Selection of Streptococcus lactis Mutants Defective in Malolactic Fermentation

An enrichment medium and a new sensitive medium were developed to detect malolactic variants in different strains of lactic bacteria. Factors such as the concentration of glucose and l-malate, pH level, and the type of indicator dye used are discussed with regard to the kinetics of malic acid conversion to lactic acid. Use of these media allowed a rapid and easier screening of mutagenized streptococcal cells unable to ferment l-malate. A collection of malolactic-negative mutants of Streptococcus lactis induced by UV, nitrosoguanidine, or transposonal mutagenesis were characterized. The results showed that several mutants were apparently defective in the structural gene of malolactic enzyme, whereas others contained mutations which may either inactivate a putative permease or affect a regulatory sequence.     

Chromatin structures of Kluyveromyces lactis centromeres in K. lactis and Saccharomyces cerevisiae

We have investigated the chromatin structure of Kluyveromyces lactis centromeres in isolated nuclei of K. lactis and Saccharomyces cerevisiae by using micrococcal nuclease and DNAse I digestion. The protected region found in K. lactis is approximately 270 bp long and encompasses the centromeric DNA elements, KlCDEI, KlCDEII, and KlCDEIII, but not KlCDE0. Halving KlCDEII to 82 bp impaired centromere function and led to a smaller protected structure (210 bp). Likewise, deletion of 5 bp from KlCDEI plus adjacent flanking sequences resulted in a smaller protected region and a decrease in centromere function. The chromatin structures of KlCEN2 and KlCEN4 present on plasmids were found to be similar to the structures of the corresponding centromeres in their chromosomal context. A different protection pattern of KlCEN2 was detected in S. cerevisiae, suggesting that KlCEN2 is not properly recognized by at least one of the centromere binding proteins of S. cerevisiae. The difference is mainly found at the KlCDEIII side of the structure. This suggests that one of the components of the ScCBF3-complex is not able to bind to KlCDEIII, which could explain the species specificity of K. lactis and S. cerevisiae centromeres.     

Heterofermentative Carbohydrate Metabolism of Lactose-Impaired Mutants of Streptococcus lactis

Two mutants of Streptococcus lactis ATCC 11454 have been isolated which possess an impaired lactose-fermenting capacity; galactose utilization is also affected, but to a lesser extent. Although the Embden-Meyerhof-Parnas pathway is the major, if not the sole, pathway of carbohydrate metabolism in the three strains, the fermentation end products of the mutants are dramatically different from the typical homolactic pattern of the wild type. Under conditions of low oxygen tension and growth-limiting lactose concentrations, mutant strain T-1 produces largely formic acid, acetic acid (2:1), and ethanol rather than lactic acid. Aerated cultures produce acetic acid, CO(2) (1:1), acetyl-methylcarbinol, and diacetyl. When the mutants use galactose as an energy source, lactic acid is the major end product, but significant heterofermentative activity is observed. The aberrations responsible for the mutant phenotypes reside in the proteins which catalyze the transport and hydrolysis of galactosides. It is hypothesized that the impaired transport system of the mutants reduces the intracellular pool of glycolytic intermediates below that of the wild type. Since fructose-1, 6-diphosphate is an activator of lactic dehydrogenase in S. lactis, lactic acid production is reduced, and pathways leading to the formation of other products are expressed.     

Glucose-induced Crypticity Toward Succinate Metabolism in Saccharomyces lactis

Saccharomyces lactis grown on glucose adapted very slowly to growth on succinate. This initial inability of glucose-grown cells to grow on succinate was paralleled by their inability to oxidize succinate. The possibility that repression by glucose of respiratory chain components was responsible for these observations was examined. Glucose-grown cells were able to respire glucose, ethyl alcohol, and lactate and were able to initiate growth on ethyl alcohol as rapidly as succinate-grown cells. Respiratory enzyme levels were essentially the same in cells grown on succinate or on glucose. Spectroscopic analysis revealed that glucose-grown cells possessed a full complement of cytochrome bands. Since by these criteria glucose-grown S. lactis appears to possess a competent respiratory system, the penetration of succinate-2,3-(14)C into succinate- and glucose-grown cells was examined directly. Glucose-grown cells exhibited a strong permeability barrier to succinate. Comparison of glucose oxidation by S. lactis and by S. cerevisiae suggests that the crypticity to succinate does not depend upon a strong Crabtree effect in S. lactis.     

Glucose Metabolism in gcr Mutants of Saccharomyces cerevisiae

A gcr2 null mutant of Saccharomyces cerevisiae grows well on glucose in spite of its lower level of glycolytic enzymes between triose phosphates and pyruvate. A quantitative analysis shows that these levels are adequate to the flux but glycerate phosphates are elevated.     

