Salt stress affects sterol biosynthesis in the halophilic black yeast Hortaea werneckii

Hortaea werneckii is a black yeast recently isolated from salterns in Slovenia. Some of the adaptations of halophilic microorganisms to increased salinity and osmolarity of the environment are alterations in membrane properties. By modulating the fluidity, sterols play an important role as a component of eukaryotic biological membranes. We studied the regulation of sterol biosynthesis in H. werneckii through the activity and amount of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG R), a key regulatory enzyme in the biosynthesis of sterols. We found some differences in the characteristics of HMG R and in its regulation by different environmental salinities in H. werneckii when compared to the mesophilic baker's yeast, Saccharomyces cerevisiae. Our results suggest that halophilic black yeast regulates sterol biosynthesis through HMG R in a different way than mesophiles, which might be a consequence of the different ecophysiology of halophilic black yeasts. From this perspective, H. werneckii is an interesting novel model organism for studies on salt stress-responsive proteins as well as on sterol biosynthesis in eukaryotes.     

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.     

Eukaryotic origin of glyceraldehyde-3-phosphate dehydrogenase genes in Clostridium thermocellum and Clostridium cellulolyticum genomes and putative fates of the exogenous gene in the subsequent genome evolution

Although lateral gene transfer (LGT) events have been frequently documented in the evolution of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), no eukaryote-to-prokaryote transfer has been reported so far. Here we describe the first case of the GAPDH gene transfer from a eukaryote to a subset of Clostridium species (Bacteria, Firmicutes). A series of phylogenetic analyses of GAPDH homologues revealed that Clostridium thermocellum and Clostridium cellulolyticum homologues have the evolutionary affinity to the eukaryotic homologues, rather than to those of bacterial species closely related to the two Clostridium species in the organismal phylogeny. These results suggest that the GAPDH genes in the two Clostridium species are of eukaryotic origin, which is the first reported case of eukaryote-to-bacterium GAPDH gene transfer. Since a previously published 16S ribosomal DNA phylogeny and our GAPDH phylogeny commonly suggest an intimate evolutionary relationship between C. thermocellum and C. cellulolyticum, a common ancestor of the two species likely acquired the eukaryotic GAPDH gene. In the C. cellulolyticum genome, the exogenous GAPDH gene was physically separated from other glycolytic genes, suggesting that this gene organization was likely achieved by a random insertion of the laterally transferred gene. On the other hand, in the C. thermocellum genome, the laterally transferred GAPDH gene clusters with other bacterial glycolytic genes. We discuss possible scenarios for the evolutionarily chimeric glycolytic gene cluster in the C. thermocellum genome.     

Autosomal Similarity Revealed by Eukaryotic Genomic Comparison

To describe eukaryotic autosomes quantitatively and determine differences between them in terms of amino acid sequences of genes, functional classification of proteins, and complete DNA sequences, we applied two theoretical methods, the Proteome-vector method and the function of degree of disagreement (FDOD) method, that are based on function and sequence similarity respectively, to autosomes from nine eukaryotes. No matter what aspect of the autosome is considered, the autosomal differences within each organism were less than that between species. Our results show that eukaryotic autosomes resemble each other within a species while those from different organisms differ. We propose a hypothesis (named intra-species autosomal random shuffling) as an explanation for our results and suggest that lateral gene transfer (LGT) did not occur frequently during the evolution of eukarya.     

Osmoadaptation-dependent activity of microsomal HMG-CoA reductase in the extremely halotolerant black yeast Hortaea werneckii is regulated by ubiquitination

We have investigated regulation of HMG-CoA reductase (HMGR) in one of the most salt-tolerant fungi, Hortaea werneckii, under different salinities and at the level of protein degradation. Two different HwHMGR isoenzymes were identified, specific to mitochondria and endoplasmic reticulum: HwHmg1 and HwHmg2, respectively. The activity of microsomal HwHmg2 is highest under hypo-saline and extremely hyper-saline conditions, and down-regulated under optimal growth conditions. We show that this is due to intense ubiquitination and proteasomal degradation of HwHmg2. The activity of the truncated mitochondrial HwHmg1 is constant under different growth conditions, suggesting an osmoadaptation-directed fate for mevalonate utilization in H. werneckii.     

