Department of Microbiology

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Assessing the occurrence and mechanisms of horizontal gene transfer during wine making
(2009-12) Barnard, Desire; Bauer, Florian; Wolfaardt, Gideon M.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Saccharomyces cerevisiae is the most commonly used organism in many fermentation-based industries including baking and the production of single cell proteins, biofuel and alcoholic beverages. In the wine industry, a consumer driven demand for new and improved products has focussed yeast research on developing strains with new qualities. Tremendous progress in the understanding of yeast genetics has promoted the development of yeast biotechnology and subsequently of genetically modified (GM) wine yeast strains. The potential benefits of such GM wine yeast are numerous, benefitting both wine makers and consumers. However, the safety considerations require intense evaluation before launching such strains into commercial production. Such assessments consider the possibility of the transfer of newly engineered DNA from the originally modified host to an unrelated organism. This process of horizontal gene transfer (HGT) creates a potential hazard in the use of such organisms. Although HGT has been extensively studied within the prokaryotic domain, there is an urgent need for similar studies on their eukaryotic counterparts. This study was therefore undertaken to help improve our understanding of this issue by investigating HGT in a model eukaryotic organism through a step-by-step approach. In a first step, this study attempted to determine whether large DNA fragments are released from fermenting wine yeast strains and, in a second step, to assess the stability of released DNA within such a fermenting background. The third step investigated in this study was to establish whether “free floating” DNA within this fermenting environment could be accepted and functionally expressed by the fermenting yeast cultures. Finally, whole plasmid transfer was also investigated as a unified event. Biofilms were also incorporated into this study as they constitute a possibly conducive environment for the observation of such HGT events. The results obtained during this study help to answer most of the above questions. Firstly, during an investigation into the possible release of large DNA fragments (>500 bp) from a GM commercial wine yeast strain (Parental strain: Vin13), no DNA could be detected within the fermenting background, suggesting that such DNA fragments were not released in large numbers. Secondly, the study revealed remarkable stability of free “floating DNA” under these fermentation conditions, identifying intact DNA of up to ~1kb in fermenting media for up to 62 days after it had been added. Thirdly, the data demonstrate the uptake and functional expression of spiked DNA by fermenting Vin13 cultures in grape must. Here, another interesting discovery was made, since it appears that the fermenting natural grape must favours DNA uptake when compared to synthetic must, suggesting the presence of carrier molecules. Additionally, we found that spiked plasmid DNA was not maintained as a circular unit, but that only the antibiotic resistance marker was maintained through genomic integration. Identification of the sites of integration showed the sites varied from one HGT event to the next, indicating that integration occurred through a process known as illegitimate recombination. Finally, we provide evidence for the direct transfer of whole plasmids between Vin13 strains. The overall outcome of this study is that HGT does indeed occur under the conditions investigated. To our knowledge, this is the first report of direct horizontal DNA transfer between organisms of the same species in eukaryotes. Furthermore, while the occurences of such events appears low in number, it cannot be assumed that HGT will not occur more frequently within an industrial scenario, making industrial scale studies similar to this one paramount before drawing further conclusions.
Carnitine in yeast and filamentous fungi
(Stellenbosch : Stellenbosch University, 2003-12) Swiegers, Jan Hendrik; Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: In the yeast Saccharomyces cerevtstee, two biochemical pathways ensure that activated cytoplasmic or peroxisomal acetyl-groups are made available for mitochondrial energy production when the cells utilise non-fermentable carbon sources. The first pathway is the glyoxylate cycle, where two activated acetyl-groups are incorporated into each cycle, which releases a C4 intermediate. This intermediate is then transported to the mitochondria where it can enter the tricarboxylic acid cycle. The second pathway is the carnitine shuttle. Activated acetyl-groups react with carnitine to form acetylcarnitine, which is then transported to the mitochondria where the acetyl group is transferred. In this study it was shown that the deletion of the glyoxylate cycle specific citrate synthase, encoded by CIT2, results in a strain that is dependent on carnitine for growth on non-fermentable carbon sources. Using a /::"cit2 strain, mutants affected in carnitine-dependent metabolic activities were generated. Complementation of the mutants with a genomic library resulted in the identification of four genes involved in the carnitine shuttle. These include: (i) the mitochondrial and peroxisomal carnitine acetyltransferase, encoded by CAT2; (ii) the outer-mitochondrial carnitine acetyltransferase, encoded by YA T1; (iii) the mitochondrial carnitine translocase, encoded by CRC1; and (iv) a newly identified carnitine acetyltransferase, encoded by YAT2. All three carnitine acetyltransferases are essential in a carnitine-dependent strain. The dependence on exogenous carnitine of the /::"cit2 strain when grown on nonfermentable carbon sources suggested that S. cerevisiae does not biosynthesise carnitine. Measurements using electrospray mass spectrometry confirmed this hypothesis. As a result an investigation was initiated into carnitine biosynthesis in order to genetically engineer a S. cerevisiae strain that could endogenously biosynthesise carnitine. The filamentous fungus, Neurospora crassa, was one of the first organisms used in the seventies to identify the precursor and intermediates of carnitine biosynthesis. However, it was only about twenty years later that the first genes encoding these enzymes where characterised. Carnitine biosynthesis is a four-step process, which starts with trimethyllysine as precursor. Trimethyllysine is converted to hydroxytrimethyllysine by the enzyme trimethyllysine hydroxylase (TMLH). Hydroxytrimethyllysine is cleaved to trimethylamino-butyraldehyde by the hydroxytrimethyllysine aldolase (HTMLA) releasing glycine. Trimethylaminobutyraldehyde is dehydrogenated to trimethylamino-butyrate (y-butyrobetaine) by trimethylamino-butyraldehyde dehydrogenase (TMABA-DH). In the last step, ybutyrobetaine is converted to t-carnltine by y-butyrobetaine hydroxylase (BBH). The N. crassa TMLH homologue was identified in the genome database based on the protein sequence homology of the human TMLH. Due to the high amount of introns predicted for this gene, the cDNA was cloned and subjected to sequencing, which then revealed that the gene indeed had seven introns. Functional expression of the gene in S. cerevisiae and subsequent enzymatic analysis revealed that the gene coded for a TMLH. It was therefore named cbs-1 for "carnitine biosynthesis gene no. 1JJ. Most of the kinetic parameters were similar to that of the human TMLH enzyme. Following this, a genomic copy of the N. crassa BBH homologue was cloned and functionally expressed in S. cerevisiae. Biochemical analysis revealed that the BBH enzyme could biosynthesise L-carnitine from y-butyrobetaine and the gene was named cbs-2. In addition, the gene could rescue the growth defect of the carnitinedependent Scii? strain on non-fermentable carbon sources when y-butyrobetaine was present. This is the first report of an endogenously carnitine biosynthesising strain of S. cerevisiae. The cloning of the remaining two biosynthesis genes presents particular challenges. To date, the HTMLA has not been characterised on the molecular level making the homology-based identification of this protein in N. crassa impossible. Although the TMABA-DH has been characterised molecularly, the protein sequence is conserved for its function as a dehydrogenase and not conserved for its function in carnitine biosynthesis, as in the case of TMLH and BBH. The reason for this is probably due to the fact that the enzyme is involved in other metabolic processes. The use of N. crassa carnitine biosynthesis mutants would probably be one way in which to overcome these obstacles. The !1cit2 mutant proved useful in studying carnitine related metabolism. We therefore searched for suppressors of !1cit2, which resulted in the cloning of RAS2. In S. cerevisiae, two genes encode Ras proteins, RAS1 and RAS2. GTP-bound Ras proteins activate