Williopsis saturnusand Williopsis beijerinckiiAre Recognized as Distinct Taxa by Means of the Polymerase Chain Reaction with Nonspecific Primers

Fifteen strains of the yeast Williopsissensu 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(Klöcker) 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. saturnusand W. beijerinckiicommonly accepted in modern yeast taxonomic manuals.     

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.     

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.     

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.     

Volatile sulphur compounds and pathways of L-methionine catabolism in Williopsis yeasts

Volatile sulphur compounds (VSCs) are important to the food industry due to their high potency and presence in many foods. This study assessed for the first time VSC production and pathways of L: -methionine catabolism in yeasts from the genus Williopsis with a view to understanding VSC formation and their potential flavour impact. Five strains of Williopsis saturnus (var. saturnus, var. subsufficiens, var. suavolens, var. sargentensis and var. mrakii) were screened for VSC production in a synthetic medium supplemented with L: -methionine. A diverse range of VSCs were produced including dimethyl disulphide, dimethyl trisulphide, 3-(methylthio)-1-propanal (methional), 3-(methylthio)-1-propanol (methionol), 3-(methylthio)-1-propene, 3-(methylthio)-1-propyl acetate, 3-(methylthio)-1-propanoic acid (methionic acid) and ethyl 3-(methylthio)-1-propanoate, though the production of these VSCs varied between yeast strains. W. saturnus var. saturnus NCYC22 was selected for further studies due to its relatively high VSC production. VSC production was characterised step-wise with yeast strain NCYC22 in coconut cream at different L: -methionine concentrations (0.00-0.20%) and under various inorganic sulphate (0.00-0.20%) and nitrogen (ammonia) supplementation (0.00-0.20%), respectively. Optimal VSC production was obtained with 0.1% of L: -methionine, while supplementation of sulphate had no significant effect. Nitrogen supplementation showed a dramatic inhibitory effect on VSC production. Based on the production of VSCs, the study suggests that the Ehrlich pathway of L: -methionine catabolism is operative in W. saturnus yeasts and can be manipulated by adjusting certain nutrient parameters to control VSC production.     

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.     

Williopsis californica, Williopsis saturnus, and Pachysolen tannophilus: novel microorganisms for stereoselective oxidation of secondary alcohols

A screening of 416 microorganisms from different taxonomical groups (bacteria, actinomycetes, yeasts, and filamentous fungi) has been performed looking for active strains in the stereoselective oxidation of secondary alcohols. The working collection was composed of 71 bacterial strains, 45 actinomycetes, 59 yeasts, 60 basidiomycetes, 33 marine fungi, and 148 filamentous fungi. All microorganisms selected were mesophilic. Yeasts were the most active microbial group in the whole-cell-catalyzed oxidation. Williopsis californica, Williopsis saturnus, and Pachysolen tannophilus were the strains of greatest interest, both as growing cells and as resting cells. The oxidation of the alcohols takes place when cells are in the stationary growth phase (after 48 h of culture). These three strains are S-stereoselective for the oxidation of racemic secondary alkanols and show stereospecificity in the oxidation of menthol or neo-menthol, whereas iso-menthol is not oxidized. In the case of the 1-tetrahydronaphtol enantiomers, only the S-enantiomer is oxidized. The three strains were immobilized by entrapment using agarose and agar from algae of the Gracilaria genus. The agarose derivatives displayed significant improvement in the stereospecificity of the reactions.     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

The Phylogenetic Relationships of the Saturn-shaped Ascospore-forming Species of the Genus Williopsis Zender and Related Genera Based on the Partial Sequences of 18S and 26S Ribosomal RNAs (Saccharomycetaceae): The Proposal of Komagataea Gen. Nov.

The partial base sequences of 18S and 26S rRNAs of strains of Williopsis and Saturnospora species were analyzed. In the three regions partially sequenced, the higher base differences were observed in the strains examined of the three species, W. californica, W. mucosa, and W. pratensis, compared with those of W. saturnus var. saturnus (type species of genus Williopsis), W. beijerinckii, W. mrakii, W. saturnus var. subsufficiens, W. suaveolens, P. membranaefaciens (type species of genus Pichia), C. matritensis (type species of genus Citeromyces), and S’spora dispora (type species of genus Saturnospora): the percent similarities were 52–82 in positions 493–622, 130 bases, of 26S rRNA, and the number of base differences was 28–6 in positions 1611–1835, 225 bases, of 26S rRNA, and the number of base differences was 25–4 in positions 1451–1618, 168 bases, of 18S rRNA. In the 18S rRNA partial base sequencings, W. mucosa had an identical base sequence with P. anomala (≡ H. anomala, type species of genus Hansenula). Based on the sequence data obtained, the taxonomic positions of the three Williopsis species mentioned above are discussed. The genus Zygowilliopsis Kudriavzev was postulated to be retained and emended, and a new genus, Komagataea was proposed for W. pratensis with a new combination, Komagataea pratensis.     

Identification and characterization of a new naringinase-producing strain, Williopsis californica Jmudeb007

A new naringinase-producing strain, Jmudeb007 was indentified by means of morphological observation, gene homogeneous analysis and physiological and biochemical test. Its characteristics in expressing naringinase were investigated by batch culture in 7 L fermentors. Jmudeb007 grown white, circular, convex, and smooth edged colonies, and single, oval-shaped and nucleus contained cells. Its gene sequences of 26S rDNA D1/D2 region and 5.8S rDNA-ITS region both showed homogeneities at 99% to Williopsis californica. It appeared positive in glucose fermentation test, negative in urease test and diazo blue B (DBB) test. Strain Jmudeb007 was identified to W. californica. Cultivation of Jmudeb007 with three media containing different amount of glucose showed it was capable of expressing naringinase which hydrolyze naringin to naringenin. The naringinase synthesis of Jmudeb007 was regulated by glucose which depended on the concentration. Jumdeb007 could express naringinase in medium containing glucose no more than 4 g/L. The present work provides a new source of naringinase, W. californica Jmudeb007, which is non-pathogenic, convenient to conduct breeding operation, easy to develop fermentation process. It is the first report about yeast that can produce naringinase, which help to explore naringinase and other glycosidase from yeast.     

Improved antiserum agar method for the serogroup differentiation of Neisseria meningitidis Y and W135

Rabbit antisera prepared to meningococcal serogroups Y and W135 strains were compared with horse antisera using the antiserum agar method (ASA) for the serogroup identification of Neisseria meningitidis. Thirty-seven group Y strains formed immunoprecipitates with the Y rabbit serum only, whereas the same Y strains formed immunoprecipitates with both the Y and W135 horse antisera. Forty-seven W135 strains formed specific immunoprecipitates with both the rabbit and horse W135 antisera by ASA. None of the 166 meningococcal isolates, representative of other meningococcal serogroups, formed immunoprecipitates with the groups Y and W135 rabbit or horse antisera. Use of specific Y and W135 rabbit antisera in ASA provides an improved technique for the serogroup differentiation of groups Y and W135 meningococci.     

Molecular genotyping of Mycobacterium tuberculosis strains isolated from patients in the Samara region by the restriction DNA fragment length polymorphism

The typing of 106 M. tuberculosis (MBT) strains isolated from patients in the Samara region by the restriction DNA fragment length polymorphism (RFLP) IS6110 revealed that most of the strains (71.7%) belonged to the W family, 5 MBT strains (4.7%) belonged to the AI family, one culture was the mixture of two strains, AI and W. In addition, 24 MBT strains (22.6%) classified with other genotypes were detected. The analysis of the sensitivity of the MBT strains to rifampicin and isoniazid, with the method of absolute concentrations and by point mutations, demonstrated that 29 MBT strains (27.3%) were sensitive to rifampicin and isoniazid and 56 MBT strains (52.9%) were resistant to rifampicin and isoniazid simultaneously. Among the MBT strains of different RFLP families, strains both sensitive and resistant to these two preparations could be detected, but strains with multiple drug resistance prevailed in the W family (61.8%).     

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.     

Identification of the female-determining region of the W chromosome in Bombyx mori

The W chromosome of the silkworm Bombyx mori is devoid of functional genes, except for the putative female-determining gene (Fem). To localize Fem, we investigated the presence of W-specific DNA markers on strains in which an autosomal fragment containing dominant marker genes was attached to the W chromosome. We produced new W-chromosomal fragments from the existing Zebra-W strain (T(W;3)Ze chromosome) by X-irradiation, and then carried out deletion mapping of these and sex-limited yellow cocoon strains (T(W;2)Y-Chu, -Abe and -Ban types) from different Japanese stock centers. Of 12 RAPD markers identified in the normal W chromosomes of most silkworm strains in Japan, the newly irradiated W(B-YL-YS)Ze chromosome contained three, the T(W;2)Y-Chu chromosome contained six, and the T(W;2)Y-Abe and -Ban chromosomes contained only one (W-Rikishi). To investigate the ability of the reduced W-chromosome translocation fragments to form heterochromatin bodies, which are found in nuclei of normal adult female sucking stomachs, we examined cells of the normal type p50 strain and the T(W;2)Y-Chu and -Abe strains. A single sex heterochromatin body was found in nuclei of p50 females, whereas we detected only small sex heterochromatin bodies in the T(W;2)Y-Chu strain and no sex heterochromatin body in the T(W;2)Y-Abe strain. Since adult females of all strains were normal and fertile, we conclude that only extremely limited region, containing the W-Rikishi RAPD sequence of the W chromosome, is required to determine femaleness. Based on a comparison of the normal W-chromosome and 7 translocation and W-deletion strains we present a map of Fem relative to the 12 W-specific RAPD markers.     

Antibiotic susceptibility of Neisseria gonorrhoeae in relation to serogroups

Susceptibility testings, by means of the agar-dilution method, were performed on nine antibiotics of 138 non-PPNG (beta-lactamase-producing = PPNG) strains, 17 of which originated from Thailand, and 88 PPNG isolates. The gonococcal strains were sero-grouped by the co-agglutination method and classified among the sero-groups W I, W II or W III. Statistically significant differences in antibiotic susceptibility between non-PPNG strains of the three sero-groups could be demonstrated with strains belonging to sero-group W I as the most sensitive and W III isolates as the most resistant. Non-PPNG strains from Thailand were significantly more resistant than the other non-PPNG isolates of the same sero-group. There was, however, no significant difference in resistance between non-PPNG strains from Thailand and PPNG isolates of the same sero-group. PPNG strains of sero-group W I were significantly more sensitive than PPNG strains of sero-group W II, whereas there was no significant difference between PPNG isolates of sero-groups W II and W III. Non-PPNG strains, not from Thailand, of sero-groups W I and W II were significantly more susceptible to non beta-lactam antibiotics than PPNG strains of corresponding sero-groups, while no such difference could be demonstrated for non-PPNG and PPNG isolates of sero-group W III.     

β-1,3-Glucanase Inhibits Activity of the Killer Toxin Produced by the Marine-Derived Yeast Williopsis saturnus WC91-2

The marine-derived Williopsis saturnus WC91-2 was found to produce very high killer toxin activity against the pathogenic yeast Metschnikowia bicuspidata WCY isolated from the diseased crab. It is interesting to observe that the purified β-1,3-glucanase from W. saturnus WC91-2 had no killer toxin activity but could inhibit activity of the WC91-2 toxin produced by the same yeast. In contrast, the WC91-2 toxin produced had no β-1,3-glucanase activity. We found that the mechanisms of the inhibition may be that the β-1,3-glucanase competed for binding to β-1,3-glucan on the sensitive yeast cell wall with the WC91-2 toxin, causing decrease in the amount of the WC91-2 toxin bound to β-1,3-glucan on the sensitive yeast cell wall and the activity of the WC91-2 toxin against the sensitive yeast cells. In order to make W. saturnus WC91-2 produce high activity of the WC91-2 toxin against the yeast disease in crab, it is necessary to delete the gene encoding β-1,3-glucanase.     

How does infection with parthenogenesis-inducing Wolbachia reduce the fitness of Trichogramma?

We analyzed the survival rate of the immature stages of Trichogramma species and lines that differed in their mode of reproduction. Specifically, we compared the mortality of arrhenotokous (W(-)), irrevertable thelytokous (W(-)), and Wolbachia-associated thelytokous (W(+)) forms. The embryonic mortality of the W(+) strains was significantly higher than that of the W(-) lines. The embryonic mortality was negligible for the arrhenotokous Trichogramma evanescens and the thelytokous Trichogramma cacoeciae, which is not infected with Wolbachia. Only 30% of the eggs of the Wolbachia-infected strains developed to adults, while the emergence rate of the Wolbachia-free strains was more than 78%, irrespective of the origin of the strains. More than 78% of the overall mortality in W(+) strains happened during the early stages of development. About 35% of embryos of W(+) strains remain in the mitotic stage even 48 h after oviposition. Most embryos of W(-) strains had already developed to cellular blastoderm after 6 h, regardless of strains. The mortality of immature stages in W(+) strains was mainly caused by the failure of the mitotic divisions.     

Discrimination between the soil yeast speciesWilliopsis saturnus andWilliopsis suaveolens by the polymerase chain reaction with the universal primer N21

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

Comparison of the proteomes of three yeast wild type strains: CEN.PK2, FY1679 and W303

Yeast deletion strains created during gene function analysis projects very often show drastic phenotypic differences depending on the genetic background used. These results indicate the existence of important molecular differences between the CEN.PK2, FY1679 and W303 wild type strains. To characterise these differences we have compared the protein expression levels between CEN.PK2, FY1679 and W303 strains using twodimensional gel electrophoresis and identified selected proteins by mass spectrometric analysis. We have found that FY1679 and W303 strains are more similar to each other than to the CEN.PK2 strain. This study identifies 62 proteins that are differentially expressed between the strains and provides a valuable source of data for the interpretation of yeast mutant phenotypes observed in CEN.PK2, FY1679 and W303 strains.