RESUMO
Many acidophilic iron-oxidizing bacteria used in the mining industry for the bioleaching of sulfidic minerals are intolerant to high chloride concentrations, resulting in problems where chloride occurs in the deposit at high concentrations or only seawater is available. In search for strains tolerating such conditions a tetrathionate- and iron-oxidizing bacterium was isolated from a tailings-contaminated beach sample at Portman Bay, Cartagena-La Union mining district, Spain, in the presence of 20 g l-1 (0.34 M) sodium chloride. The isolate was able to form spores, did not grow in the absence of NaCl, and oxidized ferrous iron in the presence of up to 1.5 M (â¼87 g l-1) NaCl. Genome sequencing based on a combination of Illumina and PacBio reads revealed two contigs, a circular bacterial chromosome of 5.2 Mbp and a plasmid of 90 kbp, respectively. The chromosome comprised seven different 16S rRNA genes. Submission of the chromosome to the Type (Strain) Genome Server (TYGS) without preselection of similar sequences revealed exclusively type strains of the genus Alicyclobacillus. In the TYGS analyses the respective most similar species were dependent on whether the final tree was derived from just 16S rRNA, from the genomes, or from the proteomes. Thus, TYGS analysis clearly showed that isolate SO9 represents a novel species of the genus Alicyclobacillus. In the presence of artificial seawater with almost 0.6 M chloride, the addition of Alicyclobacillus sp. SO9 improved copper dissolution from chalcopyrite (CuFeS2) compared to abiotic leaching without bacteria. The new isolate SO9, therefore, has potential for bioleaching at elevated chloride concentrations.
Assuntos
Alicyclobacillus , Ferro , Cobre , Alicyclobacillus/genética , Cloretos , Cloreto de Sódio , RNA Ribossômico 16S/genética , Bactérias/genética , Oxirredução , FilogeniaRESUMO
Biomining applies microorganisms to extract valuable metals from usually sulfidic ores. However, acidophilic iron (Fe)-oxidizing bacteria tend to be sensitive to chloride ions which may be present in biomining operations. This study investigates the bioleaching of pyrite (FeS2), as well as the attachment to FeS2 by Sulfobacillus thermosulfidooxidans DSM 9293T in the presence of elevated sodium chloride (NaCl) concentrations. The bacteria were still able to oxidize iron in the presence of up to 0.6M NaCl (35 g/L), and the addition of NaCl in concentrations up to 0.2M (~12 g/L) did not inhibit iron oxidation and growth of S. thermosulfidooxidans in leaching cultures within the first 7 days. However, after approximately 7 days of incubation, ferrous iron (Fe2+) concentrations were gradually increased in leaching assays with NaCl, indicating that iron oxidation activity over time was reduced in those assays. Although the inhibition by 0.1M NaCl (~6 g/L) of bacterial growth and iron oxidation activity was not evident at the beginning of the experiment, over extended leaching duration NaCl was likely to have an inhibitory effect. Thus, after 36 days of the experiment, bioleaching of FeS2 with 0.1M NaCl was reduced significantly in comparison to control assays without NaCl. Pyrite dissolution decreased with the increase of NaCl. Nevertheless, pyrite bioleaching by S. thermosulfidooxidans was still possible at NaCl concentrations as high as 0.4M (~23 g/L NaCl). Besides, cell attachment in the presence of different concentrations of NaCl was investigated. Cells of S. thermosulfidooxidans attached heterogeneously on pyrite surfaces regardless of NaCl concentration. Noticeably, bacteria were able to adhere to pyrite surfaces in the presence of NaCl as high as 0.4M. Although NaCl addition inhibited iron oxidation activity and bioleaching of FeS2, the presence of 0.2M seemed to enhance bacterial attachment of S. thermosulfidooxidans on pyrite surfaces in comparison to attachment without NaCl.
