Ferment Galactose Research Articles

Evaluation of diversity of Candida species isolated from fermented cassava during traditional small scale gari production in Nigeria

The yeast population dynamics during cassava fermentation at different time regimes was determined and the Candida species isolated from the fermented mash were identified using combined phenotypic and genomic methods. The yeast population increases as the fermentation progresses with corresponding pH reduction mediated by lactic acid bacteria and yeast co-metabolism. The phenotypic characteristic of the Candida species isolated from fermented cassava during gari production in Nigeria was determined using the sugar fermentation profile of the API 20AUX that generated the phenotypic identity of Candida species as Candida guilliermondii, Candida krusei, Candida maris, Candida galabrata. Only strain 1RB identified phenotypically as C. guilliermondii fermented all the sugars except inositol and lactose. Although, the strain 2RB identified by the API 20AUX as C. maris fermented galactose, all other strains could only ferment glucose with the presence of pseudohypae. The result of the comparison of the 18S rDNA gene sequencing with the blast database identified the strains of C. guilliermondii 100% C. krusei 98%, Candida inconspicua 100%, Candida rugopelliculosa 98%. The results also demonstrated that different strains of Candida species participated in the traditional fermentation of cassava and differentiated C. krusei from C. inconspicua using the 18S rDNA gene sequencing. C. inconspicua has not been previously reported due to its phenotypic relatedness to C. krusei. C. inconspicua may not be a good candidate to be selected as starter culture due to its medical importance; it must be genetically differentiated from the diverse strains of C. krusei that participated in the fermentation.

Genetic and physiological alterations occurring in a yeast population continuously propagated at increasing temperatures with cell recycling.

This work investigated the effects of increasing temperature from 30°C to 47°C on the physiological and genetic characteristics of Saccharomyces cerevisiae strain 63M after continuous fermentation with cell recycling in a system of five reactors in series. Steady state was attained at 30°C, and then the temperature of the system was raised so it ranged from 35°C in the last reactor to 43°C in the first reactor or feeding reactor with a 2°C difference between reactors. After 15days at steady state, the temperature was raised from 37°C to 45°C for 25days at steady state, then from 39°C to 47°C for 20 days at steady state. Starter strain 63M was a hybrid strain constructed to have a MAT a/α, LYS/lys, URA/ura genotype. This hybrid yeast showed vigorous growth on plates at 40°C, weak growth at 41°C, positive assimilation of melibiose, positive fermentation of galactose, raffinose and sucrose. Of 156 isolates obtained from this system at the end of the fermentation process, only 17.3% showed the same characteristics as starter strain 63M. Alterations in mating type reaction and in utilization of raffinose, melibiose, and sucrose were identified. Only 1.9% of the isolates lost the ability to grow at 40°C. Isolates showing requirements for lysine and uracil were also obtained. In addition, cell survival was observed at 39-47°C, but no isolates showing growth above 41°C were obtained.

Mixed sugar fermentation by Pichia stipitis, Sacharomyces cerevisiaea, and an isolated xylose-fermenting Kluyveromyces marxianus and their cocultures.

A yeast strain with higher rates and yields in the fermentation of glucose, mannose and galactose in semiaerobic conditions than Pichia stipitis and Sacharomyces cerevisiaeand ethanol tolerance than P. stipitis, was isolated from sugarcane baggase from Iranian resources. This strain that can ferment xylose with lower rates and yields than P. stipitisis characterized as Kluyveromyces marxianus. The ability of K. marxianus to ferment mixed sugars comprised of 30 g/l glucose, 30 g/l xylose, 12 g/l mannose and 8 g/l galactose (total sugar 80 g/l), as a model of many hydrolysates, were compared to P. stipitis and S. cerevisiae and then a coculture of P. stipitis and S. cerevisiae was compared with a coculture of P. stipitis and K. marxianus. In mixed sugars fermentation with individual yeasts P. stipitis shows the highest yield (0.40 gg-1) and maximum ethanol (30.23 gl-1), but K. marxianus shows the highest Qpmax (1.09 gl-1h-1) and substrate utilization efficiency (E,>99%). P. stipiti and K. marxianuscoculture shows the best results with high yield (0.42 gg-1) and maximum ethanol (31.87 gl-1), Qpmax (1.09 gl-1h-1) and substrate utilization efficiency (E,>99%). Because of the higher rates and yields of K. marxianus to ferment hexoses than P. stipitis and its higher ethanol tolerance, K. marxianus helps the P. stipitis to reach the concentration higher than 30 g/l ethanol concentration. Key words: Ethanol, Pichia stipitis, Kluyveromyces marxianus, xylose.

