Mutational analysis of centromeric DNA elements of Klayveromyces lactis and their role in determining the species specificity of the highly homologous centromeres from K. lactis and Saccharomyces cerevisiae

The centromere of Kluyveromyces lactis was delimited to a region of approximately 280 bp, encompassing KICDEI, II, and III. Removal of 6 bp from the right side of KlCDEIII plus flanking sequences abolished centromere function, and removal of 5 bp of KICDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20–80 bp from KlCDEII resulted in a decrease in plasmid stability, indicating that KlCDEII must have a certain length for proper centromere function. Centromeres of K. lactis do not function in Saccharomyces cerevisiae and vice versa. Adapting the length of K1CDEII to that of ScCDEII did not improve KlCEN function in S. cerevisiae, while doubling the ScCDEII length did not improve ScCEN function in K. lactis. Thus the difference in CDEII length is not in itself responsible for the species specificity of the centromeres from each of the two species of budding yeast. A chimeric K. lactis centromere with ScCDEIII instead of KlCDEIII was no longer functional in K. lactis, but did improve plasmid stability in S. cerevisiae, although to a much lower level then a wild-type ScCEN. This indicates that the exact CDEIII sequence is important, and suggests that the flanking AT-rich CDEII has to conform to specific sequence requirements.     

Chromatin structures of Kluyveromyces lactis centromeres in K. lactis and Saccharomyces cerevisiae

We have investigated the chromatin structure of Kluyveromyces lactis centromeres in isolated nuclei of K. lactis and Saccharomyces cerevisiae by using micrococcal nuclease and DNAse I digestion. The protected region found in K. lactis is approximately 270 bp long and encompasses the centromeric DNA elements, KlCDEI, KlCDEII, and KlCDEIII, but not KlCDE0. Halving KlCDEII to 82 bp impaired centromere function and led to a smaller protected structure (210 bp). Likewise, deletion of 5 bp from KlCDEI plus adjacent flanking sequences resulted in a smaller protected region and a decrease in centromere function. The chromatin structures of KlCEN2 and KlCEN4 present on plasmids were found to be similar to the structures of the corresponding centromeres in their chromosomal context. A different protection pattern of KlCEN2 was detected in S. cerevisiae, suggesting that KlCEN2 is not properly recognized by at least one of the centromere binding proteins of S. cerevisiae. The difference is mainly found at the KlCDEIII side of the structure. This suggests that one of the components of the ScCBF3-complex is not able to bind to KlCDEIII, which could explain the species specificity of K. lactis and S. cerevisiae centromeres.     

Structure of the Centromere Binding Factor 3 Complex from Kluyveromyces lactis

Kinetochores are the multiprotein complexes that link chromosomal centromeres to mitotic-spindle microtubules. Budding yeast centromeres comprise three sequential "centromere-determining elements", CDEI, II, and III. CDEI (8 bp) and CDEIII (∼ 25 bp) are conserved between Kluyveromyces lactis and Saccharomyces cerevisiae, but CDEII in the former is twice as long (160 bp) as CDEII in the latter (80 bp). The CBF3 complex recognizes CDEIII and is required for assembly of a centromeric nucleosome, which in turn recruits other kinetochore components. To understand differences in centromeric nucleosome assembly between K. lactis and S. cerevisiae, we determined the structure of a K. lactis CBF3 complex by electron cryomicroscopy at ∼ 4 Å resolution and compared it with published structures of S. cerevisiae CBF3. We show differences in the pose of Ndc10 and discuss potential models of the K. lactis centromeric nucleosome that account for the extended CDEII length.     

A vital function for mitochondrial DNA in the petite-negative yeastKluyveromyces lactis

Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeastKluyveromyces lactis, specific mutations in the nuclear genesMGI2 andMGI5 encoding theα- andγ-subunits of the mitochondrial F₁-ATPase, allow mtDNA to be lost. In this study we show that wild-typeK. lactis does not survive in the absence of its mitochondrial genome and that the function ofmgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing amgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned theK. lactis homologue of theSaccharomyces cerevisiae mitochondrial genome maintenance geneMGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption ofMGM101 is not lethal in a haploidmgi strain with a specific mutation in a subunit of the mitochondrial F₁-ATPase. These observations suggest that mtDNA inK. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of F₀.     

