Phylogenetic Relationships of Species of the Genus Kluyveromyces van der Walt (Saccharomycetaceae) Deduced from the Partial Sequences of 18S and 26S Ribosomal RNAs

In order to clarify the phylogenetic relationships of the species classified in the genus Kluyveromyces (Saccharomycetaceae), three partial base sequences of 18S and 26S rRNAs of eighteen strains were determined. The regions determined of the strains corresponded to positions 1451 through 1618 (168 bases) of 18S rRNA and to positions 1611 through 1835 (225 bases) and 493 through 622 (130 bases) of a strain (IFO 2376) of Saccharomyces cerevisiae. The analyses of the partial base sequences suggested that the genus Kluyveromyces is phylogenetically heterogeneous, ranging from the strains that are quite close to the strain of S. cerevisiae to the strains that are distinct enough to be classified in genera separate from the genus Saccharomyces. From our sequence data, we concluded that the extent of the genus Kluyveromyces should be restricted to only one species, K. polysporus, the type species of the genus. Kluyveromyces phaffii was also distinct enough to deserve another genus. Kluyveromyces cellobiovorus was not close to any of the strains of Kluyveromyces species examined, and should be excluded from the genus. Most of the strains of the species examined were fairly close to the strain of S. cerevisiae.     

Phylogenetic relationships among yeasts of the 'Saccharomyces complex' determined from multigene sequence analyses

Species of Saccharomyces, Arxiozyma, Eremothecium, Hanseniaspora (anamorph Kloeckera), Kazachstania, Kluyveromyces, Pachytichospora, Saccharomycodes, Tetrapisispora, Torulaspora, and Zygosaccharomyces, as well as three related anamorphic species assigned to Candida (C. castellii, C. glabrata, C. humilis), were phylogenetically analyzed from divergence in genes of the rDNA repeat (18S, 26S, ITS), single copy nuclear genes (translation elongation factor 1alpha, actin-1, RNA polymerase II) and mitochondrially encoded genes (small-subunit rDNA, cytochrome oxidase II). Single-gene phylogenies were congruent for well-supported terminal lineages but deeper branches were not well resolved. Analysis of combined gene sequences resolved the 75 species compared into 14 clades, many of which differ from currently circumscribed genera.     

Transition of the ability to generate petites in the Saccharomyces/Kluyveromyces complex

Petite-positivity - the ability to tolerate the loss of mtDNA - was examined after the treatment with ethidium bromide (EB) in over hundred isolates from the Saccharomyces/Kluyveromyces complex. The identity of petite mutants was confirmed by the loss of specific mtDNA DAPI staining patterns. Besides unequivocal petite-positive and petite-negative phenotypes, a few species exhibited temperature sensitive petite positive phenotype and petiteness of a few other species could be observed only at the elevated EB concentrations. Several yeast species displayed a mixed 'moot' phenotype, where a major part of the population did not tolerate the loss of mtDNA but several cells did. The genera from postwhole-genome duplication lineages (Saccharomyces, Kazachstania, Naumovia, Nakaseomyces) were invariably petite-positive. However, petite-positive traits could also be observed among the prewhole-genome duplication species.     

Phylogenetic analysis of ascomycete yeasts that form coenzyme Q-9 and the proposal of the new genera Babjeviella, Meyerozyma, Millerozyma, Priceomyces, and Scheffersomyces

Species assigned to the genera Debaryomyces, Lodderomyces, Spathaspora, and Yamadazyma, as well as selected species of Pichia and Candida that also form coenzyme Q-9, were phylogenetically analyzed from the combined sequences of the D1/D2 domains of the large subunit and the nearly complete small subunit rRNA genes. Species assigned to Debaryomyces partitioned into three clades and species assigned to Pichia were distributed among six clades. These well-supported clades were interpreted as genera, and from this analysis, the following new genera are proposed: Babjeviella, Meyerozyma, Millerozyma, Priceomyces, and Scheffersomyces. The genus Schwanniomyces was reinstated and emended, and the genus Yamadazyma was phylogenetically defined. From this study, 23 new combinations and 3 new ranks are proposed. The preceding genera are members of a single, large clade, and it is proposed to delineate this clade as the new family Debaryomycetaceae.     