Mitochondrial dysfunction enhances Gal4-dependent transcription

Mitochondrial dysfunction has been shown to elicit broad effects on nuclear gene expression. We show here that transcription dependent on the prototypical acidic activator Gal4 is responsive to mitochondrial dysfunction. In cells with no mitochondrial DNA, Gal4-dependent gene expression is elevated. A minimal Gal4 activator containing the DNA binding and activation domain is sufficient for this response. Transcription dependent on a fusion of Gal4 to a heterologous DNA binding domain is similarly elevated in a mitochondrial mutant. Analysis of different Gal4-dependent promoters and gel mobility shift assays suggest that the effect of mitochondrial dysfunction on Gal4 activity is related to increased DNA binding to the cognate Gal4 element. Given that fermentation is the only means to obtain energy in respiratory deficient cells, it is possible that higher Gal4 activity in cells with dysfunctional mitochondria works to promote more efficient fermentation of galactose.

Tetrapisispora namnaonensis sp. nov., a novel ascomycetous yeast species isolated from forest soil of Nam Nao National Park, Thailand

Twenty-one strains of a novel ascomycetous yeast species were isolated from soil collected in three kinds of natural forest, namely a dry dipterocarp forest, a mixed deciduous forest and a pine forest, in Nam Nao National Park, Phetchabun province, Thailand. The strains formed asci containing one to four ovoid to reniform ascospores, assimilated glucose, galactose and glycerol, fermented glucose and galactose vigorously and contained ubiquinone Q-6, indicating that they belonged to the genus Tetrapisispora. A comparative analysis of the small subunit rDNA (SSU rDNA) and the D1/D2 domain of the large subunit rDNA (LSU rDNA) of all available sequences for ascomycetous yeasts confirmed that the strains were phylogenetically related to the genus Tetrapisispora. All strains had identical nucleotide sequences in the D1/D2 domain of the LSU rDNA and differed from the nearest species, Tetrapisispora arboricola IFO 10925(T), by 6.4% nucleotide substitutions. The strains differed from Tetrapisispora arboricola by the ability to assimilate D-gluconic acid, the inability to grow on 50% glucose medium, the nuclear DNA base composition and deliquescent asci. The strains were differentiated from the other four species of Tetrapisispora on the basis of trehalose assimilation, the ability to grow on 50% glucose or 10% NaCl plus 5% glucose, vitamin requirement, the nuclear DNA base composition and the type of ascus. Based on the characteristics mentioned above, the strains are recognized as a single novel species of the genus Tetrapisispora and the name Tetrapisispora namnaonensis sp. nov. is proposed. The type strain is TN1-01(T) (=TISTR 5828(T)=JCM 12664(T)=CBS 10093(T)).

Petrimonas sulfuriphila gen. nov., sp. nov., a mesophilic fermentative bacterium isolated from a biodegraded oil reservoir

