Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota

Dietary intake of L-carnitine can promote cardiovascular diseases in humans through microbial production of trimethylamine (TMA) and its subsequent oxidation to trimethylamine N-oxide (TMAO) by hepatic flavin-containing monooxygenases. Although our microbiota are responsible for TMA formation from carnitine, the underpinning molecular and biochemical mechanisms remain unclear. In this study, using bioinformatics approaches, we first identified a two-component Rieske-type oxygenase/reductase (CntAB) and associated gene cluster proposed to be involved in carnitine metabolism in representative genomes of the human microbiota. CntA belongs to a group of previously uncharacterized Rieske-type proteins and has an unusual "bridging" glutamate but not the aspartate residue, which is believed to facilitate inter-subunit electron transfer between the Rieske centre and the catalytic mononuclear iron centre. Using Acinetobacter baumannii as the model, we then demonstrate that cntAB is essential in carnitine degradation to TMA. Heterologous overexpression of cntAB enables Escherichia coli to produce TMA, confirming that these genes are sufficient in TMA formation. Site-directed mutagenesis experiments have confirmed that this unusual "bridging glutamate" residue in CntA is essential in catalysis and neither mutant (E205D, E205A) is able to produce TMA. Together, our study reveals the molecular and biochemical mechanisms underpinning carnitine metabolism to TMA in human microbiota and assigns the role of this novel group of Rieske-type proteins in microbial carnitine metabolism

Study of Genes Relating to Degradation of Aromatic Compounds and Carbon Metabolism in Mycobacterium Sp. Strain KMS

Polycyclic aromatic hydrocarbons, produced by anthropological and natural activities, are hazardous through formation of oxidative radicals and DNA adducts. Growth of Mycobacterium sp. strain KMS, isolated from a contaminated soil, on the model hydrocarbon pyrene induced specific proteins. My work extends the study of isolate KMS to the gene level to understand the pathways and regulation of pyrene utilization. Genes encoding pyrene-induced proteins were clustered on a 72 kb section on the KMS chromosome but some also were duplicated on plasmids. Skewed GC content and presence of integrase and transposase genes suggested horizontal transfer of pyrene-degrading gene islands that also were found with high conservation in five other pyrene-degrading Mycobacterium isolates. Transcript analysis found both plasmid and chromosomal genes were induced by pyrene. These processes may enhance the survival of KMS in hydrocarbon-contaminated soils when other carbon sources are limited. KMS also grew on benzoate, confirming the functionality of an operon containing genes distinct from those in other benzoate-degrading bacteria. Growth on benzoate but not on pyrene induced a gene, benA, encoding a benzoate dioxygenase α-subunit, but not the pyrene-induced nidA encoding a pyrene dioxygenase α-subunit; the differential induction correlated with differences in promoter sequences. Diauxic growth occurred when pyrene cultures were amended with benzoate or acetate, succinate, or fructose, and paralleled delayed expression of nidA. Single phase growth and normal expression of benA was observed for benzoate single and mixed cultures. The nidA promoters had potential cAMP-CRP binding sites, suggesting that cAMP could be involved in carbon repression of pyrene metabolism. Growth on benzoate and pyrene requires gluconeogenesis. Intermediary metabolism in isolate KMS involves expression from genes encoding a novel malate:quinone oxidoreductase and glyoxylate shunt enzymes. Generation of C3 structures involves transcription of genes encoding malic enzyme, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate synthase. Carbon source modified the transcription patterns for these genes. My findings are the first to show duplication of pyrene-degrading genes on the chromosome and plasmids in Mycobacterium isolates and expression from a unique benzoate-degrading operon. I clarified the routes for intermediary metabolism leading to gluconeogenesis and established a potential role for cAMP-mediated catabolite repression of pyrene utilization

Growth of Pseudomonas aeruginosa LP5 on 2, 5-dicchlorobenzoate: Detection of aromatic ring hydroxylating dioxygenase (ARHDO) gene

Pseudomonas aeruginosa LP5 grew on 2, 5-dichlorobenzoate with doubling time (D) 6.64 d and mean growth rate (k) 0.104 d-1. The organism showed a prolonged lag period lasting 9 days followed by a sudden rise within 3 days (D= 1.1 d; k= 0.628 d-1) and death in less than 72 hours on 2, 6-dichlorobenzoate. Polymerase chain reaction (PCR) amplification of DNA of LP5 showed aromatic dihydroxylating (ARHDO) gene band with molecular weight corresponding to the targeted fragment (0.73 kb). The capability of LP5 on dichlorobenzoates and detection of dioxygenase genes is a validation of its versatility and potential for bioremediation

Diversity and catalytic potential of PAH-specific ring-hydroxylating dioxygenases from a hydrocarbon-contaminated soil.

