RESUMEN
N-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1-exo-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H· abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H· from C7 â¼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2H2O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.
Asunto(s)
Hierro , Ácidos Cetoglutáricos , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Hierro/metabolismo , Hierro/química , Hidroxilación , Ciclización , Oxigenasas/metabolismo , Oxigenasas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/químicaRESUMEN
Hyoscyamine 6ß-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples the newly installed C6 oxygen to C7 to produce the drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C-H bonds, but it remains unclear how the enzyme switches the target site and promotes (C6)O-C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen wouldâanalogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, N-acetylnorloline synthase (LolO)âcoordinate as alkoxide to the C7-H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here, we provide structural and kinetic evidence that H6H does not employ substrate coordination or repositioning for the epoxidation step but instead exploits the distinct spatial dependencies of competitive C-H cleavage (C6 vs C7) and C-O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6ß-hydroxyhyoscyamine suggest that a modest (â¼10°) shift in the Fe-O-H(C7) approach angle is sufficient to change the outcome. The 7-hydroxylation:epoxidation partition ratios of both proteins increase more than 5-fold in 2H2O, reflecting an epoxidation-specific requirement for cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished by iron coordination in advance of C-H cleavage.
Asunto(s)
Oxigenasas de Función Mixta , Hidroxilación , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/química , Especificidad por Sustrato , Biocatálisis , Compuestos Epoxi/química , Compuestos Epoxi/metabolismoRESUMEN
Acidophilic bacteria and archaea inhabit extreme geochemical "islands" that can tell us when and how geographic barriers affect the biogeography of microorganisms. Here, we describe microbial communities from extremely acidic (pH 0 to 1) biofilms, known as snottites, from hydrogen sulfide-rich caves. Given the extreme acidity and subsurface location of these biofilms, and in light of earlier work showing strong geographic patterns among snottite Acidithiobacillus populations, we investigated their structure and diversity in order to understand how geography might impact community assembly. We used 16S rRNA gene cloning and fluorescence in situ hybridization (FISH) to investigate 26 snottite samples from four sulfidic caves in Italy and Mexico. All samples had very low biodiversity and were dominated by sulfur-oxidizing bacteria in the genus Acidithiobacillus. Ferroplasma and other archaea in the Thermoplasmatales ranged from 0 to 50% of total cells, and relatives of the bacterial genera Acidimicrobium and Ferrimicrobium were up to 15% of total cells. Rare phylotypes included Sulfobacillus spp. and members of the phyla "Candidatus Dependentiae" and "Candidatus Saccharibacteria" (formerly TM6 and TM7). Although the same genera of acidophiles occurred in snottites on separate continents, most members of those genera represent substantially divergent populations, with 16S rRNA genes that are only 95 to 98% similar. Our findings are consistent with a model of community assembly where sulfidic caves are stochastically colonized by microorganisms from local sources, which are strongly filtered through environmental selection for extreme acid tolerance, and these different colonization histories are maintained by dispersal restrictions within and among caves. IMPORTANCE Microorganisms that are adapted to extremely acidic conditions, known as extreme acidophiles, are catalysts for rock weathering, metal cycling, and mineral formation in naturally acidic environments. They are also important drivers of large-scale industrial processes such as biomining and contaminant remediation. Understanding the factors that govern their ecology and distribution can help us better predict and utilize their activities in natural and engineered systems. However, extremely acidic habitats are unusual in that they are almost always isolated within circumneutral landscapes. So where did their acid-adapted inhabitants come from, and how do new colonists arrive and become established? In this study, we took advantage of a unique natural experiment in Earth's subsurface to show how isolation may have played a role in the colonization history, community assembly, and diversity of highly acidic microbial biofilms.
