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1.
Sci Total Environ ; 771: 145437, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33736182

RESUMO

Bacterial degradation is one of the most efficient ways to remove microcystins (MCs), the most frequently detected toxin in cyanobacterial blooms. Using Novosphingobium sp. ERW19 as a representative strain, our laboratory previously demonstrated that quorum sensing (QS), the cell density-dependent gene regulation system, positively regulates biodegradation of MCs via transcriptional activation of mlr-pathway-associated genes. Increasing evidence indicates that QS is involved in a wide spectrum of regulatory circuits, but it remains unclear which physiological processes in MC degradation besides the expression of MC-degrading genes are also subject to QS-dependent regulation. This study used transcriptome analysis to identify QS-regulated genes during degradation of MCs. A large percentage (up to 32.6%) of the genome of the MC-degrading bacterial strain Novosphingobium sp. ERW19 was significantly differentially expressed in the corresponding QS mutants. Pathway enrichment analysis of QS-regulated genes revealed that QS mainly influenced metabolism-associated pathways, particularly those related to amino acid metabolism, carbohydrate metabolism, and biodegradation and metabolism of xenobiotics. In-depth functional interpretation of QS-regulated genes indicated a variety of pathways were potentially associated with bacterial degradation or physiological responses to MCs, including genes involved in cell motility, cytochrome P450-dependent metabolism of xenobiotics, glutathione S-transferase (GST), envelope stress response, and ribosomes. Furthermore, QS may be involved in regulating the initial and final steps of the catabolic pathway of phenylacetic acid, an intermediate product of MC degradation. Collectively, this global survey of QS-regulated genes in a MC-degrading bacterial strain facilitates a deeper understanding of QS-controlled processes that may be important for bacterial degradation of MCs or may contribute to the physiological responses of bacteria to MCs.


Assuntos
Percepção de Quorum , Sphingomonadaceae , Biodegradação Ambiental , Perfilação da Expressão Gênica , Microcistinas , Sphingomonadaceae/genética
2.
Chemosphere ; 267: 129217, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33321275

RESUMO

Hexabromocyclododecanes (HBCDs) were used as flame-retardants until their ban in 2013. Among the 16 stereoisomers known, ε-HBCD has the highest symmetry. This makes ε-HBCD an interesting substrate to study the selectivity of biotransformations. We expressed three LinA dehydrohalogenase enzymes in E. coli bacteria, two wild-type, originating from Sphingobium indicum B90A bacteria and LinATM, a triple mutant of LinA2, with mutations of L96C, F113Y and T133 M. These enzymes are involved in the hexachlorocyclohexane (HCH) metabolism, specifically of the insecticide γ-HCH (Lindane). We studied the reactivity of those eight HBCD stereoisomers found in technical HBCD. Furthermore, we compared kinetics and selectivity of these LinA variants with respect to ε-HBCD. LC-MS data indicate that all enzymes converted ε-HBCD to pentabromocyclododecenes (PBCDens). Transformations followed Michaelis-Menten kinetics. Rate constants kcat and enzyme specificities kcat/KM indicate that ε-HBCD conversion was fastest and most specific with LinA2. Only one PBCDen stereoisomer was formed by LinA2, while LinA1 and LinATM produced mixtures of two PBCDE enantiomers at three times lower rates than LinA2. In analogy to the biotransformation of (-)ß-HBCD, with selective conversion of dibromides in R-S-configuration, we assume that 1E,5S,6R,9S,10R-PBCDen is the ε-HBCD transformation product from LinA2. Implementing three amino acids of the LinA1 substrate-binding site into LinA2 resulted in a triple mutant with similar kinetics and product specificity like LinA1. Thus, point-directed mutagenesis is an interesting tool to modify the substrate- and product-specificity of LinA enzymes and enlarge their scope to metabolize other halogenated persistent organic pollutants regulated under the Stockholm Convention.


