Glutathione S-transferase Ï´-subunit as a phenotypic suppressor of pmr1Δ strain, the Kluyveromyces lactis model for Hailey-Hailey disease.

BACKGROUND: Hailey-Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, histologically characterized by suprabasal acantholysis. HHD has been linked to mutations in ATP2C1, the gene encoding the human adenosine triphosphate (ATP)-powered calcium channel pump. METHODS: In this work, the genetically tractable yeast Kluyveromyces lactis has been used to study the molecular basis of Hailey-Hailey disease. The K. lactis strain depleted of PMR1, the orthologue of the human ATP2C1 gene, was used to screen a Madin-Darby canine kidney (MDCK) cDNA library to identify genetic interactors able to suppress the oxidative stress occurring in those cells. RESULTS: We have identified the Glutathione S-transferase Ï´-subunit (GST), an important detoxifying enzyme, which restores many of the defects associated with the pmr1Δmutant. GST overexpression in those cells suppressed the sensitivity to calcium chelating agents and partially re-established calcium (Ca2+) homeostasis by decreasing the high cytosolic Ca2+ levels in pmr1Δstrain. Moreover, we found that in the K. lactis mutant the mitochondrial dysfunction was suppressed by GST overexpression independently from calcineurin. In agreement with yeast results, a decreased expression of the human GST counterpart (GSTT1/M1) was observed in lesion-derived keratinocytes from HHD patients. CONCLUSIONS: These data highlighted the Glutathione S-transferase as a candidate gene associated with Hailey-Hailey disease. GENERAL SIGNIFICANCE: Kluyveromyces lactis can be considered a good model to study the molecular basis of this pathology.

13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis.

BACKGROUND: The construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations. METHODS: (13)C NMR spectroscopy and [1,2-(13)C2]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants. RESULTS: Rag8 showed a decrease of all (13)C glutamate fractional enrichments, except for [4-(13)C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4-(13)C]ketoglutarate pool not originating from mitochondria were suggested. (13)C lysine enrichments confirmed the presence of two compartmentalized α-ketoglutarate (α-KG) pools participating to glutamate and lysine synthesis. Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by (13)C NMR isotopomer analysis of alanine. CONCLUSIONS: (13)C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, (13)C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized α-KG pool. GENERAL SIGNIFICANCE: A compartmentalization of the α-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis. Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species.

Estimating micro area behavioural risk factor prevalence from large population-based surveys: a full Bayesian approach.

BACKGROUND: An important public health goal is to decrease the prevalence of key behavioural risk factors, such as tobacco use and obesity. Survey information is often available at the regional level, but heterogeneity within large geographic regions cannot be assessed. Advanced spatial analysis techniques are demonstrated to produce sensible micro area estimates of behavioural risk factors that enable identification of areas with high prevalence. METHODS: A spatial Bayesian hierarchical model was used to estimate the micro area prevalence of current smoking and excess bodyweight for the Erie-St. Clair region in southwestern Ontario. Estimates were mapped for male and female respondents of five cycles of the Canadian Community Health Survey (CCHS). The micro areas were 2006 Census Dissemination Areas, with an average population of 400-700 people. Two individual-level models were specified: one controlled for survey cycle and age group (model 1), and one controlled for survey cycle, age group and micro area median household income (model 2). Post-stratification was used to derive micro area behavioural risk factor estimates weighted to the population structure. SaTScan analyses were conducted on the granular, postal-code level CCHS data to corroborate findings of elevated prevalence. RESULTS: Current smoking was elevated in two urban areas for both sexes (Sarnia and Windsor), and an additional small community (Chatham) for males only. Areas of excess bodyweight were prevalent in an urban core (Windsor) among males, but not females. Precision of the posterior post-stratified current smoking estimates was improved in model 2, as indicated by narrower credible intervals and a lower coefficient of variation. For excess bodyweight, both models had similar precision. Aggregation of the micro area estimates to CCHS design-based estimates validated the findings. CONCLUSIONS: This is among the first studies to apply a full Bayesian model to complex sample survey data to identify micro areas with variation in risk factor prevalence, accounting for spatial correlation and other covariates. Application of micro area analysis techniques helps define areas for public health planning, and may be informative to surveillance and research modeling of relevant chronic disease outcomes.

Depletion of casein kinase I leads to a NAD(P)(+)/NAD(P)H balance-dependent metabolic adaptation as determined by NMR spectroscopy-metabolomic profile in Kluyveromyces lactis.

BACKGROUND: In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag(-) phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing. METHODS: A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses. RESULTS: Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced. CONCLUSIONS: The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)(+)/NAD(P)H redox balance state. GENERAL SIGNIFICANCE: The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.

Complex multipathways alterations and oxidative stress are associated with Hailey-Hailey disease.