Asunto(s)
Acidithiobacillus , Bacterias , ARN Ribosómico 16S/genética , Hibridación Fluorescente in Situ , Archaea/genética , Biopelículas , Acidithiobacillus/genética , FilogeniaRESUMEN
Iron(II)- and 2-(oxo)-glutarate-dependent (Fe/2OG) oxygenases catalyze a diverse array of oxidation reactions via a common iron(IV)-oxo (ferryl) intermediate. Although the intermediate has been characterized spectroscopically, its short lifetime has precluded crystallograhic characterization. In solution, the ferryl was first observed directly in the archetypal Fe/2OG hydroxylase, taurine:2OG dioxygenase (TauD). Here, we substitute the iron cofactor of TauD with the stable vanadium(IV)-oxo (vanadyl) ion to obtain crystal structures mimicking the key ferryl complex. Intriguingly, whereas the structure of the TauD·(VIV-oxo)·succinate·taurine complex exhibits the expected orientation of the V≡O bond-trans to the His255 ligand and toward the C-H bond to be cleaved, in what has been termed the in-line configuration-the TauD·(VIV-oxo) binary complex is best modeled with its oxo ligand trans to Asp101. This off-line-like configuration is similar to one recently posited as a means to avoid hydroxylation in Fe/2OG enzymes that direct other outcomes, though neither has been visualized in an Fe/2OG structure to date. Whereas an off-line (trans to the proximal His) or off-line-like (trans to the carboxylate ligand) ferryl is unlikely to be important in the hydroxylation reaction of TauD, the observation that the ferryl may deviate from an in-line orientation in the absence of the primary substrate may explain the enzyme's mysterious self-hydroxylation behavior, should the oxo ligand lie trans to His99. This finding reinforces the potential for analogous functional off-line oxo configurations in halogenases, desaturases, and/or cyclases.
Asunto(s)
Hierro/química , Oxigenasas de Función Mixta/química , Imitación Molecular , Vanadatos/química , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Escherichia coli/química , Enlace de Hidrógeno , Hidroxilación , Oxigenasas de Función Mixta/aislamiento & purificación , Estructura Secundaria de Proteína , Ácido Succínico/química , Taurina/química , Espectroscopía de Absorción de Rayos XRESUMEN
(S)-2-Hydroxypropylphosphonate [(S)-2-HPP, 1] epoxidase (HppE) reduces H2O2 at its nonheme-iron cofactor to install the oxirane "warhead" of the antibiotic fosfomycin. The net replacement of the C1 pro-R hydrogen of 1 by its C2 oxygen, with inversion of configuration at C1, yields the cis-epoxide of the drug [(1R,2S)-epoxypropylphosphonic acid (cis-Fos, 2)]. Here we show that HppE achieves â¼95% selectivity for C1 inversion and cis-epoxide formation via steric guidance of a radical-coupling mechanism. Published structures of the HppE·FeII·1 and HppE·ZnII·2 complexes reveal distinct pockets for C3 of the substrate and product and identify four hydrophobic residues-Leu120, Leu144, Phe182, and Leu193-close to C3 in one of the complexes. Replacement of Leu193 in the substrate C3 pocket with the bulkier Phe enhances stereoselectivity (cis:trans â¼99:1), whereas the Leu120Phe substitution in the product C3 pocket diminishes it (â¼82:18). Retention of C1 configuration and trans-epoxide formation become predominant with the bulk-reducing Phe182Ala substitution in the substrate C3 pocket (â¼13:87), trifluorination of C3 (â¼23:77), or both (â¼1:99). The effect of C3 trifluorination is counteracted by the more constrained substrate C3 pockets in the Leu193Phe (â¼56:44) and Leu144Phe/Leu193Phe (â¼90:10) variants. The ability of HppE to epoxidize substrate analogues bearing halogens at C3, C1, or both is inconsistent with a published hypothesis of polar cyclization via a C1 carbocation. Rather, specific enzyme-substrate contacts drive inversion of the C1 radical-as proposed in a recent computational study-to direct formation of the more potently antibacterial cis-epoxide by radicaloid C-O coupling.
