RESUMEN
Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.
Asunto(s)
Alteromonadaceae , Ligandos , Rhodobacteraceae , Vitamina B 12 , Océano Atlántico , Técnicas de Cocultivo , Interacciones Microbianas , Profagos/genética , Profagos/crecimiento & desarrollo , Profagos/metabolismo , Vitamina B 12/biosíntesis , Vitamina B 12/química , Vitamina B 12/metabolismo , Alteromonadaceae/crecimiento & desarrollo , Alteromonadaceae/metabolismo , Rhodobacteraceae/citología , Rhodobacteraceae/metabolismo , Rhodobacteraceae/virología , Ribonucleósidos/metabolismo , Cobamidas/metabolismo , EcosistemaRESUMEN
The human microbiome is a rich factory for metabolite production and emerging data has led to the concept that orally administered microbial strains can synthesize metabolites with neuroactive potential. Recent research from ex vivo and murine models suggests translational potential for microbes to regulate anxiety and depression through the gut-brain axis. However, so far, less emphasis has been placed on the selection of specific microbial strains known to produce the required key metabolites and the formulation in which microbial compositions are delivered to the gut. Here, we describe a double-capsule technology to deliver high numbers of metabolically active cells derived from the 24-strain probiotic product SH-DS01 to the gastrointestinal tract, including the small intestine, where immune responses and adsorption of metabolites into the bloodstream occur. Based on its genome sequence, Limosilactobacillus reuteri SD-LRE2-IT was predicted to have the genetic capacity to de novo produce a specific metabolite of interest to brain health, vitamin B12, which could be confirmed in vitro. Taken together, our data conceptualizes the importance of rationally defined microbial strain characterization based on genomics and metabolomics data, combined with carefully designed capsule technology for delivery of live cells and concomitant functionality in and beyond the gut ecosystem.
Asunto(s)
Microbioma Gastrointestinal , Limosilactobacillus reuteri/metabolismo , Probióticos/administración & dosificación , Vitamina B 12/biosíntesis , Genómica , Humanos , Análisis de Secuencia de ADNRESUMEN
The foodborne pathogen Listeria monocytogenes can form proteinaceous organelles called bacterial microcompartments (BMCs) that optimize the utilization of substrates, such as 1,2-propanediol, and confer an anaerobic growth advantage. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol, next to acetate and lactate. Rhamnose-derived 1,2-propanediol was found to link with BMCs in some human pathogens such as Salmonella enterica, but the involvement of BMCs in rhamnose metabolism and potential physiological effects on L. monocytogenes are still unknown. In this study, we first test the effect of rhamnose uptake and utilization on anaerobic growth of L. monocytogenes EGDe without or with added vitamin B12, followed by metabolic analysis. We show that vitamin B12-dependent activation of pdu stimulates metabolism and anaerobic growth of L. monocytogenes EGDe on rhamnose via 1,2-propanediol degradation into 1-propanol and propionate. Transmission electron microscopy of pdu-induced cells shows that BMCs are formed, and additional proteomics experiments confirm expression of pdu BMC shell proteins and enzymes. Finally, we discuss the physiological effects and energy efficiency of L. monocytogenes pdu BMC-driven anaerobic rhamnose metabolism and the impact on competitive fitness in environments such as the human intestine. IMPORTANCE Listeria monocytogenes is a foodborne pathogen causing severe illness and, as such, it is crucial to understand the molecular mechanisms contributing to its survival strategy and pathogenicity. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol. In our previous study, the utilization of 1,2-propanediol (pdu) in L. monocytogenes was proved to be metabolized in bacterial microcompartments (BMCs), which are self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we show that the vitamin B12-dependent activation of pdu stimulates metabolism and anaerobic growth of L. monocytogenes EGDe on rhamnose via BMC-dependent 1,2-propanediol utilization. Combined with metabolic and proteomics analysis, our discussion on the physiological effects and energy efficiency of BMC-driven rhamnose metabolism shed new light to understand the impact on L. monocytogenes competitive fitness in ecosystems such as the human intestine.
