RESUMEN
Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests that corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community- and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.
Asunto(s)
Archaea , Bacterias , Corrinoides , Microbiota , Microbiología del Suelo , Vitamina B 12 , Corrinoides/metabolismo , Vitamina B 12/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Archaea/clasificación , Archaea/genética , Archaea/metabolismo , Archaea/aislamiento & purificación , Metagenoma , Suelo/química , PraderaRESUMEN
Soil microbial communities impact carbon sequestration and release, biogeochemical cycling, and agricultural yields. These global effects rely on metabolic interactions that modulate community composition and function. However, the physicochemical and taxonomic complexity of soil and the scarcity of available isolates for phenotypic testing are significant barriers to studying soil microbial interactions. Corrinoids-the vitamin B12 family of cofactors-are critical for microbial metabolism, yet they are synthesized by only a subset of microbiome members. Here, we evaluated corrinoid production and dependence in soil bacteria as a model to investigate the ecological roles of microorganisms involved in metabolic interactions. We isolated and characterized a taxonomically diverse collection of 161 soil bacteria from a single study site. Most corrinoid-dependent bacteria in the collection prefer B12 over other corrinoids, while all tested producers synthesize B12, indicating metabolic compatibility between producers and dependents in the collection. Furthermore, a subset of producers release B12 at levels sufficient to support dependent isolates in laboratory culture at estimated ratios of up to 1000 dependents per producer. Within our isolate collection, we did not find strong phylogenetic patterns in corrinoid production or dependence. Upon investigating trends in the phylogenetic dispersion of corrinoid metabolism categories across sequenced bacteria from various environments, we found that these traits are conserved in 47 out of 85 genera. Together, these phenotypic and genomic results provide evidence for corrinoid-based metabolic interactions among bacteria and provide a framework for the study of nutrient-sharing ecological interactions in microbial communities.
Asunto(s)
Bacterias , Corrinoides , Filogenia , Microbiología del Suelo , Bacterias/genética , Bacterias/clasificación , Bacterias/metabolismo , Bacterias/aislamiento & purificación , Corrinoides/metabolismo , Microbiota , Suelo/química , Vitamina B 12/metabolismo , ARN Ribosómico 16S/genéticaRESUMEN
The 22nd genetically encoded amino acid, pyrrolysine, plays a unique role in the key step in the growth of methanogens on mono-, di-, and tri-methylamines by activating the methyl group of these substrates for transfer to a corrinoid cofactor. Previous crystal structures of the Methanosarcina barkeri monomethylamine methyltransferase elucidated the structure of pyrrolysine and provide insight into its role in monomethylamine activation. Herein, we report the second structure of a pyrrolysine-containing protein, the M. barkeri trimethylamine methyltransferase MttB, and its structure bound to sulfite, a substrate analog of trimethylamine. We also report the structure of MttB in complex with its cognate corrinoid protein MttC, which specifically receives the methyl group from the pyrrolysine-activated trimethylamine substrate during methanogenesis. Together these structures provide key insights into the role of pyrrolysine in methyl group transfer from trimethylamine to the corrinoid cofactor in MttC.
Asunto(s)
Corrinoides , Metiltransferasas , Metiltransferasas/metabolismo , Metilaminas/metabolismo , Corrinoides/metabolismoRESUMEN
Vitamin B12 (the cyanated form of cobalamin cofactors) is best known for its essential role in human health. In addition to its function in human metabolism, cobalamin also plays important roles in microbial metabolism and can impact microbial community function. Cobalamin is a member of the structurally diverse family of cofactors known as cobamides that are produced exclusively by certain prokaryotes. Cobamides are considered shared nutrients in microbial communities because the majority of bacteria that possess cobamide-dependent enzymes cannot synthesize cobamides de novo. Furthermore, different microbes have evolved metabolic specificity for particular cobamides, and therefore, the availability of cobamides in the environment is important for cobamide-dependent microbes. Determining the cobamides present in an environment of interest is essential for understanding microbial metabolic interactions. By examining the abundances of different cobamides in diverse environments, including 10 obtained in this study, we find that, contrary to its preeminence in human metabolism, cobalamin is relatively rare in many microbial habitats. Comparison of cobamide profiles of mammalian gastrointestinal samples and wood-feeding insects reveals that host-associated cobamide abundances vary and that fecal cobamide profiles differ from those of their host gastrointestinal tracts. Environmental cobamide profiles obtained from aquatic, soil, and contaminated groundwater samples reveal that the cobamide compositions of environmental samples are highly variable. As the only commercially available cobamide, cobalamin is routinely supplied during microbial culturing efforts. However, these findings suggest that cobamides specific to a given microbiome may yield greater insight into nutrient utilization and physiological processes that occur in these habitats.