Asunto(s)
Compuestos Epoxi/metabolismo , Fosfomicina/biosíntesis , Oxidorreductasas/metabolismo , Compuestos Epoxi/química , Fosfomicina/química , Radicales Libres/química , Radicales Libres/metabolismo , Conformación Molecular , Oxidorreductasas/química , Oxidorreductasas/genética , EstereoisomerismoRESUMEN
Microbial ethylene-forming enzyme (EFE) converts the C3C4 fragment of the ubiquitous primary metabolite 2-oxoglutarate (2OG) to its namesake alkene product. This reaction is very different from the simple decarboxylation of 2OG to succinate promoted by related enzymes and has inspired disparate mechanistic hypotheses. We show that EFE produces stereochemically random (equal cis and trans) 1,2-[2H2]-ethylene from (3S,4R)-[2H2]-2OG, appends an oxygen from O2 on the C1-derived (bi)carbonate, and can be diverted to ω-hydroxylated monoacid products by modifications to 2OG or the enzyme. These results implicate an unusual radical-polar hybrid mechanism involving iron(II)-coordinated acylperoxycarbonate and alkylcarbonate intermediates. The mechanism explains how EFE accesses a high-energy carboxyl radical to initiate its fragmentation cascade, and it hints at capabilities of 2OG-dependent enzymes that may await discovery and exploitation.
RESUMEN
A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H(2) and CO(2), which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H(2)-utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H(2)-utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location.
Asunto(s)
Bacterias/clasificación , Bacterias/genética , Carbón Mineral/microbiología , Metano/biosíntesis , Methanomicrobiales/clasificación , Methanomicrobiales/genética , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Dióxido de Carbono/metabolismo , Membrana Celular/química , ADN de Archaea/química , ADN de Archaea/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Hidrógeno/metabolismo , Illinois , Lípidos de la Membrana/análisis , Methanomicrobiales/aislamiento & purificación , Methanomicrobiales/metabolismo , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Compuestos Orgánicos/metabolismo , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Extremely acidic (pH 0-1.5) Acidithiobacillus-dominated biofilms known as snottites are found in sulfide-rich caves around the world. Given the extreme geochemistry and subsurface location of the biofilms, we hypothesized that snottite Acidithiobacillus populations would be genetically isolated. We therefore investigated biogeographic relationships among snottite Acidithiobacillus spp. separated by geographic distances ranging from meters to 1000s of kilometers. We determined genetic relationships among the populations using techniques with three levels of resolution: (i) 16S rRNA gene sequencing, (ii) 16S-23S intergenic transcribed spacer (ITS) region sequencing and (iii) multi-locus sequencing typing (MLST). We also used metagenomics to compare functional gene characteristics of select populations. Based on 16S rRNA genes, snottites in Italy and Mexico are dominated by different sulfur-oxidizing Acidithiobacillus spp. Based on ITS sequences, Acidithiobacillus thiooxidans strains from different cave systems in Italy are genetically distinct. Based on MLST of isolates from Italy, genetic distance is positively correlated with geographic distance both among and within caves. However, metagenomics revealed that At. thiooxidans populations from different cave systems in Italy have different sulfur oxidation pathways and potentially other significant differences in metabolic capabilities. In light of those genomic differences, we argue that the observed correlation between genetic and geographic distance among snottite Acidithiobacillus populations is partially explained by an evolutionary model in which separate cave systems were stochastically colonized by different ancestral surface populations, which then continued to diverge and adapt in situ.
