Differentiation of the yeasts Williopsis, Zygowilliopsis and Komagataea by karyotypic and PCR analyses

We used polymerase chain reaction with universal and microsatellite primers, and molecular karyotyping to evaluate the extent of divergence between the genomes of the yeasts currently assigned to the heterogeneous genus Williopsis. Pulsed-field gel electrophoresis of chromosomal DNAs indicates that Zygowilliopsis californica, Komagataea pratensis, Williopsis mucosa, Williopsis salicorniae species and Williopsis sensu stricto complex have clearly different karyotypes. In contrast, the latter six species, Williopsis saturnus, W. beijerinckii, W mrakii, W. suaveolens, W. subsufficiens and W. sargentensis, show similar banding patterns and practically cannot be differentiated on the basis of their karyotypes. The data revealed that a PCR method employing the universal primer N21 is appropriate for the distinction of Williopsis, Zygowilliopsis and Komagataea yeasts. Unique fingerprints were generated with this primer for all 10 species studied while strains of the same species showed nearly identical profiles. The data of UP-PCR are in good agreement with genetic classification and provide support for the species status of the yeasts composing the Williopsis sensu stricto complex. Microsatellite primer (GTG)5 allowing molecular typing of individual strains of the same species may be useful for investigating population structure of the saturn-spored yeasts.     

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.     

Identification of Zygowilliopsis californicaStrains of Different Origin by Means of Polymerase Chain Reaction with Universal Primers

After reevaluation of the taxonomic position of 27 yeast collection strains of different origin by UP-PCR followed by dot-hybridization, only 22 strains were assigned to the biological species Zygowilliopsis californica(Lodder) Kudriavzev. Four strains were identified as Williopsis suaveolens(Klöcker) Naumov et al. Universal primers L45 and N21 are recommended for identification of the Z. californicayeasts.     

Molecular divergence of the soil yeasts Williopsis sensu stricto

Fifty-three strains of saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA which comprised the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, Komagataea pratensis, and the Williopsis sensu stricto complex. Minisatellite primer M13 was proposed for the differentiation between twin species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.     

Molecular Genetic Analysis of the Yeast Komagataea (Williopsis) pratensisStrains Isolated from the Caucasian and Tien Shan Soils

The analysis of sixteen Komagataea (Williopsis) pratensisisolates from Caucasian and Tien Shan soils by the PCR, blot hybridization, and isoenzyme electrophoresis techniques showed that fifteen of them do belong to the species K. pratensis. The isolates from the two geographic areas differed in some physiological characteristics and in the PCR product profiles obtained with the microsatellite primers (CAC)₅and (GACA)₄.     

The genosystematics of yeast genera Willopsis zender and Zygowillopsis kudriavzev

The data have been compared on the yeast genomic divergence obtained in genetic molecular and biological studies of the yeast genera Williopsis and Zygowilliopsis taken as an example. The hybridization analysis, the enzyme electrophoresis and DNA reassociation comprise the noncontradictory estimation of the yeasts relation. Different methods demonstrate a convincing species discreteness among the yeast genomes. The reproductive isolation of the yeast taxons is defined more invariably by the genetic methods. A new combination Williopsis sargentensis (Wickerham et Kurtzman) Naumov comb. nov. has been formed.     

Linear mitochondrial DNAs of yeasts: frequency of occurrence and general features.

In most yeast species, the mitochondrial DNA (mtDNA) has been reported to be a circular molecule. However, two cases of linear mtDNA with specific termini have previously been described. We examined the frequency of occurrence of linear forms of mtDNA among yeasts by pulsed-field gel electrophoresis. Among the 58 species from the genera Pichia and Williopsis that we examined, linear mtDNA was found with unexpectedly high frequency. Thirteen species contained a linear mtDNA, as confirmed by restriction mapping, and labeling, and electron microscopy. The mtDNAs from Pichia pijperi, Williopsis mrakii, and P. jadinii were studied in detail. In each case, the left and right terminal fragments shared homologous sequences. Between the terminal repeats, the order of mitochondrial genes was the same in all of the linear mtDNAs examined, despite a large variation of the genome size. This constancy of gene order is in contrast with the great variation of gene arrangement in circular mitochondrial genomes of yeasts. The coding sequences determined on several genes were highly homologous to those of the circular mtDNAs, suggesting that these two forms of mtDNA are not of distant origins.     