Asunto(s)
Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/metabolismo , Glicoles de Propileno/metabolismo , Ramnosa/metabolismo , Vitamina B 12/metabolismo , Anaerobiosis , Proteínas Bacterianas/genética , Humanos , Intestinos/microbiología , Intestinos/fisiología , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/genética , Redes y Vías Metabólicas/efectos de los fármacos , Proteómica/métodos , Vitamina B 12/biosíntesis , Vitamina B 12/farmacologíaRESUMEN
Vitamin B12 (VB12 ) is an indispensable cofactor of metabolic enzymes and has been widely used in the food and pharmaceutical industries. In this study, the effects of medium composition on VB12 production by Propionibacterium freudenreichii were evaluated and optimized based on statistical experiments. The results showed that glucose, yeast extract, KH2 PO4 , and glycine have significant effects on VB12 production. The final titer of VB12 reached 8.32 ± 0.02 mg/L, representing a 120% increase over the non-optimized culture medium. We employed a metabolomics approach to analyze the differences of metabolite concentrations in P. freudenreichii cells cultivated in the original medium and optimized fermentation medium. Using multivariate data analysis, we identified a range of correlated metabolites, illustrating how metabolomics can be used to explain VB12 production changes by corresponding differences in the overall cellular metabolism. The concentrations of many metabolic intermediates of glycolysis, the Wood-Werkman cycle, the TCA cycle, and amino acid metabolism were increased, which contributed to the synthesis of propionic acid and VB12 due to an improved supply of energy and precursors.
Asunto(s)
Propionibacterium freudenreichii/metabolismo , Vitamina B 12/biosíntesis , Aminoácidos/metabolismo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Glucosa/metabolismoRESUMEN
Formulations with lactate as an antimicrobial and high-pressure processing (HPP) as a lethal treatment are combined strategies used to control L. monocytogenes in cooked meat products. Previous studies have shown that when HPP is applied in products with lactate, the inactivation of L. monocytogenes is lower than that without lactate. The purpose of the present work was to identify the molecular mechanisms underlying the piezo-protection effect of lactate. Two L. monocytogenes strains (CTC1034 and EGDe) were independently inoculated in a cooked ham model medium without and with 2.8% potassium lactate. Samples were pressurized at 400 MPa for 10 min at 10 °C. Samples were subjected to RNA extraction, and a shotgun transcriptome sequencing was performed. The short exposure of L. monocytogenes cells to lactate through its inoculation in a cooked ham model with lactate 1h before HPP promoted a shift in the pathogen's central metabolism, favoring the metabolism of propanediol and ethanolamine together with the synthesis of the B12 cofactor. Moreover, the results suggest an activated methyl cycle that would promote modifications in membrane properties resulting in an enhanced resistance of the pathogen to HPP. This study provides insights on the mechanisms developed by L. monocytogenes in response to lactate and/or HPP and sheds light on the understanding of the piezo-protective effect of lactate.
Asunto(s)
Membrana Externa Bacteriana/efectos de los fármacos , Ácidos Grasos/metabolismo , Ácido Láctico/farmacología , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/metabolismo , Productos de la Carne/microbiología , Animales , Antibacterianos/farmacología , ADN Bacteriano , Etanolamina/metabolismo , Manipulación de Alimentos/métodos , Microbiología de Alimentos , Industria de Procesamiento de Alimentos/métodos , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Listeria monocytogenes/genética , Listeriosis/microbiología , Membranas/efectos de los fármacos , Redes y Vías Metabólicas , Presión , Glicoles de Propileno/metabolismo , Porcinos , Temperatura , Factores de Tiempo , Vitamina B 12/biosíntesisRESUMEN
Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans, that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.
