RESUMO
Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species.IMPORTANCELactobacillus brevis is a member of the lactic acid bacteria and is often reported as the causative agent of food or beverage spoilage, in particular, that of beer. Bacterial spoilage of beer may result in product withdrawal or recall, with concomitant economic losses for the brewing industry. A very limited number of genes involved in beer spoilage have been identified and primarily include those involved in hop resistance, such as horA, hitA, and horC However, since none of these genes are universal, it is clear that there are likely (many) other molecular players involved in beer spoilage. Here, we report on the importance of a plasmid-encoded glycosyltransferase associated with beer spoilage by L. brevis that is involved in hop tolerance. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process.
Assuntos
Proteínas de Bactérias/genética , Cerveja/microbiologia , Glicosiltransferases/genética , Lactobacillales/genética , Levilactobacillus brevis/genética , Plasmídeos/genética , Proteínas de Bactérias/metabolismo , Microbiologia de Alimentos , Glicosiltransferases/metabolismo , Humulus/química , Lactobacillales/enzimologia , Levilactobacillus brevis/enzimologia , Plasmídeos/metabolismoRESUMO
Lactobacillus brevis is a lactic acid bacterium that is known as a food and beverage spoilage organism, and more specifically as a beer-spoiler. Phages of L. brevis have been described, but very limited data is available regarding temperate phages of L. brevis. Temperate phages may exert benefits to the host, while they may also be employed to combat beer spoilage. The current study reports on the incidence of prophage sequences present in nineteen distinct L. brevis genomes. Prophage induction was evaluated using mitomycin C exposure followed by genome targeted-PCR, electron microscopy and structural proteome analysis. The morphological and genome sequence analyses revealed significant diversity among L. brevis prophages, which appear to be dominated by members of the Myoviridae phage family. Based on this analysis, we propose a classification of L. brevis phages into five groups.
RESUMO
Pyruvate and acetyl-coenzyme A, located at the interface between glycolysis and TCA cycle, are important intermediates in yeast metabolism and key precursors for industrially relevant products. Rational engineering of their supply requires knowledge of compensatory reactions that replace predominant pathways when these are inactivated. This study investigates effects of individual and combined mutations that inactivate the mitochondrial pyruvate-dehydrogenase (PDH) complex, extramitochondrial citrate synthase (Cit2) and mitochondrial CoA-transferase (Ach1) in Saccharomyces cerevisiae. Additionally, strains with a constitutively expressed carnitine shuttle were constructed and analyzed. A predominant role of the PDH complex in linking glycolysis and TCA cycle in glucose-grown batch cultures could be functionally replaced by the combined activity of the cytosolic PDH bypass and Cit2. Strongly impaired growth and a high incidence of respiratory deficiency in pda1Δ ach1Δ strains showed that synthesis of intramitochondrial acetyl-CoA as a metabolic precursor requires activity of either the PDH complex or Ach1. Constitutive overexpression of AGP2, HNM1, YAT2, YAT1, CRC1 and CAT2 enabled the carnitine shuttle to efficiently link glycolysis and TCA cycle in l-carnitine-supplemented, glucose-grown batch cultures. Strains in which all known reactions at the glycolysis-TCA cycle interface were inactivated still grew slowly on glucose, indicating additional flexibility at this key metabolic junction.
Assuntos
Ciclo do Ácido Cítrico , Glicólise , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Citrato (si)-Sintase/genética , Coenzima A-Transferases/genética , Deleção de Genes , Expressão Gênica , Engenharia Metabólica , Análise do Fluxo Metabólico , Redes e Vias Metabólicas/genética , Complexo Piruvato Desidrogenase/genética , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Genomics is based on the ability to determine the transcriptome, proteome, and metabolome of a cell. These technologies only have added value when they are integrated and based on robust and reproducible workflows. This chapter describes the experimental design, sampling, sample pretreatment, data evaluation, integration, and interpretation. The actual generation of the data is not covered in this chapter since it is highly depended on available equipment and infrastructure. The enormous amount of data generated by these technologies are integrated and interpreted inorder to generate leads for strain and process improvement. Biostatistics are becoming very important for the whole work flow therefore, some general recommendations how to set up experimental design and how to use biostatistics in enhancing the quality of the data and the selection of biological relevant leads for strain engineering and target identification are described.