adenylate cyclase, Cyr1 p, which results in elevated cAMP levels. The cAMP molecules bind to the regulatory subunit of the cAMP-dependent kinase (PKA), Bcy1 p, thereby releasing the catalytic subunits Tpk1 p, Tpk2p and Tpk3p. The catalytic subunits phosphorylate a variety of regulators and enzymes involved in metabolism. Overexpression of RAS2 could suppress the growth defect of the Sclt? mutant on glycerol. In general, overexpression of RAS2 enhanced the proliferation of wild-type cells grown on glycerol. However, the enhancement of proliferation was much better for the !1cit2 strain grown on glycerol. In this respect, the retrograde response may play a role. Overexpression of RAS2 resulted in elevated levels of intracellular citrate and citrate synthase activity. It therefore appears that the suppression of !1cit2 by RAS2 overexpression is a result of the general upregulation of the respiratory capacity and possible leakage of citrate and/or citrate synthase from the mitochondria. The phenotype of RAS2 overexpression contrasts with the hyperactive RAS2val19 allele, which causes a growth defect on glycerol. However, both RAS2 overexpression and RAS2val19activate the cAMP/PKA pathway, but the RAS2val19dependent activation is more severe. Finally, this study implicated the Ras/cAMP/PKA pathway in the proliferation effect on glycerol by showing that in a Mpk1 strain, the growth effect is blocked. However, the enhanced proliferation was still observed in the Mpk2 and Mpk3 strains when RAS2 was overexpressed. Therefore, it seems that Tpk1 p plays an important role in growth on non-fermentable carbon sources, a notion that is supported by the literature.
The cloning of genes involved in carnitine-dependent activities in Saccharomyces cerevisiae
(Stellenbosch : Stellenbosch University, 2000-03) Swiegers, Jan Hendrik; Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of Science . Dept. of Microbiology.
ENGLISH ABSTRACT: L-Carnitine is a unique and important compound in eukaryotic cells. In Saccharomyces cerevisiae, L-carnitine plays a role in the transfer of acetyl groups from the peroxisomes to the mitochondria. This takes place with the help of the carnitine acetylcarnitine shuttle. The activated acyl group of acetyl-CoA in the peroxisome is transferred to carnitine with the help of a peroxisomal carnitine acetyltransferase to form an acetylcarnitine ester, releasing the CoA-SH. This ester is then transported through the peroxisomal membrane to the cytosol from where it is transported to the mitochondrion. After transport of the acetylcarnitine through the mitochondrial membranes, the reverse reaction takes place in the matrix with the help of a mitochondrial carnitine acetyltransferase, releasing carnitine and the acyl group. In S. cerevisiae, the main carnitine acetyltransferase contributing to >95% of the total carnitine acetyltransferase activity, is encoded by a single gene, CAT2. Cat2p has a peroxisomal and mitochondrial targeting signal and is located to the peroxisomal membrane and the inner-mitochondrial membrane, respectively. The reason for the activated acyl group to be transferred in the form of an acetylcarnitine, is that the peroxisomal membrane is impermeable to acetyl-CoA. This means that the acyl group needs to be transported in the form of intermediate compounds. Acetyl-CoA is formed in the peroxisome of S. cerevisiae as a result of p-oxidation of fatty acids. In yeast, the peroxisome is the sole site for p-oxidation. Fatty acids are transported to the peroxisome where they are oxidized by the p-oxidation cycle to form two-carbon acyl groups in the form of acetyl-CoA. These two-carbon acyl groups are then transferred from the peroxisome to the rest of the cell for gluconeogenesis and other anabolic pathways, or used in the tricarboxylic acid cycle (TCA) of the mitochondia to generate ATP. In this way, it is possible for the cell to use fatty acid as a sole carbon source. There is a second pathway allowing for the utilization of activated acyl groups produced in the peroxisome and that is the glyoxylate cycle. The glyoxylate cycle is a modified TCA cycle, which results in the synthesis of C4 succinate from two molecules of acetyl-CoA. In S. cerevisiae, all of the enzymes of the glyoxylate cycle are located in the peroxisome except for one, whereas in other yeasts studied, all of the glyoxylate enzymes are peroxisomal. As a result of the glyoxylate cycle, the two carbons of acetyl-CoA can leave the peroxisome in the form of succinate or other TCA intermediates like malate and citrate. These compounds are transferred through dicarboxylic acid carriers present in the peroxisomal membrane and used in further metabolic needs of the cell. To understand the role of carnitine in the cell, a strategy for the cloning of genes involved in carnitine-dependent activities in S. cerevisiae was developed. The disruption of the citrate synthetase gene, CIT2, of the glyoxylate cycle yielded a strain