A mesophilic, anaerobic, fermentative bacterium, strain BN3(T), was isolated from a producing well of a biodegraded oil reservoir in Canada. Cells were Gram-negative, non-motile rods that did not form spores. The temperature range for growth was 15-40 degrees C, with optimum growth at 37-40 degrees C. The strain grew with up 4 % NaCl, with optimum growth in the absence of NaCl. Tryptone was required for growth. Yeast extract and elemental sulfur stimulated growth. Growth was also enhanced during fermentation of glucose, arabinose, galactose, maltose, mannose, rhamnose, lactose, ribose, fructose, sucrose, cellobiose, lactate, mannitol and glycerol. Acetate, hydrogen and CO(2) were produced during glucose fermentation. Elemental sulfur and nitrate were used as electron acceptors and were reduced to sulfide and ammonium, respectively. The G + C content of the genomic DNA was 40.8 mol%. Phylogenetic analyses of the 16S rRNA gene sequence indicated that the strain was a member of the phylum 'Bacteroidetes', distantly related to the genera Bacteroides and Tannerella (similarity values of less than 90 %). The chemotaxonomic data (fatty acids, polar lipids and quinones composition) also indicated that strain BN3(T) could be clearly distinguished from its closest cultivated relatives. This novel organism possesses phenotypic, chemotaxonomic and phylogenetic traits that do not allow its classification as a member of any previously described genus; therefore, it is proposed that this isolate should be described as a member of a novel species of a new genus, Petrimonas gen. nov., of which Petrimonas sulfuriphila sp. nov. is the type species. The type strain is BN3(T) (= DSM 16547(T) = JCM 12565(T)).

A history of research on yeasts 7: enzymic adaptation and regulation.

Introduction: the scope of this article 704 Dienert’s work on adaptation of yeast to galactose 707 Enzymic adaptation or selection of mutants? 709 Galactose fermentation by yeasts 713 Monod’s work on lactose utilization by Escherichia coli 714 Gratuitous induction 716 ‘Permeases’: transport of metabolites into the cells 718 Carbon catabolite repression 720 The operon 722 The galactose pathway in yeasts: work of Leloir and others 723 Uridine diphosphate sugars 725 Genetic regulation of the galactose pathway 729 Regulation of the GAL genes 730 Genetic regulation of the utilization of other substrates 732 Maltose utilization: the MAL genes 732 Sucrose utilization: invertase and the SUC genes 733 Conclusion 733 References 733 Appendix: sugar structures 743

Sugar Utilisation and Conservation of the gal-lac Gene Cluster in Streptococcus thermophilus

The adaptation to utilise lactose as primary carbon and energy source is a characteristic for Streptococcus thermophilus. These organisms, however only utilise the glucose moiety of lactose while the galactose moiety is excreted into the growth medium. In this study we evaluated the diversity of sugar utilisation and the conservation of the gal-lac gene cluster in a collection of 18 S. thermophilus strains isolated from a variety of sources. For this purpose analysis was performed on DNA from these isolates and the results were compared with those obtained with a strain from which the complete genome sequence has been determined. The sequence, organisation and flanking regions of the S. thermophilus gal-lac gene cluster were found to be highly conserved among all strains. The vast majority of the S. thermophilus strains were able to utilize only glucose, lactose, and sucrose as carbon sources, some strains could also utilize fructose and two of these were able to grow on galactose. Molecular characterisation of these naturally occurring Gal+ strains revealed up-mutations in the galKTE promoter that were absent in all other strains. These data support the hypothesis that the loss of the ability to ferment galactose can be attributed to the low activity of the galKTE promoter, probably as a consequence of the adaptation to milk in which the lactose levels are in excess.

Characterization, expression, and mutation of the Lactococcus lactis galPMKTE genes, involved in galactose utilization via the Leloir pathway.

A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose 1-epimerase (GalM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.

Effect of Sugar Catabolite Repression in Correlation with the Structural Complexity of the Nitrogen Source on Yeast Growth and Fermentation

Biomass and ethanol production by industrial Saccharomyces cerevisiae strains were strongly affected by the structural complexity of the nitrogen source during fermentation in media containing galactose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low galactose concentrations independent of nitrogen supplementation. At high sugar concentrations altered patterns of galactose utilisation were observed. Biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for baking and brewing ale and lager strains. Baking yeast showed improved galactose fermentation performance in the medium supplemented with casamino acids. High biomass production was observed with peptone and casamino acids for the ale brewing strain, however high ethanol production was observed only in the presence of casamino acids. Conversely, peptone was the nitrogen supplement that induced higher biomass and ethanol production for the lager brewing strain. Ammonium salts always induced poor yeast performance. The results with galactose differed from those obtained with glucose and maltose which indicated that supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, suggesting that sugar catabolite repression has a central role in yeast performance in a medium containing nitrogen sources with differing levels of structural complexity.

