Mutants Of Kluyveromyces Lactis Research Articles

The Functional Specialization of Exomer as a Cargo Adaptor During the Evolution of Fungi.

Yeast exomer is a heterotetrameric complex that is assembled at the trans-Golgi network, which is required for the delivery of a distinct set of proteins to the plasma membrane using ChAPs (Chs5-Arf1 binding proteins) Chs6 and Bch2 as dedicated cargo adaptors. However, our results show a significant functional divergence between them, suggesting an evolutionary specialization among the ChAPs. Moreover, the characterization of exomer mutants in several fungi indicates that exomer's function as a cargo adaptor is a late evolutionary acquisition associated with several gene duplications of the fungal ChAPs ancestor. Initial gene duplication led to the formation of the two ChAPs families, Chs6 and Bch1, in the Saccaromycotina group, which have remained functionally redundant based on the characterization of Kluyveromyces lactis mutants. The whole-genome duplication that occurred within the Saccharomyces genus facilitated a further divergence, which allowed Chs6/Bch2 and Bch1/Bud7 pairs to become specialized for specific cellular functions. We also show that the behavior of S. cerevisiae Chs3 as an exomer cargo is associated with the presence of specific cytosolic domains in this protein, which favor its interaction with exomer and AP-1 complexes. However, these domains are not conserved in the Chs3 proteins of other fungi, suggesting that they arose late in the evolution of fungi associated with the specialization of ChAPs as cargo adaptors.

Mild Telomere Dysfunction as a Force for Altering the Adaptive Potential of Subtelomeric Genes.

Subtelomeric regions have several unusual characteristics, including complex repetitive structures, increased rates of evolution, and enrichment for genes involved in niche adaptation. The adaptive telomere failure hypothesis suggests that certain environmental stresses can induce a low level of telomere failure, potentially leading to elevated subtelomeric recombination that could result in adaptive mutational changes within subtelomeric genes. Here, we tested a key prediction of the adaptive telomere failure hypothesis-that telomere dysfunction mild enough to have little or no overall effect on cell fitness could still lead to substantial increases in the mutation rates of subtelomeric genes. Our results show that a mutant of Kluyveromyces lactis with stably short telomeres produced a large increase in the frequency of mutations affecting the native subtelomeric β-galactosidase (LAC4) gene. All lac4 mutants examined from strains with severe telomere dysfunction underwent terminal deletion/duplication events consistent with being due to break-induced replication. In contrast, although cells with mild telomere dysfunction also exhibited similar terminal deletion and duplication events, up to 50% of lac4 mutants from this background unexpectedly contained base changes within the LAC4 coding region. This mutational bias for producing base changes demonstrates that mild telomere dysfunction can be well suited as a force for altering the adaptive potential of subtelomeric genes.

Production of orotic acid by a Klura3Δ mutant of Kluyveromyces lactis

We demonstrated that a Klura3Δ, mutant of the yeast Kluyveromyces lactis is able to produce and secrete into the growth medium considerable amounts of orotic acid. Using yeast extract-peptone-glucose (YPD) based media we optimized production conditions in flask and bioreactor cultures. With cells grown in YPD 5% glucose medium, the best production in flask was obtained with a 1:12.5 ratio for flask: culture volume, 180rpm, 28°C and 200mM MOPS for pH stabilization at neutral values (initial culture pH at 8.0). The best production in a 2L bioreactor was achieved at 500rpm with 1vvm aeration, 28°C and pH 7.0. Under these optimum conditions, similar rates of orotic acid production were obtained and maximum concentration achieved after 96h was 6.7g/L in flask and bioreactor cultures. These results revealed an excellent reproducibility between both systems and provided evidence for the biotechnological potential of Klura3Δ strain to produce orotic acid since the amounts obtained are comparable to the production in flask using a similar mutant of the industrially valuable Corynebacterium glutamicum.

Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi.