Assuntos
Retardadores de Chama , Hidrocarbonetos Bromados , Sphingomonadaceae , Biotransformação , Escherichia coli , Hexaclorocicloexano , Sphingomonadaceae/genética , Estereoisomerismo
3.
Chemosphere ; 262: 128288, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33182101

RESUMO

Short-chain chlorinated paraffins (SCCPs) are listed as persistent organic pollutants (POPs) under the Stockholm Convention. Such substances are toxic, bioaccumulating, transported over long distances and degrade slowly in the environment. Certain bacterial strains of the Sphingomonadacea family are able to degrade POPs, such as hexachlorocyclohexanes (HCHs) and hexabromocyclododecanes (HBCDs). The haloalkane dehalogenase LinB, expressed in certain Sphingomonadacea, is able to catalyze the transformation of haloalkanes to hydroxylated compounds. Therefore, LinB is a promising candidate for conversion of SCCPs. Hence, a mixture of chlorinated tridecanes was exposed in vitro to LinB, which was obtained through heterologous expression in Escherichia coli. Liquid chromatography mass spectrometry (LC-MS) was used to analyze chlorinated tridecanes and their transformation products. A chloride-enhanced soft ionization method, which favors the formation of chloride adducts [M+Cl]- without fragmentation, was applied. Mathematical deconvolution was used to distinguish interfering mass spectra of paraffinic, mono-olefinic and di-olefinic compounds. Several mono- and di-hydroxylated products including paraffinic, mono-olefinic and di-olefinic compounds were found after LinB exposure. Mono- (rt = 5.9-6.9 min) and di-hydroxylated (rt = 3.2-4.5 min) compounds were separated from starting material (rt = 7.7-8.5 min) by reversed phase LC. Chlorination degrees of chlorinated tridecanes increased during LinB-exposure from nCl = 8.80 to 9.07, indicating a preferential transformation of lower chlorinated (Cl<9) tridecanes. Thus, LinB indeed catalyzed a dehalohydroxylation of chlorinated tridecanes, tridecenes and tridecadienes. The observed hydroxylated compounds are relevant CP transformation products whose environmental and toxicological effects should be further investigated.


Assuntos
Poluentes Ambientais/análise , Hidrocarbonetos Clorados/análise , Hidrolases/química , Parafina/análise , Biocatálise , Monitoramento Ambiental/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Halogenação , Hexaclorocicloexano/análise , Hidrocarbonetos Bromados/análise , Hidrolases/isolamento & purificação , Hidroxilação , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética
4.
Proc Natl Acad Sci U S A ; 117(32): 19228-19236, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32703810

RESUMO

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Sphingomonadaceae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Glutationa/química , Glutationa/metabolismo , Ferro/metabolismo , Domínios Proteicos , Sphingomonadaceae/química , Sphingomonadaceae/genética
5.
Environ Sci Technol ; 54(12): 7591-7600, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32412239

RESUMO

The chlorinated nitroaromatic antibiotic chloramphenicol (CAP) is a refractory contaminant that is widely present in various environments. However, few CAP-mineralizing bacteria have been documented, and a complete CAP catabolism pathway has yet to be identified. In this study, the bacterial strain Sphingobium sp. CAP-1 was isolated from an activated sludge sample and was shown to be capable of aerobically subsisting on CAP as the sole carbon, nitrogen, and energy source while simultaneously and efficiently degrading CAP. p-Nitrobenzoic acid (PNBA), p-nitrobenzaldehyde (PNBD), protocatechuate (PCA), and the novel side chain C3-hydroxy-oxygenated product of CAP (O-CAP) were identified during CAP degradation. Strain CAP-1 was able to convert O-CAP to intermediate product PNBA. The putative functional genes associated with PNBA catabolism into the tricarboxylic acid cycle via PCA and floc formation were also identified by genome sequencing and comparative proteome analysis. A complete pathway for CAP catabolism was proposed. The discovery of a novel CAP oxidation/detoxification process and a complete pathway for CAP catabolism enriches the fundamental understanding of the bacterial catabolism of antibiotics, providing new insights into the microbial-mediated fate, transformation, and resistance risk of CAP in the environment. The molecular basis of CAP catabolism and floc formation in strain CAP-1 also offers theoretical guidance for the enhanced bioremediation of CAP-containing environments.


Assuntos
Cloranfenicol , Sphingomonadaceae , Antibacterianos , Biodegradação Ambiental , Esgotos , Sphingomonadaceae/genética
6.
Sci Rep ; 10(1): 6662, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313127