Dosage suppression of the Kluyveromyces lactis zymocin by Saccharomyces cerevisiae ISR1 and UGP1

The Kluyveromyces lactis zymocin complex kills Saccharomyces cerevisiae cells in a process that involves tRNA cleavage by its tRNAse gamma-toxin subunit. In contrast to the gamma-toxin mode of action, the early steps of the zymocin response are less well characterized. Here, we present high-dosage suppressors of zymocin that encode a putative Pkc1-related kinase (ISR1) and UDP-glucose pyrophosphorylase (UGPase) (UGP1). Anti-UGPase Western blots and GAL10 - ISR1 overexpression suggest that zymocin suppression correlates with overproduction of UGPase or Isr1. As judged from protection against exo-zymocin and unaltered sensitivity to endogenous gamma-toxin, high-copy ISR1 and UGP1 operate in early, nontarget steps of the zymocin pathway. Consistent with a recent report on in vitro phosphorylation of Isr1 and UGPase by the CDK Pho85, high-copy ISR1 and UGP1 suppression of zymocin is abolished in a pho85 null mutant lacking CDK activity of Pho85. Moreover, suppression requires UGPase enzyme activity, and ISR1 overexpression also protects against CFW, a chitin-interfering poison. Our data agree with roles for UGPase in cell wall biosynthetic processes and for Isr1 in Pkc1-related cell wall integrity. In sum, high-copy ISR1 and UGP1 cells affect early steps of the zymocin response and potentially prevent the lethal K. lactis killer complex from establishing cell surface recognition and/or contact.     

Phylogeny of the genus Pythium and description of new genera

Phylogeny of the genus Pythium is analyzed based on sequences of the large subunit ribosomal DNA D1/D2 region and cytochrome oxidase II gene region of Pythium isolates and comprehensive species of related taxa belonging to the Oomycetes. The phylogenetic trees show that the genus Pythium is a highly divergent group and divided into five well- or moderately supported monophyletic clades. Each clade is characterized by sporangial morphology such as globose, ovoid, elongated, or filamentous shapes. Based on phylogeny and morphology, the genus Pythium (s. str.) is emended, and four new genera, Ovatisporangium, Globisporangium, Elongisporangium, and Pilasporangium, are described and segregated from Pythium s. lato.     

Reconsideration of the Phylogenetic Positions of Five Peritrich Genera,Vorticella, Pseudovorticella,Zoothamnopsis, Zoothamnium,and Epicarchesium (Ciliophora,Peritrichia, Sessilida), Based on Small Subunit rRNA Gene Sequences

ABSTRACT. In order to re‐evaluate the systematics of sessilid peritrich ciliates, small subunit (SSU) rRNA gene sequences were determined for 12 species belonging to five genera: Vorticella, Pseudovorticella, Epicarchesium, Zoothamnium, and Zoothamnopsis. Phylogenetic trees were deduced using Bayesian inference, maximum parsimony, and maximum likelihood methods. The phylogenetic analyses suggest that (1) sessilids which have stalks with continuous myonemes that contract in a zig‐zag fashion form a separate clade from those which have stalks that contract independently and in a spiral fashion, supporting the separation of the family Zoothamniidae from the family Vorticellidae and (2) Epicarchesium and Pseudovorticella, both of which have reticulate silverline systems, are more closely related to each other than to other vorticellids, suggesting that differences in the silverline system (i.e. transverse vs. reticulate) may be the result of genuine evolutionary divergence among sessilid peritrichs. However, the newly sequenced Zoothamnopsis sinica, which has a reticulate silverline pattern, nests within the unresolved Zoothamnium species that have transverse silverline patterns. Thus, there were at least two evolutions of the reticulate silverline pattern character state from a plesiomorphic transverse state in the peritrichid ciliates. The molecular work demonstrates the genus Zoothamnium to be paraphyletic in relation to morphological studies, and suggests that Astylozoon, Opisthonecta, and Vorticella microstoma possibly share a SSU rRNA secondary structure in the helix E10‐1 region.     

Why one species in New Zealand,Pugetia delicatissima (Kallymeniaceae,Rhodophyta), should become two new genera,Judithia gen. nov. and Wendya gen. nov.

