RESUMEN
Metabolomics is valuable for studying microbial metabolism, which is often used to elucidate biological functions. Effective application of metabolomics is enhanced by fundamental understanding of microbial physiology and metabolism. This review briefly highlights important aspects of metabolism that are essential for designing and executing effective metabolic and metabolomics studies. The influence of microbial physiology and metabolism on growth, energy metabolism and regulation is briefly reviewed. The chapter also evaluates factors affecting metabolic prediction.
Asunto(s)
Archaea/fisiología , Bacterias/metabolismo , Hongos/fisiología , Metabolómica/métodos , Fenómenos Fisiológicos Bacterianos , Metabolismo Energético/fisiología , Redes y Vías Metabólicas/fisiologíaRESUMEN
Isoprenoids are a highly diverse group of natural products with broad application as high value chemicals and advanced biofuels. They are synthesized using two primary building blocks, namely, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that are generated via the mevalonate (MVA) or deoxy-D-xylulose-5-phosphate (DXP) pathways. Isoprenoid biosynthetic pathways are prevalent in eukaryotes, archaea, and bacteria. Measurement of isoprenoid intermediates via standard liquid chromatography-mass spectrometry (LC-MS) protocols is generally challenging because of the hydrophilicity and complex physicochemical properties of the molecules. In addition, there is currently no reliable analytical method that can simultaneously measure metabolic intermediates from MVA and DXP pathways, including the prenyl diphosphates. Therefore, we describe a robust hydrophilic interaction liquid chromatography time-of-flight mass spectrometry (HILIC-TOF-MS) method for analyzing isoprenoid intermediates from metabolically engineered Escherichia coli strains.
Asunto(s)
Escherichia coli/metabolismo , Hemiterpenos/análisis , Espectrometría de Masas/métodos , Metabolómica/métodos , Compuestos Organofosforados/análisis , Vías Biosintéticas/genética , Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Escherichia coli/genética , Hemiterpenos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas/instrumentación , Ingeniería Metabólica/instrumentación , Ingeniería Metabólica/métodos , Metabolómica/instrumentación , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/metabolismo , Compuestos Organofosforados/metabolismoRESUMEN
Plasmid pRN1 from Sulfolobus islandicus REN1H1 is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in S. acidocaldarius, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into S. acidocaldarius E4-39, a uracil auxotroph. A 232-bp region 3' of orf904 was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated orf56 and orf904 were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking orf904 and orf56 but harboring the putative origin were transformable when orf904 and orf56 were provided in-trans; a 75-bp region 5' of the orf904 start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for Sulfolobus species.
Asunto(s)
Replicación del ADN/fisiología , Plásmidos/genética , Origen de Réplica/genética , Sulfolobus/genética , Animales , Secuencia de Bases , Vectores Genéticos/genética , Datos de Secuencia Molecular , Plásmidos/fisiologíaRESUMEN
Sulfolobus acidocaldarius utilizes glucose and xylose as sole carbon sources, but its ability to metabolize these sugars simultaneously is not known. We report the absence of diauxie during growth of S. acidocaldarius on glucose and xylose as co-carbon sources. The presence of glucose did not repress xylose utilization. The organism utilized a mixture of 1 g/liter of each sugar simultaneously with a specific growth rate of 0.079 h(-1) and showed no preference for the order in which it utilized each sugar. The organism grew faster on 2 g/liter xylose (0.074 h(-1)) as the sole carbon source than on an equal amount of glucose (0.022 h(-1)). When grown on a mixture of the two carbon sources, the growth rate of the organism increased from 0.052 h(-1) to 0.085 h(-1) as the ratio of xylose to glucose increased from 0.25 to 4. S. acidocaldarius appeared to utilize a mixture of glucose and xylose at a rate roughly proportional to their concentrations in the medium, resulting in complete utilization of both sugars at about the same time. Gene expression in cells grown on xylose alone was very similar to that in cells grown on a mixture of xylose and glucose and substantially different from that in cells grown on glucose alone. The mechanism by which the organism utilized a mixture of sugars has yet to be elucidated.