BACKGROUND: Hailey-Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesions. While a strong relationship exists between mutations in the gene that encodes the Ca(2+)/Mn(2+)-adenosine triphosphatase ATP2C1 and HHD, we still have little understanding of how these mutations affect manifestations of the disease. OBJECTIVES: This study was designed to determine early signalling events that affect epithelial cell growth and differentiation during HHD development. METHODS: Expression of key regulatory signals important for maintaining skin homeostasis were evaluated by Western blot analysis and by reverse transcriptase-polymerase chain reaction in primary keratinocytes obtained from skin biopsies of patients with HHD. Reactive oxygen species accumulation in primary keratinocytes derived from lesional skin of patients with HHD was assessed by dihydrorhodamine 123 (DHR) assay. RESULTS: HHD-derived keratinocytes showed downregulation of both Notch1 and differential regulation of different p63 isoforms. Itch and p63 are co-expressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. We found that the Itch protein was significantly decreased in HHD-derived keratinocytes whereas the expression of its target, c-Jun, remained unaffected. We also found that HHD-derived keratinocytes undergo oxidative stress, which may explain both Notch1 and Itch downregulation. CONCLUSIONS: Our attempt to explore the molecular mechanism underlying HHD indicates a complex puzzle in which multi-hit combinations of altered signal pathways may explain the wide spectrum of defects in HHD.

SOD1, a new Kluyveromyces lactis helper gene for heterologous protein secretion.

Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O(2)(-)) into oxygen and hydrogen peroxide. It is a Cu(2+)/Zn(2+) metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans. In addition, KlSOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing KlSOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses.

Cognitive and behavioural effects of genetic testing for thrombophilia.

Very few studies have examined the impact of genetic testing for thrombophilia on health behaviours, perceptions of control over risk factors for venous thromboembolism, or health services utilization. Through a postal questionnaire we compared first degree relatives with thrombophilia (carriers) most of whom had received counseling, to those without (non-carriers) with respect to: (a) perceived causes of venous thromboembolism; (b) perceived control; (c) health behaviour changes; and (d) use of health care services. 44/51 for carriers and 26/47 for non-carriers completed questionnaires. Carriers were more likely to believe their risk of venous thromboembolism 'is a little higher' or 'much higher' than average (p < 0.001) but some continued to believe their risk 'is the same as' or 'lower than' average. 16%-32% of carriers did not recognize major risk factors. Stress, worry, or depression, negative attitude, and over-exertion were over-interpreted as risks. 37.2% did not appreciate that thrombophilia increases risk. Behaviour changes were uncommon. There is a need for research on education and strategies to improve knowledge in thrombophilia carriers.

Role of nuclear genes in expression of a mitochondrial tRNA gene in Saccharomyces cerevisiae.

In yeast mitochondria, most of the isoaccepting species of tyrosyl tRNA are coded by a mitochondrial gene, tyrA. A particular isoaccepting species is coded by a second mitochondrial gene, tyrB. This gene is not expressed in certain strains of yeast which show no deficient phenotype. Genetic crosses between strains expressing or not expressing the tyrB gene demonstrate that expression is controlled by specific nuclear genes and that a mutation of the tyrA gene can be bypassed when the tyrB gene is operative.

Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae.

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.

Mutants of Kluyveromyces lactis with altered protein glycosylation are affected in cell wall morphogenesis.

We isolated spontaneous mutants resistant to sodium orthovanadate in the biotechnologically significant yeast Kluyveromyces lactis. Resistance behaved as a recessive character in all mutants analyzed. Four genes were defined by complementation analysis, from vga1 to vga4. These mutants showed defects in N-linked as well as O-linked glycosylation processes. In addition, the mutants exhibited sensitivity to the aminoglycoside hygromycin B and to calcofluor white, with the exception of vga4; this mutant grew in the presence of the antibiotic as well as the parental wild type and was resistant to calcofluor. The mutations were accompanied by alterations in the cell wall structure, as revealed by the delocalization of chitin, changes in cell shape and size and by the clumpy aspect of the cultures. The mutants isolated provide basic tools for molecular and cellular analysis of glycosylation processes in K. lactis.

KlPMR1 inactivation and calcium addition enhance secretion of non-hyperglycosylated heterologous proteins in Kluyveromyces lactis.