Development of a PCR-RFLP assay for the identification of Lactococcus lactis ssp. lactis and cremoris

The development of new starter culture of Lactococcus lactis for the manufacture of fermented dairy products with unique characteristics usually requires the isolation and identification of L. lactis up to subspecies level. Therefore, a rapid and specific PCR-RFLP assay has been developed. Forward and reverse primer sets were designed targeting the conserved house keeping gene htrA and yueF encoding a trypsin-like serine protease and a non-proteolytic protein from peptidase family M16, respectively, of L. lactis. Amplicons of 265 bp and 447 bp of htrA and yueF, respectively, were subjected to restriction fragment length polymorphism analysis. Restriction of the 265 bp amplicons with TaqI produced DNA bands of 90 bp and 175 bp with ssp. lactis, and 66 bp and 199 bp with ssp. cremoris. Similarly, restriction of PCR product of 447 bp size with AluI produced digested fragments of 125 bp and 322 bp with ssp. lactis, and 71 bp and 376 bp with ssp. cremoris. The designed primer sets were observed to be specific to L. lactis because other bacteria could not be amplified. The ssp. lactis and cremoris of L. lactis could be identified by restriction of PCR products of htrA and yueF with TaqI and AluI, respectively.     

Isolation and Characterization of the Genes Encoding Δ8-Sphingolipid Desaturase from Saccharomyces kluyveri and Kluyveromyces lactis

Saccharomyces kluyveri IFO 1685 and Kluyveromyces lactis IFO 1090 synthesize cerebroside containing 9-methyl-trans-4, trans-8-sphingadienine as a sphingoid base. From the genome of the two strains, the regions encompassing Δ⁸-sphingolipid desaturase were amplified and sequenced. The nucleotide sequences of these regions revealed single open reading frames of 1707 bp for S. kluyveri and 1722 bp for K. lactis, encoding polypeptides of 568 and 573 amino acids with molecular weights of 66.5 and 67.1 kDa, respectively. Conversion of 4-hydroxysphinganine to 4-hydroxy-trans-8-sphingenine in the cells of Saccharomyces cerevisiae was observed by the expressed gene from K. lactis and not by that from S. kluyveri. These findings may be explained by the difference in substrate specificity for the sphingoid base moiety between Δ⁸-sphingolipid desaturases of S. kluyveri and K. lactis. Received: 30 April 2002 / Accepted: 21 June 2002     

The consensus sequence of Kluyveromyces lactis centromeres shows homology to functional centromeric DNA from Saccharomyces cerevisiae

Summary The nucleotide sequences of five of the six centromeres of the yeast Kluyveromyces lactis were determined. Mutual comparison of these sequences led to the following consensus: a short highly conserved box (5-ATCACGTGA-3) flanked by an AT-rich (±90%) stretch of ± 160 by followed by another conserved box (5-TNNTTTATGTTTCCGAAAATTAATAT-3).These three elements were named K1CDEI, K1CDEII, and K1CDEIII respectively, by analogy with the situation in Saccharomyces cerevisiae. In addition, a second 100 by AT-rich (±90%) element, named K1CDE0, was found ± 150 by upstream of K1CDEI. The sequences of both K1CDEI and K1CDEIII are highly conserved between K. lactis and S. cerevisiae; however, centromeres of K. lactis do not function in S. cerevisiae and vice versa. The most obvious differences between the centromeres of the two yeast species are the length of the AT-rich CDEII, which is 161–164 by in K. lactis versus 78–86 by in S. cerevisiae and the presence in K. lactis of K1CDEO, which is not found in S. cerevisiae.     

