Zymocin, a composite chitinase and tRNase killer toxin from yeast.

Growth inhibition of Saccharomyces cerevisiae by the plasmid-encoded trimeric (alphabetagamma) zymocin toxin from dairy yeast, Kluyveromyces lactis, depends on a multistep response pathway in budding yeast. Following early processes that mediate cell-surface contact by the chitinase alpha-subunit of zymocin, later steps enable import of the gamma-toxin tRNase subunit and cleavage of target tRNAs that carry modified U34 (wobble uridine) bases. With the emergence of zymocin-like toxins, continued zymocin research is expected to yield new insights into the evolution of yeast pathosystems and their lethal modes of action.

Mutant casein kinase I (Hrr25p/Kti14p) abrogates the G1 cell cycle arrest induced by Kluyveromyces lactiszymocin in budding yeast.

Zymocin, a toxic protein complex produced by Kluyveromyces lactis, inhibits cell cycle progression in Saccharomyces cerevisiae. In studying its action, a resistant mutant ( kti14-1) was found to express the tot-phenotype typical of totDelta cells, toxin target (TOT) mutants that are impaired in RNA polymerase II Elongator function. Phenotypic analysis of a kti14-1 tot3Delta double mutant revealed a functional link between KTI14 and TOT/Elongator. Unlike totDelta cells, the kti14-1 mutant is sensitive to the drug methylmethane sulfonate (MMS), indicating that, besides being affected in TOT function, kti14-1 cells are also compromised in DNA repair. Single-copy complementation identified HRR25, which codes for casein kinase I (CKI), as KTI14. Kinase-minus hrr25 mutations (K38A and T176I) conferred zymocin resistance, while deletion of the other yeast CKI genes ( YCK1-3) had no effect. A mutation in KTI14 that truncates the P/Q-rich C-terminus of Hrr25p also dissociates MMS sensitivity from zymocin resistance; this mutant is resistant to the toxin, but shows normal sensitivity to MMS. Thus, although kinase-minus mutations are sufficient to protect yeast cells from zymocin, toxicity is also dependent on the integrity of the C-terminal region of Hrr25p, which has been implicated in determining the substrate specificity or localization of Hrr25p.

Defects in yeast RNA polymerase II transcription elicit hypersensitivity to G1 arrest induced by Kluyveromyces lactis zymocin.

The G1 cell cycle arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae requires a functional RNA polymerase II (pol II) TOT/Elongator complex. In a study of zymocin's mode of action, genetic scenarios known to impair transcription or affect the pol II machinery itself were found to elicit hypersensitivity to zymocin. Thus, mutations in components of SAGA, SWI/SNF, Mediator and Ccr4-Not, complexes involved in transcriptionally relevant functions such as nucleosome modification, chromatin remodelling and formation of the preinitiation complex, make yeast cells hypersensitive to the lethal effects of zymocin. The defects at the level of transcriptional elongation displayed by rtf1Delta, ctk1, fcp1 and rpb2 mutants also result in zymocin hypersensitivity. Intriguingly, inactivation of histone deacetylase (HDAC) activity, which is expected to reduce the demand for the histone acetyltransferase (HAT) function of TOT/Elongator, also reduces sensitivity to zymocin. Thus, zymocin interferes with pol II-dependent transcription, and this effect requires the HAT function of TOT, presumably while the Elongator complex is associated with pol II.

Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator.

The putative Kluyveromyces lactis zymocin target complex, TOT, from Saccharomyces cerevisiae comprises five Tot proteins, four of which are RNA polymerase II (RNAP II) Elongator subunits. Recently, two more Elongator subunit genes, ELP6 (TOT6) and ELP4 (TOT7), have been identified. Deletions of both TOT6 and TOT7 result in the complex tot phenotype, including resistance to zymocin, thermosensitivity, slow growth and hypersensitivity towards drugs, thus reinforcing the notion that TOT/Elongator may be crucial in signalling zymocicity. Mutagenesis of ELP3/TOT3, the Elongator histone acetyltransferase (HAT) gene, revealed that zymocin sensitivity could be uncoupled from Elongator wild-type function, indicating that TOT interacts genetically with zymocin. To test the possibility that zymocin functions by affecting RNAP II activity in a TOT/Elongator-dependent manner, global poly(A)+ mRNA levels were found to decline drastically on zymocin treatment. Moreover, cells overexpressing Fcp1p, the RNAP II carboxy-terminal domain phosphatase, acquired partial zymocin resistance, whereas cells underproducing RNAP II became zymocin hypersensitive. This suggests that zymocin may convert TOT/Elongator into a cellular poison toxic for RNAP II function and eventually leading to the observed G1 cell cycle arrest.