Asunto(s)
Acidithiobacillus/fisiología , Biopelículas , Cuevas/microbiología , Azufre/metabolismo , Acidithiobacillus/clasificación , Acidithiobacillus/genética , Acidithiobacillus/aislamiento & purificación , Italia , Metagenómica , México , Tipificación de Secuencias Multilocus , Filogenia , Filogeografía , ARN Ribosómico 16S/genéticaRESUMEN
The Frasassi and Acquasanta Terme cave systems in Italy host isolated lithoautotrophic ecosystems characterized by sulfur-oxidizing biofilms with up to 50% S(0) by mass. The net contributions of microbial taxa in the biofilms to production and consumption of S(0) are poorly understood and have implications for understanding the formation of geological sulfur deposits as well as the ecological niches of sulfur-oxidizing autotrophs. Filamentous Epsilonproteobacteria are among the principal biofilm architects in Frasassi and Acquasanta Terme streams, colonizing high-sulfide, low-oxygen niches relative to other major biofilm-forming populations. Metagenomic sequencing of eight biofilm samples indicated the presence of diverse and abundant Epsilonproteobacteria. Populations of Sulfurovum-like organisms were the most abundant Epsilonproteobacteria regardless of differences in biofilm morphology, temperature, or water chemistry. After assembling and binning the metagenomic data, we retrieved four nearly-complete genomes of Sulfurovum-like organisms as well as a Sulfuricurvum spp. Analyses of the binned and assembled metagenomic data indicate that the Epsilonproteobacteria are autotrophic and therefore provide organic carbon to the isolated subsurface ecosystem. Multiple homologs of sulfide-quinone oxidoreductase (Sqr), together with incomplete or absent Sox pathways, suggest that cave Sulfurovum-like Epsilonproteobacteria oxidize sulfide incompletely to S(0) using either O2 or nitrate as a terminal electron acceptor, consistent with previous evidence that they are most successful in niches with high dissolved sulfide to oxygen ratios. In contrast, we recovered homologs of the complete complement of Sox proteins affiliated Gammaproteobacteria and with less abundant Sulfuricurvum spp. and Arcobacter spp., suggesting that these populations are capable of the complete oxidation of sulfide to sulfate. These and other genomic data presented here offer new clues into the physiology and genetic potential of the largely uncultivated and ecologically successful cave Sulfurovum-like populations, and suggest that they play an integral role in subsurface S(0) formation.
RESUMEN
Highly acidic (pH 0-1) biofilms, known as 'snottites', form on the walls and ceilings of hydrogen sulfide-rich caves. We investigated the population structure, physiology and biogeochemistry of these biofilms using metagenomics, rRNA methods and lipid geochemistry. Snottites from the Frasassi cave system (Italy) are dominated (>70% of cells) by Acidithiobacillus thiooxidans, with smaller populations including an archaeon in the uncultivated 'G-plasma' clade of Thermoplasmatales (>15%) and a bacterium in the Acidimicrobiaceae family (>5%). Based on metagenomic evidence, the Acidithiobacillus population is autotrophic (ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), carboxysomes) and oxidizes sulfur by the sulfide-quinone reductase and sox pathways. No reads matching nitrogen fixation genes were detected in the metagenome, whereas multiple matches to nitrogen assimilation functions are present, consistent with geochemical evidence, that fixed nitrogen is available in the snottite environment to support autotrophic growth. Evidence for adaptations to extreme acidity include Acidithiobacillus sequences for cation transporters and hopanoid synthesis, and direct measurements of hopanoid membrane lipids. Based on combined metagenomic, molecular and geochemical evidence, we suggest that Acidithiobacillus is the snottite architect and main primary producer, and that snottite morphology and distributions in the cave environment are directly related to the supply of C, N and energy substrates from the cave atmosphere.
Asunto(s)
Acidithiobacillus/aislamiento & purificación , Biopelículas/clasificación , Cuevas/microbiología , Metagenómica/métodos , Azufre/metabolismo , Acidithiobacillus/clasificación , Acidithiobacillus/fisiología , Acidithiobacillus thiooxidans/genética , Acidithiobacillus thiooxidans/metabolismo , Actinobacteria/genética , Actinobacteria/metabolismo , Biopelículas/crecimiento & desarrollo , Euryarchaeota/aislamiento & purificación , Euryarchaeota/fisiología , Italia , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Procesos FototróficosRESUMEN
Symbioses involving animals and chemoautotrophic bacteria form the foundation of entire ecosystems at deep-sea hydrothermal vents and cold seeps, but have so far not been reported in terrestrial or freshwater environments. A rare example of a terrestrial ecosystem sustained by chemoautotrophy is found within the sulfide-rich Frasassi limestone cave complex of central Italy. In this study, we report the discovery of abundant filamentous bacteria on the exoskeleton of Niphargus ictus, a macroinvertebrate endemic to Frasassi. Using 16S rDNA sequencing and fluorescence in situ hybridization (FISH), we show that N. ictus throughout the large cave complex are colonized by a single phylotype of bacteria in the sulfur-oxidizing clade Thiothrix. The epibiont phylotype is distinct from Thiothrix phylotypes that form conspicuous biofilms in the cave streams and pools inhabited by N. ictus. Using a combination of 13C labeling, FISH, and secondary ion mass spectrometry (SIMS), we show that the epibiotic Thiothrix are autotrophic, establishing the first known example of a non-marine chemoautotroph-animal symbiosis. Conditions supporting chemoautotrophy, and the N. ictus-Thiothrix association, likely commenced in the Frasassi cave complex between 350,000 and 1 million years ago. Therefore, the N. ictus-Thiothrix symbiosis is probably significantly younger than marine chemoautotrophic symbioses, many of which have been evolving for tens to hundreds of million years.
