RESUMEN
In past years, lots of research has been focused on the indigenous bacteria and their mechanisms, which help in enhanced oil recovery. Most of the oil wells in Indian subcontinent have temperature higher than 60⯰C. Also, the role of methanogenic consortia from high temperature petroleum reservoir for enhanced oil recovery (EOR) has not been explored much. Hence, in the present study methanogens isolated from thermophilic oil wells (70⯰C) were evaluated for enhanced oil recovery. Methane gas is produced by methanogens, which helps in oil recovery from depleted oil wells through reservoir re-pressurization and also can be recovered from reservoir along with crude oil as alternative energy source. Therefore, in this study indigenous methanogenic consortium (TERIL146) was enriched from high temperature oil reservoir showing (12â¯mmol/l) gas production along with other metabolites. Sequencing analysis revealed the presence of Methanothermobacter sp., Thermoanaerobacter sp., Gelria sp. and Thermotoga sp. in the consortium. Furthermore, the developed indigenous consortium TERIL146 showed 8.3% incremental oil recovery in sandpack assay. The present study demonstrates successful recovery of both oil and energy (gas) by the developed indigenous methanogenic consortium TERIL146 for potential application in thermophilic depleted oil wells of Indian subcontinent.
Asunto(s)
Bacterias/aislamiento & purificación , Methanobacteriaceae/aislamiento & purificación , Consorcios Microbianos , Yacimiento de Petróleo y Gas/microbiología , Bacterias/clasificación , Bacterias/genética , Calor , Microbiología Industrial , Metano/metabolismo , Methanobacteriaceae/clasificación , Methanobacteriaceae/genética , Filogenia , Análisis de Secuencia de ADN , Thermoanaerobacter/clasificación , Thermoanaerobacter/genética , Thermoanaerobacter/aislamiento & purificación , Thermotoga maritima/clasificación , Thermotoga maritima/genética , Thermotoga maritima/aislamiento & purificaciónRESUMEN
Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H2). In this organism, the catabolism of pentose sugars such as arabinose depends on the interaction of the pentose phosphate pathway with the Embden-Myerhoff and Entner-Doudoroff pathways. Although the values for H2 yield have been determined using pentose-supplemented complex medium and predicted by metabolic pathway reconstruction, the actual effect of pathway elimination on hydrogen production has not been reported due to the lack of a genetic method for the creation of targeted mutations. Here, a spontaneous and genetically stable pyrE deletion mutant was isolated and used as a recipient to refine transformation methods for its repair by homologous recombination. To verify the occurrence of recombination and to assess the frequency of crossover events flanking the deleted region, a synthetic pyrE allele, encoding synonymous nucleotide substitutions, was used. Targeted inactivation of araA (encoding arabinose isomerase) in the pyrE mutant was accomplished using a divergent, codon-optimized Thermosipho africanus pyrE allele fused to the T. maritima groES promoter as a genetic marker. Mutants lacking araA were unable to catabolize arabinose in a defined medium. The araA mutation was then repaired using targeted recombination. Levels of synthesis of H2 using arabinose-supplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H2 production that was dependent on arabinose consumption. Development of a targeted recombination system for genetic manipulation of T. maritima provides a new strategy to explore H2 formation and life at an extremely high temperature in the bacterial domain. IMPORTANCE: We describe here the development of a genetic system for manipulation of Thermotoga maritima T. maritima is a hyperthermophilic anaerobic bacterium that is well known for its efficient synthesis of molecular hydrogen (H2) from the fermentation of sugars. Despite considerable efforts to advance compatible genetic methods, chromosome manipulation has remained elusive and hindered use of T. maritima or its close relatives as model hyperthermophiles. Lack of a genetic method also prevented efforts to manipulate specific metabolic pathways to measure their contributions to H2 yield. To overcome this barrier, a homologous chromosomal recombination method was developed and used to characterize the contribution of arabinose catabolism to H2 formation. We report here a stable genetic method for a hyperthermophilic bacterium that will advance studies on the basic and synthetic biology of Thermotogales.
Asunto(s)
Isomerasas Aldosa-Cetosa/genética , Arabinosa/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismo , Fermentación/genética , Eliminación de Gen , Hidrógeno/metabolismo , Thermotoga maritima/aislamiento & purificaciónRESUMEN
Thermotoga sp. strain RQ2 is probably a strain of Thermotoga maritima. Its complete genome sequence allows for an examination of the extent and consequences of gene flow within Thermotoga species and strains. Thermotoga sp. RQ2 differs from T. maritima in its genes involved in myo-inositol metabolism. Its genome also encodes an apparent fructose phosphotransferase system (PTS) sugar transporter. This operon is also found in Thermotoga naphthophila strain RKU-10 but no other Thermotogales. These are the first reported PTS transporters in the Thermotogales.
Asunto(s)
Genoma Bacteriano , Agua de Mar/microbiología , Thermotoga maritima/genética , Thermotoga maritima/aislamiento & purificación , Azores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Fructosa/metabolismo , Calor , Datos de Secuencia Molecular , Operón , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Thermotoga maritima/enzimología , Thermotoga maritima/metabolismoRESUMEN
The microbial community structure of beer wastewater treatment plants with hydrolyze-acidification and SBR process was studied by PCR amplification and denaturing gradient gel electrophoresis (DGGE) based 16S rDNA. Activated sludge samples were collected from both hydrolyze-acidification tank and SBR tank at different depth and disposal period. The total DNA was extracted and the 16S rDNA was amplified by universal primer. The microbial community structure was analyzed by denaturing gradient gel electrophoresis, compared with the DGGE band pattern of the screened high efficient bacteria. The PCR products were cloned and sequenced to analyze the microbial community in phylogenic evolution. The DGGE pattern showed that the microbial community in hydrolyze-acidification tank changed with the variation of depth both in microbial structure and population, and at the depth of two-meter the microbial community was relatively rich, the dominant bands were notable. The microbial community in SBR tank at different depths and different disposal periods (precipitation period, inflow period, aeration period) were consistent respectively, but the dominant bands were different. Sequences of y2, 23, 25, 31, h5, 15 yielded high homogeneity with the strains uncultured Thermotogales sp., Comamonas sp. WT OTU1, Agrobacterium tumefaciens, Bacillus subtilis, Bdellovibrio bacteriovorus, Comamonas testosteroni from GenBank. The 16S rDNA sequences of the dominant bands were not completely same with that of the high efficient bacteria, which indicated that the strains screened were not the dominant strains in actual process.
Asunto(s)
Cerveza , Reactores Biológicos/microbiología , Comamonas/aislamiento & purificación , Thermotoga maritima/aislamiento & purificación , Eliminación de Residuos Líquidos/métodos , Comamonas/genética , Electroforesis en Gel de Campo Pulsado/métodos , Reacción en Cadena de la Polimerasa/métodos , Rhizobium/genética , Rhizobium/aislamiento & purificación , Thermotoga maritima/genéticaRESUMEN
High-throughput sequencing of microbial genomes has allowed the application of functional genomics methods to species lacking well-developed genetic systems. For the model hyperthermophile Thermotoga maritima, microarrays have been used in comparative genomic hybridization studies to investigate diversity among Thermotoga species. Transcriptional data have assisted in prediction of pathways for carbohydrate utilization, iron-sulfur cluster synthesis and repair, expolysaccharide formation, and quorum sensing. Structural genomics efforts aimed at the T. maritima proteome have yielded hundreds of high-resolution datasets and predicted functions for uncharacterized proteins. The information gained from genomics studies will be particularly useful for developing new biotechnology applications for T. maritima enzymes.
Asunto(s)
Variación Genética , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Biotecnología , Metabolismo de los Hidratos de Carbono , Genoma Bacteriano , Genómica , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/genética , Calor , Hierro/metabolismo , Monosacáridos/biosíntesis , Filogenia , Polisacáridos/biosíntesis , Transducción de Señal , Azufre/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/aislamiento & purificación , Thermotoga maritima/fisiologíaRESUMEN
Sirtuins comprise a family of enzymes that catalyze the deacetylation of acetyllysine side chains in a reaction that consumes NAD+. Although several crystal structures of sirtuins bound to non-native acetyl peptides have been determined, relatively little about how sirtuins discriminate among different substrates is understood. We have carried out a systematic structural and thermodynamic analysis of several peptides bound to a single sirtuin, the Sir2 homologue from Thermatoga maritima (Sir2Tm). We report structures of five different forms of Sir2Tm: two forms bound to the p53 C-terminal tail in the acetylated and unacetylated states, two forms bound to putative acetyl peptide substrates derived from the structured domains of histones H3 and H4, and one form bound to polypropylene glycol (PPG), which resembles the apoenzyme. The structures reveal previously unobserved complementary side chain interactions between Sir2Tm and the first residue N-terminal to the acetyllysine (position -1) and the second residue C-terminal to the acetyllysine (position +2). Isothermal titration calorimetry was used to compare binding constants between wild-type and mutant forms of Sir2Tm and between additional acetyl peptide substrates with substitutions at positions -1 and +2. The results are consistent with a model in which peptide positions -1 and +2 play a significant role in sirtuin substrate binding. This model provides a framework for identifying sirtuin substrates.
Asunto(s)
Sirtuinas/química , Sirtuinas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Homología de Secuencia de Aminoácido , Sirtuinas/genética , Relación Estructura-Actividad , Especificidad por Sustrato , Termodinámica , Thermotoga maritima/enzimología , Thermotoga maritima/aislamiento & purificaciónRESUMEN
Here we characterize regions of the genomes of eight members of the hyperthermophilic genus Thermotoga. These bacteria differ from each other physiologically and by 3-20% in gene content and occupy physically distinct environments in widely disparate regions of the globe. Among the four different lineages (represented by nine different strains) that we compare, no two are closer than 96% in the average sequences of their genes. By most accepted recent definitions these are different "ecotypes" and different "species." And yet we find compelling evidence for recombination between them. We suggest that no single prokaryotic species concept can accommodate such uncoupling of ecotypic and genetic aspects of cohesion and diversity, and that without a single concept, the question of whether or not prokaryotic species might in general be cosmopolitan cannot be sensibly addressed. We can, however, recast biogeographical questions in terms of the distribution of genes and their alleles.
Asunto(s)
Genes Bacterianos , Recombinación Genética , Thermotoga maritima/genética , Thermotoga neapolitana/genética , Microbiología del Agua , Secuencia de Bases , Especiación Genética , Genómica , Genotipo , Funciones de Verosimilitud , Datos de Secuencia Molecular , Familia de Multigenes , Fenotipo , Filogenia , Alineación de Secuencia , Especificidad de la Especie , Thermotoga maritima/aislamiento & purificación , Thermotoga maritima/fisiología , Thermotoga neapolitana/aislamiento & purificación , Thermotoga neapolitana/fisiologíaRESUMEN
Membrane proteins constitute ~30% of prokaryotic and eukaryotic genomes but comprise a small fraction of the entries in protein structural databases. A number of features of membrane proteins render them challenging targets for the structural biologist, among which the most important is the difficulty in obtaining sufficient quantities of purified protein. We are exploring procedures to express and purify large numbers of prokaryotic membrane proteins. A set of 280 membrane proteins from Escherichia coli and Thermotoga maritima, a thermophile, was cloned and tested for expression in Escherichia coli. Under a set of standard conditions, expression could be detected in the membrane fraction for approximately 30% of the cloned targets. About 22 of the highest expressing membrane proteins were purified, typically in just two chromatographic steps. There was a clear correlation between the number of predicted transmembrane domains in a given target and its propensity to express and purify. Accordingly, the vast majority of successfully expressed and purified proteins had six or fewer transmembrane domains. We did not observe any clear advantage to the use of thermophilic targets. Two of the purified membrane proteins formed crystals. By comparison with protein production efforts for soluble proteins, where approximately 70% of cloned targets express and approximately 25% can be readily purified for structural studies [Christendat et al. (2000) Nat. Struct. Biol., 7, 903], our results demonstrate that a similar approach will succeed for membrane proteins, albeit with an expected higher attrition rate.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Proteínas de la Membrana/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Histidina/química , Histidina/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Homología de Secuencia , Solubilidad , Thermotoga maritima/química , Thermotoga maritima/enzimología , Thermotoga maritima/genética , Thermotoga maritima/aislamiento & purificaciónRESUMEN
A novel anaerobic, thermophilic, xylanolytic, motile rod-shaped bacterium with a sheath-like outer structure (toga) was isolated from a Mexican oil well in the Gulf of Mexico. Strain MET12T was a Gram-negative bacterium, reducing elemental sulfur, thiosulfate and sulfite to hydrogen sulfide. Its optimum growth conditions were 55 degrees C, pH 6.6, 3% NaCl and 0.15% MgCl2.6H2O. The DNA G+C content was 36.1 mol%. Phylogenetically, strain MET12T was related to members of genus Petrotoga, with similarities to Petrotoga mobilis, Petrotoga sibirica, Petrotoga miotherma and Petrotoga olearia varying from 97.6 to 98.8%. However DNA-DNA relatedness values between these species and strain MET12T were lower than 70%. As strain MET12T (=DSM 14811T=CIP 107371T) was genomically and phenotypically different from existing Petrotoga species, it is proposed as the type strain of a novel species, Petrotoga mexicana sp. nov.