An SSB encoded by and operating on linear killer plasmids from Kluyveromyces lactis.

The Kluyveromyces lactis linear plasmids k1 and k2 belong to the family of protein-primed linear DNA genomes, which includes adenoviruses. Here we identify the 18 kDa gene product of k2ORF5 as a novel putative single-stranded DNA binding protein, SSB. As judged from Western analysis using an epitope-tagged fusion protein and ssDNA-agarose affinity chromatography, the Orf5 protein preferentially binds to ssDNA in vitro. Consistently, electrophoretic mobility shift assays demonstrate that ssDNA plasmid probes from k1 and k2 are retarded by this Orf5-associated SSB activity. ORF5 gene shuffle-mediated mutagenesis in vivo results in k1/k2 plasmid instability, pointing towards a role for the Orf5 protein in plasmid replication. Consistently, the Orf5 protein protects ssDNA from exonuclease digestion and stimulates Klenow enzyme. Our findings suggest a functional role for the Orf5 protein as a putative SSB probably required during k1/k2 plasmid DNA synthesis.

Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor.

The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G(1) phase of the cell cycle. Despite its heterotrimeric (alpha beta gamma) structure, intracellular expression of its smallest subunit, the gamma-toxin, is alone responsible for the G(1) arrest. The alpha subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior. Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells. Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix. PCR-mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3 Delta), blocked export (chs7 Delta) or reduced activation (chs4 Delta), combined with mislocalization (chs4 Delta chs5 Delta; chs4 Delta chs6 Delta; chs4 Delta chs5 Delta chs6 Delta) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin. In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity. Thus, CSIII-polymerized chitin, which amounts to almost 90% of the cell's chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells.

Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin.

Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin-resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts-phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G(1) cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with ELONGATOR: Intriguingly, strains lacking non-Elongator HATs (gcn5, hat1, hpa3 and sas3) or non-Elongator transcription elongation factors TFIIS (dst1) and Spt4p (spt4) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin.

Sit4p protein phosphatase is required for sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin.

We have identified two Saccharomyces cerevisiae genes that, in high copy, confer resistance to Kluyveromyces lactis zymocin, an inhibitor that blocks cells in the G(1) phase of the cell cycle prior to budding and DNA replication. One gene (GRX3) encodes a glutaredoxin and is likely to act at the level of zymocin entry into sensitive cells, while the other encodes Sap155p, one of a family of four related proteins that function positively and interdependently with the Sit4p protein phosphatase. Increased SAP155 dosage protects cells by influencing the sensitivity of the intracellular target and is unique among the four SAP genes in conferring zymocin resistance in high copy, but is antagonized by high-copy SAP185 or SAP190. Since cells lacking SIT4 or deleted for both SAP185 and SAP190 are also zymocin resistant, our data support a model whereby high-copy SAP155 promotes resistance by competition with the endogenous levels of SAP185 and SAP190 expression. Zymocin sensitivity therefore requires a Sap185p/Sap190p-dependent function of Sit4p protein phosphatase. Mutations affecting the RNA polymerase II Elongator complex also confer K. lactis zymocin resistance. Since sit4Delta and SAP-deficient strains share in common several other phenotypes associated with Elongator mutants, Elongator function may be a Sit4p-dependent process.

Genetics and molecular physiology of the yeast Kluyveromyces lactis.

With the recent development of powerful molecular genetic tools, Kluyveromyces lactis has become an excellent alternative yeast model organism for studying the relationships between genetics and physiology. In particular, comparative yeast research has been providing insights into the strikingly different physiological strategies that are reflected by dominance of respiration over fermentation in K. lactis versus Saccharomyces cerevisiae. Other than S. cerevisiae, whose physiology is exceptionally affected by the so-called glucose effect, K. lactis is adapted to aerobiosis and its respiratory system does not underlie glucose repression. As a consequence, K. lactis has been successfully established in biomass-directed industrial applications and large-scale expression of biotechnically relevant gene products. In addition, K. lactis maintains species-specific phenomena such as the "DNA-killer system, " analyses of which are promising to extend our knowledge about microbial competition and the fundamentals of plasmid biology.

Use of gene shuffles to study the cytoplasmic transcription system operating on Kluyveromyces lactis linear DNA plasmids.