RESUMO
This study reports on the effect of inoculum history, growth substrates, and yeast extract on sodium chloride tolerance of Sulfobacillus thermosulfidooxidans DSM 9293T. The concentrations of NaCl for complete inhibition of Fe2+ oxidation by cells initially grown with ferrous iron sulfate, or tetrathionate, or pyrite as energy sources were 525 mM, 725 mM, and 800 mM, respectively. Noticeably, regardless of NaCl concentrations, oxygen consumption rates of S. thermosulfidooxidans with 20 mM tetrathionate were higher than with 50 mM FeSO4. NaCl concentrations of higher than 400 mM strongly inhibited the iron respiration of S. thermosulfidooxidans. In contrast, the presence of NaCl was shown to stimulate tetrathionate oxidation. This trend was especially pronounced in NaCl-adapted cells where respiration rates at 200 mM NaCl were threefold of those in the absence of NaCl. In NaCl-adapted cultures greater respiration rates for tetrathionate were observed than in non-NaCl-adapted cultures, especially at concentrations ≥ 200 mM NaCl. At concentrations of ≤ 200 mM NaCl, cell growth and iron oxidation were enhanced with the addition of increasing concentrations of yeast extract. Thus, cell numbers in cultures with 0.05% yeast extract were â¼5 times higher than without yeast extract addition. At NaCl concentration as high as 400 mM, however, iron oxidation rates improved compared to control assays without yeast extract, but there was no clear dependence on yeast extract concentrations. The initial growth of bacteria with and without yeast extract in the presence of different NaCl concentrations was shown to impact leaching of copper from chalcopyrite. Copper dissolution was enhanced in the presence of 200 mM NaCl and absence of yeast extract, while the addition of 0.02% yeast extract was shown to promote copper solubilization in the presence of 500 mM NaCl.
Assuntos
Reatores Biológicos/microbiologia , Clostridiales/metabolismo , Cobre/metabolismo , Tolerância ao Sal/fisiologia , Cloreto de Sódio/farmacologia , Clostridiales/efeitos dos fármacos , Clostridiales/crescimento & desenvolvimento , Compostos Ferrosos/metabolismo , Ferro/metabolismo , Fragilidade Osmótica/fisiologia , Oxirredução , Oxigênio/metabolismo , Consumo de Oxigênio/fisiologia , Sulfetos/metabolismo , Ácido Tetratiônico/metabolismoRESUMO
Biosurfactants are amphipathic molecules with relevance in biotechnology due to their structural diversity, low toxicity and biodegradability. The genus Rhodococcus has extensively been studied because of its capacity to produce trehalose-containing surfactants as well as trehalose lipids as potential pathogenic factor. Here we present the draft genome sequence of Rhodococcus erythropolis B7g isolated with toluene from fuel-contaminated soil. The genome comprises 7,175,690 bp in 121 contigs, a Gâ¯+â¯C content of 62,4% and 7,153 coding DNA sequences (CDSs), and it contains genes for trehalose biosynthesis and surfactant production. Additionally, genes for the production of trehalose-tetraester biosurfactant were identified, whose function was experimentally verified making the strain B7g a potential candidate for use in bioremediation applications or in biosurfactant exploration.
Assuntos
Genoma Bacteriano , Rhodococcus/genética , Tensoativos/metabolismo , Sequência de Bases , Anotação de Sequência Molecular , Filogenia , Metabolismo Secundário , Trealose/biossínteseRESUMO
Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1) and a two domain protein (VpStyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain. It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as NADH:FAD-oxidoreductase. A Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s-1 were determined and resulted in a catalytic efficiency (kcatKm-1) of 0.64 s-1 µM-1. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated. One mutant (AAAAA) showed 18.7-fold higher affinity for FAD (kcatKm-1 of 5.21 s-1 µM-1) while keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1, showed monooxygenase activity on styrene of 0.14 U mg-1 and 0.46 U mg-1, as well as on benzyl methyl sulfide of 1.62 U mg-1 and 3.11 U mg-1, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high substrate conversions (up to 95% in 2 h) and produced dominantly (S)-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.
Assuntos
Oxigenases/metabolismo , Flavoproteínas/metabolismo , Oxirredução , Oxirredutases/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteobactérias/enzimologia , Microbiologia do Solo , EstereoisomerismoRESUMO
Some soil bacteria are able to metabolize styrene via initial side-chain oxygenation. This catabolic route is of potential biotechnological relevance due to the occurrence of phenylacetic acid as a central metabolite. The styrene-degrading strains Rhodococcus opacus 1CP, Pseudomonas fluorescens ST, and the novel isolates Sphingopyxis sp. Kp5.2 and Gordonia sp. CWB2 were investigated with respect to their applicability to co-metabolically produce substituted phenylacetic acids. Isolates were found to differ significantly in substrate tolerance and biotransformation yields. Especially, P. fluorescens ST was identified as a promising candidate for the production of several phenylacetic acids. The biotransformation of 4-chlorostyrene with cells of strain ST was shown to be stable over a period of more than 200 days and yielded about 38 mmolproduct gcelldryweight-1 after nearly 350 days. Moreover, 4-chloro-α-methylstyrene was predominantly converted to the (S)-enantiomer of the acid with 40% enantiomeric excess.
