Nosocomial transmission of Saccharomyces cerevisiae in bone marrow transplant patients.

Saccharomyces cerevisiae is an unusual cause of clinical infection. We describe three bone marrow transplant patients on a haematology unit who developed possible invasive disease with the organism. Two patients died and both these patients appeared to have a related strain of S. cerevisiae. Screening for S. cerevisiae from throat and stool samples revealed four further patients who were carriers. Genotyping of the invasive and carriage strains demonstrated an indistinguishable strain from patients who had been on the unit at the same time, suggesting cross-infection.

Phylogenetic analysis of the psychrophobic yeast Arxiozyma telluris and the reinstatement of Candida pintolopesii (van Uden) Meyer et Yarrow and Candida slooffii van Uden et do Carmo Sousa.

A phylogenetic analysis was conducted upon ten strains of the psychrophobic yeast species Arxiozyma telluris using nuclear rDNA (18S and 26S) and mitochondrial cytochrome-c oxidase subunit II (COX2) gene sequences. Strains examined included those described originally as Candida slooffii, Torulopsis bovina (= Candida bovina) and Torulopsis pintolopesii (= Candida pintolopesii), which are all currently accepted as synonyms of Arxiozyma telluris. Comparative 18S rDNA sequence analysis showed that these strains formed a genealogically highly related group, which was phylogenetically distinct from any other ascomycetous species studied. The results showed that A. telluris, as currently described, appears to be composed of a complex of closely related but nevertheless separate taxa. rDNA and COX2 gene sequence data revealed that CBS 1787T, the type strain of C. pintolopesii, the currently recognized asexual form (anamorph) of A. telluris, along with strains CBS 2676 and CBS 2985 formed a distinct taxon that is phylogenetically separate from A. telluris. Similarly, the sequence data also showed that C. slooffii is a distinct taxon and support the reinstatement of this species. However, with regard to the relationship between the type strains of A. telluris (CBS 2685T) and C bovina (CBS 2760T), discrepancies were observed between the rDNA and COX2 sequence datasets, and these results are discussed in more detail.

Candida sorbosivorans sp. nov., a new member of the genus Candida Berkhout.

A yeast, strain NCYC 2938T, was isolated from contaminated industrial material. This material was involved in a cascade continuous process for oxidizing sorbitol (D-glucitol) to L-sorbose. The isolate is similar, although not identical, to Candida geochares and Candida magnoliae in its physiological characteristics. Sequence analysis of the 26S rDNA D1/D2 variable domain showed that it was similar to those of both Candida species, but differed sufficiently to be considered as a separate species. Both the physiological characteristics and the unique 26S rDNA D1/D2 sequence of NCYC 2938T are described here, and the yeast has been named Candida sorbosivorans sp. nov. The type strain is NCYC 2938T (= CBS 8768T).

Three new species in the Saccharomyces sensu stricto complex: Saccharomyces cariocanus, Saccharomyces kudriavzevii and Saccharomyces mikatae.

On the basis of genetic analysis, molecular karyotyping and sequence analyses of the 18S rRNA and internal transcribed spacer (ITS) region, three new Saccharomyces species are described, Saccharomyces cariocanus (with type strain NCYC 2890T), Saccharomyces kudriavzevii (with type strain NCYC 2889T) and Saccharomyces mikatae (with type strain NCYC 2888T). Genetic and molecular analyses did not confirm the previously observed conspecificity of Saccharomyces paradoxus and S. cariocanus. The latter species exhibits postzygotic isolation from representative strains from all known geographical populations of S. paradoxus: European, Far-East Asian, North American and Hawaiian.

Sorbic acid resistance: the inoculum effect.