that was dependent on carnitine when grown on the fatty acid oleic acid. This allowed for a mutagenesis strategy based on negative selection of mutants affected in carnitine-dependent activities. The ~cit2 strain was mutagenized and plated on minimal media. After replica plating on oleic acid media, mutant strains were selected that were unable to grow even in the presence of carnitine. In order to eliminate strains with defects in peroxisome biogenesis and ~-oxidation, and only select for strains with defects in carnitine-dependent activities, the mutant strains were transformed with the CIT2 gene to restore the glyoxylate cycle. Mutants that grew on oleic acid after transformation, and which are therefore not affected in activities independent of carnitine, were retained for further analysis. Transforming one of these mutants with a S. cerevisiae genomic library for functional complementation, yielded a clone carrying the YAT1 gene, coding for the carnitine acetyltransferase of the outer-mitochondrial membrane. No phenotype had previously been assigned to a mutant allele of this gene.
Genetic analysis of a signal transduction pathway : the regulation of invasive growth and starch degradation in Saccharomyces cerevisiae
(Stellenbosch : Stellenbosch University, 2004-03) Van Dyk, Dewald, 1975-; Pretorius, I. S.; Bauer, Florian; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Cells of the yeast Saccharomyces cerevisiae are able to change their morphological appearance in response to a variety of extracellular and intracellular signals. The processes involved in morphogenesis are well characterised in this organism, but the exact mechanism by which information emanating from the environment is integrated into the regulation of the actin cytoskeleton and the yeast cell cycle, is still not clearly understood. Considerable progress has, however, been made. The processes are investigated on various levels including: (i) the nature of the signals required to elicit a morphological adaptation, (ii) the mechanism by which these signals are perceived and transmitted to the nucleus for gene transcription regulation (signal transduction pathways), (iii) the role of the cytoskeleton, particularly actin, in morphogenesis, and (iv) the relationship between cell cycle regulators and factors required for alterations in cellular shape. The focus of this study was on elements involved in the regulation of one of these morphological processes, pseudohyphal formation, in S. cerevisiae. During pseudohyphal differentiation normal oval yeast cells become elongated and mother and daughter cells stay attached after cytokinesis to give rise to filaments. These filaments are able to penetrate the growth substrate, a phenomenon referred to as invasive growth. Actin remodelling is a prerequisite for the formation of elongated cells during pseudohyphal development and invasive growth. Its main contribution to this event is the directing of vesicles, containing cell wall constituents and enzymes, to specific sites of cell wall growth at the cell periphery. In order to fulfil this cellular function, actin is regulated on several levels. Signal transduction pathways that are activated in response to external nutritional signals play important roles in the regulation of the actin cytoskeleton during pseudohyphal differentiation. For this reason a literature review was compiled to introduce various aspects of actin-structure, the regulation of this structure and the functions actin performs during morphogenesis. The connection between signal transduction elements involved in morphological processes and actin remodelling is also reviewed. This study entailed the genetic analysis of numerous factors involved in the regulation of pseudohyphal differentiation, invasive growth and starch metabolism. Several transcriptional regulators playing a role in these phenomena were investigated. Apart from the transcription factors, which include Mss11p, Msn1p, Ste12p, F108p,Phd1p and Tec1p, additional elements ranging from transporters to G-proteins, were also investigated. Mutant strains deleted for one or more of these factors were constructed and tested to assess their abilities to form filaments that penetrate the growth substrate, and to utilise starch as a carbon source. Complex genetic relationships were observed for various combinations of these factors. Specifically, F108p,Msn1p and Ste12p were shown to act independently in controlling invasive growth and starch metabolism, suggesting that these factors are regulated by different signal transduction pathways. Mss11p, on the other hand, was found to play an indispensable role and seems to act as a downstream factor of Msn1 p, Fl08p, Ste12p and Tec1 p. The exception to this is Phd1 p, since multiple copies of PHD1 partially suppress the effect of a MSS11 deletion. The data suggests that Mss11 p functions at the confluence of several signalling pathways controlling the transcriptional