RESUMO
Among other factors, a distinct gene redundancy is discussed to facilitate high metabolic versatility of rhodococci. Rhodococcus opacus 1CP is a typical member in that respect and degrades a multitude of (chlorinated) aromatic compounds. In contrast to the central pathways of aromatic degradation in strain 1CP, little is known about the degree of gene redundancy and to what extent this is reflected on protein level within the steps of peripheral degradation. By means of degenerated primers deduced from tryptic peptides of a purified phenol hydroxylase component and using the amplified fragment as a labelled probe against genomic 1CP-DNA, three gene sets encoding three different two-component phenol hydroxylases pheA1/pheA2(1-3) could be identified. One of them was found to be located on the megaplasmid p1CP, which confirms the role of these elements for metabolic versatility. Protein chromatography of phenol- and 4-chlorophenol-grown 1CP-biomass gave first evidences on a functional expression of these oxygenases, which could be initially characterised in respect of their substrate specificity.
Assuntos
Oxigenases de Função Mista/genética , Rhodococcus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clorofenóis/metabolismo , Oxigenases de Função Mista/metabolismo , Família Multigênica , Fenol/metabolismo , Filogenia , Proteínas Recombinantes , Rhodococcus/genética , Especificidade por SubstratoRESUMO
Two styrene monooxygenase types, StyA/StyB and StyA1/StyA2B, have been described each consisting of an epoxidase and a reductase. A gene fusion which led to the chimeric reductase StyA2B and the occurrence in different phyla are major differences. Identification of SMOA/SMOB-ADP1 of Acinetobacter baylyi ADP1 may enlighten the gene fusion event since phylogenetic analysis indicated both proteins to be more related to StyA2B than to StyA/StyB. SMOB-ADP1 is classified like StyB and StyA2B as HpaC-like reductase. Substrate affinity and turnover number of the homo-dimer SMOB-ADP1 were determined for NADH (24 µM, 64 s(-1)) and FAD (4.4 µM, 56 s(-1)). SMOB-ADP1 catalysis follows a random sequential mechanism, and FAD fluorescence is quenched upon binding to SMOB-ADP1 (K d = 1.8 µM), which clearly distinguishes that reductase from StyB of Pseudomonas. In summary, this study confirmes made assumptions and provides phylogenetic and biochemical data for the differentiation of styrene monooxygenase-related flavin reductases.
Assuntos
Acinetobacter/enzimologia , FMN Redutase/química , FMN Redutase/metabolismo , Oxigenases/química , Oxigenases/metabolismo , Acinetobacter/genética , Sequência de Aminoácidos , Biocatálise , FMN Redutase/classificação , FMN Redutase/genética , Dados de Sequência Molecular , NAD/metabolismo , Oxirredutases/metabolismo , Oxigenases/classificação , Oxigenases/genética , Filogenia , Pseudomonas/enzimologia , Pseudomonas/genéticaRESUMO
Remediation of lead (Pb)-contaminated sites with phosphate amendments is one of the best studied and cost-effective methods for in situ immobilization. In this treatment, a very stable mineral, pyromorphite Pb5(PO4)3Cl, is formed. Several studies propose to improve this treatment method with the addition of phosphate-solubilizing bacteria (PSB). The effect of bacteria on solubilization of pyromorphite is unknown. In this study, the effect of the soil microorganisms on the stability of pyromorphite Pb5(PO4)3Cl has been investigated in a set of batch solution experiments. The mineral was reacted with Pseudomonas putida, a common soil microorganism. Dissolution of pyromorphite was enhanced by the presence of P. putida, resulting in an elevated Pb concentration in the solution. This occurred even when the bacteria were provided with an additional source of phosphate in the solution. Pyromorphite has been shown to be a potential source of nutrient phosphorus for common soil bacteria. Thus, the use of PSB in remediation treatments of Pb contaminated sites may have adverse long-term impacts on Pb immobilization. Conscious phosphate management is suggested for long-term sustainability of the in situ Pb immobilization by pyromorphite formation.
