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1.
FEMS Microbiol Ecol ; 100(9)2024 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-39118367

RESUMEN

Thermophilic acetogenic bacteria have attracted attention as promising candidates for biotechnological applications such as syngas fermentation, microbial electrosynthesis, and methanol conversion. Here, we aimed to isolate and characterize novel thermophilic acetogens from diverse environments. Enrichment of heterotrophic and autotrophic acetogens was monitored by 16S rRNA gene-based bacterial community analysis. Seven novel Moorella strains were isolated and characterized by genomic and physiological analyses. Two Moorella humiferrea isolates showed considerable differences during autotrophic growth. The M. humiferrea LNE isolate (DSM 117358) fermented carbon monoxide (CO) to acetate, while the M. humiferrea OCP isolate (DSM 117359) transformed CO to hydrogen and carbon dioxide (H2 + CO2), employing the water-gas shift reaction. Another carboxydotrophic hydrogenogenic Moorella strain was isolated from the covering soil of an active charcoal burning pile and proposed as the type strain (ACPsT) of the novel species Moorella carbonis (DSM 116161T and CCOS 2103T). The remaining four novel strains were affiliated with Moorella thermoacetica and showed, together with the type strain DSM 2955T, the production of small amounts of ethanol from H2 + CO2 in addition to acetate. The physiological analyses of the novel Moorella strains revealed isolate-specific differences that considerably increase the knowledge base on thermophilic acetogens for future applications.


Asunto(s)
Moorella , Filogenia , ARN Ribosómico 16S , ARN Ribosómico 16S/genética , Moorella/metabolismo , Moorella/genética , Moorella/crecimiento & desarrollo , Dióxido de Carbono/metabolismo , Hidrógeno/metabolismo , Fermentación , Monóxido de Carbono/metabolismo , Microbiología del Suelo , Acetatos/metabolismo , Biocatálisis , ADN Bacteriano/genética
2.
Artículo en Inglés | MEDLINE | ID: mdl-37234030

RESUMEN

Strain AMPT has been previously suggested as a strain of the species Moorella thermoacetica Jiang et al. 2009 (based on the high 16S rRNA gene identity, 98.3 %). However, genome-based phylogenetic analysis of strain AMPT reveals that this bacterium is in fact a novel species of the genus Moorella. Genome relatedness indices between strain AMPT and Moorella thermoacetica DSM 521T were below the minimum threshold values required to consider them members of the same species (digital DNA-DNA hybridization, 52.2 % (<70%); average nucleotide identity, 93.2 % (<95%)). Based on phylogenetic and phenotypic results we recommend that strain AMPT (DSM 21394T=JCM 35360T) should be classified as representing new species, for which we propose the name Moorella caeni sp. nov.


Asunto(s)
Moorella , Moorella/genética , Ácidos Grasos/química , Aguas del Alcantarillado/microbiología , Metanol , Anaerobiosis , Filogenia , ARN Ribosómico 16S/genética , Composición de Base , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Análisis de Secuencia de ADN
3.
J Biosci Bioeng ; 136(1): 13-19, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37100649

RESUMEN

Acetogens grow autotrophically and use hydrogen (H2) as the energy source to fix carbon dioxide (CO2). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H2 and CO2. We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H2 and CO2.


Asunto(s)
Dióxido de Carbono , Moorella , Dióxido de Carbono/metabolismo , Acetona/metabolismo , Electrones , Dimetilsulfóxido/metabolismo , Hidrógeno/metabolismo , Moorella/genética , Moorella/metabolismo , Oxidantes/metabolismo , Adenosina Trifosfato/metabolismo
4.
Extremophiles ; 26(3): 33, 2022 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-36352059