Zygosaccharomyces is a genus associated with the more extreme spoilage yeasts. Zygosaccharomyces spoilage yeasts are osmotolerant, fructophiles (preferring fructose), highly-fermentative and extremely preservative-resistant. Zygosaccharomyces bailii can grow in the presence of commonly-used food preservatives, benzoic, acetic or sorbic acids, at concentrations far higher than are legally permitted or organolepically acceptable in foods. An inoculum effect has been described for many micro-organisms and antimicrobial agents. The minimum inhibitory concentration (MIC) increases with the size of the inoculum; large inocula at high cell density therefore require considerably higher concentrations of inhibitors to prevent growth than do dilute cell suspensions. A substantial inoculum effect was found using sorbic acid against the spoilage yeast Zygosaccharomyces bailii NCYC 1766. The inoculum effect was not caused by yeasts metabolizing or adsorbing sorbic acid, thereby lowering the effective concentration; was not due to absence of cell-cell signals in dilute cell suspensions; and was not an artefact, generated by insufficient time for small inocula to grow. The inoculum effect appeared to be caused by diversity in the populations of yeast cells, with higher probability of sorbic acid-resistant cells being present in large inocula. It was found that individual cells of Zygosaccharomyces bailii populations, grown as single cells in microtitre plate wells, were very diverse, varying enormously in resistance to sorbic acid. 26S ribosomal DNA sequencing did not detect differences between the small fraction of resistant 'super cells' and the average population. Re-inoculation of the 'super cells' after overnight growth on YEPD showed a normal distribution of resistance to sorbic acid, similar to that of the original population. The resistance phenotype was therefore not heritable and not caused by a genetically distinct subpopulation. It was concluded that resistance of the spoilage yeast Zygosaccharomyces bailii to sorbic acid was due to the presence of small numbers of phenotypically resistant cells in the population.

Chromosomal evolution in Saccharomyces.

The chromosomal speciation model invokes chromosomal rearrangements as the primary cause of reproductive isolation. In a heterozygous carrier, chromosomes bearing reciprocal translocations mis-segregate at meiosis, resulting in reduced fertility or complete sterility. Thus, chromosomal rearrangements act as a post-zygotic isolating mechanism. Reproductive isolation in yeast is due to post-zygotic barriers, as many species mate successfully but the hybrids are sterile. Reciprocal translocations are thought to be the main form of large-scale rearrangement since the hypothesized duplication of the whole yeast genome 10(8) years ago. To test the chromosomal speciation model in yeast, we have characterized chromosomal translocations among the genomes of six closely related species in the Saccharomyces 'sensu stricto' complex. Here we show that rearrangements have occurred between closely related species, whereas more distant ones have colinear genomes. Thus, chromosomal rearrangements are not a prerequisite for speciation in yeast and the rate of formation of translocations is not constant. These rearrangements appear to result from ectopic recombination between Ty elements or other repeated sequences.

Zygosaccharomyces lentus: a significant new osmophilic, preservative-resistant spoilage yeast, capable of growth at low temperature.

Zygosaccharomyces lentus is a yeast species recently identified from its physiology and 18S ribosomal sequencing (Steels et al. 1999).The physiological characteristics of five strains of this new yeast so far isolated were investigated, particularly those of technical significance for a spoilage yeast, namely temperature range, pH range, osmotolerance, sugar fermentation, resistance to food preservatives such as sorbic acid, benzoic acid and dimethyldicarbonate (DMDC; Velcorin). Adaptation to benzoic acid, and growth in shaking and static culture were also investigated. Zygosaccharomyces lentus strains grew over a wide range of temperature (4-25 degrees C) and pH 2.2-7.0. Growth at 4 degrees C was significant. Zygosaccharomyces lentus strains grew at 25-26 degrees C in static culture but were unable to grow in aerobic culture close to their temperature maximum. All Z. lentus strains grew in 60% w/v sugar and consequently, are osmotolerant. Zygosaccharomyces lentus strains could utilize sucrose, glucose or fructose as a source of fermentable sugar, but not galactose. Zygosaccharomyces lentus strains were resistant to food preservatives, growing in sorbic acid up to 400 mg l-1 and benzoic acid to 900 mg l-1 at pH 4.0. Adaptation to higher preservative concentrations was demonstrated with benzoic acid. Resistance to DMDC was shown to be greater than that of Z. bailii and Saccharomyces cerevisiae. This study confirms that Z. lentus is an important food spoilage organism potentially capable of growth in a wide range of food products, particularly low pH, high sugar foods and drinks. It is likely to be more significant than Z. bailii in the spoilage of chilled products.

Zygosaccharomyces lentus sp. nov., a new member of the yeast genus Zygosaccharomyces Barker.