BackgroundNucleotide sugar transporters (NSTs) play an essential role in translocating nucleotide sugars into the lumen of the endoplasmic reticulum and Golgi apparatus to be used as substrates in glycosylation reactions. This intracellular transport is an essential step in the biosynthesis of glycoconjugates.ResultsWe have identified a family of 11 putative NSTs in Trypanosoma cruzi, the etiological agent of Chagas’ disease. A UDP-N-acetylglucosamine transporter, TcNST1, was identified by a yeast complementation approach. Based on a phylogenetic analysis four candidate genes were selected and used for complementation assays in a Kluyveromyces lactis mutant strain. The transporter is likely expressed in all stages of the parasite life cycle and during differentiation of epimastigotes to infective metacyclics. Immunofluorescence analyses of a GFP-TcNST1 fusion protein indicate that the transporter is localized to the Golgi apparatus. As many NSTs are multisubstrate transporters, we also tested the capacity of TcNST1 to transport GDP-Man.ConclusionsWe have identified a UDP-N-acetylglucosamine transporter in T. cruzi, which is specifically localized to the Golgi apparatus and seems to be expressed, at the mRNA level, throughout the parasite life cycle. Functional studies of TcNST1 will be important to unravel the role of NSTs and specific glycoconjugates in T. cruzi survival and infectivity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0601-7) contains supplementary material, which is available to authorized users.

Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination

Some human cancers maintain their telomeres using the alternative lengthening of telomeres (ALT) mechanism; a process thought to involve recombination. Different types of recombinational telomere elongation pathways have been identified in yeasts. In senescing yeast telomerase deletion (ter1-Δ) mutants with very short telomeres, it has been hypothesized that copying a tiny telomeric circle (t-circle) by a rolling circle mechanism is the key event in telomere elongation. In other cases more closely resembling ALT cells, such as the stn1-M1 mutant of Kluyveromyces lactis, the telomeres appear to be continuously unstable and routinely reach very large sizes. By employing two-dimensional gel electrophoresis and electron microscopy, we show that stn1-M1 cells contain abundant double stranded t-circles ranging from ∼100 to 30 000 bp in size. We also observed small single-stranded t-circles, specifically composed of the G-rich telomeric strand and tailed circles resembling rolling circle replication intermediates. The t-circles most likely arose from recombination events that also resulted in telomere truncations. The findings strengthen the possibility that t-circles contribute to telomere maintenance in stn1-M1 and ALT cells.

Impact of mitochondrial function on yeast susceptibility to antifungal compounds

Saccharomyces cerevisiae pell and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL) as well as Kluyveromyces lactis mutants impaired in the respiratory chain function (RCF) containing dysfunctional mitochondria show altered sensitivity to metabolic inhibitors. The S. cerevisiae pell mutant displayed increased sensitivity to cycloheximide, chloramphenicol, oligomycin and the cell-wall perturbing agents caffeine, caspofungin and hygromycin. On the other hand, the pel1 mutant was less sensitive to fluconazole, similarly as the K. lactis mutants impaired in the function of mitochondrial cytochromes. Mitochondrial dysfunction resulting either from the absence of PG and CL or impairment of the RCF presumably renders the cells more resistant to fluconazole. The increased tolerance of K. lactis respiratory chain mutants to amphotericin B, caffeine and hygromycin is probably related to a modification of the cell wall.

A respiratory-deficient mutation associated with high salt sensitivity inKluyveromyces lactis

A salt-sensitive mutant of Kluyveromyces lactis was isolated that was unable to grow in high-salt media. This mutant was also respiratory-deficient and temperature-sensitive for growth. The mutation mapped in a single nuclear gene that is the ortholog of BCS1 of Saccharomyces cerevisiae. The BCS1 product is a mitochondrial protein required for the assembly of respiratory complex III. The bcs1 mutation of S. cerevisiae leads to a loss of respiration, but, unlike in K. lactis, it is not accompanied by salt sensitivity. All the respiratory-deficient K. lactis mutants tested were found to be salt-sensitive compared to their isogenic wild-type strains. In the presence of the respiratory inhibitor antimycin A, the wild-type strain also became salt-sensitive. By contrast, none of the S. cerevisiae respiratory-deficient mutants tested showed increased salt sensitivity. The salt sensitivity of the Klbcs1 mutant, but not its respiratory deficiency, was suppressed by the multicopy KlVMA13 gene, a homolog of the S. cerevisiae VMA13 gene encoding a subunit of the vacuolar H(+)-ATPase. These results suggest that cellular salt homeostasis in K. lactis is strongly dependent on mitochondrial respiratory activity, and/or that the ion homeostasis of mitochondria themselves could be a primary target of salt stress.