RESUMO

The environment affects the composition and function of soil microbiome, which indirectly influences the quality of plants. In this study, 16S amplicon sequencing was used to reveal the differences in soil microbial community composition of Cistanche deserticola in three ecotypes (saline-alkali land, grassland and sandy land). Through the correlation analysis of microbial community abundance, phenylethanoid glycoside contents and ecological factors, the regulatory relationship between microbial community and the quality variation of C. deserticola was expounded. The metabolic function profile of soil microbiome was predicted using Tax4Fun. Data showed that the soil microbial communities of the three ecotypes were significantly different (AMOVA, P < 0.001), and the alpha diversity of grassland soil microbial community was the highest. Core microbiome analysis demonstrated that the soil microbial communities of C. deserticola were mostly have drought, salt tolerance, alkali resistance and stress resistance, such as Micrococcales and Bacillales. The biomarkers, namely, Oceanospirillales (saline-alkali land), Sphingomonadales (grassland) and Propionibacteriales (sandy land), which can distinguish three ecotype microbial communities, were excavated through LEfSe and random forest. Correlation analysis results demonstrated that 2'-acetylacteoside is positively correlated with Oceanospirillales in saline-alkali land soil. The metabolic function profiles displayed highly enriched metabolism (carbohydrate and amino acid metabolisms) and environmental information processing (membrane transport and signal transduction) pathways. Overall, the composition and function of soil microbiomes were found to be important factors to the quality variation of C. deserticola in different ecotypes. This work provided new insight into the regulatory relationship amongst the environment, soil microbial community and plant quality variation.


Assuntos
Bacillales/classificação , Cistanche/microbiologia , Micrococcaceae/classificação , Oceanospirillaceae/classificação , Propionibacteriaceae/classificação , Microbiologia do Solo , Sphingomonadaceae/classificação , Bacillales/genética , Bacillales/isolamento & purificação , Técnicas de Tipagem Bacteriana , China , Cistanche/fisiologia , Secas , Ecótipo , Variação Genética , Glicosídeos/biossíntese , Pradaria , Concentração de Íons de Hidrogênio , Micrococcaceae/genética , Micrococcaceae/isolamento & purificação , Oceanospirillaceae/genética , Oceanospirillaceae/isolamento & purificação , Filogenia , Propionibacteriaceae/genética , Propionibacteriaceae/isolamento & purificação , RNA Ribossômico 16S/genética , Salinidade , Tolerância ao Sal/genética , Areia/microbiologia , Solo/química , Sphingomonadaceae/genética , Sphingomonadaceae/isolamento & purificação
7.
Appl Environ Microbiol ; 86(12)2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32303545

RESUMO

Carboxylesterase PytH, isolated from the pyrethroid-degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin, and bifenthrin. To elucidate the catalytic mechanism of PytH, we report here the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported α/ß-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230, and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low-activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrate binding; both lid and core domains are involved in substrate binding, and the lid domain-induced core domain movement may make the active center correctly positioned with substrates.IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household; however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. This showed that PytH belongs to the α/ß-hydrolase fold proteins with typical catalytic Ser-His-Asp triad, though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket enabled PytH to bind bigger pyrethroid family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deepen our understanding of α/ß-hydrolase members.


Assuntos
Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/genética , Inseticidas/metabolismo , Fluoreto de Fenilmetilsulfonil/metabolismo , Piretrinas/metabolismo , Sphingomonadaceae/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Análise de Sequência de DNA , Sphingomonadaceae/metabolismo
8.
J Biosci Bioeng ; 130(1): 71-75, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32238321

RESUMO

Lignosulfonate is a by-product of the cooking process by sulfite pulping for paper manufacturing. The treatment of wood chips by various salts of sulfurous acid solubilizes lignin to produce a cellulose-rich wood pulp. Developing a technique for the conversion of lignosulfonate by-product to high value materials has an important industrial utility. Sphingobium sp. strain SYK-6, which was isolated from pulping wastewater, is one of the best enzymatically or genetically characterized bacteria for degrading lignin-derived aromatics. We have previously established a system for the production of 2-pyrone-4,6-dicarboxylic acid (PDC), a novel platform chemical that can produce a variety of bio-based polymers, by introducing of ligA, ligB, and ligC genes from SYK-6 into a mutant strain of Pseudomonas putida PpY1100. In this study, extracts from lignosulfonates, which were desulphonated and depolymerized by alkaline oxidation, were evaluated as substrates for microbiological conversion to PDC by the transgenic bacteria.