regulation of genes required for invasive growth and starch degradation. Different nutritional signals were also found to differentially regulate specific signalling elements during the invasive growth response. For example, Tec1 p requires Msn1 p activity in response to growth on media containing a limited nitrogen source. This dependency, however, was absent when invasive growth was tested on glucose and starch media. Evidence was also obtained that confirmed the transcriptional co-regulation of MUC1 and STA2. MUC1 encodes a mucin-like protein that is required for invasive growth and pseudohyphal differentiation, whereas STA2 encodes a glucoamylase required for starch degradation. Unpublished results indicated that several transcriptional regulators of invasive growth also exert an effect on starch metabolism. The data generated during this study complemented and confirmed published results. It also contributed to the compilation of a more detailed model, integrating the numerous factors involved in these signalling processes.
The molecular characterisation of Mss11p, a transcriptional activator of the Saccharomyces cerevisiae MUC1 and STA1-3 genes
(Stellenbosch : Stellenbosch University, 2002-03) Gagiano, Marco, 1971-; Pretorius, I. S.; Bauer, Florian; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Upon nutrient limitation, normal cells of the budding yeast, Saccharomyces cerevisiae, undergo a transition from ovoid cells that bud in an axial (haploid) or bipolar (diploid) fashion to elongated cells that bud in a unipolar fashion. The daughter cells stay attached to the mother cells, resulting in chains of cells referred to as pseudohyphae. These filaments can grow invasively into the growth substrate (haploid), or away from the colony (diploid), and are hypothesised to be an adaptation of yeast cells that enables them to search for nutrientrich substrates. This filamentous growth response to nutrient limitation was shown to be dependent on the expression of, amongst others, the MUC1 gene. MUC1 (also known as FL011) encodes a large, cell wall-associated, GPI-anchored threonine/serine-rich protein that bears structural resemblance to mammalian mucins and to the yeast flocculins. Deletion and overexpression studies demonstrated that it is critical for pseudohyphal differentiation and invasive growth, and that overexpression of the gene also results in strongly flocculating yeast strains. The upstream regulatory region of MUC1 comprises the largest yeast promoter identified to date and areas as far as 2.4 kb upstream of the translational start site have been shown to confer regulation on MUC1 expression. The large promoter region is not unique to MUC1, however, since it is almost identical to that of the functionally unrelated STA2 gene. The STA2 gene, as well as the identical STA1 and STA3 genes, encodes extracellular glucoamylase isozymes that enable the yeast cell to utilise starch as a carbon source. Glucoamylases liberate glucose residues from the non-reducing end of the starch molecule, thereby making it accessible to yeast cells. The high identity between the promoters of MUC1 and STA1-3 suggests that the two genes are co-regulated. In addition, several transcription factors that regulate the transcriptional levels of both MUC1 and STA2 have been identified and include Msn1p and the previously uncharacterised Mss11p. Overexpression of either Msn1p or Mss11p results in elevated levels of MUC1 and STA2 transcription and a dramatic increase in flocculation, invasive growth, pseudohyphal differentiation and the ability to utilise starch, suggesting that the two genes are indeed co-regulated. The main objective of this study was to characterise Mss11p and its role in the co-regulation of MUC1 and STA2 (as a representative member of the STA gene family). A detailed expression analysis, using Northern blots and Lacl reporter gene expression studies in different media, confirmed that these genes are indeed co-regulated to a large extent. MUC1 and STA2 are also regulated by the same transcriptional regulators, which include not only Msn1pand Mss11p, but also Ste12p, the transcription factor of the mating pheromone/filamentous growth signalling cascade, and Flo8p, a transcriptional activator of the flocculation genes. Overexpression of the genes encoding these factors results in elevated expression levels of both MUC1 and STA2 in most nutritional conditions and enhances the filamentous growth phenotypes of the strain, as well as the ability to degrade starch. On the other hand, the deletion thereof results in severe reductions in the transcription levels of MUC1 and STA2, with equally severe reductions in filamentous growth and the ability to hydrolyse starch. These expression studies also showed that the repressive effect of STA10, a previously uncharacterised negative regulator of STA2, is actually a phenotype conferred by a FLOB mutation in some laboratory strains of S. cerevisiae. The upstream