Blade-forming red algae occur worldwide and, prior to DNA sequencing, had been notoriously difficult to identify and classify, especially when lacking critical reproductive features. This, coupled in New Zealand with many longstanding assumptions that taxa were identical to non-New Zealand species or genera, resulted in many misapplied names. Pugetia delicatissima R.E. Norris, an endemic New Zealand blade-forming species of the family Kallymeniaceae, is actually comprised of one existing and one new species belonging to two distinct genera, as established by our phylogenetic analyses of DNA sequences from the rbcL gene. Analyses of combined rbcL and LSU genes showed that neither is closely related to the generitype of Pugetia, the northern-eastern Pacific, P. fragilissima Kylin. We propose the names Judithia and Wendya for these two newly revealed genera. In addition to diagnostic rbcL and LSU sequences, Judithia is morphologically and anatomically characterized by rounded to oblong blades that do not taper basally at the stipe, loosely aggregated surface cortical cells and cystocarps lacking both a pericarp and an ostiole, all features observed in the holotype of P. delicatissima. Wendya, in contrast, is characterized by blades that taper both apically and basally, compactly arranged surface cortical cells and cystocarps that have both a pericarp and a distinct ostiole. The two genera also are distinguished from one other, as well as from Pugetia by features of pre- and post-fertilization development, including the number of subsidiary cells produced on carpogonial and auxiliary branch systems, whether subsidiary cells in the carpogonial branch system fuse with the supporting cell or not, and the site of origin of gonimoblast cells. Although small in area, New Zealand hosts ten of the 27 currently recognized genera in the Kallymeniaceae and is the southern-hemisphere region of greatest generic diversification in this family.     

Zygosaccharomyces kombuchaensis,a new ascosporogenous yeast from 'Kombucha tea'

A new ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp. n. (type strain NRRL YB-4811, CBS 8849), is described; it was isolated from Kombucha tea, a popular fermented tea-based beverage. The four known strains of the new species have identical nucleotide sequences in domain D1/D2 of 26S rDNA. Phylogenetic analysis of D1/D2 and 18S rDNA sequences places Z. kombuchaensis near Zygosaccharomyces lentus. The two species are indistinguishable on standard physiological tests used for yeast identification, but can be recognized from differences in restriction fragment length polymorphism patterns obtained by digestion of 18S-ITS1 amplicons with the restriction enzymes DdeI and MboI.     

Toxin-binding properties of cytochrome P450 in Saccharomyces cerevisiae and Kluyveromyces marxianus

Microsomes from Kluyveromyces marxianus GK1005 examined by carbon monoxide difference spectroscopy showed no evidence of cytochrome P450, in contrast to microsomes isolated from a control strain of Saccharomyces cerevisiae. Benzo[a]pyrene produced a typical Type I-binding spectrum with microsomes of both yeasts, with K _s values of 82 M (S. cerevisiae) and 70 M (K. marxianus). While aflatoxin B₁ generated a typical Type I-binding spectrum with microsomes from S. cerevisiae (K _s of 178 M), the toxin did not produce a recognisable binding spectrum with microsomes from K. marxianus.     

Phylogenetic relationships among species of Pichia, Issatchenkia and Williopsis determined from multigene sequence analysis, and the proposal of Barnettozyma gen. nov., Lindnera gen. nov. and Wickerhamomyces gen. nov

Relationships among species assigned to the yeast genera Pichia, Issatchenkia and Williopsis , which are characterized by the ubiquinone CoQ-7 and inability to utilize methanol, were phylogenetically analyzed from nucleotide sequence divergence in the genes coding for large and small subunit rRNAs and for translation elongation factor-1α. From this analysis, the species separated into five clades. Species of Issatchenkia are members of the Pichia membranifaciens clade and are proposed for transfer to Pichia . Pichia dryadoides and Pichia quercuum are basal members of the genus Starmera . Williopsis species are dispersed among hat-spored taxa in each of the remaining three clades, which are proposed as the new genera Barnettozyma, Lindnera and Wickerhamomyces . Lineages previously classified as varieties of Pichia kluyveri , ' Issatchenkia ' scutulata, Starmera amethionina and ' Williopsis ' saturnus are elevated to species rank based on sequence comparisons.     

Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis

Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells. Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S. cerevisiae whether grown at 30°C (generation time 1.1 h) or 20°C (generation time 2.1 h). Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown. Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.     

Phylogenetic taxonomy of the Cercosaurini (Squamata: Gymnophthalmidae), with new genera for species of Neusticurus and Proctoporus

The tribe Cercosaurini is one of the most poorly studied groups of the lizard family Gymnophthalmidae. Recent studies have suggested that two cercosauriine genera, Neusticurus and Proctoporus , are polyphyletic. The aim of the current study was to rectify the polyphyletic relationships and construct a phylogenetic taxonomy of the Cercosaurini that is congruent with evolutionary history. Neusticurus is divided into two genera, one of them new ( Potamites ), based on the clades recovered by molecular studies and previously discussed morphological data. Proctoporus is divided into three genera, one of which is new ( Petracola ), while an older name ( Riama ) is resurrected for another. All five genera are described and defined and taxonomic keys are presented. This study represents an important advance in rectifying the taxonomy of the Cercosaurini. Many other para- and polyphyletic genera remain in the Gymnophthalmidae and much future work on this group is warranted.  © 2005 The Linnean Society of London, Zoological Journal of the Linnean Society , 2005, 143 , 405–416.     