Assuntos
Lignina/metabolismo , Extratos Vegetais/metabolismo , Pseudomonas putida/metabolismo , Pironas/metabolismo , Sphingomonadaceae/metabolismo , Celulose/metabolismo , Ácidos Dicarboxílicos/metabolismo , Pseudomonas putida/genética , Sphingomonadaceae/genética , Resíduos/análise
9.
Artif Cells Nanomed Biotechnol ; 48(1): 672-682, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32075448

RESUMO

The present study highlights the biological synthesis of silver nanoparticles (AgNPs) using Sphingobium sp. MAH-11 and also their antibacterial mechanisms against drug-resistant pathogenic microorganisms. The nanoparticle synthesis method used in this study was reliable, facile, rapid, cost-effective and ecofriendly. The AgNPs exhibited the highest absorbance at 423 nm. The TEM image expressed spherical shape of AgNPs and the size of synthesized AgNPs was 7-22 nm. The selected area diffraction (SAED) pattern and XRD spectrum revealed the crystalline structure of AgNPs. The results of FTIR analysis disclosed the functional groups responsible for the reduction of silver ion to metal nanoparticles. The biosynthesized AgNPs showed strong anti-microbial activity against drug-resistant pathogenic microorganisms. Moreover, Escherichia coli and Staphylococcus aureus were used to explore the antibacterial mechanisms of biosynthesized AgNPs. Minimal inhibitory concentrations (MICs) of E. coli and S. aureus were 6.25 µg/mL and 50 µg/mL, respectively and minimum bactericidal concentrations (MBCs) of E. coli and S. aureus were 25 µg/mL and 100 µg/mL, respectively. Results exhibited that biosynthesized AgNPs caused morphological changes and injured the membrane integrity of strains E. coli and S. aureus. The AgNPs synthesized by Sphingobium sp. MAH-11 may serve as a potent antimicrobial agent for many therapeutic applications.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Nanopartículas Metálicas/química , Prata/metabolismo , Sphingomonadaceae/metabolismo , Antibacterianos/química , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Filogenia , RNA Ribossômico 16S/genética , Prata/química , Prata/farmacologia , Sphingomonadaceae/classificação , Sphingomonadaceae/genética , Staphylococcus aureus/efeitos dos fármacos
10.
Int J Med Microbiol ; 310(2): 151396, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32005588

RESUMO

The occurrence of antibiotic resistance bacteria has become a major threat to public health. We have recently discovered a transcriptional activator that belongs to MarR family, EstR, and an esterase B (EstB) with a newly proposed de-arenethiolase activity from Sphingobium sp. SM42. De-arenethiolase activity involves the removal of the small aromatic side chain of cephalosporin antibiotics as an excellent leaving group by the enzymatic CS bond cleavage. Here, we report the regulation of estB through EstR as an activator in response to a third generation cephalosporin, cefoperazone, antibiotic. Cefoperazone induced the expression of estB in wild type Sphingobium sp., but not in the estR knockout strain, and the induction was restored in the complemented strain. Moreover, we revealed the importance of EstB localization in periplasm. Since EsB has the ability to inactivate selected ß-lactam antibiotics in vitro, it is possible that the enzyme works at the periplasmic space of Gram negative bacteria similar to ß-lactamases. EstB was genetically engineered by incorporating NlpA binding motif, or OmpA signal sequence, or SpyTag-SpyCatcher to the estB gene to mobilize it to different compartments of periplasm; inner membrane, outer membrane, and periplasmic space, respectively. Surprisingly, we found that Sphingobium sp. SM42 and E. coli expressing EstB at the periplasm were more sensitive to cefoperazone. The possible drug enhancement mechanism by enzyme was proposed. This work might lead to a novel strategy to tackle antibiotic resistance problem.


Assuntos
Cefoperazona/farmacologia , Cefalosporinas/farmacologia , Periplasma/enzimologia , Serina Endopeptidases/genética , Sphingomonadaceae/efeitos dos fármacos , Fatores de Transcrição/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Periplasma/efeitos dos fármacos , Sinais Direcionadores de Proteínas , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética
11.
Molecules ; 25(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31906348

RESUMO

The synthesis of bioplastic from marine microbes has a great attendance in the realm of biotechnological applications for sustainable eco-management. This study aims to isolate novel strains of poly-ß-hydroxybutyrate (PHB)-producing bacteria from the mangrove rhizosphere, Red Sea, Saudi Arabia, and to characterize the extracted polymer. The efficient marine bacterial isolates were identified by the phylogenetic analysis of the 16S rRNA genes as Tamlana crocina, Bacillus aquimaris, Erythrobacter aquimaris, and Halomonas halophila. The optimization of PHB accumulation by E. aquimaris was achieved at 120 h, pH 8.0, 35 °C, and 2% NaCl, using glucose and peptone as the best carbon and nitrogen sources at a C:N ratio of 9.2:1. The characterization of the extracted biopolymer by Fourier-transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (NMR), and Gas chromatography-mass spectrometry (GC-MS) proves the presence of hydroxyl, methyl, methylene, methine, and ester carbonyl groups, as well as derivative products of butanoic acid, that confirmed the structure of the polymer as PHB. This is the first report on E. aquimaris as a PHB producer, which promoted the hypothesis that marine rhizospheric bacteria were a new area of research for the production of biopolymers of commercial value.


