Search

International audience; The 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.

International audience; Ring 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.

International audience; Bacillus cereus Fnr is a member of the Crp/Fnr (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of Hbl and Nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (FnrHis) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) (StrepFnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occured as mixtures of monomers and oligomers in solution. However, apoFnrHis was mainly monomeric, while apoStrepFnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apoStrepFnr was dithiothreitol sensitive, underlining the importance of a disulphide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.

International audience; 2-Ethyhexyl nitrate (2-EHN) is a major additive of fuel which is used to comply with the cetane number of diesel. Because of its wide use and possible accidental release, 2-EHN is a potential pollutant of the environment. In this study, Mycobacterium austroafricanum IFP 2173 was selected among several strains as the best 2-EHN degrader. The 2-EHN biodegradation rate was increased in biphasic cultures where the hydrocarbon was dissolved in an inert non-aqueous phase liquid (NAPL), suggesting that the transfer of the hydrophobic substrate to the cells was a growth-limiting factor. Carbon balance calculation as well as organic carbon measurement indicated a release of metabolites in the culture medium. Further analysis by gas chromatography revealed that a single metabolite accumulated during growth. This metabolite had a molecular mass of 114 Da as determined by GC/MS and was provisionally identified as 4-ethyldihydrofuran-2(3H)-one by LC-MS/MS analysis. Identification was confirmed by analysis of the chemically synthesized lactone. Based on these results, a plausible catabolic pathway is proposed whereby 2-EHN is converted to 4-ethyldihydrofuran-2(3H)-one, which cannot be metabolised further by strain IFP 2173. This putative pathway provides an explanation for the low energetic efficiency of 2-EHN degradation and its poor biodegradability.

International audience; In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which has not so far been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas CHY-1 has been purified and characterized with respect to its biochemical and catalytic properties. The oxygenase component called PhnII, exhibited an α3β3 heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent Km for salicylate of 1.1 ± 0.2 µM. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (Km = 28 ± 6 µM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but induced a high rate of uncoupled oxidation of NADH. It also exerted a strong competitive inhibition of salicylate hydroxylation, with a Ki of 0.68 µM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and discussed in the light of our current knowledge of PAH degradation by sphingomonads.