Asunto(s)
Cobamidas , Vitamina B 12 , Animales , Bacterias/metabolismo , Cobamidas/metabolismo , Mamíferos/metabolismo , Vitamina B 12/metabolismo , Corrinoides/química , Corrinoides/metabolismoRESUMEN
The functional and genomic diversity of the human gut microbiome is shaped by horizontal transfer of mobile genetic elements (MGEs). Characterized MGEs can encode genes beneficial for their host's self-defense (e.g., antibiotic resistance) or ability to compete for essential or limited resources (e.g., vitamins). Vitamin B12 and related compounds (corrinoids) are critical nutrients that enable colonization by members of the common gut microbe phylum, the Bacteroidetes. Herein, we identify a distinct class of MGEs in the Bacteroidetes responsible for the mobilization and exchange of the genes required for transport of corrinoids, a group of cyclic tetrapyrrole cofactors including vitamin B12 (btuGBFCD). This class includes two distinct groups of conjugative transposons (CTns) and one group of phage. Conjugative transfer and vitamin B12 transport activity of two of the CTns were confirmed in vitro and in vivo, demonstrating the important role MGEs play in distribution of corrinoid transporters in the Bacteroidetes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteroidetes/metabolismo , Corrinoides/metabolismo , Microbioma Gastrointestinal , Secuencias Repetitivas Esparcidas , Proteínas de Transporte de Membrana/metabolismo , Vitamina B 12/metabolismo , Animales , Proteínas Bacterianas/genética , Bacteroidetes/crecimiento & desarrollo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Complejo Vitamínico B/metabolismoRESUMEN
Bacteria and archaea possessing the hgcAB gene pair methylate inorganic mercury (Hg) to form highly toxic methylmercury. HgcA consists of a corrinoid binding domain and a transmembrane domain, and HgcB is a dicluster ferredoxin. However, their detailed structure and function have not been thoroughly characterized. We modeled the HgcAB complex by combining metagenome sequence data mining, coevolution analysis, and Rosetta structure calculations. In addition, we overexpressed HgcA and HgcB in Escherichia coli, confirmed spectroscopically that they bind cobalamin and [4Fe-4S] clusters, respectively, and incorporated these cofactors into the structural model. Surprisingly, the two domains of HgcA do not interact with each other, but HgcB forms extensive contacts with both domains. The model suggests that conserved cysteines in HgcB are involved in shuttling HgII, methylmercury, or both. These findings refine our understanding of the mechanism of Hg methylation and expand the known repertoire of corrinoid methyltransferases in nature.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Mercurio/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Bacterianas/genética , Corrinoides/metabolismo , Desulfovibrio desulfuricans/genética , Metagenoma , Metilación , Modelos Moleculares , Complejos Multiproteicos/genética , Filogenia , Conformación Proteica , Dominios Proteicos , Espectrofotometría UltravioletaRESUMEN
Corrinoid-dependent enzyme systems rely on the super-reduced state of the protein-bound corrinoid cofactor to be functional, for example, in methyl transfer reactions. Due to the low redox potential of the [CoII ]/[CoI ] couple, autoxidation of the corrinoid cofactor occurs and leads to the formation of the inactive [CoII ]-state. For the reactivation, which is an energy-demanding process, electrons have to be transferred from a physiological donor to the corrinoid cofactor by the help of a reductive activator protein. In this study, we identified reduced flavodoxin as electron donor for the ATP-dependent reduction of protein-bound corrinoid cofactors of bacterial O-demethylase enzyme systems. Reduced flavodoxin was generated enzymatically using pyruvate:ferredoxin/flavodoxin oxidoreductase rather than hydrogenase. Two of the four flavodoxins identified in Acetobacterium dehalogenans and Desulfitobacterium hafniense DCB-2 were functional in supplying electrons for corrinoid reduction. They exhibited a midpoint potential of about -400 mV (ESHE , pH 7.5) for the semiquinone/hydroquinone transition. Reduced flavodoxin could be replaced by reduced clostridial ferredoxin. It was shown that the low-potential electrons of reduced flavodoxin are first transferred to the iron-sulfur cluster of the reductive activator and finally to the protein-bound corrinoid cofactor. This study further highlights the importance of reduced flavodoxin, which allows maintaining a variety of enzymatic reaction cycles by delivering low-potential electrons.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Corrinoides/metabolismo , Electrones , Flavodoxina/metabolismo , Hidroquinonas/metabolismo , Oxidorreductasas/metabolismo , Acetobacterium/genética , Acetobacterium/metabolismo , Proteínas Bacterianas/genética , Desulfitobacterium/genética , Desulfitobacterium/metabolismo , Flavodoxina/química , Hidroquinonas/química , Oxidación-Reducción , Oxidorreductasas/genética , EspectrofotometríaRESUMEN
ATP:Co(I)rrinoid adenosyltransferases (ACATs) catalyze the transfer of the adenosyl moiety from co-substrate ATP to a corrinoid substrate. ACATs are grouped into three families, namely, CobA, PduO, and EutT. The EutT family of enzymes is further divided into two classes, depending on whether they require a divalent metal ion for activity (class I and class II). To date, a structure has not been elucidated for either class of the EutT family of ACATs. In this work, results of bioinformatics analyses revealed several conserved residues between the C-terminus of EutT homologues and the structurally characterized Lactobacillus reuteri PduO (LrPduO) homologue. In LrPduO, these residues are associated with ATP binding and formation of an intersubunit salt bridge. These residues were substituted, and in vivo and in vitro data support the conclusion that the equivalent residues in the metal-free (i.e., class II) Listeria monocytogenes EutT (LmEutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of LmEutT variants with substitutions at phenylalanine and tryptophan residues revealed that replacement of the phenylalanine residue at position 72 affected access to the substrate-binding site and replacement of a tryptophan residue at position 238 affected binding of the Cbl substrate to the active site. Unlike the PduO family of ACATs, a single phenylalanine residue is not responsible for displacement of the α-ligand. Together, these data suggest that while EutT enzymes share a conserved ATP-binding motif and an intersubunit salt bridge with PduO family ACATs, class II EutT family ACATs utilize an unidentified mechanism for Cbl lower-ligand displacement and reduction that is different from that of PduO and CobA family ACATs.
Asunto(s)
Corrinoides/metabolismo , Listeria monocytogenes/enzimología , Aciltransferasas/metabolismo , Adenosina Trifosfato/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Aldehído Oxidorreductasas/ultraestructura , Transferasas Alquil y Aril/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Catálisis , Dominio Catalítico , Cobalto/química , Cobamidas/metabolismo , Cinética , Limosilactobacillus reuteri/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Modelos Moleculares , Mutación , Transferasas/metabolismoRESUMEN
The corrinoid B12 is synthesized only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B12 dependence on multiple occasions, and we previously demonstrated that experimental evolution of the non-B12-requiring alga Chlamydomonas reinhardtii in media supplemented with B12 generated a B12-dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B12 auxotroph. Our analyses demonstrate that B12 deprivation of metE7 disrupts C1 metabolism, causes an accumulation of starch and triacylglycerides, and leads to a decrease in photosynthetic pigments, proteins, and free amino acids. B12 deprivation also caused a substantial increase in reactive oxygen species, which preceded rapid cell death. Survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B12 limitation and an increase in B12 use efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B12-producing bacterium. Therefore, although an early B12-dependent alga would likely be poorly adapted to coping with B12 deprivation, association with B12-producers can ensure long-term survival whilst also providing a suitable environment for evolving mechanisms to tolerate B12 limitation better.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Chlamydomonas/metabolismo , Corrinoides/metabolismo , Nitrógeno/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Organisms have evolved signal transduction systems to quickly adapt their lifestyle to internal and environmental changes. While protein kinases and two-component systems are widely distributed in Bacteria, they are also found in Archaea but are less diversified and abundant. In this work, we analysed the function of the kinase RdmS and its role in a putative two-component system in the methanogenic archaeon Methanosarcina acetivorans. RdmS is encoded upstream of the regulator MsrF, which activates the expression of the corrinoid/methyltransferase fusion protein MtsD. In contrast to a typical bacterial histidine kinase, RdmS lacks a membrane domain and the conserved histidine residue for phosphorylation, indicating a different mechanism of signal transduction in comparison to bacterial counterparts. RdmS covalently binds a heme cofactor and is thereby able to bind small molecules like CO and dimethyl sulfide. Interestingly, RdmS possesses a redox-dependent autophosphorylation activity, which, however, is independent of the bound heme cofactor. In fact, our experimental data suggest a thiol-based redox sensing mechanism by RdmS. Moreover, we were able to show that RdmS interacts with the regulator protein MsrF. From these data, we conclude RdmS to be a thiol-based kinase sensing redox changes and forming an archaeal multicomponent system with the regulators MsrG/F/C.
Asunto(s)
Proteínas Arqueales/metabolismo , Histidina Quinasa/metabolismo , Methanosarcina/enzimología , Metiltransferasas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Proteínas Arqueales/genética , Corrinoides/metabolismo , Regulación de la Expresión Génica Arqueal , Hemo/metabolismo , Histidina Quinasa/genética , Methanosarcina/genética , Methanosarcina/metabolismo , Metiltransferasas/genética , Oxidación-Reducción , Fosforilación , Transducción de Señal , Sulfuros/metabolismoRESUMEN
Human gut Bacteroides use surface-exposed lipoproteins to bind and metabolize complex polysaccharides. Although vitamins and other nutrients are also essential for commensal fitness, much less is known about how commensal bacteria compete with each other or the host for these critical resources. Unlike in Escherichia coli, transport loci for vitamin B12 (cobalamin) and other corrinoids in human gut Bacteroides are replete with conserved genes encoding proteins whose functions are unknown. Here we report that one of these proteins, BtuG, is a surface-exposed lipoprotein that is essential for efficient B12 transport in B. thetaiotaomicron. BtuG binds B12 with femtomolar affinity and can remove B12 from intrinsic factor, a critical B12 transport protein in humans. Our studies suggest that Bacteroides use surface-exposed lipoproteins not only for capturing polysaccharides, but also to acquire key vitamins in the gut.
Asunto(s)
Bacteroides/metabolismo , Membrana Celular/metabolismo , Tracto Gastrointestinal/microbiología , Lipoproteínas/metabolismo , Vitamina B 12/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte Biológico/genética , Corrinoides/metabolismo , Ligamiento Genético , Vida Libre de Gérmenes , Humanos , Factor Intrinseco/metabolismo , Ratones , Modelos Moleculares , Electricidad EstáticaRESUMEN
Vitamin B12 is made by only certain prokaryotes yet is required by a number of eukaryotes such as mammals, fish, birds, worms, and Protista, including algae. There is still much to learn about how this nutrient is trafficked across the domains of life. Herein, we describe ways to make a number of different corrin analogs with fluorescent groups attached to the main tetrapyrrole-derived ring. A further range of analogs were also constructed by attaching similar fluorescent groups to the ribose ring of cobalamin, thereby generating a range of complete and incomplete corrinoids to follow uptake in bacteria, worms, and plants. By using these fluorescent derivatives we were able to demonstrate that Mycobacterium tuberculosis is able to acquire both cobyric acid and cobalamin analogs, that Caenorhabditis elegans takes up only the complete corrinoid, and that seedlings of higher plants such as Lepidium sativum are also able to transport B12.
Asunto(s)
Bacterias/metabolismo , Caenorhabditis elegans/metabolismo , Colorantes Fluorescentes/metabolismo , Lepidium sativum/metabolismo , Vitamina B 12/metabolismo , Animales , Infecciones Bacterianas/microbiología , Transporte Biológico , Corrinoides/análisis , Corrinoides/metabolismo , Colorantes Fluorescentes/análisis , Humanos , Microscopía Fluorescente , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/análisisRESUMEN
In aqueous media at neutral pH, the binding of two cyanide molecules per cobinamide can be described by two formation constants, Kf1 = 1.1 (±0.6) × 105 M-1 and Kf2 = 8.5 (±0.1) × 104 M-1, or an overall cyanide binding constant of â¼1 × 1010 M-2. In comparison, the cyanide binding constants for cobalamin and a fully oxidized form of cytochrome c oxidase, each binding a single cyanide anion, were found to be 7.9 (±0.5) × 104 M-1 and 1.6 (±0.2) × 107 M-1, respectively. An examination of the cyanide-binding properties of cobinamide at neutral pH by stopped-flow spectrophotometry revealed two kinetic phases, rapid and slow, with apparent second-order rate constants of 3.2 (±0.5) × 103 M-1 s-1 and 45 (±1) M-1 s-1, respectively. Under the same conditions, cobalamin exhibited a single slow cyanide-binding kinetic phase with a second-order rate constant of 35 (±1) M-1 s-1. All three of these processes are significantly slower than the rate at which cyanide is bound by complex IV during enzyme turnover (>106 M-1 s-1). Overall, it can be understood from these findings why cobinamide is a measurably better cyanide scavenger than cobalamin, but it is unclear how either cobalt corrin can be antidotal toward cyanide intoxication as neither compound, by itself, appears able to out-compete cytochrome c oxidase for available cyanide. Furthermore, it has also been possible to unequivocally show in head-to-head comparison assays that the enzyme does indeed have greater affinity for cyanide than both cobalamin and cobinamide. A plausible resolution of the paradox that both cobalamin and cobinamide clearly are antidotal toward cyanide intoxication, involving the endogenous auxiliary agent nitric oxide, is suggested. Additionally, the catalytic consumption of oxygen by the cobalt corrins is demonstrated and, in the case of cobinamide, the involvement of cytochrome c when present. Particularly in the case of cobinamide, these oxygen-dependent reactions could potentially lead to erroneous assessment of the ability of the cyanide scavenger to restore the activity of cyanide-inhibited cytochrome c oxidase.
Asunto(s)
Cobalto/metabolismo , Corrinoides/metabolismo , Cianuros/metabolismo , Cianuros/toxicidad , Complejo IV de Transporte de Electrones/metabolismo , Oxígeno/metabolismo , Cobalto/química , Corrinoides/química , Cianuros/química , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/química , Estructura Molecular , Oxígeno/químicaRESUMEN
A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe-S cluster-containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe-S cluster cofactors and discuss physiological implications.
Asunto(s)
Proteínas Bacterianas/química , Chloroflexi/enzimología , Coenzimas/química , Corrinoides/química , Halógenos/química , Oxidorreductasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Benzoquinonas/química , Benzoquinonas/metabolismo , Biocatálisis , Chloroflexi/química , Chloroflexi/genética , Coenzimas/metabolismo , Corrinoides/metabolismo , Transporte de Electrón , Metabolismo Energético , Expresión Génica , Halógenos/metabolismo , Cinética , Modelos Moleculares , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Filogenia , Especificidad por Sustrato , Vitamina B 12/química , Vitamina B 12/metabolismoRESUMEN
Horseradish peroxidase was reconstituted with cobalt tetradehydrocorrin, rHRP(Co(TDHC)), as a structural analog of cobalamin coordinated with an imidazolate-like His residue, which is generally seen in native enzymes. In contrast to the previously reported cobalt tetradehydrocorrin-reconstituted myoglobin, rMb(Co(TDHC)), the HRP matrix was expected to provide strong axial ligation by His170 which has imidazolate character. rHRP(CoII(TDHC)) was characterized by EPR and its reaction with reductants indicates a negative shift of its redox potential compared to rMb(Co(TDHC)). Furthermore, aqua- and CN-forms of Co(III) state were prepared. The former species was obtained by oxidation of rHRP(CoII(TDHC)) with K3[Fe(CN)6]. The cyanide-coordinated Co(III) species in the latter was prepared by ligand exchange of rHRP(CoIII(OH)(TDHC)) with exogenous cyanide upon addition of KCN. The 13C NMR chemical shift of cyanide in rHRP(CoIII(CN)(TDHC)) was determined to be 121.8 ppm. IR measurements show that the cyanide of rHRP(CoIII(CN)(TDHC)) has a stretching frequency peak at 2144 cm-1. The 13C NMR and IR measurements indicate strong coordination of cyanide to CoIII(TDHC) relative to rMb(CoIII(CN)(TDHC)). Thus, the extent of π-back donation from the cobalt ion to the cyanide ion is relatively high in rHRP(CoIII(CN)(TDHC)). The pK 1/2 values of rHRP(CoIII(OH)(TDHC)) and rHRP(CoIII(CN)(TDHC)) are the same (pK 1/2 = 3.2) as determined by a pH titration experiment, indicating that cyanide ligation does not affect Co-His ligation, whereas cyanide ligation weakens the Co-His ligation in rMb(CoIII(CN)(TDHC)). Taken together, these results indicate that HRP reconstituted with cobalt tetradehydrocorrin is a suitable cobalamin-dependent enzyme model with imidazolate-like His residue.