Asunto(s)
Vitamina B 12/biosíntesis , Vitamina B 12/genética , Vitamina B 12/metabolismo , Bacterias/metabolismo , Biotecnología/métodos , Fermentación/genética , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas , Metagenoma/genética , Metagenómica/métodos , Estudios ProspectivosRESUMEN
Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cobalto/metabolismo , Vitamina B 12/biosíntesis , Zinc/metabolismo , Escherichia coli , GTP Fosfohidrolasas , Metales/metabolismo , SalmonellaRESUMEN
Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B12) and its precursor, dicyanocobinamide ([CN]2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Metionina/biosíntesis , Mycobacterium smegmatis/metabolismo , Vitamina B 12/biosíntesis , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mycobacterium smegmatis/genética , RiboswitchRESUMEN
Sinorhizobium meliloti 320 is a vitamin B12 (VB12 ) high-producing strain that has been isolated and identified in our previous study. Because the regulatory toolbox for S. meliloti is limited, we searched for new genetic components and identified the two xylose-inducible promoters PA and PB based on a promoter-probe vector with a green fluorescent protein (GFP) as reporter. Compared with the ParaA promoter from S. meliloti, both promoters exhibited higher induced expression and lower basal expression. Subsequently, the influence of glucose or sucrose on the expression of GFP driven by these three promoters was assayed. Glucose repressed all three promoters, and the expression of ParaA was the lowest in the presence of glucose. Although sucrose repressed the expression of PA by 35% and improved the expression of ParaA by 16%, the expression level of PA was the highest and was 13% higher than that of ParaA . Lastly, we overexpressed the hemA gene in the C4 pathway using the PA promoter in S. meliloti 320, and the VB12 production of the engineered strain increased by 11%. The VB12 production was further increased by 11% by adding 0.1% sodium succinate to the culture medium.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas , Sinorhizobium meliloti , Vitamina B 12 , Xilosa , Vectores Genéticos/genética , Plásmidos/genética , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Vitamina B 12/biosíntesis , Vitamina B 12/genética , Xilosa/genética , Xilosa/metabolismo , Xilosa/farmacologíaRESUMEN
CblC is a chaperone that catalyzes removal of the ß-axial ligand of cobalamin (or B12), generating cob(II)alamin in an early step in the cofactor trafficking pathway. Cob(II)alamin is subsequently partitioned to support cellular needs for the synthesis of active cobalamin cofactor derivatives. In addition to the ß-ligand transferase activity, the Caenorhabdiitis elegans CblC (ceCblC) and clinical R161G/Q variants of the human protein exhibit robust thiol oxidase activity, converting glutathione to glutathione disulfide while concomitantly reducing O2 to H2O2. The chemical efficiency of the thiol oxidase side reaction during ceCblC-catalyzed dealkylation of alkylcobalamins is noteworthy in that it effectively scrubs ambient oxygen from the reaction mixture, leading to air stabilization of the highly reactive cob(I)alamin product. In this study, we report that the enhanced thiol oxidase activity of ceCblC requires the presence of KCl, which explains how the wasteful thiol oxidase activity is potentially curtailed inside cells where the chloride concentration is low. We have captured an unusual chlorocob(II)alamin intermediate that is formed in the presence of potassium chloride, a common component of the reaction buffer, and have characterized it by electron paramagnetic resonance, magnetic circular dichroism, and computational analyses. The ability to form a chlorocob(II)alamin intermediate could represent an evolutionary vestige in ceCblC, which is structurally related to bacterial B12-dependent reductive dehalogenases that have been proposed to form halogen cob(II)alamin intermediates in their catalytic cycle.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Glutatión Transferasa/metabolismo , Oxidorreductasas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Vitamina B 12/biosíntesis , Biocatálisis , Proteínas de Caenorhabditis elegans/química , Glutatión Transferasa/química , Modelos Moleculares , Proteínas Proto-Oncogénicas c-cbl/química , Vitamina B 12/químicaRESUMEN
BACKGROUND: Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production. RESULTS: By designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L-1, respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L-1, representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA. CONCLUSIONS: Through multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L-1 in E. coli. This lays the foundation for high-yield production of vitamin B12 in E. coli and will hopefully accelerate its industrial production.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/metabolismo , Ingeniería Metabólica , Uroporfirinas/biosíntesis , Vitamina B 12/biosíntesis , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , OperónRESUMEN
Vitamin B12 is a crucial fine chemical that is widely used in the pharmaceutical, food and chemical industries, and its production solely dependents on microbial fermentation. We previously constructed an artificial vitamin B12 biosynthesis pathway in Escherichia coli, but the yield of the engineered strains was low. Here, we removed metabolic bottlenecks of the vitamin B12 biosynthesis pathway in engineered E. coli strains. After screening cobB genes from different sources, optimizing the expression of cobN and customizing the ribosome binding sites of cobS and cobT, the vitamin B12 yield increased to 152.29 µg/g dry cell weight (DCW). Optimization of the downstream module, which converts co(II)byrinic acid a,c-diamide into adenosylcobinamide phosphate, elevated the vitamin B12 yield to 249.04 µg/g DCW. A comparison of a variety of equivalent components indicated that glucose and corn steep liquor are optimal carbon and nitrogen sources, respectively. Finally, an orthogonal array design was applied to determine the optimal concentrations of glucose and nitrogen sources including corn steep liquor and yeast extract, through which a vitamin B12 yield of 530.29 µg/g DCW was obtained. The metabolic modifications and optimization of fermentation conditions achieved in this study offer a basis for further improving vitamin B12 production in E. coli and will hopefully accelerate its industrial application.