Assuntos
Biopolímeros/biossíntese , Biopolímeros/química , Hidroxibutiratos/química , Hidroxibutiratos/metabolismo , Poliésteres/química , Poliésteres/metabolismo , Sphingomonadaceae/química , Sphingomonadaceae/metabolismo , Avicennia/microbiologia , Bacillus/química , Bacillus/genética , Bacillus/metabolismo , Biopolímeros/análise , Carbono/química , Carbono/metabolismo , Fermentação , Flavobacteriaceae/química , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Halomonas/química , Halomonas/genética , Halomonas/metabolismo , Hidroxibutiratos/análise , Espectroscopia de Ressonância Magnética , Nitrogênio/química , Nitrogênio/metabolismo , Filogenia , Poliésteres/análise , RNA Ribossômico 16S/genética , Rizosfera , Salinidade , Arábia Saudita , Água do Mar/microbiologia , Espectroscopia de Infravermelho com Transformada de Fourier , Sphingomonadaceae/genética , Sphingomonadaceae/isolamento & purificação , Temperatura
12.
Antonie Van Leeuwenhoek ; 113(5): 719-727, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31980980

RESUMO

In this study, a novel ginsenoside transforming bacterium, strain W1-2-3T, was isolated from mineral water. The 16S rRNA gene sequence analysis showed that strain W1-2-3T shares 93.7-92.2% sequence similarity with the members of the family Sphingomonadaceae and makes a group with Sphingoaurantiacus capsulatus YLT33T (93.7%) and S. polygranulatus MC 3718T (93.4%). The novel isolate efficiently hydrolyses the ginsenoside Rc to Rd. The genome comprises a single circular 2,880,809, bp chromosome with 3211 genes in total, and 1993 protein coding genes. The isolate was observed to grow at 10-37 °C and at pH 6-10 on R2A agar medium; maximum growth was found to occur at 25 °C and pH 7.0. Strain W1-2-3T was found to contain ubiquinone-10 as the predominant quinone and the fatty acids C16:1, C17:1ω6c, C14:0 2-OH, summed feature 3 (C16:1ω6c/C16:1ω7c) and summed feature 8 (C18:1ω6c/C18:1ω7c). The DNA G+C content was determined to be 65.9 mol%. Strain W1-2-3T can be distinguished from the other members of the family Sphingomonadaceae by a number of chemotaxonomic and phenotypic characteristics. The major polar lipids of strain W1-2-3T were identified as phosphatidylethanolamine, an unidentified glycolipid and an unidentified polar lipid. The major poly amine was found to be homospermidine. Based on polyphasic taxonomic analysis, strain W1-2-3T is concluded to represent a novel species within a new genus, for which the name Hankyongella ginsenosidimutans gen. nov., sp. nov. is proposed. The type strain of Hankyongella ginsenosidimutans is W1-2-3T (= KACC 18307T = LMG 28594T).


Assuntos
Ginsenosídeos/metabolismo , Águas Minerais/microbiologia , Sphingomonadaceae , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ácidos Graxos/análise , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Sphingomonadaceae/classificação , Sphingomonadaceae/genética , Sphingomonadaceae/isolamento & purificação , Sphingomonadaceae/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/análise
13.
Ecotoxicol Environ Saf ; 187: 109848, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31670182