Discrimination of the soil yeast species Williopsis saturnus and Williopsis suaveolens by the polymerase chain reaction using nonspecific primers

Thirty-five yeast strains of the genus Williopsis, analyzed by the polymerase chain reaction with the universal primer N21, were found to belong to two sibling species, W. saturnus and W. suaveolens. Such affiliation of the strains studied agrees well with the results of genetic and physiological investigations.     

Identification of Zygowillopsis californica strains of various origin by polymerase chain reaction with universal primers]

After reevaluation of the taxonomic position of 27 yeast collection strains of different origin by UP-PCR followed by dot-hybridization, only 22 strains were assigned to the biological species Zygowilliopsis californica (Lodder) Kudriavzev. Four strains were identified as Williopsis suaveolens (Kl?cker) Naumov et al. Universal primers L45 and N21 are recommended for identification of the Z. californica yeasts.     

Genomic studies on killer yeasts belonging to the genusPichia

Twenty-four species belonging to the genusPichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA.Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii andCandida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of theEcoRI andXhoI RFLP patterns withP. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis andC. albicans). However, one hybridized band, located at 3.9–4.1 Kb, seems to be peculiar to thePichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.     

Molecular divergence of the soil yeasts <Emphasis Type="Italic">Williopsis</Emphasis> sensu stricto

Fifty-three strains of Saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA comprising the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, and Komagataea pratensis and the Williopsis sensu stricto complex. The minisatellite primer M13 was proposed for differentiation between sibling species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of Saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 768–776.Original Russian Text Copyright © 2004 by Naumova, Gazdiev, Naumov.     

Molecular-genetic analysis of the yeast Komagataea (williopsis) pratensis populations from Caucasian and Tien Shan regions

The analysis of sixteen Komagataea (Williopsis) pratensis from the Caucasian and Tien Shan soils by the PCR, blot hybridization, and isoenzyme electrophoresis techniques showed that fifteen of them do belong to the species K. pratensis. The isolates from the two geographic areas differed in some physiological characteristics and in the PCR product profiles obtained with the microsatellite primers (CAC)5 and (GACA)4.     

A phylogenetic study of ubiquinone-7 species of the genus Candida based on 18S ribosomal DNA sequence divergence

To clarify phylogenetic relationships among ubiquinone 7 (Q7)-forming species of the genus Candida, we analyzed the nearly complete sequences of 18S ribosomal RNA genes (18S rDNAs) from fifty strains (including 46 type strains) of Candida species, and from 8 type strains of species/varieties of the genera Issatchenkia, Pichia and Saturnispora. Q7-forming Candida species were divided into three major groups (Group I, II, and III) and were phylogenetically distant from a group that includes the type species of the genus Candida. Group I included four clusters with basal branches that were weakly supported. The first cluster comprised C. vartiovaarae, C. maritima, C. utilis, C. freyschussii, C. odintsovae, C. melinii, C. quercuum, Williopsis saturnus var. saturnus, and W. mucosa. The second cluster comprised C. norvegica, C. montana, C. stellimalicola, C. solani, C. berthetii, and C. dendrica. Williopsis pratensis, W. californica, Pichia opuntiae and 2 related species, P. amethionina (two varieties), and P. caribaea were also included in this cluster. The third cluster comprised C. pelliculosa (anamorph of P. anomala), C. nitrativorans, and C. silvicultrix. The fourth cluster comprised C. wickerhamii and C. peltata, which were placed in the P. holstii - C. ernobii clade with Q8-containing species. Group II comprised C. pignaliae, C. nemodendra, C. methanolovescens, C. maris, C. sonorensis, C. pini, C. llanquihuensis, C. cariosilignicola, C. ovalis, C. succiphila (including its two synonyms), C. methanosorbosa, C. nitratophila, C. nanaspora, C. boidinii (including its two synonyms), W. salicorniae, and P. methanolica. Group III was composed of four clusters with strong bootstrap support. The first cluster comprised C. valida (anamorph of P. membranifaciens), C. ethanolica, C. pseudolambica, C. citrea, C. inconspicua, C. norvegensis, C. rugopelliculosa, and C. lambica. Three species and two varieties of the genus Issatchenkia were also included in this cluster. The second cluster comprised C. diversa, C. silvae, 4 Saturnispora species, and P. besseyi. The third comprised C. sorboxylosa, and the fourth comprised C. vini. Based on this 18S rDNA sequence analysis, it is evident that Q7-forming Candida species and the genera Pichia and Williopsis are polyphyletic. The genus Issatchenkia is suggested to be congeneric with the genus Pichia. The genus Saturnispora is phylogenetically definable.     