Asunto(s)
Vías Biosintéticas , Medios de Cultivo/química , Escherichia coli , Ingeniería Metabólica , Vitamina B 12 , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Vitamina B 12/biosíntesis , Vitamina B 12/genéticaRESUMEN
Vitamin B12 deficiency still persists, mainly caused by low intake of animal food products affecting vegetarians, vegans, and populations of underdeveloped countries. In this study, we investigate the biosynthesis of vitamin B12 by potential probiotic bacterium using an agroindustry residue, the liquid acid protein residue of soybean (LAPRS), as a low-cost, animal derivate-free alternative culture medium. Cultures of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 growing in LAPRS for vitamin B12 biosynthesis were studied using the Plackett-Burman experimental approach, followed by a central composite design 22 to optimize the concentration of significant variables. We also performed a proteolytic treatment of LAPRS and evaluated the optimized-hydrolyzed medium influence on the microbial growth and metabolism in shaker flask and bioreactor experiments. In this all-plant source medium, P. freudenreichii subsp. shermanii produced high concentrations of cells and high amounts of vitamin B12 (0.6 mg/g cells) after process optimization. These results suggest the possibility of producing vitamin B12 by a potential probiotic bacterium in a very cheap, animal derivate-free medium to address the needs of specific population groups, at the same time reducing the production costs of this essential vitamin.
Asunto(s)
Reactores Biológicos/microbiología , Medios de Cultivo , Propionibacterium/metabolismo , Proteínas de Soja/química , Vitamina B 12/biosíntesis , Agricultura , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Vitamina B 12/análisis , Vitamina B 12/químicaRESUMEN
Vitamin B12 (VB12) is a critical micronutrient that controls DNA metabolic pathways to maintain the host genomic stability and tissue homeostasis. We recently reported that the newly discovered commensal Propionibacterium, P. UF1, regulates the intestinal immunity to resist pathogen infection, which may be attributed in part to VB12 produced by this bacterium. Here we demonstrate that VB12 synthesized by P. UF1 is highly dependent on cobA gene-encoding uroporphyrinogen III methyltransferase, and that this vitamin distinctively regulates the cobA operon through its 5' untranslated region (5' UTR). Furthermore, conserved secondary structure and mutagenesis analyses revealed a VB12-riboswitch, cbiMCbl (140 bp), within the 5' UTR that controls the expression of downstream genes. Intriguingly, ablation of the cbiMCbl significantly dysregulates the biosynthesis of VB12, illuminating the significance of this riboswitch for bacterial VB12 biosynthesis. Collectively, our finding is an in-depth report underscoring the regulation of VB12 within the beneficial P. UF1 bacterium, through which the commensal metabolic network may improve gut bacterial cross-feeding and human health.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Propionibacterium/metabolismo , Riboswitch/genética , Vitamina B 12/biosíntesis , Regiones no Traducidas 5'/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microbioma Gastrointestinal/fisiología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutagénesis Sitio-Dirigida , Operón/genética , Probióticos/metabolismo , Propionibacterium/genéticaRESUMEN