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase

Spontaneous and ethyl methanesulfate induced mutants of Saccharomyces cerevisiae, with partial and complete deficiency of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), were isolated by selection for resistance to 8-azaadenine. Matings between totally deficient mutants and tester strain resulted in diploid heterozygotes that were sensitive to azaadenine. Upon sporulation and tetrad analysis, azaadenine resistance (and APRT deficiency) segregated as expected for a single Mendelian gene. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity in the mutants was similar to that in the wild-type cells. There was no detectable activity of adenine aminohydrolase (EC 3.5.4.2) in the wild-type or mutant cells.     

Mitotic Chromosome Transmission Fidelity Mutants in Saccharomyces Cerevisiae

We have isolated 136 independent mutations in haploid yeast strains that exhibit decreased chromosome transmission fidelity in mitosis. Eighty-five percent of the mutations are recessive and 15% are partially dominant. Complementation analysis between MATa and MAT alpha isolates identifies 11 chromosome transmission fidelity (CTF) complementation groups, the largest of which is identical to CHL1. For 49 independent mutations, no corresponding allele has been recovered in the opposite mating type. The initial screen monitored the stability of a centromere-linked color marker on a nonessential yeast chromosome fragment; the mitotic inheritance of natural yeast chromosome III is also affected by the ctf mutations. Of the 136 isolates identified, seven were inviable at 37 degrees and five were inviable at 11 degrees. In all cases tested, these temperature conditional lethalities cosegregated with the chromosome instability phenotype. Five additional complementation groups (ctf12 through ctf16) have been defined by complementation analysis of the mutations causing inviability at 37 degrees. Twenty-three of the 136 isolates exhibited growth defects at concentrations of benomyl permissive for the parent strain, and nine appeared to be tolerant of inhibitory levels of benomyl. All of the mutant strains showed normal sensitivity to ultraviolet and gamma-irradiation. Further characterization of these mutant strains will describe the functions of gene products crucial to the successful execution of processes required for aspects of the chromosome cycle that are important for chromosome transmission fidelity in mitosis.     

Fatty Acid Desaturase Mutants of Saccharomyces cerevisiae

Genetic and biochemical analyses were conducted on fatty acid mutants of yeast deficient for Δ⁹-desaturase activity in the production of palmitoleate and oleate. Two genetic loci were observed and two others are inferred; three of these were represented by respiratory-deficient (petite) strains. All strains were incapable of converting palmitate to palmitoleate and stearate to oleate whether the direct precursor or acetate was followed. All strains were capable of acylating both de novoproduced fatty acids and oleate taken up from the medium into phospholipids and neutral lipids. Two revertants were analyzed which differed in their ability to produce palmitoleate and oleate.     

Prion-Dependent Lethality of sup45 Mutants in Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae translation termination factors eRF1 (Sup45) and eRF3 (Sup35) are encoded by the essential genes SUP45 and SUP35 respectively. Heritable aggregation of Sup35 results in formation of the yeast prion [PSI⁺]. It is known that combination of [PSI⁺] with some mutant alleles of the SUP35 or SUP45 genes in one and the same haploid yeast cell causes synthetic lethality. In this study, we perform detailed analysis of synthetic lethality between various sup45 nonsense and missense mutations on one hand, and different variants of [PSI⁺] on the other hand. Synthetic lethality with sup45 mutations was detected for [PSI⁺] variants of different stringencies. Moreover, we demonstrate for the first time that in some combinations, synthetic lethality is dominant and occurs at the postzygotic stage after only a few cell divisions. The tRNA suppressor SUQ5 counteracts the prion-dependent lethality of the nonsense alleles but not of the missense alleles of SUP45, indicating that the lethal effect is due to the depletion of Sup45. Synthetic lethality is also suppressed in the presence of the C-proximal fragment of Sup35 (Sup35C) that lacks the prion domain and cannot be included into the prion aggregates. Remarkably, the production of Sup35C in a sup45 mutant strain is also accompanied by an increase in the Sup45 levels, suggesting that translationally active Sup35 up-regulates Sup45 or protects it from degradation.Key Words: Sup45, Sup35, eRF1, eRF3, amyloid, [PSI⁺], translation termination, Saccharomyces cerevisiae     

Respiratory-deficient,yeastlike mutant of Mucor

Stable mutants of Mucor bacilliformis having lost the ability to grow filamentously and to sporulate occur spontaneously with a frequency of about one in every 3000 colonies. On solid and in liquid medium these mutants have a typical yeastlike morphology and reproduce by budding. The detailed study of one of these mutants shows that the inability to form filaments and spores is accompanied by the loss of cytochrome oxidase activity. This mutant is unable to take up oxygen but has a high level of alcoholic fermentation, which appears to be the major if not the sole source of energy.This investigation was supported by Public Health Service Grant AI 07804 05.     