Assuntos
Chumbo/metabolismo , Minerais/metabolismo , Fosfatos/metabolismo , Pseudomonas putida/fisiologia , Poluentes do Solo/metabolismo , Biodegradação Ambiental , Chumbo/análise , Minerais/análise , Fosfatos/análise , Fósforo/metabolismo , Solo/química , Microbiologia do Solo , Poluentes do Solo/análiseRESUMO
Rhodococcus opacus 1CP produces trehalose dinocardiomycolates during growth on long-chained n-alkanes. Trehalose and trehalose-6-phosphate, which are synthesized via the OtsAB pathway, are probable intermediates in the biosynthesis of these biosurfactants. By molecular genetic screening for trehalose-6-phosphate synthases (TPSs and OtsAs), two chromosomal fragments of strain 1CP were obtained. Each contained an ORF whose amino acid sequence showed high similarity to TPSs. To prove the activity of the otsA1 and otsA2 gene product and to detect catalytic differences, both were expressed as His-tagged fusion proteins. Enzyme kinetics of the enriched proteins using several potential glucosyl acceptors showed an exclusive preference for glucose-6-phosphate. In contrast, both enzymes were shown to differ significantly from each other in their activity with different glucosyl nucleotides as glucosyl donors. OtsA1-His10 showed highest activity with ADP-glucose and UDP-glucose, whereas OtsA2-His10 preferred UDP-glucose. In addition, the wild-type OtsA activity of R. opacus 1CP was investigated and compared with recombinant enzymes. Results indicate that OstA2 mainly contributes to the trehalose pool of strain 1CP. OtsA1 seems to be involved in the overproduction of trehalose lipids. For the first time, a physiological role of two different OtsAs obtained of a single Rhodococcus strain was presumed.
Assuntos
Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Rhodococcus/enzimologia , Rhodococcus/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Expressão Gênica , Glucosiltransferases/química , Cinética , Dados de Sequência Molecular , Fosfatos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Especificidade por Substrato , Trealose/metabolismoRESUMO
The actinobacterium Rhodococcus opacus 1CP possesses a so far unique variant of the modified 3-oxoadipate pathway for 3-chlorocatechol degradation. One important feature is the novel dehalogenase ClcF, which converts (4R,5S)-5-chloromuconolactone to E-dienelactone. ClcF is related to muconolactone isomerase (MLI, EC 5.3.3.4). The enzyme has a ferredoxin-type fold and forms a homodecamer of 52-symmetry, typical for the MLI family. The active site is formed by residues from two monomers. The complex structure of an E27A variant with bound substrate in conjunction with mutational studies indicate that E27 acts as the proton acceptor in a univalent single-base syn-dehydrohalogenation mechanism. Despite the evolutionary specialization of ClcF, the conserved active-site structures suggest that the proposed mechanism is representative for the MLI family. Furthermore, ClcF represents a novel type of dehalogenase based on an isomerase scaffold.
Assuntos
4-Butirolactona/análogos & derivados , Hidrolases/química , Hidrolases/metabolismo , Lactonas/metabolismo , Rhodococcus/enzimologia , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Cristalização , Cristalografia por Raios X , Análise Mutacional de DNA , Hidrolases/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Rhodococcus/classificação , Rhodococcus/genética , Alinhamento de SequênciaRESUMO
Chloromuconolactone dehalogenase ClcF plays a unique role in 3-chlorocatechol degradation by Rhodococcus opacus 1CP by compensating the inability of its chloromuconate cycloisomerase ClcB2 to dechlorinate the chemically stable cycloisomerization product (4R,5S)-5-chloromuconolactone (5CML). High sequence similarities showed relatedness of ClcF to muconolactone isomerases (MLIs, EC 5.3.3.4) of the 3-oxoadipate pathway. Although both enzyme types share the ability to dechlorinate 5CML, comparison of kcat/Km indicated a significant extent of specialization of ClcF for dechlorination. This assumption was substantiated by an almost complete inability of ClcF to convert (4S)-muconolactone and the exclusive formation of cis-dienelactone from 5CML. Mutational analysis of ClcF by means of variants E27D, E27Q, Y50A, N52A, and A89S indicated relevance of some highly conserved residues for substrate binding and catalysis. Based on the putative isomerization mechanism of MLI, evidence was provided for a role of E27 in initial proton abstraction as well as of Y50 and N52 in substrate binding. In case of N52 substrate binding is likely to occur to the carboxylic group of 5CML as indicated by a significant change of product specificity. Expression in Escherichia coli BL21-CP(DE)-RIL followed by a three-step purification procedure with heat treatment is a convenient strategy to obtain recombinant ClcF and variants thereof.