Role of oxidative stress in the extremely salt-tolerant yeast Hortaea werneckii

Eukaryotic halotolerant microorganisms are important as model organisms to understand the general mechanisms of resistance to environmental salinity. The ability of the extremely halotolerant black yeast Hortaea werneckii to combat oxidative stress was addressed, using hydrogen peroxide to generate the reactive oxygen species. Increasing environmental salinity was found to have no effect on its high ability to degrade hydrogen peroxide but resulted in a decrease in viability in response to externally added hydrogen peroxide, suggesting that the latter property determines the upper limit of the salt tolerance of H. werneckii. A refinement of the model of adaptation of H. werneckii to high-salinity environments is proposed.     

Comparative genomic analysis of dha regulon and related genes for anaerobic glycerol metabolism in bacteria

The dihydroxyacetone (dha) regulon of bacteria encodes genes for the anaerobic metabolism of glycerol. In this work, genomic data are used to analyze and compare the dha regulon and related genes in different organisms in silico with respect to gene organization, sequence similarity, and possible functions. Database searches showed that among the organisms, the genomes of which have been sequenced so far, only two, i.e., Klebsiella pneumoniae MGH 78578 and Clostridium perfringens contain a complete dha regulon bearing all known enzymes. The components and their organization in the dha regulon of these two organisms differ considerably from each other and also from the previously partially sequenced dha regulons in Citrobacter freundii, Clostridium pasteurianum, and Clostridium butyricum. Unlike all of the other organisms, genes for the oxidative and reductive pathways of anaerobic glycerol metabolism in C. perfringens are located in two separate organization units on the chromosome. Comparisons of deduced protein sequences of genes with similar functions showed that the dha regulon components in K. pneumoniae and C. freundii have high similarities (80-95%) but lower similarities to those of the Clostridium species (30-80%). Interestingly, the protein sequence similarities among the dha genes of the Clostridium species are in many cases even lower than those between the Clostridium species and K. pneumoniae or C. freundii, suggesting two different types of dha regulon in the Clostridium species studied. The in silico reconstruction and comparison of dha regulons revealed several new genes in the microorganisms studied. In particular, a novel dha kinase that is phosphoenolpyruvate-dependent is identified and experimentally confirmed for K. pneumoniae in addition to the known ATP-dependent dha kinase. This finding gives new insights into the regulation of glycerol metabolism in K. pneumoniae and explains some hitherto not well understood experimental observations.     

5S rRNA Gene Arrangements in Protists: A Case of Nonadaptive Evolution

Given their high copy number and high level of expression, one might expect that both the sequence and organization of eukaryotic ribosomal RNA genes would be conserved during evolution. Although the organization of 18S, 5.8S and 28S ribosomal RNA genes is indeed relatively well conserved, that of 5S rRNA genes is much more variable. Here, we review the different types of 5S rRNA gene arrangements which have been observed in protists. This includes linkages to the other ribosomal RNA genes as well as linkages to ubiquitin, splice-leader, snRNA and tRNA genes. Mapping these linkages to independently derived phylogenies shows that these diverse linkages have repeatedly been gained and lost during evolution. This argues against such linkages being the primitive condition not only in protists but also in other eukaryote species. Because the only characteristic the diverse genes with which 5S rRNA genes are found linked with is that they are tandemly repeated, these arrangements are unlikely to provide any selective advantage. Rather, the observed high variability in 5S rRNA genes arrangements is likely the result of the fact that 5S rRNA genes contain internal promoters, that these genes are often transposed by diverse recombination mechanisms and that these new gene arrangements are rapidly homogenized by unequal crossingovers and/or by gene conversions events in species with short generation times and frequent founder events.     

Peptides encoded by short ORFs control development and define a new eukaryotic gene family

Despite recent advances in developmental biology, and the sequencing and annotation of genomes, key questions regarding the organisation of cells into embryos remain. One possibility is that uncharacterised genes having nonstandard coding arrangements and functions could provide some of the answers. Here we present the characterisation of tarsal-less (tal), a new type of noncanonical gene that had been previously classified as a putative noncoding RNA. We show that tal controls gene expression and tissue folding in Drosophila, thus acting as a link between patterning and morphogenesis. tal function is mediated by several 33-nucleotide-long open reading frames (ORFs), which are translated into 11-amino-acid-long peptides. These are the shortest functional ORFs described to date, and therefore tal defines two novel paradigms in eukaryotic coding genes: the existence of short, unprocessed peptides with key biological functions, and their arrangement in polycistronic messengers. Our discovery of tal-related short ORFs in other species defines an ancient and noncanonical gene family in metazoans that represents a new class of eukaryotic genes. Our results open a new avenue for the annotation and functional analysis of genes and sequenced genomes, in which thousands of short ORFs are still uncharacterised.     

