Ian N. Roberts

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 108 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.

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.

Engineering evolution to study speciation in yeasts

The Saccharomyces ‘sensu stricto’ yeasts are a group of species that will mate with one another, but interspecific pairings produce sterile hybrids. A retrospective analysis of their genomes revealed that translocations between the chromosomes of these species do not correlate with the groups sequence-based phylogeny (that is, translocations do not drive the process of speciation). However, that analysis was unable to infer what contribution such rearrangements make to reproductive isolation between these organisms. Here, we report experiments that take an interventionist, rather than a retrospective approach to studying speciation, by reconfiguring the Saccharomyces cerevisiae genome so that it is collinear with that of Saccharomyces mikatae. We demonstrate that this imposed genomic collinearity allows the generation of interspecific hybrids that produce a large proportion of spores that are viable, but extensively aneuploid. We obtained similar results in crosses between wild-type S. cerevisiae and the naturally collinear species Saccharomyces paradoxus, but not with non-collinear crosses. This controlled comparison of the effect of chromosomal translocation on species barriers suggests a mechanism for the generation of redundancy in the S. cerevisiae genome.

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.

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

SummaryIn 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, AB 1.18, 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.

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.

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.

Inferences of evolutionary relationships from a population survey of LTR-retrotransposons and telomeric-associated sequences in the Saccharomyces sensu stricto complex

The Saccharomyces sensu stricto complex consists of six closely related species and one natural hybrid. Intra‐ and inter‐ species variability in repetitive elements can help elucidate the population structure and evolution of these close relatives. The chromosome positions of several telomeric associated sequences (TASs) and LTR‐retrotransposons have been determined, using PFGE, in 112 isolates. Most of the repetitive elements studied are found in multiple copies in each strain, although in some subpopulations these elements are present in low copy number or are absent. Hybridization patterns and copy numbers of the repetitive elements correlate with geographic distribution. These patterns may yield interesting clues as to the origins and evolution of some TASs and retrotransposons, e.g. we can infer that Y′ originated on the left end of chromosome XIV. There is strong evidence for horizontal transfer of Ty2 between S. cerevisiae and S. mikatae. Ty1 and Ty5 are either lost easily or frequently horizontally transferred. We have also found some gross chromosomal rearrangements in isolates within species and a few new natural hybrids between species, indicating that these processes occur in the wild and are not limited to conditions of human influence. DNA sequences have been deposited with the EMBL/GenBank database under Accession Nos AJ632279–AJ632293. Copyright

Metabolic footprinting as a tool for discriminating between brewing yeasts

The characterization of industrial yeast strains by examining their metabolic footprints (exometabolomes) was investigated and compared to genome‐based discriminatory methods. A group of nine industrial brewing yeasts was studied by comparing their metabolic footprints, genetic fingerprints and comparative genomic hybridization profiles. Metabolic footprinting was carried out by both direct injection mass spectrometry (DIMS) and gas chromatography time‐of‐flight mass spectrometry (GC–TOF–MS), with data analysed by principal components analysis (PCA) and canonical variates analysis (CVA). The genomic profiles of the nine yeasts were compared by PCR–restriction fragment length polymorphism (PCR–RFLP) analysis, genetic fingerprinting using amplified fragment length polymorphism (AFLP) analysis and microarray comparative genome hybridizations (CGH). Metabolomic and genomic analysis comparison of the nine brewing yeasts identified metabolomics as a powerful tool in separating genotypically and phenotypically similar strains. For some strains discrimination not achieved genomically was observed metabolomically. Copyright

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.