RESUMO

Dimethyl terephthalate (DMT) is a primary ingredient widely used in the manufacture of polyesters and industrial plastics; its environmental fate is of concern due to its global use. Microorganisms play key roles in the dissipation of DMT from the environment; however, the enzymes responsible for the initial transformation of DMT and the possible altered toxicity due to this biotransformation have not been extensively studied. To reduce DMT toxicity, we identified the esterase gene dmtH involved in the initial transformation of DMT from the AOPP herbicide-transforming strain Sphingobium sp. C3. DmtH shows 24-41% identity with α/ß-hydrolases and belongs to subfamily V of bacterial esterases. The purified recombinant DmtH was capable of transforming DMT to mono-methyl terephthalate (MMT) and potentially transforming other p-phthalic acid esters, including diallyl terephthalate (DAT) and diethyl terephthalate (DET). Using C. elegans as an assay model, we observed the severe toxicity of DMT in inducing reactive oxygen species (ROS) production, decreasing locomotion behavior, reducing lifespan, altering molecular basis for oxidative stress, and inducing mitochondrial stress. In contrast, exposure to MMT did not cause obvious toxicity, induce oxidative stress, and activate mitochondrial stress in nematodes. Our study highlights the usefulness of Sphingobium sp. C3 and its esterase DmtH in transforming p-phthalic acid esters and reducing the toxicity of DMT to organisms.


Assuntos
Poluentes Ambientais/toxicidade , Esterases/genética , Genes Bacterianos , Ácidos Ftálicos/toxicidade , Sphingomonadaceae/metabolismo , Animais , Biodegradação Ambiental , Biotransformação , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Poluentes Ambientais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácidos Ftálicos/metabolismo , Plásticos/química , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética
14.
Environ Microbiol ; 22(1): 286-296, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31667998

RESUMO

The (R)- and (S)-enantiomers of the chiral herbicide napropamide (NAP) show different biological activities and ecotoxicities. These two enantiomers behave differently in the environment due to enantioselective catabolism by microorganisms. However, the molecular mechanisms underlying this enantioselective catabolism remain largely unknown. In this study, the genes (snaH and snpd) involved in the catabolism of NAP were cloned from Sphingobium sp. B2, which was capable of catabolizing both NAP enantiomers. Compared with (R)-NAP, (S)-NAP was much more rapidly transformed by the amidase SnaH, which initially cleaved the amide bonds of (S)/(R)-NAP to form (S)/(R)-2-(1-naphthalenyloxy)-propanoic acid [(S)/(R)-NP] and diethylamine. The α-ketoglutarate-dependent dioxygenase Snpd, showing strict stereoselectivity for (S)-NP, further transformed (S)-NP to 1-naphthol and pyruvate. Molecular docking and site-directed mutagenesis analyses revealed that when the (S)-enantiomers of NAP and NP occupied the active sites, the distance between the ligand molecule and the coordination atom was shorter than that when the (R)-enantiomers occupied the active sites, which facilitated formation of the transition state complex. This study enhances our understanding of the preferential catabolism of the (S)-enantiomer of NAP on the molecular level.


Assuntos
Amidoidrolases/metabolismo , Dioxigenases/metabolismo , Herbicidas/química , Herbicidas/metabolismo , Naftalenos/química , Naftalenos/metabolismo , Amidoidrolases/química , Amidoidrolases/genética , Dioxigenases/química , Dioxigenases/genética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética , Estereoisomerismo
15.
J Agric Food Chem ; 68(4): 1022-1029, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31884791

RESUMO

Topramezone is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor. Due to its broad-spectrum, high efficiency, and low toxicity, topramezone is a candidate herbicide for the construction of genetically modified (GM) herbicide-resistant crops. In the present study, we screened a topramezone-resistant isolate Sphingobium sp. TPM-19 and cloned a topramezone-resistant HPPD gene (SphppD) from this isolate. SpHPPD shared the highest similarity (53%) with an HPPD from Vibrio vulnificus CMCP6. SpHPPD was synthesized in Escherichia coli BL21(DE3) and purified to homogeneity using Co2+-affinity chromatography. SpHPPD was found to be a monomer. The Km and kcat of SpHPPD for 4-hydroxyphenylpyruvate (4-HPP) were 82.8 µM and 15.0 s-1, respectively. SpHPPD showed high resistance to topramezone with half maximal inhibitory concentration (IC50) and Ki values of 5.2 and 2.5 µM, respectively. Additionally, SpHPPD also showed high resistance to isoxaflutole (DKN) (IC50: 8.7 µM; Ki: 6.0 µM) and mesotrione (IC50: 4.2 µM; Ki: 1.3 µM) and moderate resistance to tembotrione (IC50: 2.5 µM; Ki: 1.0 µM). The introduction of the SphppD gene into Arabidopsis thaliana enhanced obvious resistance against topramezone. In conclusion, this study provides a novel topramezone-resistant HPPD gene for the genetic engineering of GM herbicide-resistant crops.


Assuntos
4-Hidroxifenilpiruvato Dioxigenase/química , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/química , Pirazóis/química , Sphingomonadaceae/enzimologia , 4-Hidroxifenilpiruvato Dioxigenase/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Resistência a Herbicidas , Herbicidas/química , Herbicidas/metabolismo , Herbicidas/farmacologia , Cinética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Pirazóis/metabolismo , Pirazóis/farmacologia , Sphingomonadaceae/química , Sphingomonadaceae/genética
16.
Appl Microbiol Biotechnol ; 104(3): 1125-1134, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31832710

RESUMO

Sphingobium sp. strain TCM1 can significantly degrade chlorinated organophosphorus flame retardants, such as tris(2-chloroethyl) phosphate. The PhoK of strain TCM1 (Sb-PhoK) is the main alkaline phosphatase (APase) that catalyzes the last step in the degradation pathway. Here, we purified and characterized Sb-PhoK produced in E. coli, and analyzed the regulation of Sb-phoK gene expression in strain TCM1. The recombinant Sb-PhoK was produced in the mature form, lacking a putative signal peptide, and formed a homodimer. Purified Sb-PhoK exhibited 384 U/mg of specific activity at 37 °C. The optimum temperature was 50 °C, and Sb-PhoK was completely inactivated when incubated at 60 °C for 10 min. The optimum pH was 10, with stability observed at pH 6.0-10.5. Sb-PhoK was suggested to contain two Ca2+ and one Zn2+ per subunit, but excess addition of Zn2+ into the reaction mixture markedly inhibited the enzyme activity. Sb-PhoK showed phosphatase activity against various phosphorylated compounds, except for bis(p-nitrophenyl) phosphate, indicating that it is a phosphomonoesterase with broad substrate specificity. The Km and kcat for p-nitrophenyl phosphate were 2.31 mM and 1270 s-1, respectively, under optimal conditions. The enzyme was strongly inhibited by vanadate, dithiothreitol, and SDS, but was highly resistant to urea and Triton X-100. Sb-phoK gene expression was regulated by the inorganic phosphate concentration in culture medium, and was induced at a low inorganic phosphate concentration. The deletion of Sb-phoB gene resulted in no induction of Sb-phoK gene even at a low inorganic phosphate concentration, confirming that Sb-PhoK is a member of Pho regulon.


Assuntos
Fosfatase Alcalina/biossíntese , Regulação Bacteriana da Expressão Gênica , Sphingomonadaceae/genética , Fosfatase Alcalina/genética , Biocatálise , Escherichia coli/genética , Retardadores de Chama/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Organofosfatos/metabolismo , Proteínas Recombinantes/biossíntese , Sphingomonadaceae/enzimologia
17.
Genes (Basel) ; 10(11)2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31683600

RESUMO

In this work, we have shown that Novosphingobium tardaugens NBRC 16725 (strain ARI-1), a bacterial strain that was isolated due to its capacity to mineralize the estrogenic endocrine compound 17ß-estradiol, is also able to mineralize testosterone, the androgenic endocrine compound. Using in silico analysis, we predicted a new putative steroid degradation (SD) gene cluster in strain ARI-1, which resembles genes involved in testosterone degradation in Comamonas testosteroni and other testosterone degrading bacteria like Actinobacteria (like Rhodococcus and Mycobacteria genera) although with significant differences in gene organization. A whole transcriptomic analysis of N. tardaugens revealed that testosterone produces a limited induction of the genes of the SD cluster that show a high basal expression in its absence. The 3ß/17ß-hydroxysteroid dehydrogenase involved in the first metabolic step of testosterone degradation was identified by using genetic and biochemical approaches. The construction of knockout mutant strains in the genes of the SD cluster together with in silico analyses suggests the existence of gene redundancy in the genome of N. tardaugens. This work will expand the knowledge about the metabolic pathways and biotransformation capabilities of a Gram-negative bacterium that could become a new model system in the bacterial steroid degradation field.


Assuntos
Androgênios/metabolismo , Proteínas de Bactérias/metabolismo , Hidroxiesteroide Desidrogenases/metabolismo , Sphingomonadaceae/enzimologia , Testosterona/metabolismo , Proteínas de Bactérias/genética , Biotransformação , Hidroxiesteroide Desidrogenases/genética , Sphingomonadaceae/genética
18.
J Agric Food Chem ; 67(44): 12228-12236, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31638826

RESUMO

Zeaxanthin is a value-added carotenoid with wide applications. This study aims to manipulate a generally recognized as safe and carotenoid-producing bacterium, Sphingobium sp., for enhanced production of zeaxanthin and exopolysaccharides. First, whole-genome sequencing and analysis of pathway genes were applied to define the carotenoid pathway in Sphingobium sp. Second, a Sphingobium transformation system was established to engineer metabolite flux into zeaxanthin. By a combination of chemical mutagenesis and removal of bottlenecks of carotenoid biosynthesis via overexpression of three rate-limiting enzymes, the genetically modified Sphingobium DIZ strain produced 21.26 mg/g dry cell weight of zeaxanthin, which was about 4-fold higher than the wild type. Upon optimization of culture conditions, the DIZ strain produced 479.5 mg/L of zeaxanthin with the productivity of 4.99 mg/L/h and 21.9 g/L of exopolysaccharides using a fed-batch fermentation strategy. This study represents the first genetic manipulation of Sphingobium sp., a biotechnologically important bacterium, for high-yield production of value-added metabolites.


Assuntos
Proteoglicanas/biossíntese , Sphingomonadaceae/genética , Sphingomonadaceae/metabolismo , Zeaxantinas/biossíntese , Técnicas de Cultura Celular por Lotes , Meios de Cultura/metabolismo , Fermentação , Engenharia Metabólica
19.
Genes (Basel) ; 10(9)2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500174

RESUMO

Sphingopyxis inhabit diverse environmental niches, including marine, freshwater, oceans, soil and anthropogenic sites. The genus includes 20 phylogenetically distinct, valid species, but only a few with a sequenced genome. In this work, we analyzed the nearly complete genome of the newly described species, Sphingopyxis lindanitolerans, and compared it to the other available Sphingopyxis genomes. The genome included 4.3 Mbp in total and consists of a circular chromosome, and two putative plasmids. Among the identified set of lin genes responsible for γ-hexachlorocyclohexane pesticide degradation, we discovered a gene coding for a new isoform of the LinA protein. The significant potential of this species in the remediation of contaminated soil is also correlated with the fact that its genome encodes a higher number of enzymes potentially involved in aromatic compound degradation than for most other Sphingopyxis strains. Additional analysis of 44 Sphingopyxis representatives provides insights into the pangenome of Sphingopyxis and revealed a core of 734 protein clusters and between four and 1667 unique proteins per genome.


Assuntos
Genoma Bacteriano , Hexaclorocicloexano/metabolismo , Praguicidas/metabolismo , Sphingomonadaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Sphingomonadaceae/enzimologia , Sphingomonadaceae/metabolismo
20.
Toxins (Basel) ; 11(10)2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31547007

RESUMO

Biodegradation is efficient for removing cyanobacterial toxins, such as microcystins (MCs) and nodularin (NOD). However, not all the microbial strains with the microcystin-biodegrading enzymes MlrA and MlrC could biodegrade NOD. Studies on genes and enzymes for biodegrading NOD can reveal the function and the biodegradation pathway of NOD. Based on successful cloning and expression of the USTB-05-A and USTB-05-C genes from Sphingopyxis sp. USTB-05, which are responsible for the biodegradation of MCs, the pathway for biodegrading NOD by these two enzymes was investigated in this study. The findings showed that the enzyme USTB-05-A converted cyclic NOD (m/z 825.4516) into its linear type as the first product by hydrolyzing the arginine and Adda peptide bond, and that USTB-05-C cut off the Adda and glutamic acid peptide bond of linearized NOD (m/z 843.4616) and produced dimeric Adda (m/z 663.4377) as the second product. Further, based on the homology modeling of enzyme USTB-05-A, site-directed mutants of USTB-05-A were constructed and seven crucial sites for enzyme USTB-05-A activity were found. A complete enzymatic mechanism for NOD biodegradation by USTB-05-A in the first step was proposed: glutamic acid 172 and histidine 205 activate a water molecule facilitating a nucleophilic attack on the arginine and Adda peptide bond of NOD; tryptophan 176 and tryptophan 201 contact the carboxylate side chain of glutamic acid 172 and accelerate the reaction rates; and histidine 260 and asparagine 264 function as an oxyanion hole to stabilize the transition states.


Assuntos
Peptídeos Cíclicos/metabolismo , Sphingomonadaceae/metabolismo , Biodegradação Ambiental , Clonagem Molecular , Redes e Vias Metabólicas , Peptídeos Cíclicos/química , Sphingomonadaceae/genética
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