regulatory regions of MUC1 and STA2 are the largest promoters in the yeast genome. By sequencing the upstream areas of STA2 and STA3 and comparing them to the sequence of MUC 1, it was shown that these upstream areas are 99.7%identical over more than 3 900 base pairs (bp) upstream of the translational start. With the exception of a few minor substitutions, the only significant difference between the MUC1 and STA2 promoters is the presence of a 20-bp and a 64-bp sequence found in the MUC1 promoter, but not in the promoters of any of the STA1-3 genes. Through a promoter-deletion analysis, it was shown that Mss11p, Msn1pand Flo8p exert their control over the transcription of MUC1 and STA2 from an 90-bp sequence located at -1 160 to -1 070 in the STA2 and -1 210 to -1 130 in the MUC1 promoters. This sequence also mediates the effect of carbon catabolite repression on the transcription of STA2 and MUC1. Despite the similarities in the expression patterns of MUC1 and STA2, some discrepancies also exist. The most significant difference is that, in wild-type cells and under all nutritional conditions tested, MUC1 transcription is reduced significantly if compared to the transcription levels of STA2. This was attributed to the presence of the 20- and 64-bp sequences, that are present in the promoter region of MUC1, but absent from that of STA2. To place the transcriptional regulators of MUC1 and STA2 in the context of known signal transduction pathways, an epistasis analysis was conducted between MSN1, MSS11 and components of the mating pheromone/filamentous response MAPkinase cascade and cAMPPKA pathway that were shown to be required for the filamentous growth response. This analysis revealed that Msn1p functions in a third, as yet uncharacterised, signal transduction pathway, also downstream of Ras2p,but independent of the two identified pathways, i.e. the cAMP-PKA and pheromone response/filamentous growth response MAP kinase pathways. However, Mss11p seems to function downstream of all three the identified pathways. This suggestsa critical and central role for Mss11p in determining the transcription levels of MUC1 and STA2. To further characterise Mss11p and its role in the transcriptional regulation of MUC1 and STA2, it was also subjected to a detailed deletion and mutation analysis. Mss11p was shown to harbour two distinct activation domains required for the activation of MUC1 and STA2, but also able to activate a reporter gene expressed from under the GALl promoter. The more prominent of the activation domains of Mss11p was shown to be one of the domains with homology to Flo8p, designated H2. The H2 domain has significant homology to a number of proteins of unknown function from a range of different organisms. A multi-sequence alignment allowed the identification of conserved amino acids in this domain. Mutations in two of the four conserved amino acid pairs in the H2 domain completely eliminated the activation function of Mss11p. The poly-glutamine and poly-asparagine domains of Mss11p are not required for its activation function. The deletion of these domains has no impact on the ability of Mss11p to activate MUC1 or STA2 or of the Gal4p-Mss11p fusion to activate the lacl reporter gene expressed from under the GAL7 promoter. Gal4p fusions of either of these domains were also unable to trans-activate the PGAL7-lacl reporter gene. As such, it was concluded that neither of these domains performs a function in the role of Mss11p as a transcriptional activator. We also demonstrated that the putative ATP/GTP-binding domain (P-loop) is not required for the transcriptional activation function of Mss11p. In an attempt to identify other target genes of Mss11p, the use of micro-arrays was employed to assessthe impact of the overexpression and deletion of MSS11 on the total yeast transcriptome. These results showed that MUC1 and STA2 are the only two genes in the ISP15 genetic background that are significantly (more than 15-fold) enhanced by the overexpression of MSS11. Mss11p therefore seemsto playa very specific or dedicated role in MUC1 and STA2 transcription. This analysis also identified several genes (DBP2, ROM2, YPLOBOC, YGR053C, YNL179C, YGR066C) that are repressed by overexpression of MSS11 and activated when MSS11 is deleted. To integrate all the results, three possible models for the activation of MUC1 and STA2 transcription by Mss11p are proposed: (i) Mss11p performs the role of a transcriptional mediator, possibly in a protein complex, to convey information from upstream regulatory elements to the transcription machinery assembledat the core promoters of MUC1 and STA2; (ii) Mss11p plays a more direct role in transcriptional activation, possibly as a transcription factor itself; and (iii) Mss11p facilitates transcription of the MUC1 and STA2 promoters as part of a larger complex that removes or releases the chromatin barrier over the MUC1 and STA2 promoters in responseto specific nutritional signals.