Unusual growth characteristics of a spoilage yeast, originally isolated from spoiled whole-orange drink and previously identified as Zygosaccharomyces bailii, prompted careful re-examination of its taxonomic position. Small-subunit rRNA gene sequences were determined for this strain and for four other strains also originally described as Z. bailii but which, in contrast to other strains of this species, grew poorly or not at all under aerobic conditions with agitation, failed to grow in the presence of 1% acetic acid and failed to grow at 30 degrees C. Comparative sequence analysis revealed that these strains represented a phylogenetically distinct taxon closely related to, but distinct from, Z. bailii and Zygosaccharomyces bisporus. Furthermore, sequence analysis of the internal transcribed spacer (ITS) region showed that, while all five strains had identical ITS2 sequences, they could be subdivided into two groups based on ITS1 sequences. Despite such minor inter-strain sequence variation, these yeasts could readily be distinguished from all other currently described Zygosaccharomyces species by using ITS sequences. On the basis of the phylogenetic results presented, a new species comprising the five strains, Zygosaccharomyces lentus sp. nov., is described and supporting physiological data are discussed, including a demonstration that growth of this species is particularly sensitive to the presence of oxygen. The type strain of Z. lentus is NCYC D2627T.

Phylogenetic heterogeneity of the genus Williopsis as revealed by 18S rRNA gene sequences.

A phylogenetic investigation of the ascomycetous yeast genus Williopsis was performed by using 18S rRNA gene sequence analysis. Comparative sequence analysis revealed the genus to be phylogenetically heterogeneous. The five varieties of Williopsis saturnus [var. mrakii, var. sargentensis, var. saturnus (type), var. suaveolens and var. subsufficiens] were found to have identical 18S rRNA gene sequences and formed a distinct group, quite separate from all other Williopsis and non-Williopsis species examined. Williopsis mucosa was found to be the closet phylogenetic relative to the Williopsis saturnus group, however a sequence divergence of approximately 2.3% suggests this species may belong to a separate genus. The recently described species Williopsis salicorniae was found to exhibit a relatively close association with Ogataea minuta (identical to Pichia minuta), the type species of the genus Ogataea. The remaining two members of the genus, Williopsis californica and Williopsis pratensis, were found to form distinct lineages, displaying no specific association with any other Williopsis or non-Williopsis species. Based on comparative analysis of 18S rRNA genes it is apparent that the genus Williopsis as presently constituted is not monophyletic, and that the five currently recognized species form separate sublines each potentially worthy of separate generic status. The genus Williopsis should be restricted to the type species Williopsis saturnus and its five varieties. Despite the five varieties of Williopsis saturnus being genealogically indistinguishable at the 18S rRNA gene level, sequence analysis of the Internal transcribed spacer (ITS) region revealed that the five varieties could be differentiated on both their ITS1 and their ITS2 sequences, providing further evidence of the value of ITS sequences for discrimination of yeasts at the subspecies level.

A phylogenetic analysis of the genus Saccharomyces based on 18S rRNA gene sequences: description of Saccharomyces kunashirensis sp. nov. and Saccharomyces martiniae sp. nov.

A phylogenetic investigation of the ascomycetous yeast genus Saccharomyces was performed by using 18S rRNA gene sequence analysis. Comparative sequence analysis showed that the genus is phylogenetically very heterogeneous. Saccharomyces species were found to be phylogenetically interdispersed with members of other ascomycetous genera (e.g., the genera Kluyveromyces, Torulaspora, and Zygosaccharomyces). The four species of the Saccharomyces sensu stricto complex (viz., Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces paradoxus, and Saccharomyces pastorianus) were found to be phylogenetically closely related to one another, displaying exceptionally high levels of sequence similarity (> or = 99.9%). These four species formed a natural group that was quite separate from the other Saccharomyces and non-Saccharomyces species examined. Saccharomyces exiguus and its anamorph, Candida holmii, were found to be genealogically almost identical and, along with Saccharomyces barnettii, formed a stable group closely related to, but nevertheless distinct from, Kluyveromyces africanus, Kluyveromyces lodderae, Saccharomyces rosinii, Saccharomyces spencerorum, and Saccharomyces sp. strain CBS 7662T (T = type strain). Saccharomyces spencerorum and Kluyveromyces lodderae displayed a particularly close genealogical affinity with each other, as did Saccharomyces castellii and Saccharomyces dairensis. Similarly, Saccharomyces servazzii, Saccharomyces unisporus, and Saccharomyces sp. strain CBS 6904 were found to be genotypically highly related and to form a phylogenetically distinct lineage. The recently reinstated species Saccharomyces transvaalensis was found to form a distinct lineage and displayed no specific association with any other Saccharomyces or non-Saccharomyces species. Saccharomyces kluyveri formed a very loose association with a group which included Kluyveromyces thermotolerans, Kluyveromyces waltii, Zygosaccharomyces cidri, and Zygosaccharomyces fermentati. Saccharomyces spp. strain CBS 6334T, on the other hand, displayed no specific association with any of the other Saccharomyces spp. studied, although a neighbor-joining analysis did reveal that this strain exhibited a loose phylogenetic affinity with Kluyveromyces polysporus and Kluyveromyces yarrowii. On the basis of the phylogenetic findings, two new Saccharomyces species, Saccharomyces kunashirensis (with type strain CBS 7662) and Saccharomyces martiniae (with type strain CBS 6334), are described.