Asunto(s)
Anfípodos/microbiología , Anfípodos/fisiología , Fenómenos Fisiológicos Bacterianos , Simbiosis , Thiothrix/clasificación , Thiothrix/aislamiento & purificación , Animales , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Agua Dulce , Hibridación Fluorescente in Situ , Compuestos Inorgánicos/metabolismo , Italia , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Thiothrix/genética , Thiothrix/metabolismoRESUMEN
The sulfidic Frasassi cave system affords a unique opportunity to investigate niche relationships among sulfur-oxidizing bacteria, including epsilonproteobacterial clades with no cultivated representatives. Oxygen and sulfide concentrations in the cave waters range over more than two orders of magnitude as a result of seasonally and spatially variable dilution of the sulfidic groundwater. A full-cycle rRNA approach was used to quantify dominant populations in biofilms collected in both diluted and undiluted zones. Sulfide concentration profiles within biofilms were obtained in situ using microelectrode voltammetry. Populations in rock-attached streamers depended on the sulfide/oxygen supply ratio of bulk water (r=0.97; P<0.0001). Filamentous epsilonproteobacteria dominated at high sulfide to oxygen ratios (>150), whereas Thiothrix dominated at low ratios (<75). In contrast, Beggiatoa was the dominant group in biofilms at the sediment-water interface regardless of sulfide and oxygen concentrations or supply ratio. Our results highlight the versatility and ecological success of Beggiatoa in diffusion-controlled niches, and demonstrate that high sulfide/oxygen ratios in turbulent water are important for the growth of filamentous epsilonproteobacteria.
Asunto(s)
Epsilonproteobacteria/clasificación , Epsilonproteobacteria/aislamiento & purificación , Azufre/metabolismo , Microbiología del Agua , Agua/análisis , Biopelículas/crecimiento & desarrollo , ADN Bacteriano/genética , ADN Ribosómico/genética , Epsilonproteobacteria/genética , Epsilonproteobacteria/metabolismo , Biblioteca de Genes , Italia , Oxígeno/análisis , Oxígeno/metabolismo , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Azufre/análisisRESUMEN
Recent data on the physiology of inorganic carbon acquisition by the model marine diatom Thalassiosira weissflogii (Grunow) demonstrate the importance of the catalytic equilibration of HCO3-and CO2by carbonic anhydrases located in the periplasm and in the cytoplasm. These enzymes can use Zn, Co or Cd as their metal centre, and their activity increases at low ambient CO2. The silica frustule provides buffering for extracellular CA activity, The transmembrane transport of CO2 may occur by passive diffusion. Under CO2 limitation, the cytoplasmic HCO3-is used to form malate and oxaloacetic acid via phosphoenolpyruvate carboxylase. It appears that subsequent decarboxylation of these compounds in the chloroplast regenerates CO2 near the site of Rubisco, and thus provides the organism with an effective unicellular C4 photosynthetic pathway. These results, together with other published data, bring up two major questions regarding inorganic carbon acquisition in diatoms: What is the major species of inorganic carbon (CO2 or HCO3-) transported across the membrane under natural conditions? And what is the form of carbon (inorganic or organic) accumulated by the cells?