The Kluyveromyces lactis KlPMR1 gene is the functional homologue of Saccharomyces cerevisiae PMR1 which encodes a Ca(2+)-ATPase localized in the Golgi apparatus. We studied the effects of KlPMR1 inactivation on the glycosylation and secretion of native and heterologous proteins in K. lactis. We used acid phosphatase, recombinant human serum albumin and alpha-glucoamylase from Arxula adeninivorans as reporter proteins. The Klpmr1Delta strain showed enhanced secretion of the heterologous proteins analyzed; the improved rHSA production did not result from enhanced transcription but rather involved increased translation and/or secretion efficiency. The growth rate of mutant cells resulted slower as compared to that of wild-type strain. The addition of 10mM calcium to the culture medium, however, not only completely relieved the growth defect of the mutant cells but also improved the rate of heterologous proteins production. Moreover, the addition of this ion in the culture medium of K. lactis did not suppress the glycosylation defects; this is an important difference with respect to S. cerevisiae where the glycosylation is partially restored by Ca(2+) addition. The Klpmr1Delta strain as a host offers thus an additional advantage for those cases requiring that the produced recombinant protein would not result hyperglycosylated.

Biotreatment of Cr(VI) contaminated waters by sulphate reducing bacteria fed with ethanol.

Biological treatment of Cr(VI) contaminated waters was performed in fixed bed reactors inoculated with SRB (sulphate-reducing bacteria) growing on ethanol. Treatment efficiency was evaluated by checking chemical abatement of Cr(VI) and by ecotoxicological tests using the nematode Caenorhabditis elegans. A preliminary comparison between ethanol and lactate was performed, denoting that using ethanol, the same values of final sulphate abatement were obtained. In addition ethanol showed to be a substrate more competitive than lactate in kinetic terms. Fixed bed column reactors were continuously fed with a solution containing sulphates (3 g L(-1)), ethanol (1.5 g L(-1)) and Cr(VI) (50 mg L(-1)). At steady state the column inoculated with SRB removed 65 ± 5% of sulphate and 95 ± 5% of chromium. Bioactive removal mechanisms predominated over biosorption. Diminution of Cr(VI) toxicity was assessed by using the nematode C. elegans as a test organism showing that the survival of nematodes was 20% in the presence of the untreated influent and raised up to 53% when the nematodes were exposed to the treated effluent.

Biotreatment and bioassessment of heavy metal removal by sulphate reducing bacteria in fixed bed reactors.

In this work a batch-optimised mixture (w/w %: 6% leaves, 9% compost, 3% Fe(0), 30% silica sand, 30% perlite, 22% limestone) was investigated in a continuous fixed bed column reactor for the treatment of synthetic acid-mine drainage (AMD). A column reactor was inoculated with sulphate-reducing bacteria and fed with a solution containing sulphate and heavy metals (As(V), Cd, Cr(VI), Cu and Zn). At steady state, sulphate abatement was 50+/-10%, while metals were totally removed. A degradation rate constant (k) of 0.015+/-0.001h(-1) for sulphate removal was determined from column data by assuming a first order degradation rate. Reduction of AMD toxicity was assessed by using the nematode Caenorhabditis elegans as a test organism. A lethality assay was performed with the toxicants before and after the treatment, showing that only 5% of the animals were still alive after 48h in presence of the contaminants, while the percentage increased to 73% when the nematodes were exposed to the solution eluted from the column.

The Kluyveromyces lactis CPY homologous genes: cloning and characterization of the KlPCL1 gene.

A 3.85-kb genomic fragment containing the KlPCL1 gene, with an open reading frame (ORF) of 1359 bp, was isolated from Kluyveromyces lactis genomic library by heterologous colony hybridization using the Saccharomyces cerevisiae PRC1 (ScPRC1) gene as a probe. The KlPCL1 nucleotide sequence was identical to the KLLAOC17490g ORF of K. lactis and showed >55 % identity with S. cerevisiae YBR139w and PRC1 genes encoding carboxypeptidases. The deduced KlPcl1p amino acid sequence displayed strong similarities to yeast and higher eukaryotic carboxypeptidases. In silico analyses revealed that KlPcl1p contained several highly conserved regions characteristic of the serine-type carboxypeptidases, such as the catalytic triad in the active site and the LNGGPGCSS, FHIAGESYAGHYIP and ICNWLGN motifs involved in the substrate binding. All this suggests that the KlPCL1 gene product belongs to the serine carboxypeptidase family. Sporulation and ascus dissection of a diploid strain heterozygous for single-copy disruption of KlPCL1 revealed that this gene is not essential in K. lactis. Further analyses of haploid and diploid deletion mutants demonstrated that disruption of the KlPCL1 gene neither impaired sporulation nor affected growth abilities of K. lactis cells under a variety of physiological conditions, e.g., growth on different carbon sources, at various temperatures or pH of the medium, and under nitrogen depletion.

APY-1, a novel Caenorhabditis elegans apyrase involved in unfolded protein response signalling and stress responses.