Akkermansia muciniphila is a mucin-degrading bacterium found in the gut of most humans and is considered a "next-generation probiotic." However, knowledge of the genomic and physiological diversity of human-associated Akkermansia sp. strains is limited. Here, we reconstructed 35 metagenome-assembled genomes and combined them with 40 publicly available genomes for comparative genomic analysis. We identified at least four species-level phylogroups (AmI to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B12) biosynthesis within the AmII and AmIII phylogroups. To verify these predictions, 10 Akkermansia strains were isolated from adults and screened for vitamin B12 biosynthesis genes via PCR. Two AmII strains were positive for the presence of cobalamin biosynthesis genes, while all 9 AmI strains tested were negative. To demonstrate vitamin B12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B12 was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B12 production by the AmII phylogroup. This novel physiological trait of human-associated Akkermansia strains may affect how these bacteria interact with the human host and other members of the human gut microbiome.IMPORTANCE There is significant interest in the therapeutic and probiotic potential of the common gut bacterium Akkermansia muciniphila However, knowledge of both the genomic and physiological diversity of this bacterial lineage is limited. Using a combination of genomic, molecular biological, and traditional microbiological approaches, we identified at least four species-level phylogroups with differing functional potentials that affect how these bacteria interact with both their human host and other members of the human gut microbiome. Specifically, we identified and isolated Akkermansia strains that were able to synthesize vitamin B12 The ability to synthesize this important cofactor broadens the physiological capabilities of human-associated Akkermansia strains, fundamentally altering our understanding of how this important bacterial lineage may affect human health.
Asunto(s)
Genoma Bacteriano , Verrucomicrobia/genética , Vitamina B 12/biosíntesis , Vitaminas/biosíntesis , Niño , Preescolar , Genómica , Humanos , Verrucomicrobia/metabolismo , Vitamina B 12/genética , Vitaminas/genéticaRESUMEN
A network of components from different metabolic pathways is the building scaffold of an indispensable compound in the human organism-vitamin B12 . The biosynthesis of this compound is restricted to a limited number of representatives of bacteria and archaea, while vitamin B12 -dependent enzymes are spread through several domains of life. Different attempts have been performed to increase vitamin B12 levels in dietary products, particularly in vegetarian and vegan dietary regimes. The integration of vitamin B12 in microalgae through symbiosis with microorganisms generally recognized as safe, for example the probiotic Lactobacillus reuteri, can even increase the nutritional value of the microalgal biomass. This study reviews the microbial production of vitamin B12 based on genetic analyses and chemical studies. Recent genetic approaches are focused, particularly potential metabolic engineering targets to increase vitamin B12 production. The bioincorporation of vitamin B12 in microalgae as an attempt to provide a superfood is also reviewed. PRACTICAL APPLICATIONS: Novel food habits (i.e., vegan lifestyle) may lack relevant nutrients, including vitamin B12 . Therefore, there is an increased demand for dietary products rich in vitamin B12 . Of potential interest is the provision of microbial-based superfood rich in numerous nutrients, including this vitamin. This manuscript provides an in-depth and timely overview on vitamin B12 biosynthesis and the major advances on metabolic engineering for improved vitamin B12 production by probiotic bacteria and other microorganisms generally recognized as safe. A relevant advance would result from the bioincorporation of vitamin B12 in alternative microorganisms (non-vitamin B12 producers) increasingly recognized as superfood, that is microalgae.