RESUMEN

In hydrothermal ecosystems, the dissolution of sulfur dioxide in water results in the formation of sulfite, which can be used in microbial metabolism. A limited number of thermophiles have been isolated using sulfite as an electron acceptor. From a terrestrial thermal spring, Sakhalin Island, Russia, we isolated a thermophilic anaerobic bacterium (strain SLA38T). Cells of strain SLA38T were spore-forming straight rods. Growth was observed at temperatures 45-65 °C (optimum at 60 °C) and pH 5.5-9.0 (optimum at pH 6.5-7.0). The novel isolate was capable of anaerobic respiration with sulfite, thiosulfate, fumarate and perchlorate or fermentative growth. Strain SLA38T utilized glycerol, lactate, pyruvate and yeast extract. It grew lithoautotrophically on carbon monoxide with thiosulfate as electron acceptor, producing acetate. The genome size of the isolate was 2.9 Mbp and genomic DNA G + C content was 53.6 mol%. Analysis of the 16S rRNA gene sequences revealed that strain SLA38T belongs to the genus Moorella. Based on the physiological features and phylogenetic analysis, we propose to assign strain SLA38T to a new species of the genus Moorella, as Moorella sulfitireducens sp. nov. The type strain is SLA38T (= DSM 111068T = VKM B-3584T).


Asunto(s)
Manantiales de Aguas Termales , Moorella , Moorella/genética , ARN Ribosómico 16S/genética , Filogenia , Manantiales de Aguas Termales/microbiología , Composición de Base , Anaerobiosis , Tiosulfatos , ADN Bacteriano/química , Técnicas de Tipificación Bacteriana , Ecosistema , Análisis de Secuencia de ADN , Bacterias Anaerobias/genética , Sulfitos
5.
Environ Microbiol ; 24(4): 2000-2012, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35278024

RESUMEN

Moorella thermoacetica is one of the well-studied thermophilic acetogenic bacteria. It grows by oxidation of organic substrates, CO or H2 coupled to CO2 reduction to acetate. Here, we describe that M. thermoacetica can also use dimethyl sulfoxide as terminal electron acceptor. Growth of M. thermoacetica on glucose or H2  + CO2 was stimulated by dimethyl sulfoxide (DMSO). Membranes showed a DMSO reductase activity, that was induced by growing cells in presence of DMSO. The enzyme used reduced anthraquinone-2,6-disulfonate, benzyl- and methyl viologen as electron donor, but not NAD(P)H. Activity was highest at pH 5 and 60°C, the Km for DMSO was 2.4 mM. Potential DMSO reductase subunits were identified by peptide mass fingerprinting; they are encoded in a genomic region that contains three potential dmsA genes, three dmsB genes and one dmsC gene. Transcriptome analysis revealed that two different dmsAB gene clusters were induced in the presence of DMSO. The function of these two and their predicted biochemical features are discussed. In addition, the data are in line with the hypothesis that M. thermoacetica can use DMSO alongside CO2 as electron acceptor and DMSO reduction is catalysed by an energy-conserving, membrane-bound electron transport chain with DMSO as final electron acceptor.


Asunto(s)
Dimetilsulfóxido , Moorella , Bacterias , Dióxido de Carbono , Moorella/genética
6.
J Biosci Bioeng ; 132(6): 569-574, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34518108

RESUMEN

Gas fermentation is a promising biological process for the conversion of CO2 or syngas into valuable chemicals. Homoacetogens are microorganisms growing autotrophically using CO2 and H2 or CO and metabolizing them to form acetate coupled with energy conservation. The challenge in the metabolic engineering of the homoacetogens is divergence of the acetate formation, whose intermediate is acetyl-CoA, to a targeted chemical with sufficient production of adenosine triphosphate (ATP). In this study, we report that an engineered strain of the thermophilic homoacetogen Moorella thermoacetica, in which a pool of acetyl-CoA is diverted to ethanol without ATP production, can maintain autotrophic growth on syngas. We estimated the ATP production in the engineered strains under different gaseous compositions by considering redox-balanced metabolism for ethanol and acetate formation. The culture test showed that the combination of retaining a level of acetate production and supplying the energy-rich CO allowed maintenance of the autotrophic growth during ethanol production. In contrast, autotrophy was collapsed by complete elimination of the acetate pathway or supplementation of H2-CO2. We showed that the intracellular level of ATP was significantly lowered on H2-CO2 in consistent with the incompetence. In the meantime, the complete disruption of the acetate pathway resulted in the redox imbalance to produce ethanol from CO, albeit a small loss in the ATP production. Thus, preservation of a fraction of acetate formation is required to maintain sufficient ATP and balanced redox in CO-containing gases for ethanol production.