Functional redundancy of Caenorhabditis elegans nucleotide‐sugar transporters

There are several examples of the pathophysiological relevance of nucleotide-sugar transporters (NSTs) in yeast, protozoal parasites, C.elegans and mammals. Nevertheless, the physiological roles of NSTs may be hampered by redundant gene function. C.elegans is a genetically and developmentally well characterized organism for which only 2 out of 16 NST-like proteins have been described. In the present work, C03H5.2 –a cDNA encoding a putative NST- was functionally expressed in S.cerevisiae. The corresponding Golgi vesicle enriched fraction transported UDP-GalNAc and UDP-GlcNAc in a temperature-dependent and saturable manner. UDP-GlcNAc transport activity was also demonstrated in vivo by complementation of a Kluyveromyces lactis mutant defective in this transport. Inactivation of C03H5.2 in wild type worms by RNAi didn’t show any visual phenotype. Srf-3, a previously described NST in C.elegans also has UDP-GlcNAc transport activity and srf-3 mutants have a very mild visual phenotype. Therefore, a possible biochemical redundancy in this activity was investigated. Inactivation of C03H5.2 by RNAi in srf-3 mutants showed a striking phenotype characterized by abnormal positions of eggs, oocyte accumulation and body constriction. Moreover, transgenic animals for C03H5.2 promoter::GFP fusion were examined and compared to the expression pattern of srf-3, a partial overlapping localization was revealed. This provides the first evidence for biochemical redundancy of NSTs in any organism and constitutes a starting point to understand regulation of these transporters in multicellular organisms. NIH –GM30365

TheKluyveromyces lactisα1,6-mannosyltransferase KlOch1p is required for cell-wall organization and proper functioning of the secretory pathway

Mutants of Kluyveromyces lactis denominated vga (vanadate glycosylation affected) bear various combinations of glycosylation and cell-wall defects. The vga3 mutation of K. lactis was mapped in the KlOCH1 gene, encoding the functional homologue of the Saccharomyces cerevisiaealpha1,6-mannosyltransferase. Quantitative analysis of cell-wall components indicated a noticeable increase of chitin and beta1,6-glucans and a severe decrease of mannoproteins in the mutant cells as compared with the wild-type counterparts. Fine-structure determination of the beta1,6-glucan polymer indicated that, in the vga3-1 strain, the beta1,6-glucans are shorter and have more branches than in the wild-type strain. This suggests that cell-wall remodelling changes take place in K. lactis in the presence of glycosylation defects. Moreover, the vga3 cells showed a significantly improved capability of secreting heterologous proteins. Such a capability, accompanied by the highly reduced N-glycosylation, may be of biotechnological interest, especially when hyper-glycosylation of recombinant products must be avoided.

Screening for telomeric recombination in wild-typeKluyveromyces lactis

Both subtelomeric and telomeric recombination events can be greatly enhanced in Kluyveromyces lactis mutants lacking telomerase and having abnormally short telomeres. In this study, we utilized cells containing a single telomere composed of mutant repeats carrying a phenotypically silent mutation to test whether the exchange of telomeric repeats was a frequent event in mitotic and meiotic wild-type K. lactis cells. Amongst more than 100 subclones followed during multiple passages of mitotic growth, one instance of a terminal duplication extending into a subtelomeric sequence was observed, but no occurrences of intertelomeric recombination were found. This suggests that intertelomeric recombination is not an important contributor to telomere maintenance in normal K. lactis cells. Rare recombination events resulting in the replacement of a subtelomeric marker with a sequence from another chromosome end also led to the replacement of the telomeric repeat tract. This is consistent with these events being a result of break-induced replication. Movement of a subtelomeric or telomeric sequence from one chromosome end to another was not observed in haploid cells derived from mating and sporulation. This suggests that the exchange of telomeric repeats is not a routine occurrence in K. lactis meiosis.

A history of research on yeasts 9: regulation of sugar metabolism1

A history of research on yeasts 9: regulation of sugar metabolism1

Disruption of the MNN10 gene enhances protein secretion in Kluyveromyces lactis and Saccharomyces cerevisiae.