Protein glycosylation modulates a wide variety of intracellular events and dysfunction of the glycosylation pathway has been reported in a variety of human pathologies. Endo-apyrases have been suggested to have critical roles in protein glycosylation and sugar metabolism. However, deciphering the physiological relevance of Endo-apyrases activity has actually proved difficult, owing to their complexity and the functional redundancy within the family. We report here that a UDP/GDPase, homologous to the human apyrase Scan-1, is present in the membranes of Caenorhabditis elegans, encoded by the ORF F08C6.6 and hereinafter-named APY-1. We showed that ER stress induced by tunicamycin or high temperature resulted in increased transcription of apy-1. This increase was not observed in C. elegans mutants defective in ire-1 or atf-6, demonstrating the requirement of both ER stress sensors for up-regulation of apy-1. Depletion of APY-1 resulted in constitutively activated unfolded protein response. Defects in the pharynx and impaired organization of thin fibers in muscle cells were observed in adult worms depleted of APY-1. Some of the apy-1(RNAi) phenotypes are suggestive of premature aging, because these animals also showed accumulation of lipofuscin and reduced lifespan that was not dependent on the functioning of DAF-2, the receptor of the insulin/IGF-1 signaling pathway.

Characterization of the superoxide dismutase SOD1 gene of Kluyveromyces marxianus L3 and improved production of SOD activity.

Superoxide dismutase (SOD) activity is one major defense line against oxidative stress for all of the aerobic organisms, and industrial production of this enzyme is highly demanded. The Cu/Zn superoxide dismutase gene (KmSOD1) of Kluyveromyces marxianus L3 was cloned and characterized. The deduced KmSod1p protein shares 86% and 71% of identity with Kluyveromyces lactis and Saccharomyces cerevisiae Sod1p, respectively. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc and in enzymatic function were conserved. To the aim of developing a microbial production of Cu/Zn superoxide dismutase, we engineered the K. marxianus L3 strain with the multicopy plasmid YG-KmSOD1 harboring the KmSOD1 gene. The production of KmSOD1p in K. marxianus L3 and K. marxianus L3 (pYG-KmSOD1) in response to different compositions of the culture medium was evaluated. The highest specific activity (472 U(SOD) mg(prot) (-1)) and the highest volumetric yield (8.8 x 10(5) U(SOD) l(-1)) were obtained by the recombinant strain overexpressing KmSOD1 in the presence of Cu(2+) and Zn(2+) supplements to the culture media. The best performing culture conditions were positively applied to a laboratory scale fed-batch process reaching a volumetric yield of 1.4 x 10(6) U(SOD) l(-1).

Transcripts of mitochondrial tRNA genes in Saccharomyces cerevisiae.

The transcription of a group of tRNA genes from the large tRNA gene cluster of mitochondrial DNA from Saccharomyces cerevisiae has been investigated by hybridization with DNA probes carrying tRNA coding sequences and small portions of the A + T rich intergenic regions. Results have shown that in some rho- mutants (DS502, F11) mature tRNA was absent, but a few transcripts could be detected. Some high molecular weight species actually hybridized with DNA probes carrying different tRNA coding sequences. Low molecular weight transcripts (100-150 nucleotides, carrying one tRNA sequence) were also present in these mutants. A high molecular weight transcript was also observed in the wild type, though in much more limited amount. The low molecular weight transcripts were analysed by the S1 mapping technique and found to include both a tRNA sequence and the upstream 5' flanking region extending as far as the 3' end of the preceding tRNA gene. The results suggest the existence of a common transcript bearing several tRNA sequences and indicate a possible mechanism of processing, which might be defective in mutants.

The KlPMR1 gene of Kluyveromyces lactis encodes for a P-type Ca(2+)-ATPase.

A novel P-type Ca(2+)-ATPase gene has been cloned and sequenced in the yeast Kluyveromyces lactis. The gene has been named KlPMR1 and is localized on chromosome I. The putative gene product contains 936 residues and has a calculated molecular weight of 102,437 Da. Analysis of deduced amino acid sequence (KlPmr1p) indicated that the encoded protein retains all the highly conserved domains characterizing the P-type ATPases. KlPmr1p shares 71% amino acid identity with Pmr1p of S. cerevisiae, 62% with HpPmr1p of Hansenula polymorpha, 56% with Y1Pmr1p of Yarrowia lipolytica and 52% with the Ca(2+)-ATPase encoded for by the SPCA1 gene of Rattus norvegicus; these similarities place KlPmr1p in the SPCA group (secretory pathway Ca(2+)-ATPase) of the P-type ATPases. The K. lactis strain harbouring the Klpmr1 disrupted gene is not able to grow in presence of low calcium concentrations and shows hypersensitivity to high concentrations of EGTA in the medium. These defects are relieved by PMR1 of S. cerevisiae on a centromeric plasmid, demonstrating that KlPMR1 encodes for a functional Pmr1p homologue.

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.