Assuntos
Biocatálise , Hidrolases/genética , Hidrolases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodococcus/enzimologia , Rhodococcus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Domínio Catalítico , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Cupriavidus necator/enzimologia , Análise Mutacional de DNA , Expressão Gênica , Hidrolases/química , Hidrolases/isolamento & purificação , Cinética , Lactonas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Chloroaromatic compounds are often very persistent environmental pollutants. Nevertheless, numerous bacteria are able to metabolize these compounds and to utilize them as sole energy and carbon sources. Rhodococcus opacus 1CP is able to degrade several chloroaromatic compounds, some of them via a variation of the 3-chlorocatechol branch of the modified ortho-cleavage pathway. This branch in R. opacus differs from that in Proteobacteria in the inability of the chloromuconate cycloisomerase to dehalogenate. Instead, a unique enzyme designated as chloromuconolactone dehalogenase (ClcF) is recruited. ClcF dehalogenates 5-chloromuconolactone to cis-dienelactone and shows a high similarity to muconolactone isomerases (EC 5.3.3.4). However, unlike the latter enzymes, it is unable to catalyse the isomerization of muconolactone to 3-oxoadipate enollactone. In order to characterize the catalytic mechanism of this unusual dehalogenase, the enzyme was crystallized and subjected to X-ray structural analysis. Data sets to up to 1.65â Å resolution were collected from two different crystal forms using synchrotron radiation. Crystal form I (space group P2(1)) contained 40 subunits in the asymmetric unit, whereas ten subunits were present in crystal form II (space group P2(1)2(1)2(1)). The self-rotation function revealed the orientations of the molecular symmetry axes of the homodecamer of 52 symmetry.
Assuntos
Hidrolases/química , Rhodococcus/enzimologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Cristalização , Hidrolases/metabolismoRESUMO
Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg(-1) represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 µmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process.
Assuntos
Isomerases/metabolismo , Proteínas de Membrana/metabolismo , Rhodococcus/enzimologia , Coenzimas/metabolismo , Estabilidade Enzimática , Compostos de Epóxi/metabolismo , Concentração de Íons de Hidrogênio , Isomerases/química , Isomerases/isolamento & purificação , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Fenilacetatos/metabolismo , Rhodococcus/química , Especificidade por Substrato , TemperaturaRESUMO
Styrene monooxygenases (SMOs) are catalysts for the enantioselective epoxidation of terminal alkenes. Most representatives comprise a reductase and a monooxygenase which are encoded by separate genes (styA, styB). Only six presumed self-sufficient one-component SMOs (styA2B) have previously been submitted to databases, and one has so far been characterized. StyA2B can be supported by another epoxidase (StyA1) encoded by styA1, a gene in direct neighborhood of styA2B. The present report describes the identification of a further styA1/styA2B-like SMO, which was detected in Rhodococcus opacus MR11. Based on the initially available sequences of styA2B-type SMOs, primers directed at conserved sequences were designed and a 7,012-bp genomic fragment from strain MR11 was obtained after PCRs and subsequent genome walking. Six open reading frames (ORFs) were detected and compared to genomic fragments of strains comprising either two- or one-component SMOs. Among the proteins encoded by the ORFs, the monooxygenase StyA1/StyA2B showed the highest divergence on amino acid level when comparing proteins from different sources. That finding, a rare distribution of styA2B genes among bacteria, and the general observation of evolution from simple to complex systems indicate that one-component SMOs evolved from two-component ancestors. Analysis of gene products from styA/styB- and styA1/styA2B-like SMOs revealed that a fusion of styA/styB to styA2B might have happened at least twice among microorganisms. This points to a convergent evolution of one-component SMOs.
Assuntos
Evolução Molecular , Flavoproteínas/genética , Flavoproteínas/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Sequência de Aminoácidos , Biocatálise , Flavoproteínas/química , Genômica , Dados de Sequência Molecular , Oxigenases/química , Reação em Cadeia da Polimerase , Rhodococcus/enzimologia , Rhodococcus/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
SiteFinding-PCR has been recently reported to be a useful technique in order to identify unknown DNA fragments located adjacent to available sequences. However, this method has so far only been applied to few DNA sources including plants, samples from bioleaching communities, and a Pseudomonas strain. In order to complete the sequence information of two gene clusters in Gram-positive rhodococci the original protocol was applied yielding amplicons of insufficient size. The binding site of the previously published SiteFinder-2 oligo proved to be unsuitable for Rhodococcus and other members of the Actinobacteria since the binding motif occurred too frequently. Available genome sequences of different Actinobacteria were analysed and the binding site of the SiteFinder oligo modified. Moreover, PCR conditions were adapted to the high GC content of the template DNA allowing the successful adaptation of this method to two members of the Actinobacteria.
Assuntos
Actinobacteria/genética , Passeio de Cromossomo/métodos , Reação em Cadeia da Polimerase/métodos , Composição de Bases/genética , DNA Bacteriano/genéticaRESUMO
Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH2 provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH2 is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH2, resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH2-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view.
Assuntos
Proteínas de Bactérias/metabolismo , FMN Redutase/metabolismo , Oxigenases/metabolismo , Rhodococcus/metabolismo , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , FMN Redutase/genética , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Molecular , Oxigenases/genética , Rhodococcus/genéticaRESUMO
The subjects of the article are investigations concerning the ability of both Rhodococcus opacus 1CP and mixed bacterial cultures to use selected surfactants as sole carbon and energy source. In a comparative manner the biosurfactants rhamnolipid, sophorolipid and trehalose tetraester, and the synthetic surfactant Tween 80 were examined. Particular emphasis was put on a combinatorial approach to determine quantitatively the degree of surfactant degradation by applying calorimetry, thermodynamic calculations and mass spectrometry, HPLC as well as determination of biomass. The pure bacterial strain R. opacus was only able to metabolize a part of the synthetic surfactant Tween 80, whereas the mixed bacterial cultures degraded all of the applied surfactants. Exclusive for the biosurfactant rhamnolipid a complete microbial degradation could be demonstrated. In the case of the other surfactants only primary degradation was observed.
Assuntos
Calorimetria/métodos , Rhodococcus/metabolismo , Tensoativos/metabolismo , Glicolipídeos/química , Glicolipídeos/metabolismo , Polissorbatos/química , Polissorbatos/metabolismo , Rhodococcus/química , Rhodococcus/crescimento & desenvolvimento , Tensoativos/química , Trealose/química , Trealose/metabolismoRESUMO
Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His(10)-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.
Assuntos
Proteínas de Bactérias/fisiologia , Oxigenases/fisiologia , Rhodococcus/enzimologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Genoma Bacteriano/genética , Genoma Bacteriano/fisiologia , Modelos Biológicos , Dados de Sequência Molecular , Oxigenases/classificação , Oxigenases/genética , Oxigenases/metabolismo , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodococcus/genética , Rhodococcus/metabolismo , Estireno/química , Estireno/metabolismoRESUMO
Motivated by the finding that Pseudomonas knackmussii B13 but not Rhodococcus opacus 1CP grows in the absence of externally provided CO(2), we investigated the assimilation of (13)CO(2) into active cells cultivated with non-labelled glucose as sole energy substrate. (13)C found in the bulk biomass indicated a substantial but different CO(2) assimilation by Pseudomonas and Rhodococcus, respectively (3500 per thousand and 2600 per thousand). Cellular fatty acids were labelled from -15 per thousand to 470 per thousand and amino acids from 500 per thousand to 24,000 per thousand demonstrating clear differences between various compound classes. 'You are what you eat plus 1 per thousand' is therefore only valid for the average bulk C without specific isotope signature deviation of the external CO(2) or carbonates. Odd-numbered and 10-methyl fatty acids, which are much more abundant in Rhodococcus or other Gram-positive bacteria, were up to fivefold higher enriched in (13)C relative to the Pseudomonas fatty acids. A high-level growth-phase-independent, labelling of the oxaloacetate-derived amino acids indicated heterotrophic CO(2) fixation by anaplerotic reactions known to replenish the tricarboxylic acid cycle. Although both strains assimilate CO(2) via similar general pathways, Rhodococcus depends to a much higher extent on carboxylations reactions with external CO(2) owing to the formation of odd-numbered fatty acids. As a general consequence, heterotrophic fixation of CO(2) should be taken into account in investigations of degradation experiments using isotope tracer compounds.