Screening for genes affecting super-secreting phenotype of the over-secreting mutant of Kluyveromyces lactis resulted in isolation of the gene named KlMNN10, sharing high homology with Saccharomyces cerevisiae MNN10. The disruption of the KlMNN10 in Kluyveromyces lactis, as well as of MNN10 and MNN11 in Saccharomyces cerevisiae, conferred the super-secreting phenotype. MNN10 isolated from Saccharomyces cerevisiae suppressed the super-secretion phenotype in Kluyveromyces lactis klmnn10, as did the homologous KlMNN10. The genes MNN10 and MNN11 of Saccharomyces cerevisiae encode mannosyltransferases responsible for the majority of the alpha-1,6-polymerizing activity of the mannosyltransferase complex. These data agree with the view that the structure of glycoproteins in a yeast cell wall strongly influences the release of homologous and heterologous proteins in the medium. The set of genes namely the suppressors of the over-secreting phenotype, could be attractive for further analysis of gene functions, over-secreting mechanisms and for construction of new strains optimized for heterologous protein secretion. KlMNN10 has EMBL accession no. AJ575132.

The deletion of the succinate dehydrogenase gene KlSDH1 in Kluyveromyces lactis does not lead to respiratory deficiency.

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.

Isolation of a Kluyveromyces lactis Mutant Enriched in S-Adenosyl-L-Methionine and Growing in Whey Medium

The ability of six lactose-digesting yeast strains to accumulate S-adenosyl-L-methionine (AdoMet) when grown on whey medium was studied.Kluyveromyces lactis ATCC 8585 accumulated the greatest amount of AdoMet (15.2 mg per gram biomass) and was chosen for the development of an Ado-Met-enriched mutant. The Ado-Met-enriched mutant AM-65 was selected from mutants induced with N-methyl-N-nitro-N"-nitrosoguanidine. The strain accumulated 61.6 mg AdoMet per gram biomass. The effect of concentrations of medium components on AdoMet biosynthesis was studied. The ability of the mutant to accumulate AdoMet remained stable after multiple reinoculations.

Isolation and study of KlLSM4, a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae.

We have isolated the KlLSM4 gene as a multicopy suppressor of a Kluyveromyces lactis mutant which shows a rag(-) phenotype (resistance to antimycin A on glucose). This gene is homologous to the ScLSM4 of Saccharomyces cerevisiae, which codes for an essential 187 amino acid protein containing Sm-like domains. These motifs are present in the evolutionarily conserved family of the Sm-like proteins, which are involved in a large number of cellular processes, including pre-mRNA splicing and mRNA decapping. We demonstrated that the first 72 amino acids of KlLsm4p, which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells lacking the wild-type protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of cell viability in the stationary phase. The KlLSM4 sequence has been deposited in the EMBL Data library under Accession No. AJ311719.

RAG4 gene encodes a glucose sensor in Kluyveromyces lactis.

The rag4 mutant of Kluyveromyces lactis was previously isolated as a fermentation-deficient mutant, in which transcription of the major glucose transporter gene RAG1 was affected. The wild-type RAG4 was cloned by complementation of the rag4 mutation and found to encode a protein homologous to Snf3 and Rgt2 of Saccharomyces cerevisiae. These two proteins are thought to be sensors of low and high concentrations of glucose, respectively. Rag4, like Snf3 and Rgt2, is predicted to have the transmembrane structure of sugar transporter family proteins as well as a long C-terminal cytoplasmic tail possessing a characteristic 25-amino-acid sequence. Rag4 may therefore be expected to have a glucose-sensing function. However, the rag4 mutation was fully complemented by one copy of either SNF3 or RGT2. Since K. lactis appears to have no other genes of the SNF3/RGT2 type, we suggest that Rag4 of K. lactis may have a dual function of signaling high and low concentrations of glucose. In rag4 mutants, glucose repression of several inducible enzymes is abolished.

Amphotericin B resistance and membrane fluidity in Kluyveromyces lactis strains.

