Anne-Marie Zeeman

The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae.

The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs.

Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359

Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose‐limited chemostat cultures (D=0·05–0·40 h−1) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose‐grown shake‐flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake‐flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady‐state chemostat cultures (D=0·10 h−1) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose‐limited chemostat cultures (D=0·10 h−1) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady‐state growth at μ=0·10 h−1 to growth at μmax. It is concluded that in K. lactis, as in other Crabtree‐negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation.

Analysis of gene- and strand-specific repair in the moderately UV-sensitive Saccharomyces cerevisiae rad23 mutant

The RAD23 gene of Saccharomyces cerevisiae is involved in nucleotide excision repair (NER) and mutations in this gene confer a moderate sensitivity to UV irradiation. However, no repair of either cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, the major types of lesions formed upon UV irradiation, was detectable during the first 4 h post UV irradiation in a rad23 mutant. rad23, like the rad7 and rad16 mutants, is not as UV sensitive as completely NER-deficient mutants. The rad7 and rad16 mutants are only partly defective in NER: non-transcribed strands are completely refractory to repair while transcription-coupled repair is not affected. To investigate whether the rad23 mutant has similar strand-specific repair characteristics we analyzed gene-specific CPD removal from several loci using strand-specific probes but did not detect any repair. The moderate UV sensitivity of rad23 mutants as compared to completely NER-deficient mutants is therefore not due to gene- or strand-specific removal of lesions, indicating that rad23 mutants do not have a similar repair defect as rad7 or rad16 mutants, but are presumably defective in general NER. The rad23 mutation does not suppress the high UV sensitivity of completely NER-deficient rad1 or rad14 strains. This demonstrates that the relatively high survival of rad23 mutants in not due to an increased tolerance for the lesions that seem to persist in the genome but rather requires some NER function.

Regulation of pyruvate metabolism in chemostat cultures of Kluyveromyces lactis CBS 2359

Regulation of currently identified genes involved in pyruvate metabolism of Kluyveromyces lactis strain CBS 2359 was studied in glucose‐limited, ethanol‐limited and acetate‐limited chemostat cultures and during a glucose pulse added to a glucose‐limited steady‐state culture. Enzyme activity levels of the pyruvate dehydrogenase complex, pyruvate decarboxylase, alcohol dehydrogenase, acetyl‐CoA synthetase and glucose‐6‐phosphate dehydrogenase were determined in all steady‐state cultures. In addition, the mRNA levels of KlADH1‐4, KlACS1, KlACS2, KlPDA1, KlPDC1 and RAG1 were monitored under steady‐state conditions and during glucose pulses. In K. lactis, as in Saccharomyces cerevisiae, enzymes involved in glucose utilization (glucose‐6‐phosphate dehydrogenase, pyruvate dehydrogenase, pyruvate decarboxylase) showed the highest expression levels on glucose, whereas enzymes required for ethanol or acetate consumption (alcohol dehydrogenase, acetyl‐CoA synthetase) showed the highest enzyme activities on ethanol. In cases where mRNA levels were determined, these corresponded well with the corresponding enzyme activities, suggesting that regulation is mostly achieved at the transcriptional level. Surprisingly, the activity of the K. lactis pyruvate dehydrogenase complex appeared to be regulated at the level of KlPDA1 transcription. The conclusions from the steady‐state cultures were corroborated by glucose pulse experiments. Overall, expression of the enzymes of pyruvate metabolism in the Crabtree‐negative yeast K. lactis appeared to be regulated in the same way as in Crabtree‐positive S. cerevisiae, with one notable exception: the PDA1 gene encoding the E1α subunit of the pyruvate dehydrogenase complex is expressed constitutively in S. cerevisiae. Copyright

Inactivation of the Kluyveromyces lactis KIPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation

The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.

The acetyl co‐enzyme A synthetase genes of Kluyveromyces lactis

Two Kluyveromyces lactis genes encoding acetyl co‐enzyme A synthetase isoenzymes were isolated. One we named KlACS1, as it has high similarity to the ACS1 gene of Saccharomyces cerevisiae. The other gene, KlACS2, showed more similarity to S. cerevisiae ACS2 than to KlACS1 or ScACS1. This suggests that divergence of the two isogenes occurred before the evolutionary separation of the species and that the different functions have been conserved. In line with this idea is the regulation of transcription of the genes. The mode of regulation appeared to be maintained between ScACS1 and KlACS1 and between ScACS2 and KlACS2. The KlACS1 transcript was absent in glucose‐grown cells, whereas transcription levels in ethanol‐ and acetate‐grown cells were high. Disruption of the KlACS1 gene did not result in growth defects on glucose or ethanol. The growth rate on acetate, however, was reduced by a factor of two. KlACS2 was expressed at similar levels during growth on glucose and acetate, whereas expression on ethanol was slightly higher. A null mutant in this gene showed a reduced growth rate on all three carbon sources. Taken together, these data suggest that KlACS2 is used during growth on glucose and that KlACS1 is most dominant during growth on acetate. Strains in which both ACS genes are deleted could only be retrieved when a plasmid containing the ACS2 gene was present, suggesting that the double mutant is lethal. Tetrad analysis confirmed that non‐viable spores with a deduced Klacs1Klacs2 genotype germinated but could not divide further. It therefore appears that, as in S. cerevisiae, the pyruvate dehydrogenase bypass formed by the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl co‐enzyme A synthetase is essential for growth. These results are in apparent contradiction with the growth on glucose of a strain with a disruption in the only structural pyruvate decarboxylase gene of K. lactis. Residual enzyme activity might, however, account for this discrepancy, or Acs fulfils an additional as yet unknown function, separate from its enzymatic activity. The sequences of KlACS1 and KlACS2 have been deposited in the EMBL database under Accession Nos AF061265 and AF134491, respectively. Copyright

Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii

A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl‐CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210–219) and RVDDVVNVSG (ScAcs1p; 574–583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl‐CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1–0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid‐grown cells. The nucleotide sequence of a common 5269 bp fragment has been deposited in the EMBL Data Library under Accession No. AJ314837. Copyright