Killer-yeast strains with a broad spectrum of action: a search among collection strains and preliminary classification

Killer strains were looked for among certain yeast species belonging to five spore-forming genera (Hansenula, Pichia, Kluyveromyces, Williopsis and Zygowilliopsis) and one imperfect genus (Candida). 171 killers were found among 272 strains, and many of them had a high activity and a broad action spectrum. These as well as S. cerevisiae K1 and K2 killers (173 killers all in ail) were classified according to differences in their activity and the spectra of action on susceptible strains. Thirty-four groups belonging to 13 classes were isolated.     

Williopsis saturnus and Williopsis beijerinckii-different taxons from polymerase chain reaction data with nonspecific primers]

Fifteen strains of the yeast Williopsis sensu stricto were analyzed by means of UP-PCR. With the N21 universal primer, this approach showed that the strains could be clearly divided into two groups corresponding to the species W. saturnus (Kl?cker) Zender and W. beijerinckii (van der Walt) Naumov et Vustin. The results obtained are in good agreement with data of genetic and isoenzyme analyses and provide no support for the conspecificity of W. saturnus and W. beijerinckii commonly accepted in modern determination manuals.     

The incidence of killer activity of non-Saccharomyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer-sensitive interactions were observed at pH 4.5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3.5, the incidence of killer-sensitive interactions was reduced by 700% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.     

Influence of <Emphasis Type="Italic">Lumbricus terrestris</Emphasis> earthworms on the structure of the yeast community of forest litter

The taxonomic structure of yeast communities was studied in forest litter and soil, as well as in substrates transformed by the activity of Lumbricus terrestris earthworms (leaves in heaps, the gut contents, and coproliths). The activity of L. terrestris has a weak effect on the total yeast abundance but results in substantial changes in the community taxonomic composition. The share of ascomycetous yeasts is significantly higher in the substrates associated with the activity of earthworms. The teleomorphic ascomycetes Williopsis saturnus were isolated from the gut contents. The effect of earthworms on the composition of the yeast community in the process of forest litter destruction is more pronounced than seasonal changes.     

Cider fermentation with three Williopsis saturnus yeast strains and volatile changes

Williopsis saturnus var. subsufficiens NCYC 2728, W. saturnus var. saturnus NCYC 22 and W. saturnus var. mrakii NCYC 500 were used to carry out cider fermentation to assess their impact on the volatile composition of cider. The changes of yeast cell population, °Brix and pH were similar among the three yeasts. Strain NCYC 500 grew best, with the highest cell population of 1.14 × 108 CFU ml−1, followed by strains NCYC 2728 and NCYC 22 (8 × 107 CFU ml−1 and 3.19 × 107 CFU ml−1 respectively). Esters were the most abundant volatiles produced, followed by alcohols. Among the esters, ethyl acetate, 2-phenylethyl acetate, isoamyl acetate, cis-3-hexenyl acetate and hexyl acetate were the major volatiles. The major alcohols were ethanol, isoamyl alcohol, 2-phenylethyl alcohol and isobutyl alcohol. The three Williopsis yeasts transformed volatile compounds during cider fermentation with significant variations in terms of volatile production and degradation. This study implied that fermentation with Williopsis yeasts could result in cider with a more complex yet fruity aroma.     

Linear mitochondrial DNAs of yeasts: closed-loop structure of the termini and possible linear-circular conversion mechanisms.

The terminal structure of the linear mitochondrial DNA (mtDNA) from three yeast species has been examined. By enzymatic digestion, alkali denaturation, and sequencing of cloned termini, it was shown that in Pichia pijperi and P. jadinii, both termini of the linear mtDNA were made of a single-stranded loop covalently joining the two strands, as in the case of vaccinia virus DNA. The left and right loop sequences were in either of two orientations, suggesting the existence of a flip-flop inversion mechanism. Contiguous to the terminal loops, inverted terminal repeats were present. The mtDNA from Williopsis mrakii seems to have an analogous structure, although terminal loops could not be directly demonstrated. Electron microscopy revealed the presence, among linear molecules, of a small number of circular DNAs, mostly of monomer length. Linear and circular models of replication are considered, and possible conversion mechanisms between linear and circular forms are discussed. A flip-flop inversion mechanism between the inverted repeat sequences within a circular intermediate may be involved in the generation of the linear form of mtDNA.     

Use of sensitivity to antibiotics produced by representatives of the genera Williopsis and Zygowilliopsis in the identification of yeasts

Yeast strains belonging to the genera Candida and Hansenula were shown to differ in their susceptibility to the action of protein antibiotics produced by the yeasts Williopsis and Zygowilliopsis. This finding can be used as an additional criterion for yeast identification.