Phylogenetic relationships among members of the ascomycetous yeast genera Brettanomyces, Debaryomyces, Dekkera, and Kluyveromyces deduced by small-subunit rRNA gene sequences.

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.

Use of an rRNA internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Zygosaccharomyces and Torulaspora.

Analyses of the sequences of the small-subunit (18S) rRNA gene and two internal transcribed spacers (ITSs), ITS1 and ITS2, revealed that members of the yeast genera Torulaspora and Zygosaccharomyces are phylogenetically intermixed. Despite some minor differences in 18S rRNA-, ITS1-, and ITS2-derived trees, in general the patterns of the relationships inferred from the three chronometers were in good agreement. The ITS sequences of Torulaspora and Zygosaccharomyces species exhibited far greater interspecies differences than the 18S rRNA sequences and were better than 18S rRNA sequences for measuring close genealogical relationships. Despite the existence of interstrain ITS sequence variation in some species, it is possible to identify conserved regions in both ITSs that are useful in species differentiation.

The isolation of Ant1, a transposable element from Aspergillus niger.

A transposable element has been isolated from the industrially important fungus Aspergillus niger (strain N402). The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. It had inserted at a TA site and appeared to have duplicated the target site upon insertion. The isolated element was found to be 4798 bp in length and contained 37-bp inverted, imperfect, terminal repeats (ITRs). The sequence of the central region of the element revealed an open reading frame (designated ORF1) which showed similarity, at the amino acid level, to the transposase of the Tc1/mariner class of DNA transposons. Another sequence within the central region of the element showed similarity to the 3' coding and downstream untranslated region of the amyA gene of A. niger. Sequence homology and structural features indicate that this element, which has been named Ant1 (A. niger transposon 1), is related to the Tc1/mariner group of DNA transposons. Ant1 is apparently present as a single copy in strain N402 of A. niger.

A novel approach for discovering retrotransposons: characterization of a long terminal repeat element in the spoilage yeast Pichia membranaefaciens and its use in strain identification.

A novel PCR-based approach designed to detect retrotransposon long terminal repeat (LTR) elements via their association with tRNA genes was applied to Pichia membranaefaciens, an industrially important food spoilage yeast. A single primer based on tRNA gene sequences was used to amplify DNA fragments from different strains, and an observed fragment size difference among strains was found to correspond to the expected size of an integrated LTR. A 289-bp element was cloned as part of the larger fragment and shown to be present in a high copy number and variable genomic location in all strains examined. Sequence analysis revealed the element to be bounded by nucleotides TG at the 5' end and CA at the 3' end and to exhibit target site duplication and other sequence motifs diagnostic of retrotransposon LTRs. LTR sequence data enabled the development of a rapid identification method which distinguished among different strains. The novel method for LTR isolation and the strain identification system are both likely to prove generally applicable for a wide range of other organisms.

The genetic relationship of Lodderomyces elongisporus to other ascomycete yeast species as revealed by small-subunit rRNA gene sequences.

The 18S rRNA gene sequence of the ascomycete yeast Lodderomyces elongisporus was determined by PCR-direct sequencing. The phylogenetic inter-relationship of Lodderomyces elongisporus and other ascomycete yeast species was examined by comparative sequence analysis. Lodderomyces elongisporus was found to be most closely related to Candida parapsilosis, C. tropicalis and C. albicans, exhibiting sequence similarity values of greater than 97.5%. The relationship between L. elongisporus and Candida parapsilosis in particular is discussed with regard to the possibility that L. elongisporus is the teleomorph (sexual form) of C. parapsilosis.

Genetic interrelationship among species of the genus Zygosaccharomyces as revealed by small-subunit rRNA gene sequences.

The phylogenetic interrelationship of species of the genus Zygosaccharomyces was examined by 18S rRNA gene sequencing. Comparative analysis of the sequence data revealed the genus to consist of a number of distinct subdivisions. The most prevalent species associated with food spoilage, Z. bailii, Z. bisporus and Z. rouxii, along with Z. mellis were found to form one subdivision. Zygosaccharomyces cidri and Z. fermentati formed a distinct species pair, as did Z. microellipsoides and Z. mrakii. Zygosaccharomyces florentinus formed a separate line displaying no specific relationship with any of the other Zygosaccharomyces species examined. Comparison with nine published ascosporogenous yeast 18S rRNA gene sequences showed that Z. microellipsoides and Z. mrakii were genealogically very close to Torulaspora delbrueckii (both displaying 99.8% 18S rRNA sequence similarity), raising the possibility that these two Zygosaccharomyces species should be moved to the genus Torulaspora. The topologies of trees derived from complete 18S rRNA gene sequences and from individual domains within the gene were compared and the implications of using partial sequence data for inferring phylogenetic relationships discussed.

A pyruvate decarboxylase gene from Aspergillus parasiticus.

A gene encoding a putative pyruvate decarboxylase (EC 4.1.1.1) was isolated from a genomic library of the filamentous fungus Aspergillus parasiticus strain SU-1. The deduced amino acid sequence showed 37% homology to PDC1 from Saccharomyces cerevisiae. Although A. parasiticus has an obligate growth requirement for oxygen, it produced ethanol in shake flask cultures indicating a response to anoxic conditions mediated by pyruvate decarboxylase.

Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme.

The cDNA gene encoding porcine pancreatic prophospholipase A2 (proPLA2) was cloned into an Aspergillus niger expression vector downstream of the glucoamylase (glaA) gene promoter region. When this construct was transformed into A. niger, no detectable PLA2 was produced. Evidence was obtained showing that the PLA2 gene was transcribed and that PLA2 is extremely susceptible to both intracellular and extracellular proteases of A. niger, thus indicating that translation products would be rapidly degraded. By fusing the proPLA2-encoding sequence to the entire glaA gene, secreted yields of PLA2 up to 10 micrograms/ml were obtained from a transformed protease-deficient strain of A. niger. PLA2 was secreted in young cultures as a fusion protein, but in older cultures, it was processed from the glucoamylase carrier protein. Secreted PLA2 was shown to be enzymatically active and to have the correct N-terminal amino acid (aa) sequence, although another form of processed PLA2 was also produced. This form included two aa of the proregion from PLA2. The potential for improving yields of secreted heterologous proteins from A. niger still further is discussed.

Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases.

In the present study, the extracellular protease activity in a strain of the filamentous fungus Aspergillus niger was investigated and mutant strains deficient in the production of extracellular proteases were isolated. The major protease, which is responsible for 80-85% of the total activity, is aspergillopepsin A, a protein of ca. 43 kDa, the activity of which is inhibited by pepstatin. In addition, a second protease, aspergillopepsin B, is produced, which is much less sensitive to inhibition by pepstatin. Several protease-deficient mutants were obtained by in vivo UV mutagenesis. In addition, a mutant lacking aspergillopepsin A was constructed by an in vitro gene replacement strategy. In this mutant, AB1.1, the entire coding region of the gene for aspergillopepsin A (pepA) is deleted. In three UV-induced mutants, aspergillopepsin A is also missing. One of these mutants, AB1.18, is mutated in the pepA gene, which is located on chromosome I. One of the other mutants, AB1.13, which has only 1-2% of the extracellular protease activity in the parent strain, is deficient in both aspergillopepsin A and aspergillopepsin B. The mutation involved, prt-13, has been localized to chromosome VI, and is probably a mutation in a regulatory gene. Another mutation involved in loss of protease function, prt-39, is located on chromosome VIII. Degradation of various heterologous proteins in culture media of the mutants is reduced but, even in strain AB1.13, not completely abolished.