Genetic diversity and technological properties of Streptococcus thermophilus strains isolated from dairy products

To evaluate the genetic diversity and the technological properties of 44 strains of Streptococcus thermophilus isolated from dairy products. Methods Strains were analysed for some relevant technological properties, i.e. exopolysaccharide (EPS) production, growth kinetic in skim milk medium, urease activity and galactose fermentation. The EPS production, determined by evaluating the colour of the colonies grown in ruthenium red milk agar, was observed in 50% of the analysed strains. Urease activity, determined by colorimetric and conductimetric methods, showed that 91% of the isolates, all except four, could hydrolyse urea. A conductimetric approach was also used for the evaluation of the overall metabolic behaviour in milk of Strep. thermophilus strains and the differences observed allowed grouping of the strains in seven different clusters. A total of 11 strains were able to produce acid in presence of galactose. Genetic diversity of Streptococcus thermophilus strains, evaluated by Random Amplified Polymorphic DNA fingerprinting (RAPD) and amplified epsC-D restriction analysis, allowed the identification of 21 different genotypes. Comparison between the genotypic and phenotypic data highlights an interesting correlation between some important technological properties and well-defined genotypes. The genetic and technological characterization carried out on several Strep. thermophilus strains of dairy origin should expand the knowledge on this important lactic acid bacteria species and lead to a simple, rapid, and reliable identification of strains on the basis of well-defined biotechnological properties.

Identification of Pichia anomala isolated from yoghurt by RFLP of the ITS region

Several packs of swollen retailed plain and flavoured yoghurt were examined. The most commonly found species was Pichia anomala, identified both by physiological tests and RFLP analysis of the rDNA internal transcribed spacer (ITS) region. The isolated strains did not ferment lactose and were positive for galactose fermentation, confirming the hypothesis that galactose-fermenting yeast could be the cause of spoilage in yoghurt.

Reflections on glycobiology.

Reflections on glycobiology.

Activation of silent gal genes in the lac-gal regulon of Streptococcus thermophilus.

Streptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galM gene and the lactose operon lacSZ. Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal(+) mutant NZ302G, the transcription of the galR gene was induced by growth on lactose. Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis of the gal promoter regions of NZ302G and nine other independently isolated Gal(+) mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions -9 and -15 as well as a single-base-pair insertion at position -37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose-negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.

Analysis of yeasts derived from natural fermentation in a Tokaj winery.

The diversity of yeast flora was investigated in a spontaneously fermenting sweet white wine in a Tokaj winery. The non-Saccharomyces yeasts dominating the first phase of fermentation were soon replaced by a heterogeneous Saccharomyces population, which then became dominated by Saccharomyces bayanus. Three Saccharomyces sensu stricto strains isolated from various phases of fermentation were tested for genetic stability, optimum growth temperature, tolerance to sulphur dioxide, copper and ethanol as well as for the ability to produce hydrogen sulphide and various secondary metabolites known to affect the organoleptic properties of wines. The analysis of the single-spore cultures derived from spores of dissected asci revealed high stability of electrophoretic karyotypes and various degrees of heterozygosity for mating-types, the fermentation of galactose and the production of metabolic by-products. The production levels of the by-products did not segregate in a 2:2 fashion, suggesting that the synthesis of these compounds is under polygenic control.

Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae

We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24(s) monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24(s) monomer, is transcribed under the control of the GAL 10p on a chimeric 2-microm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h(-1) to washout (0.143 h(-1)). Both p24(s) monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24(s) monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-microm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h(-1). Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h(-1)), even though HBsAg expression was maximal. Total p24(s) monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h(-1).

Lactobacillus nagelii sp. nov., an organism isolated from a partially fermented wine.

A Gram-positive rod was isolated from a commercial grape wine undergoing a sluggish/stuck alcoholic fermentation. The organism produced DL-lactic acid from glucose without gas formation, produced dextran from sucrose, hydrolysed aesculin and fermented galactose, D-glucose, D-fructose, D-mannose, L-sorbose, rhamnose, mannitol, sorbitol, methyl alpha-D-glucoside, N-acetylglucosamine, amygdalin, salicin, cellobiose, maltose, sucrose, trehalose and beta-gentiobiose. 16S rRNA gene sequence analysis revealed that the isolate was phylogenetically a member of the genus Lactobacillus and formed a distinct subline within the Lactobacillus casei cluster of species. On the basis of phenotypic and phylogenetic evidence, Lactobacillus nagelii sp. nov. ATCC 700692T is proposed as a new species.

