Isolation and molecular characterization of KlCOX14, a gene of Kluyveromyces lactis encoding a protein necessary for the assembly of the cytochrome oxidase complex

The yeast Kluyveromyces lactis was mutagenized with ethyl methane sulphonate and mutants unable to grow on respiratory carbon sources were isolated. Functional complementation of one of these mutants led to the isolation of KlCOX14, a gene encoding a 64 amino acid protein which is the functional homologue of Saccharomyces cerevisiae Cox14p, a protein necessary for the assembly of the cytochrome oxidase holoenzyme (Glerum et al., 1995). The disruption of KlCOX14 resulted in the absence of the absorption bands relative to cytochromes a and a(3) and in the complete loss of respiratory activity. Klcox14 mutants display the typical phenotype of pet mutants and have a reduced growth rate. In addition, unlike the wild-type, Klcox14 mutants are able to grow by fermentation also in the presence of low glucose. The nucleotide sequence of KlCOX14 has been deposited in the EMBL databank with Accession No. AJ238801.     

Disruption of genes encoding pyruvate dehydrogenase kinases leads to retarded growth on acetate and ethanol in Saccharomyces cerevisiae

Two open reading frames, YIL042c (PKP1) and YGL059w, with 25% sequence similarity to human pyruvate dehydrogenase kinases, were shown to have protein kinase activity. Using GFP fusions, it was demonstrated that the proteins localize in discrete submitochondrial regions. Strains with a null mutation in these loci grew poorly on acetate and ethanol as carbon sources. Doubling times increased from ca. 4 h in the wild-type to > 6 h for the mutants. Growth rates of the mutants could be restored to wild-type levels by simultaneous disruption of the PDA1 gene, encoding the E1alpha subunit of the pyruvate dehydrogenase complex. This observation and the pyruvate dehydrogenase activities measured in the mutant strains and the wild-type grown on glucose or acetate suggest that the slow growth phenotype on C2 carbon sources is caused by a futile cycle in which phosphoenolpyruvate is converted back to acetyl coenzyme A.     

感染青枯病与黑胫病烟株的根际土壤、根及茎秆微生物代谢特征分析

为明确感染青枯病、黑胫病烟株的根际土壤、根及茎秆中微生物代谢特征,采用Biolog-ECO代谢表型技术研究了微生物群落对6类31种碳源的代谢情况。结果表明：(1)同一烟株的根际土壤、根、茎秆中微生物的碳源代谢能力依次减弱;烟株感病后根和根际土壤中微生物对6类碳源的利用率无显著变化,茎秆微生物对多聚物类、氨基酸类和酚类碳源的利用率提高。(2)感染青枯病、黑胫病烟株根和根际土壤微生物均能代谢31种碳源;感染青枯病烟株茎秆微生物对除2-羟基苯甲酸以外的30种碳源均能正常代谢;感染黑胫病烟株茎秆微生物不能代谢D-甘露醇、葡萄糖-1-磷酸盐、D-葡萄糖胺酸等10种碳源。(3)感染黑胫病烟株的根和根际土壤微生物的平均颜色变化率（Average well color development,AWCD）显著低于健康烟株,茎秆微生物AWCD值略高于健康烟株;感染青枯病烟株茎秆微生物的AWCD值显著高于健康烟株,根和根际土壤微生物AWCD值与健康烟株无显著差异。(4)感病烟株的根和根际土壤微生物的香农-威纳指数（Shannon-Wiener）、辛普森指数（Simpson）和皮诺（Pielou）指数较健康烟...     

碳源对manA基因突变大肠杆菌代谢的影响

ManA基因编码的甘露糖-6-磷酸异构酶在大肠杆菌中催化D-甘露糖和D-果糖的异构化,促进大肠杆菌对碳源的代谢吸收。本文通过研究manA基因突变大肠杆菌对碳源的利用和编码糖代谢基因情况,探讨甘露糖-6-磷酸异构酶对大肠杆菌糖代谢的影响。采用Ⅱ型内含子逆转录突变方法构建manA基因突变大肠杆菌,分析manA基因突变大肠杆菌对不同碳源的利用情况和manA基因突变对大肠杆菌糖代谢相关基因表达的影响,结果显示,大肠杆菌BL21(DE3)ΔmanA以甘露糖、果糖为碳源时,菌株生长受到显著抑制;以淀粉为碳源时,BL21(DE3)ΔmanA菌株的生长显著优于野生型大肠杆菌;以葡萄糖为碳源时,manA基因突变对大肠杆菌的生长无显著影响。通过基因表达分析,发现大肠杆菌BL21(DE3)ΔmanA中甘露糖代谢相关基因的表达显著性降低;果糖代谢途径中6-磷酸果糖激酶Ⅰ亚基的编码基因(pfkA)显著下调表达;水解淀粉的α-淀粉酶编码基因(malS)显著性上调表达。ManA基因突变影响大肠杆菌甘露糖、果糖和淀粉代谢途径中相关基因的表达,从而影响大肠杆菌对碳源的利用。     

Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources

Expression of the peroxisome-deficient (Per^?) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6^ts and per14-11^ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D -alanine (for both mutants) or methylamine (for per14-11^ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D -alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.     

Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis

Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.     