Characterization of Glycogen-Deficient Glc Mutants of Saccharomyces Cerevisiae

Forty-eight mutants of Saccharomyces cerevisiae with defects in glycogen metabolism were isolated. The mutations defined eight GLC genes, the functions of which were determined. Mutations in three of these genes activate the RAS/cAMP pathway either by impairment of a RAS GTPase-activating protein (GLC1/IRA1 and GLC4/IRA2) or by activating Ras2p (GLC5/RAS2). SNF1 protein kinase (GLC2) was found to be required for normal glycogen levels. Glycogen branching enzyme (GLC3) was found to be required for significant glycogen synthesis. GLC6 was shown to be allelic to CIF1 (and probably FDP1, BYP1 and GGS1), mutations in which were previously found to prevent growth on glucose; this gene is also the same as TPS1, which encodes a subunit of the trehalose-phosphate synthase. Mutations in GLC6 were capable of increasing or decreasing glycogen levels, at least in part via effects on the regulation of glycogen synthase. GLC7 encodes a type 1 protein phosphatase that contributes to the dephosphorylation (and hence activation) of glycogen synthase. GLC8 encodes a homologue of type 1 protein phosphatase inhibitor-2. The genetic map positions of GLC1/IRA1, GLC3, GLC4/IRA2, GLC6/CIF1/TPS1 (and the adjacent VAT2/VMA2), and GLC7 were clarified. From the data on GLC3, there may be a suppression of recombination near the chromosome V centromere, at least in some strains.     

利用一步基因中断法构建克鲁氏乳酸酵母leu2突变体

将克鲁氏乳酸酵母ＬＥＵ2　基因编码区的部分序列用酿酒酵母的ＵＲＡ３基因替换,然后用此段转化两株克鲁氏乳酸酵母。通过体内同源重组,部分缺失的外源leu2　片段取代下了酵母染色体上的正常的ＬＥＵ2基因,由此得到leu2转化子。经过这些转化子在非选择条件下的稳定性测定,没有发现回复子。结果表明,用一步基因中断法成功地建立了稳定的ＬＥＵ2基因突变体。这为在克鲁氏乳酸酵母中构建有效的宿主-载体系统提供了一个有用的营养缺陷型选择标记。Acstract: TheＬＥＵ2 gene of the yeast Kluyveromyces lactis was disrupted by replacing a part of the coding sequence with URA3 gene of the yeast S.cerevisiae. Transformation of two k.lactis strains with the disrupted leu2 fragment resulted in the substitution og partilly deleted LEU2 gene for the with-type LEU2 gene on the chromosome.Thus, two leu2 mutants were generated and no reversion could be detected after prolonged growth in the non-selective medium. The results show that the stable leu2 mutants have been constructed successfully by one-step gene disruption. The isolation of these mutants would provide a useful auxotrophic marker to facilitate the development of an efficient host-vector system in K.lactis.     

Genome-Wide Screen for Oxalate-Sensitive Mutants of Saccharomyces cerevisiae

Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. In order to discover yeast genes whose orthologs in the pathogen may confer self-tolerance and whose plant orthologs may protect the host, a Saccharomyces cerevisiae deletion library consisting of 4,827 haploid mutants harboring deletions in nonessential genes was screened for growth inhibition and survival in a rich medium containing 30 mM oxalic acid at pH 3. A total of 31 mutants were identified that had significantly lower cell yields in oxalate medium than in an oxalate-free medium. About 35% of these mutants had not previously been detected in published screens for sensitivity to sorbic or citric acid. Mutants impaired in endosomal transport, the rgp1Δ, ric1Δ, snf7Δ, vps16Δ, vps20Δ, and vps51Δ mutants, were significantly overrepresented relative to their frequency among all verified yeast open reading frames. Oxalate exposure to a subset of five mutants, the drs2Δ, vps16Δ, vps51Δ, ric1Δ, and rib4Δ mutants, was lethal. With the exception of the rib4Δ mutant, all of these mutants are impaired in vesicle-mediated transport. Indirect evidence is provided suggesting that the sensitivity of the rib4Δ mutant, a riboflavin auxotroph, is due to oxalate-mediated interference with riboflavin uptake by the putative monocarboxylate transporter Mch5.