International audienceRing-hydroxylating dioxygenases (RHDs) catalyze the initial oxidation step of a range of aromatic hydrocarbons including polycyclic aromatic hydrocarbons (PAHs). As such, they play a key role in the bacterial degradation of these pollutants in soil. Several polymerase chain reaction (PCR)-based methods have been implemented to assess the diversity of RHDs in soil, allowing limited sequence-based predictions on RHD function. In the present study, we developed a method for the isolation of PAH-specific RHD gene sequences of Gram-negative bacteria, and for analysis of their catalytic function. The genomic DNA of soil PAH degraders was labeled in situ by stable isotope probing, then used to PCR amplify sequences specifying the catalytic domain of RHDs. Sequences obtained fell into five clusters phylogenetically linked to RHDs from either Sphingomonadales or Burkholderiales. However, two clusters comprised sequences distantly related to known RHDs. Some of these sequences were cloned in-frame in place of the corresponding region of the phnAIa gene from Sphingomonas CHY-1 to generate hybrid genes, which were expressed in Escherichia. coli as chimerical enzyme complexes. Some of the RHD chimeras were found to be competent in the oxidation of two- and three-ring PAHs, but other appeared unstable. Our data are interpreted in structural terms based on 3D modeling of the catalytic subunit of hybrid RHDs. The strategy described herein might be useful for exploring the catalytic potential of the soil metagenome and recruit RHDs with new activities from uncultured soil bacteria

Functional diversity of bacterial genes associated with aromatic hydrocarbon degradation in anthropogenic dark earth of Amazonia.

The objective of this work was to evaluate the catabolic gene diversity for the bacterial degradation of aromatic hydrocarbons in anthropogenic dark earth of Amazonia (ADE) and their biochar (BC). Functional diversity analyses in ADE soils can provide information on how adaptive microorganisms may influence the fertility of soils and what is their involvement in biogeochemical cycles. For this, clone libraries containing the gene encoding for the alpha subunit of aromatic ring-hydroxylating dioxygenases (a-ARHD bacterial gene) were constructed, totaling 800 clones. These libraries were prepared from samples of an ADE soil under two different land uses, located at the Caldeirão Experimental Station - secondary forest (SF) and agriculture (AG) -, and the biochar (SF_BC and AG_BC, respectively). Heterogeneity estimates indicated greater diversity in BC libraries; and Venn diagrams showed more unique operational protein clusters (OPC) in the SF_BC library than the ADE soil, which indicates that specific metabolic processes may occur in biochar. Phylogenetic analysis showed unidentified dioxygenases in ADE soils. Libraries containing functional gene encoding for the alpha subunit of the aromatic ring-hydroxylating dioxygenases (ARHD) gene from biochar show higher diversity indices than those of ADE under secondary forest and agriculture

Characterization of novel PAH dioxygenases from the bacterial metagenomic DNA of a contaminated soil.

International audienceRing hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using (13)C-phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria, especially Rhodocyclaceae and Burkholderiales, consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in E. coli, but full activity required the co-expression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a Sphingomonad. The four RHDs showed markedly different preferences for 2- and 3-ring PAHs, but were poorly active on 4-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3, C-4 positions, suggesting that degradation occurred through an alternate pathway

Substrate Specificity and Structural Characteristics of the Novel Rieske Nonheme Iron Aromatic Ring-Hydroxylating Oxygenases NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1

The Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1 have been implicated in the initial oxidation of high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs), forming cis-dihydrodiols. To clarify how these two RHOs are functionally different with respect to the degradation of HMW PAHs, we investigated their substrate specificities to 13 representative aromatic substrates (toluene, m-xylene, phthalate, biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, carbazole, and dibenzothiophene) by enzyme reconstitution studies of Escherichia coli. Both Nid systems were identified to be compatible with type V electron transport chain (ETC) components, consisting of a [3Fe-4S]-type ferredoxin and a glutathione reductase (GR)-type reductase. Metabolite profiles indicated that the Nid systems oxidize a wide range of aromatic hydrocarbon compounds, producing various isomeric dihydrodiol and phenolic compounds. NidAB and NidA3B3 showed the highest conversion rates for pyrene and fluoranthene, respectively, with high product regiospecificity, whereas other aromatic substrates were converted at relatively low regiospecificity. Structural characteristics of the active sites of the Nid systems were investigated and compared to those of other RHOs. The NidAB and NidA3B3 systems showed the largest substrate-binding pockets in the active sites, which satisfies spatial requirements for accepting HMW PAHs. Spatially conserved aromatic amino acids, Phe-Phe-Phe, in the substrate-binding pockets of the Nid systems appeared to play an important role in keeping aromatic substrates within the reactive distance from the iron atom, which allows each oxygen to attack the neighboring carbons