Asunto(s)
Complejos de Coordinación/química , Corrinoides/química , Hemo/química , Histidina/química , Peroxidasa de Rábano Silvestre/química , Imidazoles/química , Cobalto/química , Complejos de Coordinación/metabolismo , Corrinoides/metabolismo , Hemo/metabolismo , Histidina/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Imidazoles/metabolismo , Modelos Moleculares , Conformación MolecularRESUMEN
The aim of this study is to obtain a systems-level understanding of the interactions between Dehalococcoides and corrinoid-supplying microorganisms by analyzing community structures and functional compositions, activities, and dynamics in trichloroethene (TCE)-dechlorinating enrichments. Metagenomes and metatranscriptomes of the dechlorinating enrichments with and without exogenous cobalamin were compared. Seven putative draft genomes were binned from the metagenomes. At an early stage (2 days), more transcripts of genes in the Veillonellaceae bin-genome were detected in the metatranscriptome of the enrichment without exogenous cobalamin than in the one with the addition of cobalamin. Among these genes, sporulation-related genes exhibited the highest differential expression when cobalamin was not added, suggesting a possible release route of corrinoids from corrinoid producers. Other differentially expressed genes include those involved in energy conservation and nutrient transport (including cobalt transport). The most highly expressed corrinoid de novo biosynthesis pathway was also assigned to the Veillonellaceae bin-genome. Targeted quantitative PCR (qPCR) analyses confirmed higher transcript abundances of those corrinoid biosynthesis genes in the enrichment without exogenous cobalamin than in the enrichment with cobalamin. Furthermore, the corrinoid salvaging and modification pathway of Dehalococcoides was upregulated in response to the cobalamin stress. This study provides important insights into the microbial interactions and roles played by members of dechlorinating communities under cobalamin-limited conditions.IMPORTANCE The key chloroethene-dechlorinating bacterium Dehalococcoides mccartyi is a cobalamin auxotroph, thus acquiring corrinoids from other community members. Therefore, it is important to investigate the microbe-microbe interactions between Dehalococcoides and the corrinoid-providing microorganisms in a community. This study provides systems-level information, i.e., taxonomic and functional compositions and dynamics of the supportive microorganisms in dechlorinating communities under different cobalamin conditions. The findings shed light on the important roles of Veillonellaceae species in the communities compared to other coexisting community members in producing and providing corrinoids for Dehalococcoides species under cobalamin-limited conditions.
Asunto(s)
Chloroflexi/genética , Chloroflexi/metabolismo , Perfilación de la Expresión Génica , Metagenómica , Consorcios Microbianos , Vitamina B 12/metabolismo , Bacterias/metabolismo , Biodegradación Ambiental , Vías Biosintéticas/genética , Chloroflexi/efectos de los fármacos , Corrinoides/metabolismo , Genoma Bacteriano , Halogenación , Consorcios Microbianos/efectos de los fármacos , Consorcios Microbianos/genética , Tricloroetileno/metabolismo , Veillonellaceae/genética , Veillonellaceae/metabolismo , Vitamina B 12/farmacologíaRESUMEN
A cobalamin (Cbl) cofactor in corrinoid iron-sulfur protein (CoFeSP) is the primary methyl group donor and acceptor in biological carbon oxide conversion along the reductive acetyl-CoA pathway. Changes of the axial coordination of the cobalt ion within the corrin macrocycle upon redox transitions in aqua-, methyl-, and cyano-Cbl bound to CoFeSP or in solution were studied using X-ray absorption spectroscopy (XAS) at the Co K-edge in combination with density functional theory (DFT) calculations, supported by metal content and cobalt redox level quantification with further spectroscopic methods. Calculation of the highly variable pre-edge X-ray absorption features due to core-to-valence (ctv) electronic transitions, XANES shape analysis, and cobalt-ligand bond lengths determination from EXAFS has yielded models for the molecular and electronic structures of the cobalt sites. This suggested the absence of a ligand at cobalt in CoFeSP in α-position where the dimethylbenzimidazole (dmb) base of the cofactor is bound in Cbl in solution. As main species, (dmb)CoIII(OH2), (dmb)CoII(OH2), and (dmb)CoIII(CH3) sites for solution Cbl and CoIII(OH2), CoII(OH2), and CoIII(CH3) sites in CoFeSP-Cbl were identified. Our data support binding of a serine residue from the reductive-activator protein (RACo) of CoFeSP to the cobalt ion in the CoFeSP-RACo protein complex that stabilizes Co(II). The absence of an α-ligand at cobalt not only tunes the redox potential of the cobalamin cofactor into the physiological range, but is also important for CoFeSP reactivation.