Expression of 17beta-hydroxysteroid dehydrogenases in mesophilic and extremophilic yeast

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are enzymes responsible for reversible interconversions of biologically active 17-hydroxy and inactive 17-keto steroids. We have performed a survey of 17beta-HSD activity in yeast. Constitutive 17beta-HSD activity was found in three mesophilic yeast species: Candida tropicalis, Cryptococcus tsukubaensis, and Saccharomyces cerevisiae as well as in three extremophilic black yeast species: Hortaea werneckii, Trimmatostroma salinum, and Phaeotheca triangularis, indicating that 17beta-HSD activity is widely distributed among yeast. In extremophilic black yeast, NaCl modulated enzyme activity. Enzymes resembling 17beta-HSD from the filamentous fungus Cochliobolus lunatus were detected in Trimmatostroma salinum and Phaeotheca triangularis. Sequences with identity to the Saccharomyces cerevisiae YBR159w gene were not observed in other yeast species possessing a similar enzyme activity. The results suggest the existence of at least three different types of 17beta-HSD in yeast.     

OrthoParaMap: Distinguishing orthologs from paralogs by integrating comparative genome data and gene phylogenies

Background  

In eukaryotic genomes, most genes are members of gene families. When comparing genes from two species, therefore, most genes in one species will be homologous to multiple genes in the second. This often makes it difficult to distinguish orthologs (separated through speciation) from paralogs (separated by other types of gene duplication). Combining phylogenetic relationships and genomic position in both genomes helps to distinguish between these scenarios. This kind of comparison can also help to describe how gene families have evolved within a single genome that has undergone polyploidy or other large-scale duplications, as in the case of Arabidopsis thaliana – and probably most plant genomes.     

Foraminifera and Cercozoa are related in actin phylogeny: two orphans find a home?

In recent years, the increased sampling of protein-coding genes from diverse eukaryotes has revealed that many aspects of each gene tree are at odds with other phylogenies. This has led to the belief that each gene tree has unique strengths and weaknesses, suggesting that an accurate picture of eukaryotic relationships will be achieved only through comparative phylogeny using several different genes. To this end, actin genes were characterized from two genera of chlorarachniophytes, Chlorarachnion and Lotharella, and three species of the cercomonad flagellate Cercomonas: Phylogenetic trees including these new actin genes confirm the recently proposed relationship between chlorarachniophytes and cercomonads (Cercozoa) and, more importantly, also show a close relationship between Cercozoa and Foraminifera. Both of these are major eukaryotic groups encompassing extremely diverse organisms, yet there is no strong evidence for the evolutionary position of either from morphological or molecular data. The union of Cercozoa and Foraminifera suggested by actin phylogeny represents a novel step in the long process of determining the broad relationships between all major eukaryotic groups.     

Taxonomy of Exophiala werneckii and its relationship to Microsporum mansonii

Study of the neotype culture of Exophiala werneckii (Cladosporium werneckii), as well as additional isolates, has shown that the yeast-like and hyphal conidiogenous cells are annellides. E. werneckii cannot be accommodated in Cladosporium, a genus characterized by catenulate holoblastic coanidia. E. werneckii and the dematiaceous hyphomycete that Castellani misidentified as Microsporum mansonii (Cladosporium mansonii) are two entirely different fungi. M. mansonii is considered to be a synonym of the Malassezia furfur since both names were used to describe in tissue the etiologic agent of pityriasis versicolor.     

Novel alternative splicing of mRNAs encoding poly(A) polymerases in Arabidopsis

The Arabidopsis thaliana genome possesses four genes whose predicted products are similar to eukaryotic poly(A) polymerases from yeasts and animals. These genes are all expressed, as indicated by RT/PCR and Northern blot analysis. The four Arabidopsis PAPs share a conserved N-terminal catalytic core with other eukaryotic enzymes, but differ substantially in their C-termini. Moreover, one of the four Arabidopsis enzymes is significantly shorter than the other three, and is more divergent even within the conserved core of the protein. Nonetheless, the protein encoded by this gene, when produced in and purified from E. coli, possesses nonspecific poly(A) polymerase activity. Genes encoding these Arabidopsis PAPs give rise to a number of alternatively spliced mRNAs. While the specific nature of the alternative splicing varied amongst these three genes, mRNAs from the three "larger" genes could be alternatively spliced in the vicinity of the 5th and 6th introns of each gene. Interestingly, the patterns of alternative splicing vary in different tissues. The ubiquity of alternative splicing in this gene family, as well as the differences in specific mechanisms of alternative processing in the different genes, suggests an important function for alternatively spliced PAP mRNAs in Arabidopsis.