Ring-hydroxylating dioxygenases involved in PAH biodegradation : structure, function, biodiversity

International audienceThe first step in the biodegradation of PAHs by aerobic bacteria is catalyzed by metalloenzymes known as ring-hydroxylating dioxygenases (RHDs). Because of the hydrophobic nature and chemical resistance of PAHs, their initial attack by RHDs is a difficult reaction, which is critical to the whole degradation process. This chapter gives an overview of the current knowledge on the genetics, structure, catalytic mechanism and diversity of RHDs involved in PAH degradation. In the past decade, the crystal structures of 10 RHDs have been determined, giving insights into the mechanism of substrate recognition and regioselectivity of dioxygenation. The reaction catalyzed by the archetypal naphthalene dioxygenase has been investigated in detail, thus providing a better understanding of the RHD catalytic mechanism. Studies on the catabolic genes responsible for PAH degradation in several bacterial taxa have highlighted the great phylogenetic diversity of RHDs. The implementation of culture-independent methods has afforded means to further explore the environmental diversity of PAH-degrading bacteria and RHDs. Recent advances in this field now allow the in situ identification of bacteria responsible for pollutant removal. Further biotechnological developments based on microarrays and functional metagenomics should lead to the conception of molecular tools useful for the bioremediation of PAH-contaminated ecosystems

From Functional Potential of Soil Bacterial Communities Towards Petroleum Hydrocarbons Bioremediation

Molecular ecology researches are rapidly advancing the knowledge of microorganisms associated with petroleum hydrocarbon degradation, one of the major large-scale pollutants in terrestrial ecosystems. The design and monitoring of bioremediation techniques for hydrocarbons rely on a thorough understanding of the diversity of enzymes involved in the processes of hydrocarbon degradation and the microbes that harbor their allocated genes. This review describes the impact of hydrocarbon pollution on soil microbial communities, the state of the art of detecting functional genes, and functional groups. We will focus on i) the structure, function and succession behavior of microbial communities exposed to hydrocarbons, ii) key genes and pathways, iii) future prospect into bioremediation of petroleum hydrocarbons in aerobic environments. The aim is to get a fundamental insight in these issues to ultimately improve petroleum hydrocarbons bioremediation. Keywords: Petroleum hydrocarbons, microbial communities, functional genes, oil degradation, bioremediation. DOI: 10.7176/JBAH/9-10-01 Publication date:May 31st 201

Sequencing and functional analysis of a multi-component dioxygenase from PAH-degrading Sphingomonas paucimobilis EPA505

Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic organic compounds consisting of two or more fused benzene rings. PAHs derive from many different sources including petroleum refining, wood treatment, and coal coking industries. Because of their structural stability and water insolubility, PAHs are extremely resistant to degradation. These compounds are also believed to have mutagenic, carcinogenic, and teratogenic effects. Therefore, there are currently 16 PAH compounds on the EPA\u27s list of priority pollutants. Many species of bacteria have the ability to breakdown these persistent pollutants. However, bioremediation strategies using these organisms have many unresolved issues. While laboratory experiments can easily demonstrate the ability of these organisms to breakdown pollutants, environmental factors may reduce degradation abilities in situ. Within the prokaryotes, members of the genus Sphingomonas have demonstrated a greater ability to breakdown PAHs. Spingomonas paucimobilis EPA505, for example, was shown to degrade a wide range of PAHs including the high molecular weight PAH fluoranthene, which it could also use as a sole carbon source. Because of its potential as a bioremediation tool, it is important to study the molecular basis of PAH catabolism in EPA505. A genomic library of EPA505 was constructed and probed for genes involved in PAH degradation. Complete gene sequences were obtained for four subunits which are involved in the first step of the PAH catabolism. This step is catalyzed by a dioxygenase enzyme and yields a dihydrodiol intermediate. Two of the gene sequences encode an alpha and beta subunit of the dioxygenase. The third gene encodes a ferredoxin subunit and the fourth gene codes for a ferredoxin reductase subunit. The four genes were cloned for expression. Expression host cells were induced to test the activity of the four recombinant proteins on various PAHs. When cells expressing all four subunits were incubated with naphthalene and phenanthrene, the corresponding dihydrodiol product was detected using GC-MS. No dihydrodiol product was detected when fluoranthene was tested. In addition, no dihydrodiol products were detected for any substrate when cells lacking the two ferredoxin subunits were tested. This study identified and showed functional analysis of one enzyme, a PAH degrading dioxygenase in the PAH catabolic pathway of Sphingomonas paucimobilis EPA505. There is still much to learn in order to fully appreciate and take advantage of this organism as an efficient tool for bioremediation