Gene Responses to Oxygen Availability in Kluyveromyces lactis: an Insight on the Evolution of the Oxygen-Responding System in Yeast

The whole-genome duplication (WGD) may provide a basis for the emergence of the very characteristic life style of Saccharomyces cerevisiae—its fermentation-oriented physiology and its capacity of growing in anaerobiosis. Indeed, we found an over-representation of oxygen-responding genes in the ohnologs of S. cerevisiae. Many of these duplicated genes are present as aerobic/hypoxic(anaerobic) pairs and form a specialized system responding to changing oxygen availability. HYP2/ANB1 and COX5A/COX5B are such gene pairs, and their unique orthologs in the ‘non-WGD’ Kluyveromyces lactis genome behaved like the aerobic versions of S. cerevisiae. ROX1 encodes a major oxygen-responding regulator in S. cerevisiae. The synteny, structural features and molecular function of putative KlROX1 were shown to be different from that of ROX1. The transition from the K. lactis-type ROX1 to the S. cerevisiae-type ROX1 could link up with the development of anaerobes in the yeast evolution. Bioinformatics and stochastic analyses of the Rox1p-binding site (YYYATTGTTCTC) in the upstream sequences of the S. cerevisiae Rox1p-mediated genes and of the K. lactis orthologs also indicated that K. lactis lacks the specific gene system responding to oxygen limiting environment, which is present in the ‘post-WGD’ genome of S. cerevisiae. These data suggested that the oxygen-responding system was born for the specialized physiology of S. cerevisiae.     

Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae

Heterologous expression of Pleurotus ostreatus POXC and POXA1b laccases in two yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae, was performed. Both transformed hosts secreted recombinant active laccases, although K. lactis was much more effective than S. cerevisiae. rPOXA1b transformants always had higher secreted activity than rPOXC transformants did. The lower tendency of K. lactis with respect to S. cerevisiae to hyperglycosylate recombinant proteins was confirmed. Recombinant laccases from K. lactis were purified and characterised. Specific activities of native and recombinant POXA1b are similar. On the other hand, rPOXC specific activity is much lower than that of the native protein, perhaps due to incomplete or incorrect folding. Both recombinant laccase signal peptides were correctly cleaved, with rPOXA1b protein having two C-terminal amino acids removed. The availability of the established recombinant expression system provides better understanding of laccase structure–function relationships and allows the development of new oxidative catalysts through molecular evolution techniques.     

Identification of Kluyveromyces lactis Telomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain

Telomeres in the budding yeast Kluyveromyces lactis consist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactis telomerase RNA, encoded by the TER1 gene, is ~1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactis whole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactis telomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactis telomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactis telomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.     

Transformation of Kluyveromyces lactis cells by plasmid DNA does not require alkaline cations

Summary A procedure for the transformation ofKluyveromyces lactis based on the Li salt method for introducing plasmid DNA into intact yeast cells is described. Contrary toSaccharomyces cerevisiae, lithium salts are dispensable for inducing competence inK. lactis. 2-Mercaptoethanol, a compound that stimulates transformation inS. cerevisiae, showed an opposite effect. inK. lactis. On the other hand, the presence of PEG 4000 and a heat shock were absolutely required to obtain high transformation efficiency.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

Regulation of cytokinesis in the milk yeast Kluyveromyces lactis

Cytokinesis in yeast and mammalian cells is a highly coordinated process mediated by the constriction of an actomyosin ring. In yeasts, it is accompanied by the formation of a chitinous primary septum. Although much is known about the regulation of cytokinesis in budding yeast, overlapping functions of redundant genes complicates genetic analyses. Here, we investigated the effects of various deletion mutants on cytokinesis in the milk yeast Kluyveromyces lactis. To determine the spatiotemporal parameters of cytokinesis components, live-cell imaging of fluorophor-tagged KlMyo1 and a new Lifeact probe for KlAct1 was employed. In contrast to Saccharomyces cerevisiae, where deletion of ScMYO1 is lethal, Klmyo1 deletion was temperature-sensitive. Transmission and scanning electron microscopy demonstrated that the Klmyo1 deletion cells had a defect in the formation of the primary septum and in cell separation; this result was confirmed by FACS analyses. Deletion of KlCYK3 was lethal, whereas in S. cerevisiae a cyk3 deletion is synthetically lethal with hof1 deletion. Growth of Klhof1 mutants was osmoremedial at 25 °C, as it is in S. cerevisiae. CYK3 and HOF1 genes cross-complemented in both species, suggesting that they are functional homologs. Inn1, a common interactor for these two regulators, was essential in both yeasts and the encoding genes did not cross-complement. The C2 domain of the Inn1 homologs conferred species specificity. Thus, our work establishes K. lactis as a model yeast to study cytokinesis with less genetic redundancy than S. cerevisiae. The viability of Klmyo1 deletions provides an advantage over budding yeast to study actomyosin-independent cytokinesis. Moreover, the lethality of Klcyk3 null mutants suggests that there are fewer functional redundancies with KlHof1 in K. lactis.     