Asunto(s)
Etanol , Moorella , Acetatos , Procesos Autotróficos , Moorella/genética
7.
Sci Rep ; 11(1): 2139, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33495538

RESUMEN

Biohybrids composed of microorganisms and nanoparticles have emerged as potential systems for bioenergy and high-value compound production from CO2 and light energy, yet the cellular and metabolic processes within the biological component of this system are still elusive. Here we dissect the biohybrid composed of the anaerobic acetogenic bacterium Moorella thermoacetica and cadmium sulphide nanoparticles (CdS) in terms of physiology, metabolism, enzymatics and transcriptomic profiling. Our analyses show that while the organism does not grow on L-cysteine, it is metabolized to acetate in the biohybrid system and this metabolism is independent of CdS or light. CdS cells have higher metabolic activity, despite an inhibitory effect of Cd2+ on key enzymes, because of an intracellular storage compound linked to arginine metabolism. We identify different routes how cysteine and its oxidized form can be innately metabolized by the model acetogen and what intracellular mechanisms are triggered by cysteine, cadmium or blue light.


Asunto(s)
Carbono/metabolismo , Cisteína/metabolismo , Metabolismo Energético , Acetatos/metabolismo , Transporte Biológico/efectos de los fármacos , Cadmio/farmacología , Isótopos de Carbono , Mezclas Complejas , Cisteína/farmacología , Metabolismo Energético/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Luz , Espectroscopía de Resonancia Magnética , Moorella/genética , Moorella/crecimiento & desarrollo , Moorella/efectos de la radiación , Moorella/ultraestructura , Oxidación-Reducción , Transcriptoma/genética
8.
J Biol Chem ; 295(31): 10522-10534, 2020 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-32503839

RESUMEN

Vitamin B12 and other cobamides are essential cofactors required by many organisms and are synthesized by a subset of prokaryotes via distinct aerobic and anaerobic routes. The anaerobic biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12, involves five reactions catalyzed by the bza operon gene products, namely the hydroxybenzimidazole synthase BzaAB/BzaF, phosphoribosyltransferase CobT, and three methyltransferases, BzaC, BzaD, and BzaE, that conduct three distinct methylation steps. Of these, the methyltransferases that contribute to benzimidazole lower ligand diversity in cobamides remain to be characterized, and the precise role of the bza operon protein CobT is unclear. In this study, we used the bza operon from the anaerobic bacterium Moorella thermoacetica (comprising bzaA-bzaB-cobT-bzaC) to examine the role of CobT and investigate the activity of the first methyltransferase, BzaC. We studied the phosphoribosylation catalyzed by MtCobT and found that it regiospecifically activates 5-hydroxybenzimidazole (5-OHBza) to form the 5-OHBza-ribotide (5-OHBza-RP) isomer as the sole product. Next, we characterized the domains of MtBzaC and reconstituted its methyltransferase activity with the predicted substrate 5-OHBza and with two alternative substrates, the MtCobT product 5-OHBza-RP and its riboside derivative 5-OHBza-R. Unexpectedly, we found that 5-OHBza-R is the most favored MtBzaC substrate. Our results collectively explain the long-standing observation that the attachment of the lower ligand in anaerobic cobamide biosynthesis is regiospecific. In conclusion, we validate MtBzaC as a SAM:hydroxybenzimidazole-riboside methyltransferase (HBIR-OMT). Finally, we propose a new pathway for the synthesis and activation of the benzimidazolyl lower ligand in anaerobic cobamide biosynthesis.