Phylogenetic Analysis of Coccidia Based on 18S rDNA Sequence Comparison Indicates that Isospora Is Most Closely Related to Toxoplasma and Neospora

The phylogenetic relationships and taxonomic affinities of coccidia with isosporan-type oocysts have been unclear as overlapping characters, recently discovered life cycle features, and even recently discovered taxa. continue to be incorporated into biological classifications of the group. We determined the full or partial 18S ribosomal RNA gene sequences of three mammalian Isospora spp., Isospora felis, Isospora ohioensis and Isospora suis , and a Sarcocystis sp. of a rattlesnake, and used these sequences for a phylogenetic analysis of the genus Isospora and the cyst-forming coccidia. Various alveolate 18S rDNA sequences were aligned and analyzed using maximum parsimony to obtain a phylogenetic hypothesis for the group. The three Isospora spp. were found to be most closely related to Toxoplasma gondii and Neospora caninum. This clade in turn formed the sister group to the Sarcocystis spp. included in the analysis. The results confirm that the genus Isospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae. Furthermore, there appear to be two lineages within the Sarcocystidae. One lineage comprises Isospora and the Toxoplasma/Neospora clade which share the characters of having a proliferative phase of development preceding gamogony in the definitive host and an exogenous phase of sporogony. The other lineage comprises the Sarcocystis spp. which have no proliferative phase in the definitive host and an endogenous phase of sporogony.     

Phylogenetic position of South American Cheilanthes (Cheilanthoideae,Pteridaceae): Advances in the generic circumscription and segregation of the new genus Mineirella

This analysis corroborates and expands our previous results regarding the phylogenetic position of Cheilanthes species from South America. We sequenced three plastid genetic regions, one genic (rbcL) and two genic plus intergenic spacers (trnL + trnL-F and rps4 + rps4-trnS) from 25 South American cheilanthoid species. This allowed us to elucidate phylogenetic relationships that have been historically unresolved or were lowly supported. Here, we analyzed 45 Cheilanthes species (23 from South America) and circumscribed Cheilanthes s.s. in a strongly supported clade that contains three subclades: (i) exclusively from South America, (ii) from Australasia + South America, and (iii) from Africa. The position of three South American species, previously referred to the informal “Cheilanthes geraniifolia group”, is confirmed as a highly supported group outside Cheilanthes s.s. and within the Adiantopsis–Doryopteris clade. This group is described here as the new genus Mineirella. The new combinations for the genus and illustrations are included. Additionally, we discuss the morphological innovations that provide evidence to support the different clades.     

Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

Genetic and fermentation properties of the K2 killer yeast, Saccharomyces cerevisiae T206

Saccharomyces cerevisiae T206 K⁺R⁺, a K₂ killer yeast, was differentiated from other NCYC killer strains of S. cerevisiae on the basis of CHEF-karyotyping and mycoviral RNA separations. Genomic DNA of strain T206 was resolved into 13 chromosome bands, ranging from approximately 0.2 to 2.2 Mb. The resident virus in strain T206 yielded L and M RNA species of approximately 5.1 kb and 2.0 kb, respectively. In micro-scale vinifications, strain T206 showed a lethal effect on a K^-R^- mesophilic wine yeast. Metabolite accumulation and toxin activity were measured over a narrow pH range of 3.2 to 3.5. Contrary to known fermentation trends, the challenged fermentations were neither stuck nor protracted although over 70% of the cell population was killed. Toxin-sensitive cells showed cytosolic efflux.     

Growth and fermentation characteristics of new selected strains of Saccharomyces at high temperatures and high cell densities

Maintenance of high cell viability was the main characteristic of our new strains of thermotolerant Saccharomyces. Total sugar conversion to ethanol was observed for sugarcane juice fermentation at 38-40 degrees C in less than 10 h and without continuous aeration of the culture. Invertase activity differed among the selected strains and increased during fermentation but was not dependent on cell viability. Invertase activity of the cells and optimum temperature for growth, as well as velocity of ethanol formation, were dependent on medium composition and the type of strain used. At high sugarcane syrup concentrations, the best temperature for ethanol formation by strain 781 was 35 degrees C. Distinct differences among the velocities of ethanol production using selected strains were also observed in sugarcane syrup at 35-38 degrees C.     

Identification and functional expression of a new xylose isomerase from the goat rumen microbiome in Saccharomyces cerevisiae

The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions.