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Kluyveromyces marxianus is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for K. marxianus. However, the complexity of the kefir microbiota makes the traits of K. marxianus difficult to study. In this research, we focused on K. marxianus cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC-MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of K. marxianus had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the GAP1 gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of K. marxianus, releasing proteins and peptides. These findings can be fundamental for K. marxianus probiotic and kefir studies in the future., (© 2021. The Author(s).)

tRNAs are encoded by a large gene family, usually with several isogenic tRNAs interacting with the same codon. Mutations in the anticodon region of other tRNAs can overcome specific tRNA deficiencies. Phylogenetic analysis suggests that such mutations have occurred in evolution, but the driving force is unclear. We show that in yeast suppressor mutations in other tRNAs are able to overcome deficiency of the essential TRT2 -encoded tRNA Thr CGU at high temperature (40°C). Surprisingly, these tRNA suppressor mutations were obtained after whole-genome transformation with DNA from thermotolerant Kluyveromyces marxianus or Ogataea polymorpha strains but from which the mutations did apparently not originate. We suggest that transient presence of donor DNA in the host facilitates proliferation at high temperature and thus increases the chances for occurrence of spontaneous mutations suppressing defective growth at high temperature. Whole-genome sequence analysis of three transformants revealed only four to five nonsynonymous mutations of which one causing TRT2 anticodon stem stabilization and two anticodon mutations in non-threonyl-tRNAs, tRNA Lys CUU and tRNA eMet CAU , were causative. Both anticodon mutations suppressed lethality of TRT2 deletion and apparently caused the respective tRNAs to become novel substrates for threonyl-tRNA synthetase. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) data could not detect any significant mistranslation, and reverse transcription-quantitative PCR results contradicted induction of the unfolded protein response. We suggest that stress conditions have been a driving force in evolution for the selection of anticodon-switching mutations in tRNAs as revealed by phylogenetic analysis. IMPORTANCE In this work, we have identified for the first time the causative elements in a eukaryotic organism introduced by applying whole-genome transformation and responsible for the selectable trait of interest, i.e., high temperature tolerance. Surprisingly, the whole-genome transformants contained just a few single nucleotide polymorphisms (SNPs), which were unrelated to the sequence of the donor DNA. In each of three independent transformants, we have identified a SNP in a tRNA, either stabilizing the essential tRNA Thr CGU at high temperature or switching the anticodon of tRNA Lys CUU or tRNA eMet CAU into CGU, which is apparently enough for in vivo recognition by threonyl-tRNA synthetase. LC-MS/MS analysis indeed indicated absence of significant mistranslation. Phylogenetic analysis showed that similar mutations have occurred throughout evolution and we suggest that stress conditions may have been a driving force for their selection. The low number of SNPs introduced by whole-genome transformation may favor its application for improvement of industrial yeast strains., (Copyright © 2021 Deparis et al.)

Robiola di Roccaverano, from the Piedmont region of Italy, is a Protected Designation of Origin soft cheese made with raw goat milk. The peculiarity of this cheese is that during the manufacturing process, a natural starter culture (NC) is added to raw milk. This study examined the viable microorganisms of technological interest, including lactic acid bacteria and fungal populations, in samples of raw milk, NC, and fresh and ripened cheese collected from one dairy using culture-dependent techniques. First, the isolated colonies were analyzed using random amplification of polymorphic DNA (RAPD) PCR, and strains with similar fingerprints were clustered together. Further, representative isolates of each group were subjected to 16S or 26S ribosomal DNA sequencing. Finally, species-specific PCR was conducted to distinguish the Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris. Among the studied lactic acid bacteria, 13 RAPD profiles were obtained, corresponding to 9 different bacterial species or subspecies. Concerning mold and yeast isolates, 5 species were found that coincided with 5 RAPD types. Observing the strains isolated in the study, Lc. lactis was the most prevalent species in raw milk and NC samples, and Leuconostoc mesenteroides was the predominant species identified in 5- and 15-d cheese isolates. Furthermore, whereas only these 2 species were detected in NC, Enterococcus and Lactobacillus genera were found in raw milk and cheese, respectively. Concerning the mold and yeast isolates, in NC Kluyveromyces spp. was mainly found, and in cheese samples the representative species were Geotrichum candidum and Yarrowia lipolytica. Finally, raw milk and cheese safety were evaluated, and the samples complied with the standard required by European Commission regulation number 2073/2005., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Dysbiosis, or imbalance in the gut microbiome, has been implicated in auto-immune, inflammatory, neurological diseases as well as in cancers. More recently it has also been shown to be associated with ocular diseases. In the present study, the association of gut microbiome dysbiosis with bacterial Keratitis, an inflammatory eye disease which significantly contributes to corneal blindness, was investigated. Bacterial and fungal gut microbiomes were analysed using fecal samples of healthy controls (HC, n = 21) and bacterial Keratitis patients (BK, n = 19). An increase in abundance of several antiinflammatory organisms including Dialister, Megasphaera, Faecalibacterium, Lachnospira, Ruminococcus and Mitsuokella and members of Firmicutes, Veillonellaceae, Ruminococcaceae and Lachnospiraceae was observed in HC compared to BK patients in the bacterial microbiome. In the fungal microbiome, a decrease in the abundance of Mortierella, Rhizopus, Kluyveromyces, Embellisia and Haematonectria and an increase in the abundance of pathogenic fungi Aspergillus and Malassezia were observed in BK patients compared to HC. In addition, heatmaps, PCoA plots and inferred functional profiles also indicated significant variations between the HC and BK microbiomes, which strongly suggest dysbiosis in the gut microbiome of BK patients. This is the first study demonstrating the association of gut microbiome with the pathophysiology of BK and thus supports the gut-eye axis hypothesis. Considering that Keratitis affects about 1 million people annually across the globe, the data could be the basis for developing alternate strategies for treatment like use of probiotics or fecal transplantation to restore the healthy microbiome as a treatment protocol for Keratitis.

Cheese is a typical handcrafted fermented food in Kazak minority from the Uighur Autonomy Region in China and Central Asia. Among the microbial community that is responsible for Kazak cheese fermentation, yeasts play important role in flavor formation during ripening. To develop ripening cultures, we isolated 123 yeasts from 25 cheese products in Kazak, and identified 87 isolates by the D1/D2 domain of the large subunit rRNA gene sequence. Pichia kudriavzevii was the dominant yeast in Kazak cheese, followed by Kluyveromyces marxianus and Kluyveromyces lactis. Of these, the ability to exhibit enzyme of dominant isolates and contribution to the typical flavor of cheeses was assessed. Enzyme producing yeast strains were inoculated in Hazak cheese-like medium and volatile compounds were identified by head space solid phase micro extraction coupled to gas chromatography and mass spectroscopy. Pichia kudriavzevii N-X displayed the strongest extracellular proteolytic and activity on skim milk agar and produced a range of aroma compounds (ethanol, ethyl acetate, 3-methylbutanol, and acetic acid) for Kazak cheese flavor, could be explored as ripening cultures in commercial production of Kazak cheeses., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The yeast Kluyveromyces marxianus possesses advantageous traits like rapid growth, GRAS (generally regarded as safe) status and thermotolerance that make it very suitable for diverse biotechnological applications. Although physiological studies demonstrate wide phenotypic variation within the species, there is only limited information available on the genetic diversity of K. marxianus. The aim of this work was to develop a multilocus sequence typing (MLST) method for K. marxianus to improve strain classification and selection. Analysis of housekeeping genes in a number of sequenced strains led to the selection of five genes, IPP1, TFC1, GPH1, GSY2 and SGA1, with sufficient polymorphic sites to allow MLST analysis. These loci were sequenced in an additional 76 strains and used to develop the MLST. This revealed wide diversity in the species and separation of the culture collection and wild strains into multiple distinct clades. Two subsets of strains that shared sources of origin were subjected to MLST and split decomposition analysis. The latter revealed evidence of recombination, indicating that this yeast undergoes mating in the wild. A public access web-based portal was established to allow expansion of the database and application of MLST to additional K. marxianus strains. This will aid understanding of the genetic diversity of the yeast and facilitate biotechnological exploitation.

Kluyveromyces marxianus is a safe yeast used in the food and biotechnology sectors. One of the important traits that sets it apart from the familiar yeasts, Saccharomyces cerevisiae, is its capacity to grow using lactose as a carbon source. Like in its close relative, Kluyveromyces lactis, this requires lactose transport via a permease and intracellular hydrolysis of the disaccharide. Given the importance of the trait, it was intriguing that most, but not all, strains of K. marxianus are reported to consume lactose efficiently. In this study, primarily through heterologous expression in S. cerevisiae and K. marxianus, it was established that a single gene, LAC12, is responsible for lactose uptake in K. marxianus. Strains that failed to transport lactose showed variation in 13 amino acids in the Lac12p protein, rendering the protein non-functional for lactose transport. Genome analysis showed that the LAC12 gene is present in four copies in the subtelomeric regions of three different chromosomes but only the ancestral LAC12 gene encodes a functional lactose transporter. Other copies of LAC12 may be non-functional or have alternative substrates. The analysis raises some interesting questions regarding the evolution of sugar transporters in K. marxianus., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Targeted gene replacement in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555 has been hampered by its propensity to non-homologous end joining (NHEJ). To enhance homologous recombination (HR) by blocking NHEJ, we identified and disrupted the K. marxianus KU80 gene. The ku80 deletion mutant strain (Kmku80∆) of K. marxianus KCTC 17555 did not show apparent growth defects under several conditions with the exception of exposure to tunicamycin. The targeted disruption of the three model genes, KmLEU2, KmPDC1, and KmPDC5, was increased by 13-70 % in Kmku80∆, although the efficiency was greatly affected by the length of the homologous flanking fragments. In contrast, the double HR frequency was 0-13.7 % in the wild-type strain even with flanking fragments 1 kb long. Therefore, Kmku80∆ promises to be a useful recipient strain for targeted gene manipulation.

Background: Ashbya gossypii is an industrially relevant microorganism traditionally used for riboflavin production. Despite the high gene homology and gene order conservation comparatively with Saccharomyces cerevisiae, it presents a lower level of genomic complexity. Its type of growth, placing it among filamentous fungi, questions how close it really is from the budding yeast, namely in terms of metabolism, therefore raising the need for an extensive and thorough study of its entire metabolism. This work reports the first manual enzymatic genome-wide re-annotation of A. gossypii as well as the first annotation of membrane transport proteins., Results: After applying a developed enzymatic re-annotation pipeline, 847 genes were assigned with metabolic functions. Comparatively to KEGG's annotation, these data corrected the function for 14% of the common genes and increased the information for 52 genes, either completing existing partial EC numbers or adding new ones. Furthermore, 22 unreported enzymatic functions were found, corresponding to a significant increase in the knowledge of the metabolism of this organism. The information retrieved from the metabolic re-annotation and transport annotation was used for a comprehensive analysis of A. gossypii's metabolism in comparison to the one of S. cerevisiae (post-WGD - whole genome duplication) and Kluyveromyces lactis (pre-WGD), suggesting some relevant differences in several parts of their metabolism, with the majority being found for the metabolism of purines, pyrimidines, nitrogen and lipids. A considerable number of enzymes were found exclusively in A. gossypii comparatively with K. lactis (90) and S. cerevisiae (13). In a similar way, 176 and 123 enzymatic functions were absent on A. gossypii comparatively to K. lactis and S. cerevisiae, respectively, confirming some of the well-known phenotypes of this organism., Conclusions: This high quality metabolic re-annotation, together with the first membrane transporters annotation and the metabolic comparative analysis, represents a new important tool for the study and better understanding of A. gossypii's metabolism.

Est1 and Ebs1 in Saccharomyces cerevisiae are paralogous proteins that arose through whole-genome duplication and that serve distinct functions in telomere maintenance and translational regulation. Here we present our functional analysis of the sole Est1/Ebs1 homologue in the related budding yeast Kluyveromyces lactis (named KlEst1). We show that similar to other Est1s, KlEst1 is required for normal telomere maintenance in vivo and full telomerase primer extension activity in vitro. KlEst1 also associates with telomerase RNA (Ter1) and an active telomerase complex in cell extracts. Both the telomere maintenance and the Ter1 association functions of KlEst1 require its N-terminal domain but not its C terminus. Analysis of clusters of point mutations revealed residues in both the N-terminal TPR subdomain and the downstream helical subdomain (DSH) that are important for telomere maintenance and Ter1 association. A UV cross-linking assay was used to establish a direct physical interaction between KlEst1 and a putative stem-loop in Ter1, which also requires both the TPR and DSH subdomains. Moreover, similar to S. cerevisiae Ebs1 (ScEbs1) (but not ScEst1), KlEst1 confers rapamycin sensitivity and may be involved in nonsense-mediated decay. Interestingly, unlike telomere regulation, this apparently separate function of KlEst1 requires its C-terminal domain. Our findings provide insights on the mechanisms and evolution of Est1/Ebs1 homologues in budding yeast and present an attractive model system for analyzing members of this multifunctional protein family.

Horizontal gene transfer has been occasionally mentioned in eukaryotic genomes, but such events appear much less numerous than in prokaryotes, where they play important functional and evolutionary roles. In yeasts, few independent cases have been described, some of which corresponding to major metabolic functions, but no systematic screening of horizontally transferred genes has been attempted so far. Taking advantage of the synteny conservation among five newly sequenced and annotated genomes of Saccharomycetaceae, we carried out a systematic search for HGT candidates amidst genes present in only one species within conserved synteny blocks. Out of 255 species-specific genes, we discovered 11 candidates for HGT, based on their similarity with bacterial proteins and on reconstructed phylogenies. This corresponds to a minimum of six transfer events because some horizontally acquired genes appear to rapidly duplicate in yeast genomes (e.g. YwqG genes in Kluyveromyces thermotolerans and serine recombinase genes of the IS607 family in Saccharomyces kluyveri). We show that the resulting copies are submitted to a strong functional selective pressure. The mechanisms of DNA transfer and integration are discussed, in relation with the generally small size of HGT candidates. Our results on a limited set of species expand by 50% the number of previously published HGT cases in hemiascomycetous yeasts, suggesting that this type of event is more frequent than usually thought. Our restrictive method does not exclude the possibility that additional HGT events exist. Actually, ancestral events common to several yeast species must have been overlooked, and the absence of homologs in present databases leaves open the question of the origin of the 244 remaining species-specific genes inserted within conserved synteny blocks.

Seven strains of a novel Kluyveromyces species were isolated from seven water samples collected from a mangrove forest. Analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene sequences revealed that the sequences of five strains (RS8(T), RS20, RS54, RV42 and RV89) were identical and differed from the other two strains (RS65 and RV153) by only one nucleotide substitution in 544 nucleotides (nt). The closest species in terms of pairwise sequences similarity was Kluyveromyces aestuarii, but the level of nucleotide substitution (six to seven nucleotide substitutions in 544 nt) was sufficient to justify the description of a separate species. The phylogenetic tree based on the sequence of the D1/D2 domain of the LSU rRNA gene rather suggested that the new species is a sister species of K. aestuarii and forms a clade with the other six recognized species of Kluyveromyces. Sequence analysis of the internal transcribed spacer (ITS1-5.8S rRNA gene-ITS2) region supported their distinct status as a species. The phenotypic characteristics of the seven strains were typical of the genus Kluyveromyces. On this basis, the seven strains were assigned to a single novel species of the genus Kluyveromyces, for which the name Kluyveromyces siamensis sp. nov. is proposed. The type strain is RS8(T) (BCC 25962(T)=NBRC 103859(T)=CBS 10860(T)).

Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.

The Kluyveromyces lactis ter1-16T strain contains mutant telomeres that are poorly bound by Rap1, resulting in a telomere-uncapping phenotype and significant elongation of the telomeric DNA. The elongated telomeres of ter1-16T allowed the isolation and examination of native yeast telomeric DNA by electron microscopy. In the telomeric DNA isolated from ter1-16T, looped molecules were observed with the physical characteristics of telomere loops (t-loops) previously described in mammalian and plant cells. ter1-16T cells were also found to contain free circular telomeric DNA molecules (t-circles) ranging up to the size of an entire telomere. When the ter1-16T uncapping phenotype was repressed by overexpression of RAP1 or recombination was inhibited by deletion of rad52, the isolated telomeric DNA contained significantly fewer t-loops and t-circles. These results suggest that disruption of Rap1 results in elevated recombination at telomeres, leading to increased strand invasion of the 3' overhang within t-loop junctions and resolution of the t-loop junctions into free t-circles.

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.

A brief outline of the current taxonomic status of the genus Kluyveromyces is presented. Noteworthy are the transfer of several former Kluyveromyces species to other genera, the retention of the name Kluyveromyces for K. lactis, K. marxianus, and four related species, and some recent attempts to clarify the variety status of strains assigned to K. lactis.

Genome-wide analysis of transcriptional regulation is generally carried out on well-characterized reference laboratory strains; hence, the characteristics of industrial isolates are therefore overlooked. In a previous study on the major cheese yeast Kluyveromyces lactis, we have shown that the reference strain and an industrial strain used in cheese making display a differential gene expression when grown on a single carbon source. Here, we have used more controlled conditions, i.e., growth in a fermentor with pH and oxygen maintained constant, to study how these two isolates grown in glucose reacted to an addition of lactose. The observed differences between sugar consumption and the production of various metabolites, ethanol, acetate, and glycerol, correlated with the response were monitored by the analysis of the expression of 482 genes. Extensive differences in gene expression between the strains were revealed in sugar transport, glucose repression, ethanol metabolism, and amino acid import. These differences were partly due to repression by glucose and another, yet-unknown regulation mechanism. Our results bring to light a new type of K. lactis strain with respect to hexose transport gene content and repression by glucose. We found that a combination of point mutations and variation in gene regulation generates a biodiversity within the K. lactis species that was not anticipated. In contrast to S. cerevisiae, in which there is a massive increase in the number of sugar transporter and fermentation genes, in K. lactis, interstrain diversity in adaptation to a changing environment is based on small changes at the level of key genes and cell growth control.

The strong LAC4 promoter (P(LAC4)) from Kluyveromyces lactis has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of P(LAC4) to promote gene expression in Escherichia coli. This can interfere with the process of assembling expression constructs in E. coli cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of P(LAC4) variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the E. coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with P(LAC4). Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by approximately 87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in K. lactis cells to levels comparable to wild-type P(LAC4). We created a novel integrative expression vector (pKLAC1) containing the P(LAC4) variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in E. coli cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in E. coli prior to their expression in K. lactis.

Using the Genolevures sequencing data, we developed an expression micro-array for the yeast Kluyveromyces lactis consisting of 482 genes, mainly involved in central metabolism, compound transport facilitators and stress response. The array was validated using the LAC/GAL system. By comparing gene expression in the laboratory reference strain CBS2359 and in an industrial strain B1, we demonstrated the influence of two carbon sources, glucose and lactose, on the expression of genes involved in the respiratory and in the fermentative metabolic pathways. We also showed that the two strains, although both originating from dairies, display unexpected differences in gene expression on each type of carbon source.

The currently accepted formal division of the species Kluyveromyces lactis into two taxonomic varieties, Kl. lactis var. lactis and Kl. lactis var. drosophilarum, is based arbitrarily on phenotypic and ecological characters. On the other hand, the genetic hybridisation analysis and molecular karyotyping of its synonyms allowed us [FEMS Yeast Res. 2 (2002) 39] to reinstate them in the genus Zygofabospora Kudriavzev emend G. Naumov (=Kluyveromyces Kurtzman et al., 2001) as the varieties Zf. lactis var. lactis, Zf. lactis var. krassilnikovii, Zf. lactis var. drosophilarum, Zf. lactis var. phaseolospora and Zf. lactis var. vanudenii. In the present work, we studied forty Kl. lactis strains of different geographic and ecological origins by means of restriction analysis of the PCR-amplified non-coding nrDNA regions encompassing the intergenic spacer 2 (IGS2) and the internal transcribed spacers (ITS1 and ITS2). The results confirmed the complex structure of Kl. lactis. Moreover, four additional genetic populations were identified: three in North America ('aquatic', 'pseudovanudenii' and 'new') and one in Far-East Asia ('oriental'). Comparative sequence analysis of the 5.8S-rRNA gene and the two internal transcribed spacers revealed that the populations 'aquatic' and 'oriental' formed distinct taxa which are phylogenetically separate from the five known populations. However, some discrepancies were observed between the restriction and sequencing data. Genetic hybridisation analysis needs to be done to further elucidate the genetic relationships between the populations of Kl. lactis.

Genera currently assigned to the Saccharomycetaceae have been defined from phenotype, but this classification does not fully correspond with species groupings determined from phylogenetic analysis of gene sequences. The multigene sequence analysis of Kurtzman and Robnett [FEMS Yeast Res. 3 (2003) 417-432] resolved the family Saccharomycetaceae into 11 well-supported clades. In the present study, the taxonomy of the Saccharomyctaceae is evaluated from the perspective of the multigene sequence analysis, which has resulted in reassignment of some species among currently accepted genera, and the proposal of the following five new genera: Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora.

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.

Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free beta-galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating. Immobilization of the beta-galactosidase on to Duolite A-568 increased the synthesis of GOS. GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media. The advantage of microwave heating on GOS formation was also examined. Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS. The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized beta-glucosidase and with added co-solvents such as hexanol.

The wealth of comparative genomics data from yeast species allows the molecular evolution of these eukaryotes to be studied in great detail. We used "proximity plots" to visually compare chromosomal gene order information from 14 hemiascomycetes, including the recent Génolevures survey, to Saccharomyces cerevisiae. Contrary to the original reports, we find that the Génolevures data strongly support the hypothesis that S. cerevisiae is a degenerate polyploid. Using gene order information alone, 70% of the S. cerevisiae genome can be mapped into "sister" regions that tile together with almost no overlap. This map confirms and extends the map of sister regions that we constructed previously by using duplicated genes, an independent source of information. Combining gene order and gene duplication data assigns essentially the whole genome into sister regions, the largest gap being only 36 genes long. The 16 centromere regions of S. cerevisiae form eight pairs, indicating that an ancestor with eight chromosomes underwent complete doubling; alternatives such as segmental duplications can be ruled out. Gene arrangements in Kluyveromyces lactis and four other species agree quantitatively with what would be expected if they diverged from S. cerevisiae before its polyploidization. In contrast, Saccharomyces exiguus, Saccharomyces servazzii, and Candida glabrata show higher levels of gene adjacency conservation, and more cases of imperfect conservation, suggesting that they split from the S. cerevisiae lineage after polyploidization. This finding is confirmed by sequences around the C. glabrata TRP1 and IPP1 loci, which show that it contains sister regions derived from the same duplication event as that of S. cerevisiae.

The rDNA sequencing data obtained during the last 5 years in several laboratories clearly demonstrate that within the heterogeneous genus Kluyveromyces there is a group of highly related species, which we refer to as the genus Zygofabospora Kudriavzev 1960 emend. G. Naumov 2002. This genus includes four hybridizing species, Zf. marxiana, Zf. dobzhanskii, Zf. lactis, Zf. wickerhamii (Zygofabospora sensu stricto), and two taxonomically related species, Zf. aestuarii, Zf. nonfermentans (Nagahama et al.) G. Naumov comb. nov. (Zygofabospora sensu lato). We studied the relationships of the yeasts composing the Zf. lactis complex. Genetic hybridization analysis and molecular karyotyping revealed partial genetic-isolation varieties, Zf. lactis var. drosophilarum (Shehata et al.) G. Naumov comb. nov. and Zf. lactis var. phaseolospora (Shehata et al.) G. Naumov comb. nov. from North America, and Zf. lactis var. krassilnikovii (Kudriavzev) G. Naumov comb. nov. from Europe. The dairy yeast Zf. lactis var. lactis G. Naumov comb. nov. yields highly fertile hybrids with its wild ancestor Zf. lactis var. krassilnikovii and semi-sterile hybrids with North American taxa. Besides, Zf. lactis var. lactis and Zf. lactis var. krassilnikovii formed fertile hybrids with the South African yeast Zf. lactis var. vanudenii (van der Walt et Nel) G. Naumov comb. nov. The reinstatement of the latter yeast at the variety level has been done taking into account the results of the present study and the literature data on its geographic isolation, high divergence of the karyotype and mitochondrial DNA.

Significant substratum damage can occur when plasticized PVC (pPVC) is colonized by microorganisms. We investigated microbial colonization of pPVC in an in situ, longitudinal study. Pieces of pPVC containing the plasticizers dioctyl phthalate and dioctyl adipate (DOA) were exposed to the atmosphere for up to 2 years. Fungal and bacterial populations were quantified, and colonizing fungi were identified by rRNA gene sequencing and morphological characteristics. Aureobasidium pullulans was the principal colonizing fungus, establishing itself on the pPVC between 25 and 40 weeks of exposure. A group of yeasts and yeast-like fungi, including Rhodotorula aurantiaca and Kluyveromyces spp., established themselves on the pPVC much later (after 80 weeks of exposure). Numerically, these organisms dominated A. pullulans after 95 weeks, with a mean viable count +/- standard error of 1,000 +/- 200 yeast CFU cm(-2), compared to 390 +/- 50 A. pullulans CFU cm(-2). No bacterial colonization was observed. We also used in vitro tests to characterize the deteriogenic properties of fungi isolated from the pPVC. All strains of A. pullulans tested could grow with the intact pPVC formulation as the sole source of carbon, degrade the plasticizer DOA, produce extracellular esterase, and cause weight loss of the substratum during growth in vitro. In contrast, several yeast isolates could not grow on pPVC or degrade DOA. These results suggest that microbial succession may occur during the colonization of pPVC and that A. pullulans is critical to the establishment of a microbial community on pPVC.

Translation of mitochondrially coded mRNAs in Saccharomyces cerevisiae depends on membrane-bound mRNA-specific activator proteins, whose targets lie in the mRNA 5'-untranslated leaders (5'-UTLs). In at least some cases, the activators function to localize translation of hydrophobic proteins on the inner membrane and are rate limiting for gene expression. We searched unsuccessfully in divergent budding yeasts for orthologs of the COX2- and COX3-specific translational activator genes, PET111, PET54, PET122, and PET494, by direct complementation. However, by screening for complementation of mutations in genes adjacent to the PET genes in S. cerevisiae, we obtained chromosomal segments containing highly diverged homologs of PET111 and PET122 from Saccharomyces kluyveri and of PET111 from Kluyveromyces lactis. All three of these genes failed to function in S. cerevisiae. We also found that the 5'-UTLs of the COX2 and COX3 mRNAs of S. kluyveri and K. lactis have little similarity to each other or to those of S. cerevisiae. To determine whether the PET111 and PET122 homologs carry out orthologous functions, we deleted them from the S. kluyveri genome and deleted PET111 from the K. lactis genome. The pet111 mutations in both species prevented COX2 translation, and the S. kluyveri pet122 mutation prevented COX3 translation. Thus, while the sequences of these translational activator proteins and their 5'-UTL targets are highly diverged, their mRNA-specific functions are orthologous.

Eleven strains of a new species of the genus Kluyveromyces, characterized as having evanescent asci and Q-6 as the major ubiquinone, were isolated from sediments, a clam and a crab collected at depths of 1000-2000 m in Suruga Bay and Sagami Bay, Japan. A phylogenetic tree based on small-subunit (18S) rRNA gene sequences placed these isolates into a cluster of Kluyveromyces. DNA complementarity and phylogenetic trees of internal transcribed spacer (ITS) regions and 5.8S rRNA genes showed that the isolates are closely related to Kluyveromyces aestuarii, but that these two species are genetically distinct. The isolates are described as Kluyveromyces nonfermentans sp. nov. Because this species lacks the fermentative ability considered to be an important criterion for the genus Kluyveromyces, the definition of the genus has been emended. The type strain of K. nonfermentans is strain SY-33T (= JCM 10232T).

Seven strains of three new yeast species were isolated from soil, flowers and leaves in the Nansei Islands, Japan. These isolates most closely resembled Kluyveromyces phaffii in physiological characteristics and nuclear DNA base composition (30-32 mol% G + C), but on the basis of DNA-DNA hybridization and electrophoretic karyotyping they were categorized into three new species different from K. phaffii. Phylogenetic analysis using 18S rRNA gene sequences showed that the three new species and K. phaffii were highly related to one another and phylogenetically separate from the members of other species. On the basis of phylogeny and physiological characters, it is proposed that the three new species represent novel taxa and should be designated Tetrapisispora iriomotensis gen. nov., sp. nov. (type strain IFO 10929T), Tetrapisispora nanseiensis gen. nov., sp. nov. (type strain IFO 10899T) and Tetrapisispora arboricola gen. nov., sp. nov. (type strain IFO 10925T), while Kluyveromyces phaffii becomes Tetrapisispora phaffii comb. nov.

The accuracy of the Microbial Identification System (MIS; MIDI, Inc. ) for identification of yeasts to the species level was compared by using 438 isolates grown on prepoured BBL Sabouraud dextrose agar (SDA) and prepoured Remel SDA. Correct identification was observed for 326 (74%) of the yeasts cultured on BBL SDA versus only 214 (49%) of yeasts grown on Remel SDA (P < 0.001). The commercial source of the SDA used in the MIS procedure significantly influences the system's accuracy.

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.

A molecular systematic investigation of members of the ascomycetous yeast genera Brettanomyces, Debaryomyces, Dekkera, and Kluyveromyces was performed by using 18S rRNA gene sequence analysis. Our comparative sequence analysis revealed that Brettanomyces anomalus and Brettanomyces bruxellensis were closely related to one another and also to their teleomorphs, Dekkera anomala and Dekkera bruxellensis, respectively. Together with Dekkera custersiana and Dekkera naardenensis, these four species formed a stable and distinct phylogenetic group. The three representative species of the genus Debaryomyces examined (viz., Debaryomyces castellii, Debaryomyces hansenii, and Debaryomyces udenii) were found to be genealogically highly related to each other and exhibited a specific phylogenetic affinity (level of sequence similarity, approximately 99.2%) with Candida guilliermondii (teleomorph, Pichia guilliermondii). Debaryomyces species and C. guilliermondii formed a distinct phylogenetic group, which displayed a significant association with a phylogenetically coherent cluster encompassing Lodderomyces elongisporus, Candida albicans, and four other Candida species. In contrast to the situation with the genera Brettanomyces and Debaryomyces, the genus Kluyveromyces displayed very marked phylogenetic heterogeneity. Kluyveromyces polysporus, the type species of the genus Kluyveromyces, and six other Kluyveromyces species (viz., Kluyveromyces africanus, Kluyveromyces delphensis, Kluyveromyces lodderae, Kluyveromyces thermotolerans, Kluyveromyces waltii, and Kluyveromyces yarrowii) were phylogenetically intermixed with species of the genera Zygosaccharomyces, Saccharomyces, and Torulaspora. In contrast, Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces wickerhamii, and three Kluyveromyces marxianus varieties, along with their anamorph, Candida kefyr, formed a highly stable monophyletic group worthy of separate generic status. Kluyveromyces blattae and Kluyveromyces phaffii formed two distinct phylogenetic lines that did not exhibit particularly close affinity with each other or other ascomycetous yeast genera. Our phylogenetic findings are discussed in the context of the results of other genotypic and phenotypic studies.

An analytical method in which we used the selective adsorption of several fluorophores by yeast cells is described. The suitability of using binary mixtures of 1-pyrene butyric acid, 3,6-dimethylamino acridine, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, and rhodamine B isothiocyanate for the characterization and identification of microorganisms was tested with 98 yeast strains belonging to the genera Candida, Hansenula, Kluyveromyces, Pichia, Rhodotorula, and Saccharomyces. The application of multivariate statistical methods and pattern recognition methods to the allocation of the yeast strains into genus-species-strain structures and to a comparison of fluorescence data sets for differentiation and identification purposes showed the usefulness of the method.