Assuntos
Interpretação Estatística de Dados , Fungos/genética , Fungos/metabolismo , Perfilação da Expressão Gênica/métodos , Engenharia Metabólica , Metaboloma , Modelos Estatísticos , Proteoma/genética , Proteoma/metabolismo , Proteômica , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , RNA Fúngico/metabolismo , Biologia de SistemasRESUMO
An essential property of all cells is the ability to exit from active cell division and persist in a quiescent state. For single-celled microbes this primarily occurs in response to nutrient deprivation. We studied the genetic requirements for survival of Saccharomyces cerevisiae when starved for either of two nutrients: phosphate or leucine. We measured the survival of nearly all nonessential haploid null yeast mutants in mixed populations using a quantitative sequencing method that estimates the abundance of each mutant on the basis of frequency of unique molecular barcodes. Starvation for phosphate results in a population half-life of 337 hr whereas starvation for leucine results in a half-life of 27.7 hr. To measure survival of individual mutants in each population we developed a statistical framework that accounts for the multiple sources of experimental variation. From the identities of the genes in which mutations strongly affect survival, we identify genetic evidence for several cellular processes affecting survival during nutrient starvation, including autophagy, chromatin remodeling, mRNA processing, and cytoskeleton function. In addition, we found evidence that mitochondrial and peroxisome function is required for survival. Our experimental and analytical methods represent an efficient and quantitative approach to characterizing genetic functions and networks with unprecedented resolution and identified genotype-by-environment interactions that have important implications for interpretation of studies of aging and quiescence in yeast.
Assuntos
Genes Fúngicos/genética , Leucina/deficiência , Fosfatos/deficiência , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Biologia de Sistemas/métodos , Mutação , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNARESUMO
Microbes tailor their growth rate to nutrient availability. Here, we measured, using liquid chromatography-mass spectrometry, >100 intracellular metabolites in steady-state cultures of Saccharomyces cerevisiae growing at five different rates and in each of five different limiting nutrients. In contrast to gene transcripts, where approximately 25% correlated with growth rate irrespective of the nature of the limiting nutrient, metabolite concentrations were highly sensitive to the limiting nutrient's identity. Nitrogen (ammonium) and carbon (glucose) limitation were characterized by low intracellular amino acid and high nucleotide levels, whereas phosphorus (phosphate) limitation resulted in the converse. Low adenylate energy charge was found selectively in phosphorus limitation, suggesting the energy charge may actually measure phosphorus availability. Particularly strong concentration responses occurred in metabolites closely linked to the limiting nutrient, e.g., glutamine in nitrogen limitation, ATP in phosphorus limitation, and pyruvate in carbon limitation. A simple but physically realistic model involving the availability of these metabolites was adequate to account for cellular growth rate. The complete data can be accessed at the interactive website http://growthrate.princeton.edu/metabolome.
Assuntos
Carbono/farmacologia , Espaço Intracelular/metabolismo , Metaboloma , Nitrogênio/farmacologia , Fósforo/farmacologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Análise por Conglomerados , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Espaço Intracelular/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Modelos Biológicos , Fosfatos/farmacologia , Compostos de Amônio Quaternário/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
Starvation of yeast cultures limited by auxotrophic requirements results in glucose wasting and failure to achieve prompt cell-cycle arrest when compared with starvation for basic natural nutrients like phosphate or sulfate. We measured the survival of yeast auxotrophs upon starvation for different nutrients and found substantial differences: When deprived of leucine or uracil, viability declined exponentially with a half-life of <2 days, whereas when the same strains were deprived of phosphate or sulfate, the half-life was approximately 10 days. The survival rates of nongrowing auxotrophs deprived of uracil or leucine depended on the carbon source available during starvation, but were independent of the carbon source during prior growth. We performed an enrichment procedure for mutants that suppress lethality during auxotrophy starvation. We repeatedly found loss-of-function mutations in a number of functionally related genes. Mutations in PPM1, which methylates protein phosphatase 2A, and target of rapamycin (TOR1) were characterized further. Deletion of PPM1 almost completely suppressed the rapid lethality and substantially suppressed glucose wasting during starvation for leucine or uracil. Suppression by a deletion of TOR1 was less complete. We suggest that, similar to the Warburg effect observed in tumor cells, starving yeast auxotrophs wastes glucose as a consequence of the failure of conserved growth control pathways. Furthermore, we suggest that our results on condition-dependent chronological lifespan have important implications for the interpretation and design of studies on chronological aging.
