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Killer plasmids pGKL1 and pGKL2 of double-stranded linear DNAs were transferred from Kluyveromyces lactis to strains of Kluyveromyces fragilis and Candida pseudotropicalis. The resultant killer strains produced 17-fold and 6-fold larger amounts of killer toxin than K. lactis did, respectively. The killer toxin produced by each species appeared to be a glycoprotein.
A selective and differential solid medium, called Kluyveromyces Differential Medium (KDM), is described for the isolation of Kluyveromyces marxianus and K. lactis from dairy products. Its discriminative potential is based on the detection of the enzyme beta-galactosidase, in the absence of lactose. Of the more than 95 strains tested, including yeasts, bacteria, and filamentous fungus, only the strains of K. marxianus and K. lactis produced blue colonies on the medium due to the presence of X-Gal/ IPTG. The bacterial strains were not able to grow in KDM. On this basis, the medium was very satisfactory when testing naturally or experimentally contaminated dairy food products. When quality assessment tests were performed, optimal values of productivity (growth and color) and selectivity were obtained for K. marxianus and K. lactis.
A comparative molecular genetic study of 37 Kluyveromyces strains of different origin has made it possible to find molecular markers that can differentiate between the dairy yeast Kluyveromyces lactis var. lactis and the genetically close wild Kl. lactis strains from the European "krassilnikovii" population, which are unable to ferment lactose. A restriction fragment length polymorphism analysis of the IGS2 region of the strains' rDNA reveals two different AluI profiles, one of which corresponds to Kl. lactis var. lactis while the other corresponds to yeasts from the "krassilnikovii" population. The AluI restriction profile of the IGS2 region of the rDNA also makes it possible to differentiate between the physiologically similar species Kl. marxianus and Kl. lactis. The origin of clinical Kl. lactis var. lactis isolates is discussed.
The degree of sensitivity of the yeasts Kluyveromyces bulgaricus and K. lactis to amphotericin B is linked to a difference in the sterol composition of their membranes. No direct proportionality was found between sensitivity and the quantity of sterols present. At sublethal doses, amphotericin B perturbed sterol synthesis, resulting in ergosterol precursor accumulation. An ergosterol pathway is proposed for Kluyveromyces.
Two fragments containing both an autonomous replicating sequence (ARS) and a centromere have been isolated and sequenced from the yeast Kluyveromyces marxianus. The ARS and centromeric core sequences are only 500 bp apart, but ARS activity could be separated from the centromeric sequences. Centromeric sequences are organized in a similar way to those of budding yeasts: two well-conserved elements: CDEI (5' TCACGTG 3') and CDEIII (5' TNTTCCGAAAGTWAAA 3'), are separated by a 165 bp AT-rich (+/- 90%) CDEII element whose length is twice that of Saccharomyces cerevisiae CDEII but almost identical to that of K. lactis. The ARS-core consensus sequence (5' TTTATTGTT 3') is also similar to that of K. lactis. Both ARS and centromeric elements function in this strain, albeit inefficiently, but not in S. cerevisiae. A third ARS-containing fragment with a different organization has been isolated and sequenced.
Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen.
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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 17 species belonging to the genus Kluyveromyces and 12 reference and outgroup species was performed using mitochondrial cytochrome-c oxidase II gene sequences. The genus Kluyveromyces appears as a polyphyletic taxon formed by species included within the following four main groups. The Kluyveromyces phaffii group encompasses the species Kluyveromyces blattae, K. phaffii and Kluyveromyces yarrowii. The Kluyveromyces marxianus group is a monophyletic group consisting of the species Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, K. marxianus and Kluyveromyces wickerhamii. The monophyletic Kluyveromyces thermotolerans group is formed by K. thermotolerans, Kluyveromyces waltii and Saccharomyces kluyveri (which appears in the mitochondrial tree as the sister clade of the K. marxianus group). Finally, the Saccharomyces cerevisiae group contains the remaining Kluyveromyces species, as well as the reference Saccharomyces species (sensu lato and sensu stricto) and Candida glabrata (the phylogenetic relationships within this group are unclear according to the bootstrap test). The phylogenetic relationships obtained for this mitochondrial gene are, for the most part, congruent with previous trees based on nuclear rRNA sequences, except for the position of K. yarrowii and the close relationship between the K. marxianus and K. thermotolerans groups. These differences, as well as the existence of these groups, are discussed in the context of previous studies based on phenotypic, genetic and molecular data. Although additional studies are required to decipher the phylogenetic relationships between the genus Kluyveromyces and the closely related genera Saccharomyces, Torulaspora and Zygosaccharomyces, future changes to their taxonomic status should take account of the existence of these four groups of Kluyveromyces species.