Asunto(s)
Proteínas Bacterianas/metabolismo , Bencimidazoles/metabolismo , Cobamidas/biosíntesis , Metiltransferasas/metabolismo , Moorella/metabolismo , Pentosiltransferasa/metabolismo , Anaerobiosis , Proteínas Bacterianas/genética , Cobamidas/genética , Metilación , Metiltransferasas/genética , Moorella/genética , Pentosiltransferasa/genética
9.
J Biosci Bioeng ; 129(2): 160-164, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31506242

RESUMEN

Bioconversion from inexpensive renewable resource, such as biomass, to liquid fuel is one of the promising technologies to reduce the use of petroleum. We previously reported the genetically engineered Moorella thermoacetica could produce ethanol from the lignocellulosic feedstock. However, it was still unclear which carbon source in the substrate was preferentially consumed to produce ethanol. To identify the hierarchy of the sugar utilization during ethanol fermentation of this strain, we analyzed the sugar composition of lignocellulosic feedstock, and consumption rate of sugars during the fermentation process. The hydrolysates after acid pretreatment and enzymatic saccharification contained glucose, xylose, galactose, arabinose, and mannose. Time course data suggested that xylose was the most preferred carbon source among those sugars during ethanol fermentation. Ethanol yield was 0.40 ± 0.06 and 0.40 ± 0.12 g/g-total sugar, from lignocellulosic hydrolysates of Japanese cedar (Cryptomeria japonica) and rice straw (Oryza sativa), respectively. The results demonstrated that the genetically engineered M. thermoacetica is a promising candidate for thermophilic ethanol fermentation of lignocellulosic feedstocks, especially hemicellulosic sugars.


Asunto(s)
Etanol/metabolismo , Lignina/metabolismo , Moorella/metabolismo , Azúcares/metabolismo , Fermentación , Ingeniería Genética , Calor , Hidrólisis , Moorella/genética
10.
Nat Commun ; 10(1): 3311, 2019 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-31427571

RESUMEN

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.


Asunto(s)
Acetobacterium/enzimología , Proteínas Bacterianas/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Metiltransferasas/metabolismo , Moorella/enzimología , Acetobacterium/genética , Secuencias de Aminoácidos/genética , Proteínas Bacterianas/genética , Metilación de ADN , ADN Bacteriano/metabolismo , Epigénesis Genética , Genoma Bacteriano , Moorella/genética , Análisis de Secuencia de ADN
11.
Proc Natl Acad Sci U S A ; 115(15): 3846-3851, 2018 04 10.
Artículo en Inglés | MEDLINE | ID: mdl-29581263

RESUMEN

Pyruvate:ferredoxin oxidoreductase (PFOR) is a microbial enzyme that uses thiamine pyrophosphate (TPP), three [4Fe-4S] clusters, and coenzyme A (CoA) in the reversible oxidation of pyruvate to generate acetyl-CoA and carbon dioxide. The two electrons that are generated as a result of pyruvate decarboxylation are used in the reduction of low potential ferredoxins, which provide reducing equivalents for central metabolism, including the Wood-Ljungdahl pathway. PFOR is a member of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily, which plays major roles in both microbial redox reactions and carbon dioxide fixation. Here, we present a set of crystallographic snapshots of the best-studied member of this superfamily, the PFOR from Moorella thermoacetica (MtPFOR). These snapshots include the native structure, those of lactyl-TPP and acetyl-TPP reaction intermediates, and the first of an OFOR with CoA bound. These structural data reveal the binding site of CoA as domain III, the function of which in OFORs was previously unknown, and establish sequence motifs for CoA binding in the OFOR superfamily. MtPFOR structures further show that domain III undergoes a conformational change upon CoA binding that seals off the active site and positions the thiolate of CoA directly adjacent to the TPP cofactor. These structural findings provide a molecular basis for the experimental observation that CoA binding accelerates catalysis by 105-fold.