Assuntos
Viabilidade Microbiana , Fenômenos Fisiológicos da Nutrição , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos , Contagem de Colônia Microbiana , Meios de Cultura , Alimentos , Redes Reguladoras de Genes , Genótipo , Glucose/metabolismo , Leucina/deficiência , Mutação/genética , Fosfatos/deficiência , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/isolamento & purificação , Uracila/metabolismoRESUMO
We studied the relationship between growth rate and genome-wide gene expression, cell cycle progression, and glucose metabolism in 36 steady-state continuous cultures limited by one of six different nutrients (glucose, ammonium, sulfate, phosphate, uracil, or leucine). The expression of more than one quarter of all yeast genes is linearly correlated with growth rate, independent of the limiting nutrient. The subset of negatively growth-correlated genes is most enriched for peroxisomal functions, whereas positively correlated genes mainly encode ribosomal functions. Many (not all) genes associated with stress response are strongly correlated with growth rate, as are genes that are periodically expressed under conditions of metabolic cycling. We confirmed a linear relationship between growth rate and the fraction of the cell population in the G0/G1 cell cycle phase, independent of limiting nutrient. Cultures limited by auxotrophic requirements wasted excess glucose, whereas those limited on phosphate, sulfate, or ammonia did not; this phenomenon (reminiscent of the "Warburg effect" in cancer cells) was confirmed in batch cultures. Using an aggregate of gene expression values, we predict (in both continuous and batch cultures) an "instantaneous growth rate." This concept is useful in interpreting the system-level connections among growth rate, metabolism, stress, and the cell cycle.
Assuntos
Ciclo Celular , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Análise por Conglomerados , Meios de Cultura , Etanol/metabolismo , Alimentos , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Glucose/metabolismo , Modelos Biológicos , Análise de Regressão , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae grown with six different nitrogen sources were subjected to transcriptome analysis. The use of chemostats enabled an analysis of nitrogen-source-dependent transcriptional regulation at a fixed specific growth rate. A selection of preferred (ammonium and asparagine) and nonpreferred (leucine, phenylalanine, methionine and proline) nitrogen sources was investigated. For each nitrogen source, distinct sets of genes were induced or repressed relative to the other five nitrogen sources. In total, 131 such 'signature transcripts' were identified in this study. In addition to signature transcripts, genes were identified that showed a transcriptional coresponse to two or more of the six nitrogen sources. For example, 33 genes were transcriptionally upregulated in leucine-grown, phenylalanine-grown and methionine-grown cultures; this was partly attributed to the involvement of common enzymes in the dissimilation of these amino acids. In addition to specific transcriptional responses elicited by individual nitrogen sources, their impact on global regulatory mechanisms such as nitrogen catabolite repression (NCR) were monitored. NCR-sensitive gene expression in the chemostat cultures showed that ammonium and asparagine were 'rich' nitrogen sources. By this criterion, leucine, proline and methionine were 'poor' nitrogen sources, and phenylalanine showed an 'intermediate' NCR response.
Assuntos
Saccharomyces cerevisiae/metabolismo , Aminoácidos/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Nitrogênio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Compostos de Amônio Quaternário/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transcrição GênicaRESUMO
Saccharomyces cerevisiae can use a broad range of compounds as sole nitrogen source. Many amino acids, such as leucine, tyrosine, phenylalanine and methionine, are utilized through the Ehrlich pathway. The fusel acids and alcohols produced from this pathway, along with their derived esters, are important contributors to beer and wine flavor. It is unknown how these compounds are exported from the cell. Analysis of nitrogen-source-dependent transcript profiles via microarray analysis of glucose-limited, aerobic chemostat cultures revealed a common upregulation of PDR12 in cultures grown with leucine, methionine or phenylalanine as sole nitrogen source. PDR12 encodes an ABC transporter involved in weak-organic-acid resistance, which has hitherto been studied in the context of resistance to exogenous organic acids. The hypothesis that PDR12 is involved in export of natural products of amino acid catabolism was evaluated by analyzing the phenotype of null mutants in PDR12 or in WAR1, its positive transcriptional regulator. The hypersensitivity of the pdr12Delta and war1Delta strains for some of these compounds indicates that Pdr12p is involved in export of the fusel acids, but not the fusel alcohols derived from leucine, isoleucine, valine, phenylalanine and tryptophan.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Aminoácidos/metabolismo , Ácidos Carboxílicos/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Deleção de Genes , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
