RESUMEN
Wickerhamomyces anomalus LBCM1105 is a yeast isolated from cachaça distillery fermentation vats, notable for exceptional glycerol consumption ability. We report its draft genome with 20.5x in-depth coverage and around 90% extension and completeness. It harbors the sequences of proteins involved in glycerol transport and metabolism.
RESUMEN
The pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H+-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.
Asunto(s)
Kluyveromyces/metabolismo , Transporte Biológico , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactosa/metabolismo , Glucosa/metabolismo , Cinética , Kluyveromyces/química , Kluyveromyces/genética , Lactosa/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismoRESUMEN
This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.
Asunto(s)
Membrana Celular/enzimología , Membrana Celular/metabolismo , Proteínas Quinasas/análisis , ATPasas de Translocación de Protón/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Glucosa/metabolismo , Mutación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we investigated the effects of carbon sources on internalization of Lac12 using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and is localized intracellularly. Surprisingly, either galactose or lactose in the new media caused the internalization, and cells responded differently to these two sugars. Our results reveal that this process is dependent on sugar species and also sugar concentration. Lac12-GFP internalization causes reduction of [C(14) ]lactose uptake rates and also occurs in a Klsnf1 mutant strain; it is therefore independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, as internalization was induced by 2-deoxyglucose, and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in the absence of protein synthesis.
Asunto(s)
Represión Catabólica , Galactosa/metabolismo , Glucosa-6-Fosfato/metabolismo , Kluyveromyces/metabolismo , Lactosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Isótopos de Carbono/análisis , Membrana Celular/enzimología , Desoxiglucosa/metabolismo , Desoxiglucosa/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactosa/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Glucosa-6-Fosfato/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Espacio Intracelular/enzimología , Kluyveromyces/enzimología , Kluyveromyces/genética , Lactosa/farmacología , Litio/farmacología , Microscopía Fluorescente , Proteínas de Transporte de Monosacáridos/genética , Fenotipo , Fosfoglucomutasa/antagonistas & inhibidores , Fosfoglucomutasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
BACKGROUND: Corynebacterium pseudotuberculosis, a gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. METHODOLOGY AND FINDINGS: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. CONCLUSIONS: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829.
Asunto(s)
Corynebacterium pseudotuberculosis/patogenicidad , Evolución Molecular , Genoma Bacteriano , Virulencia/genética , Corynebacterium pseudotuberculosis/genéticaRESUMEN
The plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is an enzyme that plays a very important role in the yeast physiology. The addition of protonophores, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), also triggers a clear in vivo activation of this enzyme. Here, we demonstrate that CCCP-induced activation of the plasma membrane H(+)-ATPase shares some similarities with the sugar-induced activation of the enzyme. Phospholipase C and protein kinase C activities are essential for this activation process while Gpa2p, a G protein involved in the glucose-induced activation of the ATPase, is not required. CCCP also induces a phospholipase C-dependent increase in intracellular calcium. Moreover, we show that the availability of extracellular calcium is required for CCCP stimulation of H(+)-ATPase, suggesting a possible connection between calcium signaling and activation of ATPase.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/análogos & derivados , Membrana Celular/enzimología , Ionóforos/farmacología , ATPasas de Translocación de Protón/metabolismo , Saccharomyces cerevisiae/metabolismo , Calcio/análisis , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Citosol/química , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteína Quinasa C/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
In this work, we show that glucose-induced activation of plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is strongly dependent on calcium metabolism and that the glucose sensor Snf3p works in a parallel way with the G protein Gpa2p in the control of the pathway. The role of Snf3p is played by the Snf3p C-terminal tail, since in a strain with the deletion of the SNF3 gene, but also expressing a chimera protein formed by Hxt1p (a glucose transporter) and the Snf3p C-terminal tail, a normal glucose-activation process can be observed. We present evidences indicating that Snf3p would be the sensor for the internal signal (phosphorylated sugars) of this pathway that would connect calcium signaling and activation of the plasma membrane ATPase. We also show that Snf3p could be involved in the control of Pmc1p activity that would regulate the calcium availability in the cytosol.
Asunto(s)
Señalización del Calcio/fisiología , Membrana Celular/enzimología , ATPasas de Translocación de Protón/fisiología , Saccharomyces cerevisiae/fisiología , ATPasas Transportadoras de Calcio/metabolismo , Activación Enzimática , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Glucosa/fisiología , Proteínas Facilitadoras del Transporte de la Glucosa , Proteínas de Transporte de Monosacáridos/metabolismo , Fosforilación , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Protein kinase C is apparently involved in the control of many cellular systems: the cell wall integrity pathway, the synthesis of ribosomes, the appropriated reallocation of transcription factors under specific stress conditions and also the regulation of N-glycosylation activity. All these observations suggest the existence of additional targets not yet identified. In the context of the control of carbon metabolism, previous data had demonstrated that Pkc1p might play a central role in the control of cellular growth and metabolism in yeast. In particular, it has been suggested that it might be involved in the derepression of genes under glucose-repression by driving an appropriated subcellular localization of transcriptional factors, such as Mig1p. In this work, we show that a pkc1Delta mutant is unable to grow on glycerol because it cannot perform the derepression of the GUT1 gene that encodes glycerol kinase. Additionally, active transport is also partially affected. Using this phenotype, we were able to isolate a new pkc1Delta revertant. We also isolated two transformants identified as the nuclear exportin Msn5 and the histone deacetylase Hos2 extragenic suppressors of this mutation. Based on these results, we postulate that Pkc1p may be involved in the control of the cellular localization and/or regulation of the activity of nuclear proteins implicated in gene expression.
Asunto(s)
Glicerol/metabolismo , Proteína Quinasa C/deficiencia , Saccharomyces cerevisiae/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Glicerol Quinasa/genética , Proteína Quinasa C/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
An ecto-NTP diphosphohydrolase (NTPDase) activity, insensitive to inhibitors of ATPases and phosphatases, was characterized on the surface of live Trypanosoma cruzi intact parasites. The enzyme exhibits broad substrate specificity, typical of NTPDases, and a high hydrolysis rate for GTP. A 2282 bp message encoding a full-length NTPDase was cloned by RT-PCR using epimastigote mRNA. A single protein was immunoprecipitated from [(35)S]methionine-labeled parasites using antibodies against Toxoplasma gondii NTPase I. This antibody localized an NTPDase on the external surface of all forms of T. cruzi, as seen by confocal immuno-fluorescence microscopy. The NTPDase could be part of the parasite's purine salvage pathway. Additionally, trypomastigotes (infective form) presented a 2:1 ATP/ADP hydrolysis ratio, while epimastigotes (non-infective form) presented a 1:1 ratio, suggesting a possible role for the NTPDase in the parasite's virulence mechanisms.
Asunto(s)
Pirofosfatasas/análisis , Pirofosfatasas/metabolismo , Trypanosoma cruzi/enzimología , Animales , Clonación Molecular , Microscopía Fluorescente , Datos de Secuencia Molecular , Pruebas de Precipitina , Pirofosfatasas/genética , Pirofosfatasas/inmunología , Análisis de Secuencia , Especificidad por Sustrato , Trypanosoma cruzi/citología , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
In this paper, comparative molecular studies between authentic Saccharomyces cerevisiae strains, related species, and the strain described as Saccharomyces boulardii were performed. The response of a S. boulardii strain and a S. cerevisiae strain (W303) to different stress conditions was also evaluated. The results obtained in this study show that S. boulardii is genetically very close or nearly identical to S. cerevisiae. Metabolically and physiologically, however, it shows a very different behavior, particularly in relation to growth yield and resistance to temperature and acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.