Asunto(s)
Proteínas Bacterianas/química , Coenzima A/metabolismo , Moorella/enzimología , Piruvato-Sintasa/química , Piruvato-Sintasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Coenzima A/química , Cristalografía por Rayos X , Ferredoxinas/química , Ferredoxinas/metabolismo , Cinética , Moorella/química , Moorella/genética , Piruvato-Sintasa/genética , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo
12.
Appl Environ Microbiol ; 83(15)2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28526793

RESUMEN

Calderihabitans maritimus KKC1 is a thermophilic, hydrogenogenic carboxydotroph isolated from a submerged marine caldera. Here, we describe the de novo sequencing and feature analysis of the C. maritimus KKC1 genome. Genome-based phylogenetic analysis confirmed that C. maritimus KKC1 was most closely related to the genus Moorella, which includes well-studied acetogenic members. Comparative genomic analysis revealed that, like Moorella, C. maritimus KKC1 retained both the CO2-reducing Wood-Ljungdahl pathway and energy-converting hydrogenase-based module activated by reduced ferredoxin, but it lacked the HydABC and NfnAB electron-bifurcating enzymes and pyruvate:ferredoxin oxidoreductase required for ferredoxin reduction for acetogenic growth. Furthermore, C. maritimus KKC1 harbored six genes encoding CooS, a catalytic subunit of the anaerobic CO dehydrogenase that can reduce ferredoxin via CO oxidation, whereas Moorella possessed only two CooS genes. Our analysis revealed that three cooS genes formed known gene clusters in other microorganisms, i.e., cooS-acetyl coenzyme A (acetyl-CoA) synthase (which contained a frameshift mutation), cooS-energy-converting hydrogenase, and cooF-cooS-FAD-NAD oxidoreductase, while the other three had novel genomic contexts. Sequence composition analysis indicated that these cooS genes likely evolved from a common ancestor. Collectively, these data suggest that C. maritimus KKC1 may be highly dependent on CO as a low-potential electron donor to directly reduce ferredoxin and may be more suited to carboxydotrophic growth compared to the acetogenic growth observed in Moorella, which show adaptation at a thermodynamic limit.IMPORTANCECalderihabitans maritimus KKC1 and members of the genus Moorella are phylogenetically related but physiologically distinct. The former is a hydrogenogenic carboxydotroph that can grow on carbon monoxide (CO) with H2 production, whereas the latter include acetogenic bacteria that grow on H2 plus CO2 with acetate production. Both species may require reduced ferredoxin as an actual "energy equivalent," but ferredoxin is a low-potential electron carrier and requires a high-energy substrate as an electron donor for reduction. Comparative genomic analysis revealed that C. maritimus KKC1 lacked specific electron-bifurcating enzymes and possessed six CO dehydrogenases, unlike Moorella species. This suggests that C. maritimus KKC1 may be more dependent on CO, a strong electron donor that can directly reduce ferredoxin via CO dehydrogenase, and may exhibit a survival strategy different from that of acetogenic Moorella, which solves the energetic barrier associated with endergonic reduction of ferredoxin with hydrogen.


Asunto(s)
Monóxido de Carbono/metabolismo , Genoma Bacteriano , Sedimentos Geológicos/microbiología , Hidrógeno/metabolismo , Moorella/genética , Moorella/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ferredoxinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genómica , Calor , Hidrogenasas/genética , Hidrogenasas/metabolismo , Moorella/clasificación , Moorella/aislamiento & purificación , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Filogenia
13.
Metab Eng ; 41: 173-181, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28433737

RESUMEN

Mono-ethylene glycol (MEG) is an important petrochemical with widespread use in numerous consumer products. The current industrial MEG-production process relies on non-renewable fossil fuel-based feedstocks, such as petroleum, natural gas, and naphtha; hence, it is useful to explore alternative routes of MEG-synthesis from gases as they might provide a greener and more sustainable alternative to the current production methods. Technologies of synthetic biology and metabolic engineering of microorganisms can be deployed for the expression of new biochemical pathways for MEG-synthesis from gases, provided that such promising alternative routes are first identified. We used the BNICE.ch algorithm to develop novel and previously unknown biological pathways to MEG from synthesis gas by leveraging the Wood-Ljungdahl pathway of carbon fixation of acetogenic bacteria. We developed a set of useful pathway pruning and analysis criteria to systematically assess thousands of pathways generated by BNICE.ch. Published genome-scale models of Moorella thermoacetica and Clostridium ljungdahlii were used to perform the pathway yield calculations and in-depth analyses of seven (7) newly developed biological MEG-producing pathways from gases, including CO2, CO, and H2. These analyses helped identify not only better candidate pathways, but also superior chassis organisms that can be used for metabolic engineering of the candidate pathways. The pathway generation, pruning, and detailed analysis procedures described in this study can also be used to develop biochemical pathways for other commodity chemicals from gaseous substrates.


Asunto(s)
Dióxido de Carbono/metabolismo , Monóxido de Carbono/metabolismo , Clostridium , Glicol de Etileno/metabolismo , Hidrógeno/metabolismo , Ingeniería Metabólica/métodos , Moorella , Clostridium/genética , Clostridium/metabolismo , Moorella/genética , Moorella/metabolismo
14.
Appl Environ Microbiol ; 83(8)2017 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-28159797

RESUMEN

For the efficient production of target metabolites from carbohydrates, syngas, or H2-CO2 by genetically engineered Moorella thermoacetica, the control of acetate production (a main metabolite of M. thermoacetica) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica, this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 (pduL1) and Moth_1181 (pduL2), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 (T-ldh). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate.IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform.


Asunto(s)
Acetatos/metabolismo , Fermentación , Ingeniería Genética , Moorella/genética , Moorella/metabolismo , Acetilcoenzima A/metabolismo , Anaerobiosis , Carbono/metabolismo , L-Lactato Deshidrogenasa/genética , Moorella/enzimología , Fosfato Acetiltransferasa/metabolismo , Glicoles de Propileno/metabolismo , Thermoanaerobacter/genética
15.
Extremophiles ; 21(1): 15-26, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27623994

RESUMEN

Thermophilic microorganisms as well as acetogenic bacteria are both considered ancient. Interestingly, only a few species of bacteria, all belonging to the family Thermoanaerobacteraceae, are described to conserve energy from acetate formation with hydrogen as electron donor and carbon dioxide as electron acceptor. This review reflects the metabolic differences between Moorella spp., Thermoanaerobacter kivui and Thermacetogenium phaeum, with focus on the biochemistry of autotrophic growth and energy conservation. The potential of these thermophilic acetogens for biotechnological applications is discussed briefly.


Asunto(s)
Aclimatación , Ciclo del Carbono , Moorella/metabolismo , Thermoanaerobacter/metabolismo , Metabolismo Energético , Calor , Moorella/genética , Moorella/fisiología , Thermoanaerobacter/genética , Thermoanaerobacter/fisiología
16.
Extremophiles ; 20(5): 653-61, 2016 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-27338272

RESUMEN

Moorella thermoacetica is one of the model acetogenic bacteria for the resolution of the Wood-Ljungdahl (acetyl-CoA) pathway in which CO2 is autotrophically assimilated yielding acetyl-CoA as central intermediate. Its further conversion into acetate relies on subsequent phosphotransacetylase (PTA) and acetate kinase reactions. However, the genome of M. thermoacetica contains no pta homologous gene. It has been speculated that the moth_0864 and moth_1181 gene products sharing similarities with an evolutionarily distinct phosphotransacylase involved in 1,2-propanediol utilization (PDUL) of Salmonella enterica act as PTAs in M. thermoacetica. Here, we demonstrate specific PTA activities with acetyl-CoA as substrate of 9.05 and 2.03 U/mg for the recombinant enzymes PDUL1 (Moth_1181) and PDUL2 (Moth_0864), respectively. Both showed maximal activity at 65 °C and pH 7.6. Native proteins (90 kDa) are homotetramers composed of four subunits with apparent molecular masses of about 23 kDa. Thus, one or both PDULs of M. thermoacetica might act as PTAs in vivo catalyzing the penultimate step of the Wood-Ljungdahl pathway toward the formation of acetate. In silico analysis underlined that up to now beside of M. thermoacetica, only Sporomusa ovata contains only PDUL like class(III)-PTAs but no other phosphotransacetylases or phosphotransbutyrylases (PTBs).