Transcriptional regulation of branched-chain amino-acid metabolism in Saccharomyces cerevisiae involves two key regulator proteins, Leu3p and Gcn4p. Leu3p is a pathway-specific regulator, known to regulate six genes involved in branched-chain amino-acid metabolism and one gene in nitrogen assimilation. Gcn4p is a global regulator, involved in the general response to amino-acid and purine starvation. To investigate the contribution of Leu3p in regulation of gene expression, a leu3Delta strain was compared to an isogenic reference strain using DNA-microarray analysis. This comparison was performed for both glucose-grown/ammonium-limited and ethanol-limited/ammonium-excess chemostat cultures. In ethanol-limited cultures, absence of Leu3p led to reduced transcript levels of six of the seven established Leu3p target genes, but did not affect key physiological parameters. In ammonium-limited cultures, absence of Leu3p caused a drastic decrease in storage carbohydrate content. mRNA levels of genes involved in storage carbohydrate metabolism were also found reduced. Under N-limited conditions, the leu3Delta genotype elicited an amino-acid starvation response, leading to increased transcript levels of many amino-acid biosynthesis genes. By combining the transcriptome data with data from earlier studies that measured DNA binding of Leu3p both in vitro and in vivo, BAT1, GAT1 and OAC1 were identified as additional Leu3p-regulated genes. This study demonstrates that unravelling of transcriptional regulation networks should preferably include several cultivation conditions and requires a combination of experimental approaches.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transativadores/genética , Meios de Cultura , Etanol , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Compostos de Amônio Quaternário , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.
Assuntos
Carboxiliases/metabolismo , Leucina/metabolismo , Metionina/metabolismo , Fenilalanina/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Meios de Cultura , Descarboxilação , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Especificidade por Substrato , Regulação para CimaRESUMO
Genome-wide analysis of transcriptional regulation is generally studied by determining sets of "signature transcripts" that are up- or down-regulated relative to a reference situation when a single culture parameter or genetic modification is changed. This approach is especially relevant for defining small subsets of transcripts for use in high throughput, cost-effective diagnostic analyses. However, this approach may overlook the simultaneous control of transcription by more than one environmental parameter. This study represents the first quantitative assessment of the impact of transcriptional cross-regulation by different environmental parameters. As a model, we compared the response of aerobic as well as anaerobic chemostat cultures of the yeast Saccharomyces cerevisiae to growth limitation by four different macronutrients (carbon, nitrogen, phosphorus, and sulfur). The identity of the growth-limiting nutrient was shown to have a strong impact on the sets of transcripts that responded to oxygen availability and vice versa. We concluded that identification of reliable signature transcripts for specific environmental parameters can be obtained only by combining transcriptome data sets obtained under several sets of reference conditions. Furthermore, the two-dimensional approach to transcriptome analysis is a valuable new tool to study the interaction of different transcriptional regulation systems.
Assuntos
Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Saccharomyces cerevisiae/genética , Transcrição Gênica , Aerobiose , Anaerobiose , Meios de Cultura , Oxigênio/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Profiles of genome-wide transcriptional events for a given environmental condition can be of importance in the diagnosis of poorly defined environments. To identify clusters of genes constituting such diagnostic profiles, we characterized the specific transcriptional responses of Saccharomyces cerevisiae to growth limitation by carbon, nitrogen, phosphorus, or sulfur. Microarray experiments were performed using cells growing in steady-state conditions in chemostat cultures at the same dilution rate. This enabled us to study the effects of one particular limitation while other growth parameters (pH, temperature, dissolved oxygen tension) remained constant. Furthermore, the composition of the media fed to the cultures was altered so that the concentrations of excess nutrients were comparable between experimental conditions. In total, 1881 transcripts (31% of the annotated genome) were significantly changed between at least two growth conditions. Of those, 484 were significantly higher or lower in one limitation only. The functional annotations of these genes indicated cellular metabolism was altered to meet the growth requirements for nutrient-limited growth. Furthermore, we identified responses for several active transcription factors with a role in nutrient assimilation. Finally, 51 genes were identified that showed 10-fold higher or lower expression in a single condition only. The transcription of these genes can be used as indicators for the characterization of nutrient-limited growth conditions and provide information for metabolic engineering strategies.