Derepression of galactose metabolism in melibiase producing bakers’ and distillers’ yeast

Beet molasses is widely used as a growth substrate for bakers’ and distillers’ yeast in the production of biomass and ethanol. Most commercial yeasts do not fully utilise the carbohydrates in molasses since they are incapable of hydrolysing the disaccharide melibiose to glucose and galactose. Also, expression of genes encoding enzymes for the utilisation of carbon sources that are alternatives to glucose is tightly regulated, sometimes limiting rates of yeast growth and/or ethanol production. The GAL genes are regulated by specific induction by galactose and repression during growth on glucose. In an industrial distillers’ yeast, two genes interacting synergistically in glucose repression of galactose utilisation, MIG1 and GAL80, have been disrupted with MEL1, encoding melibiase. The physiology of the wild-type strain and the recombinant strains was investigated on mixtures of glucose and galactose and on molasses. The recombinant strain started to ferment galactose when 9.7 g l −1 glucose was still present during a batch fermentation, whereas the wild-type strain did not consume any galactose in the presence of glucose. The ethanol yield in the recombinant strain was 0.50 g ethanol g sugar −1 in an ethanol fermentation on molasses, compared with 0.48 g ethanol g sugar −1 for the wild-type strain. The increased ethanol yield was due to utilisation of melibiose in the molasses.

Actinobacillus actinomycetemcomitans serotype e--biotypes, genetic diversity and distribution in relation to periodontal status.

Actinobacillus actinomycetemcomitans isolates from 356 individuals were screened for identification of serotype e in order to investigate its distribution in relation to periodontal status. From subjects with serotype e, 1-6 isolates per subject (n = 61) were genotyped using arbitrarily primed-polymerase chain reaction (AP-PCR) and apaH gene polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis to determine the genetic heterogeneity within the serotype. Furthermore, one serotype e strain per subject was tested for fermentation of 8 carbohydrates for biotyping. Among patients with adult periodontitis (n = 219), localized juvenile periodontitis (n = 55) and other forms of early-onset periodontitis (n = 18) serotypes b, a and c, respectively, were the most frequently detected serotypes. Non-periodontitis subjects (n = 64) were predominantly colonized with serotype c. Serotype e was found in 30 (14%) adult periodontitis patients, 2 (11%) early-onset periodontitis patients and in 5 (8%) non-periodontitis individuals, but in none of the 55 localized juvenile periodontitis patients. AP-PCR distinguished 3 and apaH gene PCR-RFLP analysis 2 genotypes among the 61 A. actinomycetemcomitans serotype e isolates, one genotype per subject. The AP-PCR genotypes 1 and 3 represented the apaH genotype 1 and the AP-PCR genotype 2 the apaH genotype 2. On the basis of variable fermentation of galactose and xylose, 3 biotypes among A. actinomycetemcomitans serotype e were established. Contrary to the absence of A. actinomycetemcomitans serotype e in localized juvenile periodontitis patients, its detection frequency was comparable among other forms of periodontitis and periodontal health. Clinical serotype e isolates form at least 2 genetic types and 3 biotypes.

Haemophilus paragallinarum in chickens in Indonesia : II . Physiological and biochemical properties of Haemophilus spp . from diseased chickens

A total of 46 isolates of Haemophilus spp . consisted of 42 local isolates from diseased chickens and four reference strains of Haemophilus paragallinarum were examined for their physiological and biochemical properties . These isolates consisted of 2 groups. One group consisted of 21 local isolates and reference strains of H. paragallinarum were catalase negative and identified as Haemophilus paragallinarum and they did not ferment galactose and trehalose . The other group was catalase positive and suspected as Haemophilus avium . This catalase positive group of these 21 avian Haemophili should be examined with other carbohydrates in order to be identified accurately . Keywords : Haemophilus spp ., physiological, biochemical, chickens