Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast Hortaea werneckii

<p>Abstract</p> <p>Background</p> <p>Fluctuations in external salinity force eukaryotic cells to respond by changes in the gene expression of proteins acting in protective biochemical processes, thus counteracting the changing osmotic pressure. The high-osmolarity glycerol (HOG) signaling pathway is essential for the efficient up-regulation of the osmoresponsive genes. In this study, the differential gene expression of the extremely halotolerant black yeast <it>Hortaea werneckii </it>was explored. Furthermore, the interaction of mitogen-activated protein kinase HwHog1 and RNA polymerase II with the chromatin in cells adapted to an extremely hypersaline environment was analyzed.</p> <p>Results</p> <p>A cDNA subtraction library was constructed for <it>H. werneckii</it>, adapted to moderate salinity or an extremely hypersaline environment of 4.5 M NaCl. An uncommon osmoresponsive set of 95 differentially expressed genes was identified. The majority of these had not previously been connected with the adaptation of salt-sensitive <it>S. cerevisiae </it>to hypersaline conditions. The transcriptional response in hypersaline-adapted and hypersaline-stressed cells showed that only a subset of the identified genes responded to acute salt-stress, whereas all were differentially expressed in adapted cells. Interaction with HwHog1 was shown for 36 of the 95 differentially expressed genes. The majority of the identified osmoresponsive and HwHog1-dependent genes in <it>H. werneckii </it>have not been previously reported as Hog1-dependent genes in the salt-sensitive <it>S. cerevisiae</it>. The study further demonstrated the co-occupancy of HwHog1 and RNA polymerase II on the chromatin of 17 up-regulated and 2 down-regulated genes in 4.5 M NaCl-adapted <it>H. werneckii </it>cells.</p> <p>Conclusion</p> <p>Extremely halotolerant <it>H. werneckii </it>represents a suitable and highly relevant organism to study cellular responses to environmental salinity. In comparison with the salt-sensitive <it>S. cerevisiae</it>, this yeast shows a different set of genes being expressed at high salt concentrations and interacting with HwHog1 MAP kinase, suggesting atypical processes deserving of further study.</p

Sensing and responding to hypersaline conditions and the HOG signal transduction pathway in fungi isolated from hypersaline environments

Sensing and responding to changes in NaCl concentration in hypersaline environments is vital for cell survival. In this paper, we identified and characterized key components of the high-osmolarity glycerol (HOG) signal transduction pathway, which is crucial in sensing hypersaline conditions in the extremely halotolerant black yeast Hortaea werneckii and in the obligate halophilic fungus Wallemia ichthyophaga. Both organisms were isolated from solar salterns, their predominating ecological niche. The identified components included homologous proteins of both branches involved in sensing high osmolarity (SHO1 and SLN1) and the homologues of mitogen-activated protein kinase module (MAPKKK Ste11, MAPKK Pbs2, and MAPK Hog1). Functional complementation of the identified gene products in S. cerevisiae mutant strains revealed some of their functions. Structural protein analysis demonstrated important structural differences in the HOG pathway components between halotolerant/halophilic fungi isolated from solar salterns, salt-sensitive S. cerevisiae, the extremely salt-tolerant H. werneckii, and halophilic W. ichthyophaga. Known and novel gene targets of MAP kinase Hog1 were uncovered particularly in halotolerant H. werneckii. Molecular studies of many salt-responsive proteins confirm unique and novel mechanisms of adaptation to changes in salt concentration

Reconstruction of the High-Osmolarity Glycerol (HOG) Signaling Pathway from the Halophilic Fungus Wallemia ichthyophaga in Saccharomyces cerevisiae

The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like other fungi, W. ichthyophaga detects changes in environmental salinity mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with protein modeling data, our study reveals conserved and non-conserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus

Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast -0

<p><b>Copyright information:</b></p><p>Taken from "Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast "</p><p>http://www.biomedcentral.com/1471-2164/8/280</p><p>BMC Genomics 2007;8():280-280.</p><p>Published online 16 Aug 2007</p><p>PMCID:PMC2034391.</p><p></p>aline stress at indicated time points (Hyperosmotic stress). 26S rRNA () was used as an internal control for template normalization. Genes whose expression was not affected by salt-stress are presented in (A) and those that responded to salt stress are presented in (B)

Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast -2

<p><b>Copyright information:</b></p><p>Taken from "Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast "</p><p>http://www.biomedcentral.com/1471-2164/8/280</p><p>BMC Genomics 2007;8():280-280.</p><p>Published online 16 Aug 2007</p><p>PMCID:PMC2034391.</p><p></p>enes are presented in red boxes and down-regulated genes are in green boxes. HwHog1-ChIP positive genes are underlined