Application of comet assay for the assessment of DNA damage caused by chemical genotoxins in the dairy yeast Kluyveromyces lactis

Kluyveromyces lactis, also known as dairy yeast, has numerous applications in scientific research and practice. It has been approved as a GRAS (Generally Recognized As Safe) organism, a probiotic, a biotechnological producer of important enzymes at industrial scale and a bioremediator of waste water from the dairy industry. Despite these important practical applications the sensitivity of this organism to genotoxic substances has not yet been assessed. In order to evaluate the response of K. lactis cells to genotoxic agents we have applied several compounds with well-known cyto- and genotoxic activity. The method of comet assay (CA) widely used for the assessment of DNA damages is presented here with new special modifications appropriate for K. lactis cells. The comparison of the response of K. lactis to genotoxins with that of Saccharomyces cerevisiae showed that both yeasts, although considered close relatives, exhibit species-specific sensitivity toward the genotoxins examined.     

Isolation, Nucleotide Sequence, and Physiological Relevance of the Gene Encoding Triose Phosphate Isomerase from Kluyveromyces lactis

Lack of triose phosphate isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae ΔTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.     

Controlled Integration into the Lactococcus Chromosome of the pCI829-Encoded Abortive Infection Gene from Lactococcus lactis subsp. lactis UC811

The phage insensitivity gene of lactococcal plasmid pCI829 which encodes an abortive infection defense mechanism (Abi) was inserted into the Lactococcus lactis subsp. lactis CH919 chromosome by utilizing the integration plasmid pCI194, which contains 4.2 kb of homology with the conjugative transposon Tn919. Chloramphenicol-resistant transformants expressed phage insensitivity to the prolate-headed phage c2 and the small isometric-headed phage 712, and hybridization analysis indicated that transformants contained pCI194 integrated in single copy. The level of phage insensitivity expressed by the transformants was reduced from that observed when the abi gene was located on a replicating plasmid, as determined by plaque assay and burst size analysis. Amplification of the integrated structure after growth in increased concentrations of chloramphenicol resulted in an increase in the expression of phage insensitivity. Hybridization analysis revealed that while pCI194 was stably maintained in an integrated state over 100 generations in the absence of selective pressure, the ability to express phage insensitivity was lost. Hybridization analysis also revealed that DNA flanking the abi gene contains homology to the CH919 chromosome.     

PCR Amplification of the Gene acmA Differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.     

Centromere parC of plasmid R1 is curved

The centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by its migration anomaly in polyacrylamide gels. The 159 bp long parC is strongly curved with anomaly values (k-factors) close to 2. The properties of the parC curvature agree with those of other curved DNA sequences. parC contains two regions of 5-fold repeated iterons separated by 39 bp. We modified 4 bp within this intermediate sequence so that we could analyse the two 5-fold repeated regions independently. The analysis shows that the two repeat regions are not independently curved parts of parC but that the overall curvature is a property of the whole fragment. Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate that curvature might be a general property of centromeres.     

Rapid PCR-Based Method Which Can Determine Both Phenotype and Genotype of Lactococcus lactis Subspecies

A highly efficient, rapid, and reliable PCR-based method for distinguishing Lactococcus lactis subspecies (L. lactis subsp. lactis and L. lactis subsp. cremoris) is described. Primers complementary to positions in the glutamate decarboxylase gene have been constructed. PCR analysis with extracted DNA or with cells of different L. lactis strains resulted in specific fragments. The length polymorphism of the PCR fragments allowed a clear distinction of the L. lactis subspecies. The amplified fragment length polymorphism with the primers and the restriction fragment length polymorphism of the amplified products agreed perfectly with the identification based on genotypic and phenotypic analyses, respectively. Isolates from cheese starters were investigated by this method, and amplified fragments of genetic variants were found to be approximately 40 bp shorter than the typical L. lactis subsp. cremoris fragments.