The transcription of the majority of the ribosomal protein (rp) genes of Saccharomyces cerevisiae is activated by cis-acting elements, designated RPG boxes, which specifically bind the multifunctional protein RAP1 in vitro. To investigate to what extent this global system of transcription regulation has been conserved, we have isolated a number of rp genes of the related yeast species Kluyveromyces lactis and Kluyveromyces marxianus, whose counterparts in Saccharomyces are controlled by RAP1. The coding regions of these genes showed a sequence similarity of about 90% when compared to their Saccharomyces counterparts. In contrast, little or no sequence similarity was found between the upstream regions and the intervening sequences of Kluyveromyces and Saccharomyces homologs. However, the occurrence and the position of the introns is conserved. The sequence data also show that the physical linkage that exists in S. cerevisiae between the rp genes encoding RP59 (CRY1), S24 and L46 is conserved in Kluyveromyces. Northern analysis demonstrated that each of the isolated Kluyveromyces genes is transcriptionally active. By sequence comparison we identified a number of conserved sequences in the upstream region of each of the Kluyveromyces rp genes, which we designated the X, Z and RPGK boxes. The last one is highly similar, though not identical, to the S. cerevisiae RPG box. Functional analysis of the intergenic region between the genes encoding Kluyveromyces ribosomal proteins S24 and L46 showed that the RPGK box (+Z box) functions as a transcriptional activator, while the X box acts as a transcriptional repressor. Band-shift assays confirmed the existence of a RAP1-like protein in Kluyveromyces that binds to the RPGK box but not to the S. cerevisiae RPG box. In contrast, S. cerevisiae RAP1 did recognize the RPGK box.
The abundant multifunctional protein ABF1 of Saccharomyces cerevisiae binds to the upstream region of several genes, including some ribosomal protein genes like the one encoding protein S33. Deletion of the ABF1-binding sequence lowers the transcription of these genes three- to more than ten-fold. We have isolated the S33 genes of two related yeast species, Kluyveromyces lactis and Kluyveromyces marxianus. Comparison of the nucleotide sequences of these S33 genes with their counterpart from S. cerevisiae shows a strong sequence similarity covering the whole of the coding regions. In contrast, little or no sequence similarity is found in the 5'-flanking regions of the three genes. Also the trailer regions differ considerably in both length and sequence from one species to another. An ABF1-binding site is present in the upstream region of the S33 gene of K. marxianus. Retardation analyses showed that this sequence is able to bind a protein present in Kluyveromyces cells with a molecular mass somewhat lower than that of S. cerevisiae ABF1. Functional analyses, using a beta-glucuronidase reporter system, showed that the ABF1-binding site is indeed involved in transcription activation of the K. marxianus S33 gene in Kluyveromyces cells. A S. cerevisiae ABF1-gene-specific probe showed only weak hybridization with Kluyveromyces DNA and Northern blots did not show a signal. These results indicate that S. cerevisiae and Kluyveromyces contain functionally related but structurally dissimilar ABF1-type proteins.
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.
Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from L-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher L-methionine demethiolating activity, resulting in a direct conversion of L-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.
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).
The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 A resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106-113 and 292-301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105-113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105-113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase.