RESUMEN
Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with â¼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.
Asunto(s)
Proteínas Bacterianas , Colorantes Fluorescentes , Bacterias Gramnegativas Quimiolitotróficas , Sideróforos , Acinetobacter baumannii , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Caulobacter crescentus , Enterobactina/análisis , Enterobactina/metabolismo , Escherichia coli/metabolismo , Colorantes Fluorescentes/química , Bacterias Gramnegativas Quimiolitotróficas/química , Bacterias Gramnegativas Quimiolitotróficas/genética , Bacterias Gramnegativas Quimiolitotróficas/metabolismo , Humanos , Hierro/metabolismo , Klebsiella pneumoniae , Sideróforos/análisis , Sideróforos/metabolismoRESUMEN
A new carbonic anhydrase (CA, EC 4.2.1.1) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 was identified and characterized. The bacterial carbonic anhydrase gene was expressed in Escherichia coli yielding an active enzyme, which was purified in large amounts. The recombinant protein (SspCA) was found to belong to the α-CA class and displays esterase activity. The kinetic parameters were determined by using CO(2) and p-nitrophenylacetate (p-NpA) as substrates. The bacterial enzyme presented specific activity comparable to that of bovine carbonic anhydrase (bCA II) but it showed biochemical properties never observed for the mammalian enzyme. The thermophilic enzyme, in fact, was endowed with high thermostability and with unaltered residual activity after prolonged exposure to heat up to 100°C. SspCA and the bovine carbonic anhydrase (bCA II) were immobilized within a polyurethane (PU) foam. The immobilized bacterial enzyme was found to be active and stable at 100°C up to 50 h.
Asunto(s)
Proteínas Bacterianas/química , Dióxido de Carbono/química , Anhidrasas Carbónicas/química , Bacterias Gramnegativas Quimiolitotróficas/química , Nitrofenoles/química , Animales , Proteínas Bacterianas/aislamiento & purificación , Anhidrasa Carbónica II/química , Anhidrasas Carbónicas/aislamiento & purificación , Bovinos , Electroforesis en Gel de Poliacrilamida , Pruebas de Enzimas , Estabilidad de Enzimas , Escherichia coli/genética , Bacterias Gramnegativas Quimiolitotróficas/enzimología , Calor , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/aislamiento & purificación , Cinética , Poliuretanos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por SustratoRESUMEN
The genomic organization of acidophilic chemolithotrophic bacteria belonging to the genus Thiobacillus, Thiomonas and Leptospirillum was studied using pulsed field gel electrophoresis techniques (PFGE). The electrophoretic analysis of intact DNA prepared from different strains showed that all have a circular chromosome, with sizes ranging from 1.9 Mb for Leptospirillum ferrooxidans ATCC 49879, the smallest genome for an acidophilic strict chemolithoautotrophic microorganism, to 3.8 Mb for Thiomonas cuprina DSM 5495, the largest in this study. The number of extrachromosomal elements present varied from none, as observed in several isolates of Leptospirillum ferrooxidan, to five in Thiobacillus thiooxidans ATCC 8085. The mixotroph Thiomonas cuprina DSM 5495 was found to have a linear 50 kb megaplasmid which was inducible when the bacteria was grown in chemolithotrophic conditions. Low-frequency restriction fragment analysis (LFRFA) of different acidophilic chemolithotrophs and related species was carried out by PFGE to determine macrorestriction patterns for rare cutters (SpeI, XbaI, SwaI, PmeI), which were then used for taxonomic identification (karyotyping), genome size determination, and generation of physical and genetic maps.
Asunto(s)
Electroforesis en Gel de Campo Pulsado/métodos , Genoma Bacteriano , Bacterias Gramnegativas Quimiolitotróficas/química , Bacterias Gramnegativas Quimiolitotróficas/genética , Mapeo Cromosómico , Herencia Extracromosómica , Mapeo Físico de Cromosoma , Mapeo Restrictivo , Thiobacillus/genéticaRESUMEN
BACKGROUND INFORMATION: Lucina pectinata is a large tropical lucinid known to harbour sulphide-oxidizing bacteria in specialized gill cells. Conventional TEM (transmission electron microscopy) has shown that bacteriocytes also harbour visibly 'empty' vesicles whose chemical content remains, to date, only roughly determined. RESULTS: In the present study, L. pectinata gill tissues were cryo-fixed as fast as possible by performing high-pressure freezing before a freeze-substitution process and finally performing a cryo-embedding in Lowicryl. Ultrathin sections were then used for a cryo-EFTEM (where EFTEM stands for energy-filtered TEM) microanalysis. Results show that bacteriocytes within the gill tissues contain elemental sulphur in small vesicles produced by the host itself. In instances of sporadic depletion of sulphur in the environment, such structures may act as energy sources for bacterial endosymbionts. CONCLUSIONS: The cryo-EFTEM techniques represent (i) the only method used to date to locate and preserve sulphur at the cellular level and (ii) a powerful tool for sulphur metabolism analysis in thioautotrophic symbiont relationships.
Asunto(s)
Crioultramicrotomía/métodos , Bacterias Gramnegativas Quimiolitotróficas/química , Energía Filtrada en la Transmisión por Microscopía Electrónica/métodos , Azufre/análisis , Simbiosis , Animales , Bivalvos/microbiología , Congelación , Bacterias Gramnegativas Quimiolitotróficas/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
Novel tertiary branched tetra-amines, quaternary branched penta-amines, linear penta-amines and linear hexa-amines were distributed as the major polyamines in six obligately extremely thermophilic eubacteria belonging to Thermoleophilum, Bacillus or Hydrogenobacter. The major polyamine of Thermoleophilum album and Thermoleophilum minutum was identified as a quaternary branched penta-amine, 4,4-bis(3-aminopropyl)-1,8-diamino-4-azaoctane (NH2[CH2]3N+([CH2]3NH2)2[CH2]4NH2) by h.p.l.c., t.l.c. and g.c.-m.s. Hydrogenobacter thermophilus and Hydrogenobacter halophilus contained another quaternary branched penta-amine, 4,4-bis(3-aminopropyl)-1,7-diamino-4-azaheptane (NH2[CH2]3N([CH2]3NH2)2[CH2]3NH2) as the major polyamine, and tertiary branched tetra-amines (4-(3-aminopropyl)-1,7-diamino-4-azaheptane (NH2[CH2]3N([CH2]3NH2)[CH2]3NH2), 4-(3-aminopropyl)-1,8-diamino-4-azaoctane (NH2[CH2]3N([CH2]3NH2)[CH2]4NH2)) and 4,4-bis(3-aminopropyl)-1,8-diamino-4-azaoctane were confirmed as minor components. Bacillus schlegelii contained a branched tetra-amine, 4-(3-aminopropyl)-1,8-diamino-4-azaoctane, a branched penta-amine, 4,4-bis(3-aminopropyl)-1,8-diamino-4-azaoctane, a linear penta-amine, 1,16-diamino-4,8,13-triazahexadecane (NH2[CH2]3NH[CH2]3NH[CH2]4NH[CH2]3NH2) and linear hexa-amine(s), 1,20-diamino-4,8,12,17-tetra-azaeicosane (NH2[CH2]3NH[CH2]3NH[CH2]3NH[CH2]4NH[CH2]3NH2 ) and/or 1,20-diamino-4,8,13,17-tetra-azaeicosane (NH2[CH2]3NH[CH2]3NH[CH2]4NH[CH2]3NH[CH2]3NH2 ).
Asunto(s)
Bacillus/química , Bacterias Gramnegativas/química , Bacterias Gramnegativas Quimiolitotróficas/química , Poliaminas/análisis , Cromatografía de Gases , Cromatografía Líquida de Alta Presión , Calor , Poliaminas/química , Poliaminas/aislamiento & purificación , Especificidad de la EspecieRESUMEN
The phospholipid composition of Hydrogenobacter thermophilus strain TK-6, an obligately chemolithoautotrophic, extremely thermophilic hydrogen bacterium, was analyzed. Two of four phospholipids detected from the strain were assumed to be phosphatidylinositol and phosphatidylglycerol. An aminophospholipid named PX, whose content among the phospholipids was 65%, was found to have a novel chemical structure by analysis of the dilyso form with nuclear magnetic resonance and fast atom bombardment-mass spectrometry (FAB-MS) and by analysis of the intact PX with FAB-MS as 1,2-diacyl-3-O-(phospho-2'-O-(1'-amino)-2',3',4',5'-pentanetetrol)-sn-glycerol. Structurally similar phospholipids have been identified in Methanospirillum hungatei, Methanolacinia paynteri, and Methanogenium cariaci, which all belong to the Archaea.
Asunto(s)
Amino Azúcares/química , Bacterias Gramnegativas Quimiolitotróficas/química , Ácidos Fosfatidicos/química , Amino Azúcares/aislamiento & purificación , Archaea/química , Ácidos Fosfatidicos/aislamiento & purificación , Fosfolípidos/químicaRESUMEN
The structure of pseudoazurin from Thiosphaera pantotropha has been determined and compared to structures of both soluble and membrane-bound periplasmic redox proteins. The results show a matching set of unipolar, but promiscuous, docking motifs based on a positive hydrophobic surface patch on the electron shuttle proteins pseudoazurin and cytochrome c550 and a negative hydrophobic patch on the surface of their known redox partners. The observed electrostatic handedness is argued to be associated with the charge-asymmetry of the membrane-bound components of the redox chain due to von Heijne's 'positives-inside' principle. We propose a 'positives-in-between' rule for electron shuttle proteins, and expect a negative hydrophobic patch to be present on both the highest and lowest redox potential species in a series of electron carriers.
Asunto(s)
Azurina/análogos & derivados , Transporte de Electrón , Azurina/química , Azurina/metabolismo , Cristalografía por Rayos X , Grupo Citocromo c/química , Grupo Citocromo c/metabolismo , Citocromos/química , Citocromos/metabolismo , Bacterias Gramnegativas Quimiolitotróficas/química , Modelos Moleculares , Nitrito Reductasas/química , Nitrito Reductasas/metabolismo , Conformación ProteicaRESUMEN
Paramagnetic NMR and optical studies of the oxidized forms of mesophile Pseudomonas aeruginosa cytochrome c(551) and its quintuple mutant (F7A/V13M/F34Y/E43Y/V78I), and thermophile Hydrogenobacter thermophilus cytochrome c(552) demonstrated that the amino acid side chain packings in the protein interior influence the coordination bond between the heme iron and the axial methionine in the proteins. The strength of heme axial coordinations was found to correlate with the overall protein thermostability.