A new role for carnitine in yeasts
(Stellenbosch : Stellenbosch University, 2006-04) Font-Sala, Candide; Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: L-Carnitine (3-0H-4-N-trimethylaminobutanoic acid), also called vitamin Br, is required for the metabolism of fatty acids. Only one specific metabolic activity has been ascribed to L-carnitine in eukaryotic organisms, the transfer of activated acyl residues. In the case of yeast, this process involves the transfer of activated acetyl residues from the peroxisomes or the cytoplasm to the mitochondria. In Saccharomyces cerevisiae, 13-oxidation of fatty acids takes place exclusively in the peroxisomes. The process generates peroxisomal acetyl-CoA, and the activated acetyl-residue has to be transferred to the mitochondria for energy production. Acetyl-CoA and other acyl-CoAs however can not be transferred across intracellular membranes. The activated acetyl residue is therefore transferred to a molecule of carnitine to form acetyl carnitine, which can be shuttled across membranes. The reverse reaction, the transfer of the activated acetyl to free CoA-SH and the liberation of carnitine takes place in the mitochondria. This process is also referred to as the carnitine shuttle. Most organisms, including some yeast, fungi, plants and all mammals, but not S. cerevisiae, can synthesize carnitine from lysine and S-adenosyl-methionine. However, in humans, carnitine synthesis is insufficient to satisfy carnitine requirements, and dietary contributions are essential. Various diseases linked to carnitine deficiencies have been described. Such deficiencies include those found in neonates who, in the absence of carnitine, are unable to assimilate fatty acids from milk, or genetically inborn errors of metabolism, frequently linked to a defective transport of carnitine into cells. More recent literature suggests that carnitine supplementation can have beneficial effects in a number of pathologies, and can also provide some protection against diabetes and liver disease. It has furthermore been suggested that carnitine can contribute to slowing brain aging and to improve conditions of patients suffering from neurodegenerative diseases such as Alzheimer's disease. The accumulation of such data may suggest that carnitine plays additional, as yet unrecognized roles in cellular physiology. In the study reported here, the yeast S. cerevisiae was used to identify possible additional roles for carnitine in cellular metabolism. The study furthermore attempted to identify genes that may be associated with such additional roles. The data show that carnitine supplementation of the growth substrate can protect yeast cells from hyper osmotic and high temperature stress. These protective effects are independent of the metabolic role of carnitine, since deletion of genes that are essential for the carnitine shuttle does not reduce the protective effect. The investigation also suggests that there are no other metabolic roles for carnitine in yeast than the carnitine shuttle, and that it therefore may act as a compatible solute in osmo-protection. The data also indicate a role for PH087, previously identified as a low affinity inorganic phosphate carrier, in the protective effect of carnitine. PH087 overexpression strains accumulate higher concentrations of carnitine, whereas pho87t:. strains contain less carnitine than the corresponding wild type strain. The data therefore suggest either a direct or a regulatory role of the protein in carnitine uptake.

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