Extremophilic yeasts

We report the first identification and characterisation of the glycerol-3-phosphate dehydrogenase (GPD) genes from extremely halophilic fungi. The black ascomycetous yeast Hortaea werneckii and the non-melanised basidiomycetous fungus Wallemia ichthyophaga inhabit similar hypersaline environments, yet they have two different strategies of haloadaptation throughGpd1-regulated glycerol synthesis. The extremely halotolerant H. werneckii codes for two salt-inducible GPD1 genes that show similar gene transcription regulation and have 98% amino-acid sequence identity between paraloguesč however, they have distinct effects when expressed heterologouslyin Saccharomyces cerevisiae gpd mutants. Only the HwGpd1B isoform complements the function of Gpd in the gpd1 mutant, whereas none of the Gpd1 isoforms can rescue the salt sensitivity of the gpd1gpd2 double mutant. The obligate halophile W. ichthyophaga codes for only one GPD1 orthologue, the transcription of which is less affected by salt when compared to the H. werneckii homologues. Heterologous expression of WiGPD1 in S. cerevisiae recovers halotolerance of the gpd1 and gpd1gpd2 mutant strains, which is probably due to the overall high amino-acid similarity of the Gpd1 protein in W. ichthyophaga and S. cerevisiae. Phylogenetic analysis of amino-acid sequences reveals that the evolutionary origins of all of these three novel enzymes correspond to the phylogeny of the fungal species from which the genes were identified

The Halophilic Fungus Hortaea werneckii and the Halotolerant Fungus Aureobasidium pullulans Maintain Low Intracellular Cation Concentrations in Hypersaline Environments

Hortaea werneckii and Aureobasidium pullulans, black yeast-like fungi isolated from hypersaline waters of salterns as their natural ecological niche, have been previously defined as halophilic and halotolerant microorganisms, respectively. In the present study we assessed their growth and determined the intracellular cation concentrations of salt-adapted and non-salt-adapted cells of both species at a wide range of salinities (0 to 25% NaCl and 0 to 20% NaCl, respectively). Although 5% NaCl improved the growth of H. werneckii, even the minimal addition of NaCl to the growth medium slowed down the growth rate of A. pullulans, confirming their halophilic and halotolerant nature. Salt-adapted cells of H. werneckii and A. pullulans kept very low amounts of internal Na(+) even when grown at high NaCl concentrations and can be thus considered Na(+) excluders, suggesting the existence of efficient mechanisms for the regulation of ion fluxes. Based on our results, we can conclude that these organisms do not use K(+) or Na(+) for osmoregulation. Comparison of cation fluctuations after a hyperosmotic shock, to which nonadapted cells of both species were exposed, demonstrated better ionic homeostasis regulation of H. werneckii compared to A. pullulans. We observed small fluctuations of cation concentrations after a hyperosmotic shock in nonadapted A. pullulans similar to those in salt-adapted H.werneckii, which additionally confirmed better regulation of ionic homeostasis in the latter. These features can be expected from organisms adapted to survival within a wide range of salinities and to occasional exposure to extremely high NaCl concentrations, both characteristic for their natural environment

Adaptation of the glycerol-3-phosphate dehydrogenase Gpd1 to high salinities in the extremely halotolerant Hortaea werneckii and halophilic Wallemia ichthyophaga

We report the first identification and characterisation of the glycerol-3-phosphate dehydrogenase (GPD) genes from extremely halophilic fungi. The black ascomycetous yeast Hortaea werneckii and the non-melanised basidiomycetous fungus Wallemia ichthyophaga inhabit similar hypersaline environments, yet they have two different strategies of haloadaptation throughGpd1-regulated glycerol synthesis. The extremely halotolerant H. werneckii codes for two salt-inducible GPD1 genes that show similar gene transcription regulation and have 98% amino-acid sequence identity between paraloguesč however, they have distinct effects when expressed heterologouslyin Saccharomyces cerevisiae gpd mutants. Only the HwGpd1B isoform complements the function of Gpd in the gpd1 mutant, whereas none of the Gpd1 isoforms can rescue the salt sensitivity of the gpd1gpd2 double mutant. The obligate halophile W. ichthyophaga codes for only one GPD1 orthologue, the transcription of which is less affected by salt when compared to the H. werneckii homologues. Heterologous expression of WiGPD1 in S. cerevisiae recovers halotolerance of the gpd1 and gpd1gpd2 mutant strains, which is probably due to the overall high amino-acid similarity of the Gpd1 protein in W. ichthyophaga and S. cerevisiae. Phylogenetic analysis of amino-acid sequences reveals that the evolutionary origins of all of these three novel enzymes correspond to the phylogeny of the fungal species from which the genes were identified

Desmosome Assembly and Cell-Cell Adhesion Are Membrane Raft-dependent Processes

The aim of our study was to investigate the association of desmosomal proteins with cholesterol-enriched membrane domains, commonly called membrane rafts, and the influence of cholesterol on desmosome assembly in epithelial Madin-Darby canine kidney cells (clone MDc-2). Biochemical analysis proved an association of desmosomal cadherin desmocollin 2 (Dsc2) in cholesterol-enriched fractions that contain membrane raft markers caveolin-1 and flotillin-1 and the novel raft marker ostreolysin. Cold detergent extraction of biotinylated plasma membranes revealed that ∼60% of Dsc2 associates with membrane rafts while the remainder is present in nonraft and cholesterol-poor membranes. The results of immunofluorescence microscopy confirmed colocalization of Dsc2 and ostreolysin. Partial depletion of cholesterol with methyl-β-cyclodextrin disturbs desmosome assembly, as revealed by sequential recordings of live cells. Moreover, cholesterol depletion significantly reduces the strength of cell-cell junctions and partially releases Dsc2 from membrane rafts. Our data indicate that a pool of Dsc2 is associated with membrane rafts, particularly with the ostreolysin type of membrane raft, and that intact membrane rafts are necessary for desmosome assembly. Taken together, these data suggest cholesterol as a potential regulator that promotes desmosome assembly