast high-osmolarity glycerol (HOG) stress-activated protein kinase Hog1 is activated in response to hyperosmotic stress, inducing the production and retention of glycerol to restore osmotic balance. Hog1 promotes retention of glycerol through closure of the plasma-membrane glycerol channel Fps1. Treatment of yeast with the toxic trivalent metalloid arsenite (As(III)) also activates Hog1 as part of a protective response in which Hog1 closes Fps1, the main entry port for As(III). In this study, we investigated how cells treated with As(III) avoid creating a new stress caused by the accumulation of glycerol in the absence of hyperosmotic stress conditions. We found that As(III) treatment did not induce glycerol accumulation and, in fact, blocked the accumulation of glycerol induced by constitutive Hog1 activity. We demonstrated that As(III) blocks glycerol production indirectly after its metabolic activation to methylarsenite (MAs(III)), which is a potent inhibitor of glycerol-3-phosphate dehydrogenase. Finally, we used a biotinylated arsenic probe to establish that Cys306 of yeast Gpd1, a highly conserved residue within the active site, is the key target of MAs(III). Conservative mutations at this residue greatly diminished Gpd1 activity. This study offers insight into mechanisms by which SAPK outputs are tailored to specific stressors.

An Osmoregulatory Mechanism Operating through OmpR and LrhA Controls the Motile-Sessile Switch in the Plant Growth-Promoting Bacterium Pantoea alhagi.

Adaptation to osmotic stress is crucial for bacterial growth and survival in changing environments. Although a large number of osmotic stress response genes have been identified in various bacterial species, how osmotic changes affect bacterial motility, biofilm formation, and colonization of host niches remains largely unknown. In this study, we report that the LrhA regulator is an osmoregulated transcription factor that directly binds to the promoters of the flhDC, eps, and opgGH operons and differentially regulates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production, synthesis of osmoregulated periplasmic glucans (OPGs), biofilm formation, and root colonization of the plant growth-promoting bacterium Pantoea alhagi LTYR-11Z. Further, we observed that the LrhA-regulated OPGs control RcsCD-RcsB activation in a concentration-dependent manner, and a high concentration of OPGs induced by increased medium osmolarity is maintained to achieve the high level of activation of the Rcs phosphorelay, which results in enhanced EPS synthesis and decreased motility in P. alhagi Moreover, we showed that the osmosensing regulator OmpR directly binds to the promoter of lrhA and promotes its expression, while lrhA expression is feedback inhibited by the activated Rcs phosphorelay system. Overall, our data support a model whereby P. alhagi senses environmental osmolarity changes through the EnvZ-OmpR two-component system and LrhA to regulate the synthesis of OPGs, EPS production, and flagellum-dependent motility, thereby employing a hierarchical signaling cascade to control the transition between a motile lifestyle and a biofilm lifestyle.IMPORTANCE Many motile bacterial populations form surface-attached biofilms in response to specific environmental cues, including osmotic stress in a range of natural and host-related systems. However, cross talk between bacterial osmosensing, swimming, and biofilm formation regulatory networks is not fully understood. Here, we report that the pleiotropic regulator LrhA in Pantoea alhagi is involved in the regulation of flagellar motility, biofilm formation, and host colonization and responds to osmotic upshift. We further show that this sensing relies on the EnvZ-OmpR two-component system that was known to detect changes in external osmotic stress. The EnvZ-OmpR-LrhA osmosensing signal transduction cascade is proposed to increase bacterial fitness under hyperosmotic conditions inside the host. Our work proposes a novel regulatory mechanism that links osmosensing and motile-sessile lifestyle transitions, which may provide new approaches to prevent or promote the formation of biofilms and host colonization in P. alhagi and other bacteria possessing a similar osmoregulatory mechanism.

Osmoprotective effect of ubiquinone in lipid vesicles modelling the E. coli plasma membrane

Bacteria need to be able to adapt to sudden changes in their environment, including drastic changes in the surrounding osmolarity. As part of this adaptation, the cells adjust the composition of their cytoplasmic membrane. Recent studies have shown that ubiquinones, lipid soluble molecules involved in cell respiration, are overproduced by bacteria grown in hyperosmotic conditions and it is thus believed that these molecules can provide with osmoprotection. Hereby we explore the mechanisms behind these observations. Liposomes with a lipid headgroup composition mimicking that of the cytoplasmic membrane of E. coli are used as suitable models. The effect of ubiquinone-10 (Q10) on water transport across the membranes is characterized using a custom developed fluorescence-based experimental approach to simultaneously determine the membrane permeability coefficient and estimate the elastic resistance of the membrane towards deformation. It is shown that both parameters are affected by the presence of ubiquinone-10. Solanesol, a molecule similar to Q10 but lacking the quinone headgroup, also provides with osmoprotection although it only improves the resistance of the membrane against deformation. The fluorescence experiments are complemented by cryogenic transmission electron microscopy studies showing that the E. coli membrane mimics tend to flatten into spheroid oblate structures when osmotically stressed, suggesting the possibility of lipid segregation. In agreement with its proposed osmoprotective role, the flattening process is hindered by the presence of Q10.