A modified double selection approach for manipulation of the cytoplasmic plasmids k1 and k2 from dairy yeast Kluyveromyces lactis has been exploited to investigate promoter and gene function. Using TRP1-mediated integration of a LEU2 gene fusion, we have shown that expression of the selection marker is strictly dependent on the k2 promoter UCS5. Also, k2ORF6, the gene encoding the RNA polymerase specific for UCS recognition, is functional when shuffled between the plasmids. Once transplaced onto k1 by means of gene shuffling, the hybrid ORF6 complemented an orf6 deletion created on plasmid k2 eventually yielding yeast strains that contained only two recombinant plasmids: a k2 derivative (rk2/6) with a k2orf6::TRP1 gene deletion, and a k1 derivative (rk1/6) carrying the transplaced ORF6 allele along with the LEU2 marker. This interchangeability of both UCS promoter activity and gene function between k2 and k1 supports the concept of an autonomous transcription system that operates on these nonconventional yeast plasmids.

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles.

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

Microbial linear plasmids.

While plasmids were originally considered to be generally circular until almost two decades ago, linear elements were reported to exist as well. They are now known to be common genetic elements in both, pro- and eukaryotes. Two types of linear plasmids exist, the so-called hairpin plasmids with covalently closed ends and those with proteins bound to their 5' termini. Hairpin plasmids are common in human-pathogenic Borrelia spirochetes, in which they are instrumental in escape from the immunological response; cryptic hairpin elements are present in mitochondria of the plant pathogenic fungus Rhizoctonia solani. Plasmids with 5' attached proteins constitute the largest group. In actinomycetous bacteria they are conjugative and usually confer advantageous phenotypes, e.g. formation of antibiotics, degradation of xenobiotics, heavy-metal resistance and growth on hydrogen as the sole energy source. In contrast, the majority of linear plasmids from eukaryotes are cryptic, with only a few exceptions. In some yeasts a killer phenotype may be associated, the most thoroughly investigated elements being those from Kluyveromyces lactis killer strains. In Neurospora spp. and in Podospora anserina, senescence and longevity respectively are correlated with linear plasmids. This review focuses on the biology of linear plasmids, their environmental significance and their use as tools in molecular and applied microbiology.

ORF7 of yeast plasmid pGKL2: analysis of gene expression in vivo.

ORF7 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a putative RNA polymerase subunit of 16 kDa. RNA analysis detected a single, plasmid-dependent ORF7 transcript of 550 nt indicating that the gene is transcribed mono-cistronically. Attempted one-step gene disruption of ORF7 resulted in chromosomal integration of the marker gene rather than the formation of stable recombinant k2ORF7(0) deletion plasmids. Thus, ORF7 appears to be a potential cis-dominant locus the integrity of which is indispensable for plasmid stability. The ORF7 gene product was over-produced as a c-myc-tagged fusion protein in Escherichia coli. Western-blot analysis of total yeast protein extracts using an antibody against this Orf7-c-myc fusion product identified a protein band with an apparent molecular weight of 17 kDa. This protein corresponds in size to the predicted product and is only detectable in plasmid-carrying killer yeasts.

A cytoplasmic gene-shuffle system in Kluyveromyces lactis: use of epitope tagging to detect a killer plasmid-encoded gene product.

A novel gene shuffle approach has been developed for investigating the functions of genes on the cytoplasmic linear DNA killer plasmids of Kluyveromyces lactis. By transplacing k2ORF5 from larger plasmid pGKL2(k2) onto pGKL1(k1) we have shown this gene to be essential and functionally interchangeable between plasmids. Once transferred onto k1, k2ORF5 is fully able to complement a k2ORF5(0) deletion on k2 in trans, giving rise to yeast strains containing only the two recombinant plasmid forms. Additionally, the in vivo product of k2ORF5 has been identified as a 19.5 kDa protein by transplacing an epitope-tagged k2ORF5 allele from k2 to k1. The ease of detection of the tagged ORF5 product in comparison to TRF1, the gene product of k2ORF10, indicates that Orf5p is one of the most abundant k2 products, implying structural rather than regulatory function.

Yeast killer plasmid pGKL2: molecular analysis of UCS5, a cytoplasmic promoter element essential for ORF5 gene function.

A k2/k1 plasmid gene shuffle system has been used to investigate linear plasmid promoter function in Kluyveromyces lactis. By transplacing various ORF5 deletion constructs from the larger plasmid k2 onto k1, and analysing trans-complementation of an ORF5(0) deletion on k2, a 40 bp k2 fragment, including the UCS motif of ORF5 (UCS5), has been identified as a cis-acting promoter element essential for ORF5 gene function. Qualitative and quantitative transcript analyses of a UCS5-ScLEU2 fusion gene using Northern blot analysis and phosphor image technology revealed a plasmid-dependent LEU2 transcript distinct in size (1.55 kb) and regulation from its nuclear counterpart (1.35 kb): cytoplasmic, UCS5-driven expression of the marker gene was non-repressible by leucine and reduced five- to eight-fold compared to fully derepressed nuclear K1LEU2 mRNA levels. Thus, the killer plasmids k2 and k1 appear to express low levels of transcript overall, when relative gene copy numbers (one for the nuclear allele versus 50-100 copies for the plasmid-borne LEU2 gene) are taken into account.