The membrane fluidity of reduced-amphotericin B (AmB)-sensitivity Kluyveromyces lactis mutant strain is higher than that of the wild-type K. lactis strain. After culture of the K. lactis and K. lactis mutant cells in the presence of subinhibitory doses of AmB (10 and 125 mg/liter, respectively), the plasma membranes of both yeast strains also showed a higher fluidity than did those of control cells. High membrane fluidity was associated with changes in the structural properties of the membranes. Culture of the K. lactis and K. lactis mutant cells in the presence of AmB induced changes in membrane lipid contents. In particular, phospholipid contents were increased in both strains treated with AmB, compared with their corresponding counterparts. As a result, the sterol/phospholipid ratio decreased. The relative proportion of monounsaturated fatty acids also increased after AmB treatment. The saturated fatty acid/monounsaturated fatty acid ratio decreased in K. lactis and K. lactis mutant cells treated with AmB but also in K. lactis mutant control cells compared to that in the K. lactis wild strain. These changes in lipid composition explain the higher fluidity, which could represent a process of metabolic resistance of the yeasts to AmB.

Vga Mutants of Kluyveromyces lactis show cell integrity defects.

We studied the cell wall alterations that occur in mutants of Kluyveromyces lactis impaired in glycosylation. The mutants belong to four complementation groups named vga1 to vga4 (vanadate glycosylation affected), characterized by sodium orthovanadate resistance and alteration of the glycosylation profile of native invertase. A drastic reduction of the alkali-soluble fraction of the beta-D-glucan was observed in vga1, vga2 and vga3 cells, accompanied by an increase in the chitin content of the cell wall. In vga4 cells, both beta-D-glucan fractions (alkali-soluble and alkali-insoluble) were reduced to about half of the corresponding wild-type value but the chitin content was normal. A protein related to Fks1p, the catalytic subunit of the major 1,3-beta-D-glucan synthase of S. cerevisiae, was detected in K. lactis. The amount of this Fks1p-like protein increased 7-10 times in vga1, vga2 and vga3 mutants as compared to wild-type cells; the same strains released significant amounts of beta-D-glucan in the culture supernatant. These mutations also resulted in abnormally thick cell walls with conspicuous irregularities in the structure, as revealed by electron microscopy and by an altered resistance to Zymolyase. The observed high responsiveness of cell wall phenotypes to alterations of glycosylation make K. lactis an attractive system for studying the interconnections between these processes.

A Klaac null mutant of Kluyveromyces lactis is complemented by a single copy of the Saccharomyces cerevisiae AAC1 gene.

The KlAAC gene, encoding the ADP/ATP carrier in Kluveromyces lactis, has previously been cloned by complementation of the op1(aac2) mutation of Saccharomyces cerevisiae. We examined the effect of a null mutation of this gene on the phenotype of K. lactis. The consequence of this mutation was found to be multiple. The mutant was respiratory deficient, had an undetectable level of cytochrome a-a3 and b and did not grow on glycerol. The mitochondrial D-lactate ferricytochrome c oxidoreductase activity, as well as the lactate-induced transcription of its gene, KlDLD, was severely reduced. Furthermore, the mutant was unable to grow on galactose, maltose and raffinose. Transcript analysis showed that KlAAC was the only ADP/ATP carrier gene present in K. lactis. The Klaac mutation was fully complemented not only by AAC2, the major gene for the ADP/ATP carrier in S. cerevisiae, but also by AAC1, a gene which is poorly expressed in S. cerevisiae. AAC1 introduced in K. lactis was transcribed to a high level consistent with normal growth on glycerol being restored in the transformed mutant. KlAAC was not subject to control by KlHap2, in contrast to AAC2 which is regulated by the Hap2 complex in S. cerevisiae.

Impaired growth on glucose of a pyruvate dehydrogenase-negative mutant of Kluyveromyces lactis is due to a limitation in mitochondrial acetyl-coenzyme A uptake.

A Kluyveromyces lactis mutant with a disruption in the KlPDA1 gene, encoding the E1 alpha subunit of the pyruvate dehydrogenase complex, exhibited a four-fold reduced specific growth rate on glucose in minimal medium. Growth of the Klpda1 mutant on glucose in complex medium was not affected. Its growth on defined media could be restored by adding amino acids that require mitochondrial acetyl-CoA for their biosynthesis as nitrogen sources. This, together with the observation that low-concentrations of L-carnitine also restored growth on glucose, indicates that the slow-growth phenotype of the Klpda1 mutant is due to a limited capacity of the mitochondria for import of cytosolic acetyl-CoA.