Asunto(s)
Microalgas/metabolismo , Vitamina B 12/biosíntesis , Animales , Tecnología de Alimentos , Fenómenos Fisiológicos de la Nutrición , Vitamina B 12/químicaRESUMEN
Helicobacter pylori infection (HPI) is a prevalent infectious disease associated with gastric ulcer, gastric cancer, and many nongastrointestinal disorders. To identify genes that may serve as microbial markers for HPI, we performed shotgun metagenomic sequencing of fecal samples from 313 Chinese volunteers who had undergone a C14 breath test. Through comparing differences in intestinal microbial community structure between H. pylori-positive and H. pylori-negative individuals, we identified 58 HPI-associated microbial species (P < 0.05, Wilcoxon test). A classifier based on microbial species markers showed high diagnostic ability for HPI (AUC = 0.84). Furthermore, levels of gut microbial vitamin B12 (VB12) biosynthesis and plasma VB12 were significantly lower in H. pylori-positive individuals compared with H. pylori-negative individuals (P < 0.05, Wilcoxon test). This study reveals that certain alterations in gut microbial species and functions are associated with HPI and shows that gut microbial shift in HPI patients may indirectly elevate the risk of VB12 deficiency.
Asunto(s)
Microbioma Gastrointestinal/genética , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Adulto , Anciano , China , Femenino , Helicobacter pylori/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Vitamina B 12/biosíntesis , Vitamina B 12/sangre , Adulto JovenRESUMEN
The spirochete species Leptospira interrogans is the most common cause of leptospirosis, producing acute to chronic infections in most mammalian species. This pathogenic bacterium has an ability to evolve in many ways to occupy various environments and hosts. In this study, we performed chronology analysis to look for insight into the emergence of Leptospira species, focusing on L. interrogans, and investigated gene gain and loss related to their adaptation in strains isolated from asymptomatic dogs. Chronology analysis revealed that the emergence of L. interrogans was around 53.7 million years ago (MYA), corresponding to the Paleogene period that coincided with an optimal climatic temperature and the evolution of suitable mammalian hosts. Gene families encoding for vitamin B2, B12 biosynthesis, cell adhesion and external encapsulating structure were found to be enriched in L. interrogans isolated from the urine of asymptomatic dogs. The activity of these gene families may have favored adaptations resulting in chronic infections.
Asunto(s)
Enfermedades de los Perros/microbiología , Leptospira/fisiología , Leptospirosis/veterinaria , Familia de Multigenes , Riboflavina/biosíntesis , Vitamina B 12/biosíntesis , Animales , Adhesión Bacteriana , Perros , Evolución Molecular , Genoma Bacteriano , Genómica/métodos , Leptospira/aislamiento & purificación , Cadenas de Markov , Filogenia , Secuenciación Completa del GenomaRESUMEN
Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis's Vitamin B12 metabolism and related pathways to reduce the pathogen's virulence in its hosts.
Asunto(s)
Corynebacterium pseudotuberculosis/metabolismo , Vitamina B 12/metabolismo , Adenina/química , Adenina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cinética , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Estructura Secundaria de Proteína , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Espectrometría de Fluorescencia , Homología Estructural de Proteína , Suramina/química , Suramina/metabolismo , Vitamina B 12/biosíntesis , Vitamina B 12/químicaRESUMEN
Only a few cobalamin-producing bacterial species are known which are suitable for food fermentations. The strain of Acetobacter pasteurianus DSM 3509 was found to have the capability to synthesize cobalamin. A survival test and a preliminary genetic study of the gene of uroporphyrinogen-III synthase indicated the ability to synthesize cobalamin. By a modified microbiological assay based on Lactobacillus delbrueckii ssp. lactis DSM 20355, 4.57 ng/mL of cyanocorrinoids and 0.75 ng/mL of noncorrinoid growth factors were detected. The product extracted and isolated by immunoaffinity chromatography in its cyanide form had the similar UV spectrum as standard cyanocobalamin and Coα-[α-(7-adenyl)]-(Coß-cyano) cobamide also known as pseudovitamin B12 produced by Lactobacillus reuteri DSM 20016. The chromatographically separated product of A. pasteurianus was subjected to mass spectrometrical analysis. There, its fragmentation pattern turned out to be equivalent to that of cyanocobalamin also produced by Propionibacterium freudenreichii ssp. freudenreichii DSM 20271 and clearly differs from pseudovitamin B12. Due to the presence of this species in several food applications, there might be cobalamin residues in food fermented with these bacteria.