The DNA and RNA polymerase genes of yeast plasmid pGKL2 are essential loci for plasmid integrity and maintenance.

Novel recombinant plasmids derived from the Kluyveromyces lactis killer plasmid k2 have been constructed to study plasmid biology and gene function. In vivo recombination between native resident k2 and suitable disruption vectors, employing the KITRP1 gene fused to a plasmid promoter as selection marker, yielded ORF2 and ORF6 deletion plasmids at high frequencies. As judged from Southern hybridization and plasmid restriction mapping analyses, these novel hybrids, termed rk2/2 and rk2/6, respectively, carry deletions in their putative DNA (ORF2) and RNA (ORF6) polymerase structural genes with central regions replaced by the input marker DNA. Long-term selection for TRP1 over 350 generations of growth did not favour maintenance of hybrids over wild-type k2. Thus, neither rk2/2 nor rk2/6 was fully functional and able to displace parental k2, indicating that both target genes are essential for plasmid integrity or maintenance. Recombinant plasmids were reduced in copy number relative to k2 with rk2/2 more drastically affected than rk2/6 implying a direct involvement of the ORF2 product in plasmid replication and an indirect maintenance function for the ORF6 gene product.

Kluyveromyces lactis killer plasmid pGKL2: molecular analysis of an essential gene, ORF5.

The ORF5 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One-step gene disruption of ORF5, via in vivo homologous recombination between native plasmid k2 and a transfer vector employing the Saccharomyces cerevisiae LEU2 gene fused to the k2 UCS5 element, yielded Leu+ transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by the LEU2 marker, termed rk2, in addition to the wild-type plasmids k1 and k2. Northern analysis detected a plasmid-dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+ selective growth; however rk2 was displaced by k2 during non-selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over-expression of an epitope-tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predicted product.

Kluyveromyces lactis killer system: ORF1 of pGKL2 has no function in immunity expression and is dispensable for killer plasmid replication and maintenance.

To functionally characterize the genes encoded by the larger killer plasmid pGKL2 from Kluyveromyces lactis a previously developed in-vivo recombination system was exploited. An in-vitro modified version of the cytoplasmically expressible LEU2 gene cartridge (LEU2*) flanked by appropriate pGKL2 segments was used to replace the central part of the ORF1 region of pGKL2. Transformation of a Leu- killer strain resulted in the expected disruption of ORF1 in the resident pGKL2. The Leu+ transformants obtained can be assigned to three classes. Class I carries both killer plasmids, pGKL1/2, and the recombinant pGKL2 derivative termed pRKL2. Class II and III additionally harbor palindrome and hairpin-like plasmids, respectively. Upon subculturing of class I transformants under selective pressure, segregation of the native pGKL2 and the recombinant pRKL2 eventually occurs resulting in total loss of pGKL2. No differences concerning killer and immunity phenotype between a pRKL2-harboring strain and the native pGKL2-carrying recipient could be detected. Thus pGKL2 ORF1 is dispensable for both expression of killer/immunity phenotypes and for the replication and maintenance of the K. lactis killer plasmids.

[Hearing handicap in childhood--responsibilities of public health, early diagnosis]. / Hörbehinderung im Kindesalter--Aufgaben des öffentlichen Gesundheitswesens, Frühdiagnostik.

Serious congenital hearing impairment occur in 1 per 1,000 people. Hereditary disease, perinatal complications, and postnatal disease present particular risk factors. Children, who do not hear or are extremely hard of hearing are slow in their language development and thus become retarded in their mental growth if not given appropriate stimulation. The Counseling Center for the hearing impaired was stablished as a multidisciplinary institution of the Health Office of Berlin-Neukölln for early recognition of and therapy for hearing impaired children. The average age at initial diagnosis of children with hearing impairment in the Federal Republic of Germany is between 3.3-3.4 years according to statistics from 1981. An effort should be made, however, to use the time before the completion of the first year of on's life for an optimal early therapy. Statistics from the Counseling Center for the hearing impaired indicate that at the present time all children with distinct hearing impairments, born in the FRG and West-Berlin, are diagnosed before they reach 32 months of age and are then assigned to special education programs. The average age of recognizing hearing impairments is 13.4 months. This favorable, however not optimal result can be attributed to the situation of a metropolis and its population informed by special professional events for target groups including employees of theYouth Health Services, established pediatricians and ENT specialists, teachers, social workers, educators, and also to the model of the "Berlin-Neukölln Counseling Center" which has proven successful over the past 30 years.