Asunto(s)
Proteínas Bacterianas/metabolismo , Genes Bacterianos , Moorella/enzimología , Fosfato Acetiltransferasa/metabolismo , Propilenglicol/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Estabilidad de Enzimas , Calor , Concentración de Iones de Hidrógeno , Moorella/genética , Fosfato Acetiltransferasa/química , Fosfato Acetiltransferasa/genética , Multimerización de Proteína
17.
Int J Syst Evol Microbiol ; 66(8): 3249-3251, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27199008

RESUMEN

Based on the results of DNA-DNA hybridization and 16S rRNA gene sequence analyses, it was ascertained that the type strain of Moorella thermoautotrophica does not exist in any established culture collection or with the authors who originally described this species. Therefore, this species cannot be included in any further scientific studies. It is proposed that the Judicial Commission place the name Moorella thermoautotrophica on the list of rejected names if a suitable type strain is not found or a neotype is not proposed within two years following the publication of this Request for an Opinion.


Asunto(s)
Moorella/clasificación , Filogenia , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Moorella/genética , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN
18.
Proc Natl Acad Sci U S A ; 112(34): 10792-7, 2015 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-26246619

RESUMEN

Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B12 itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism.


Asunto(s)
Bencimidazoles/metabolismo , Eubacterium/metabolismo , Vitamina B 12/biosíntesis , Anaerobiosis , Archaea/genética , Archaea/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Corrinoides/biosíntesis , ADN Recombinante/genética , Escherichia coli/metabolismo , Eubacterium/genética , Genes Arqueales , Genes Bacterianos , Geobacter/genética , Geobacter/metabolismo , Redes y Vías Metabólicas , Datos de Secuencia Molecular , Estructura Molecular , Moorella/genética , Moorella/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Riboswitch/genética , Salmonella typhimurium/crecimiento & desarrollo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico
19.
J Food Prot ; 78(7): 1392-6, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26197294

RESUMEN

A quantitative real-time PCR assay was developed to specifically detect and quantify Moorella thermoacetica and/or Moorella thermoautotrophica from canned coffee beverages. Six different combinations of newly designed primers were examined, and primer pair v1-1F/v4R was found to specifically amplify M. thermoacetica and M. thermoautotrophica. The minimum detection sensitivity was 15 fg of pure culture DNA from M. thermoacetica. Twenty commercial canned coffee beverages were then screened for the presence of M. thermoacetica, and two were shown to contain >1.3 and >1.0 CFU/ml, respectively. Therefore, the assay developed in this study may be useful for accurately tracking and quantifying M. thermoacetica and M. thermoautotrophica in beverage samples.


Asunto(s)
Bebidas/microbiología , Contaminación de Alimentos/análisis , Alimentos en Conserva/microbiología , Moorella/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Esporas Bacterianas/aislamiento & purificación , Cartilla de ADN/genética , Moorella/genética , Moorella/crecimiento & desarrollo , Esporas Bacterianas/genética , Esporas Bacterianas/crecimiento & desarrollo
20.
J Bacteriol ; 196(18): 3303-14, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25002540

RESUMEN

Moorella thermoacetica can grow with H2 and CO2, forming acetic acid from 2 CO2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown.


Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Moorella/enzimología , Proteínas Bacterianas/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Moorella/genética , Moorella/metabolismo
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