Asunto(s)
Proteínas Bacterianas/química , Cobalto/química , Corrinoides/química , Proteínas Hierro-Azufre/química , Vitamina B 12/química , Espectroscopía de Absorción de Rayos X/métodos , Proteínas Bacterianas/metabolismo , Fenómenos Químicos , Cobalto/metabolismo , Corrinoides/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Firmicutes/metabolismo , Iones/química , Iones/metabolismo , Proteínas Hierro-Azufre/metabolismo , Ligandos , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Soluciones , Vitamina B 12/metabolismoRESUMEN
The microbial mechanisms and key metabolites that shape the composition of the human gut microbiota are largely unknown, impeding efforts to manipulate dysbiotic microbial communities toward stability and health. Vitamins, which by definition are not synthesized in sufficient quantities by the host and can mediate fundamental biological processes in microbes, represent an attractive target for reshaping microbial communities. Here, we discuss how vitamin B12 (cobalamin) impacts diverse host-microbe symbioses. Although cobalamin is synthesized by some human gut microbes, it is a precious resource in the gut and is likely not provisioned to the host in significant quantities. However, this vitamin may make an unrecognized contribution in shaping the structure and function of human gut microbial communities.
Asunto(s)
Tracto Gastrointestinal/microbiología , Microbiota , Vitamina B 12/metabolismo , Animales , Corrinoides/análisis , Corrinoides/metabolismo , Tracto Gastrointestinal/metabolismo , Humanos , Vitamina B 12/análisisRESUMEN
The role of the corrinoid cofactor in reductive dehalogenation catalysis by tetrachloroethene reductive dehalogenase (PceA) of Sulfurospirillum multivorans was investigated using isotope analysis of carbon and chlorine. Crude extracts containing PceA--harboring either a native norpseudo-B12 or the alternative nor-B12 cofactor--were applied for dehalogenation of tetrachloroethene (PCE) or trichloroethene (TCE), and compared to abiotic dehalogenation with the respective purified corrinoids (norpseudovitamin B12 and norvitamin B12), as well as several commercially available cobalamins and cobinamide. Dehalogenation of TCE resulted in a similar extent of C and Cl isotope fractionation, and in similar dual-element isotope slopes (εC/εCl) of 5.0-5.3 for PceA enzyme and 3.7-4.5 for the corrinoids. Both observations support an identical reaction mechanism. For PCE, in contrast, observed C and Cl isotope fractionation was smaller in enzymatic dehalogenation, and dual-element isotope slopes (2.2-2.8) were distinctly different compared to dehalogenation mediated by corrinoids (4.6-7.0). Remarkably, εC/εCl of PCE depended in addition on the corrinoid type: εC/εCl values of 4.6 and 5.0 for vitamin B12 and norvitamin B12 were significantly different compared to values of 6.9 and 7.0 for norpseudovitamin B12 and dicyanocobinamide. Our results therefore suggest mechanistic and/or kinetic differences in catalytic PCE dehalogenation by enzymes and different corrinoids, whereas such differences were not observed for TCE.
Asunto(s)
Cloro/análisis , Corrinoides/metabolismo , Epsilonproteobacteria/enzimología , Halogenación , Hidrolasas/metabolismo , Tetracloroetileno/metabolismo , Tricloroetileno/metabolismo , Isótopos de Carbono , Fraccionamiento Químico , Corrinoides/química , Marcaje IsotópicoRESUMEN
Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I).