RAG1 and RAG2: nuclear genes involved in the dependence/independence on mitochondrial respiratory function for growth on sugars

The analysis of five independent isolates of Kluyveromyces lactis shows that CBS 2359, CBS 683 and CBS 4574 could grow in the presence of mitochondrial inhibitors (antimycin A, oligomycin or erythromycin) and that CBS 2360 and CBS 141 were unable to grow in the presence of drugs. The resistant growth was observed only on glucose and not on other fermentable carbon sources (galactose, lactose). The phenotype 'growth on glucose in the presence of mitochondrial inhibitors' was called Rag+. This phenotype was found to be controlled by two unlinked nuclear genes: RAG1 and RAG2. Either of their recessive alleles, rag1 and rag2, led to the Rag- phenotype (i.e. the failure of growth on glucose in the presence of antimitochondrial drugs). Rag- strains represent the case in which fermentative growth becomes absolutely dependent on the functioning of the normal respiratory chain.     

A mutation in the COX5 gene of the yeast Scheffersomyces stipitis alters utilization of amino acids as carbon source, ethanol formation and activity of cyanide insensitive respiration

Scheffersomyces stipitis PJH was mutagenized by random integrative mutagenesis and the integrants were screened for lacking the ability to grow with glutamate as sole carbon source. One of the two isolated mutants was damaged in the COX5 gene, which encodes a subunit of the cytochrome c oxidase. BLAST searches in the genome of Sc. stipitis revealed that only one singular COX5 gene exists in Sc. stipitis, in contrast to Saccharomyces cerevisiae, where two homologous genes are present. Mutant cells had lost the ability to grow with the amino acids glutamate, proline or aspartate and other non-fermentable carbon sources, such as acetic acid and ethanol, as sole carbon sources. Biomass formation of the mutant cells in medium containing glucose or xylose as carbon source was lower compared with the wild-type cells. However, yields and specific ethanol formation of the mutant were much higher, especially under conditions of higher aeration. The mutant cells lacked both cytochrome c oxidase activity and cyanide-sensitive respiration, whereas ADH and PDC activities were distinctly enhanced. SHAM-sensitive respiration was obviously essential for the fermentative metabolism, because SHAM completely abolished growth of the mutant cells with both glucose or xylose as carbon source.     

Isolation and characterization of peroxisomal protein import (Pim−) mutants of Hansenula polymorpha

In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim^? phenotype). Genetic analysis of back-crossed mutants revealed five different complementation groups, which were designated PERI–PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim^? phenotypes were determined by single recessive nuclear mutations. The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome-borne enzymes in wild-type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild-type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.     

非酿酒酵母对有机酸类碳源代谢特征的研究

以酿酒酵母（Saccharomyces cerevisiae）RV002为对照,将发酵毕赤酵母（Pichia fermentans）JT-1-3和季也蒙毕赤酵母（Meyerozyma guilliermondii）JP-4-2分别接种于以L-苹果酸、柠檬酸、葡萄糖和乙醇为单一碳源或双碳源的培养基中,测定酵母生物量及碳源代谢动态变化,研究两株非酿酒酵母（non-Saccharomyces）对碳源尤其是有机酸类碳源的利用情况。结果表明,发酵毕赤酵母JT-1-3对L-苹果酸的代谢率最高（49.74%）,季也蒙毕赤酵母JP-4-2对柠檬酸的代谢率最高（28.30%）;除发酵毕赤酵母JT-1-3在L-苹果酸与乙醇共存的培养基中L-苹果酸消耗量（58.88%）增加外,其余情况下,乙醇或葡萄糖的存在会抑制非酿酒酵母对L-苹果酸或柠檬酸的利用,这将为进一步利用两株非酿酒酵母酿造酸度适宜的果酒提供理论基础。     

Rapamycin pre-treatment preserves viability, ATP level and catabolic capacity during carbon starvation of Saccharomyces cerevisiae

Saccharomyces cerevisiae growing exponentially in anaerobic batch cultures that are suddenly exposed to carbon starvation will rapidly lose almost all ATP. This will cause an energy deficiency and adaptation to starvation conditions is prohibited. As a result, viability and fermentative capacity will be drastically reduced during prolonged starvation. However, if the cells are incubated in the presence of rapamycin (which will inactivate the TOR pathway) before carbon starvation ATP levels, viability and fermentative capacity will be preserved to a much larger extent compared to untreated cells. The beneficial effect of rapamycin cannot be explained by induction of a stationary phase phenotype. In fact, under these anaerobic well-controlled growth conditions, rapamycin-treated cells were still metabolically active and continued to grow, albeit not exponentially and with a reduced protein content. It is hypothesized that the loss of ATP during carbon starvation occurs because protein synthesis does not make an immediate arrest at the onset of starvation. Since there are no external or internal energy sources, this will rapidly deplete the cells of ATP. Rapamycin-treated cells, on the other hand, have already downregulated the protein-synthesizing machinery and are thus better suited to cope with a sudden carbon starvation condition. This hypothesis is strengthened by the fact that treating the cells with the protein synthesis inhibitor cycloheximide also improves the carbon starvation tolerance, although not to the same extent as rapamycin. The even better effect of rapamycin is explained by accumulation of storage carbohydrates, which is not observed for cycloheximide-treated cells.     

Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identification of a suppressor of the pyc phenotype

We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source. Growth was possible with aspartate or glutamate as nitrogen source. The gene PpPYC1 expressd from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. In a P. pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions. The sequence of the PpPYC1 gene has been entered in the EMBL nucleotide sequence databank: Accession Number Y11106. © 1998 John Wiley & Sons, Ltd.