Yves Jouanneau

Identification of Pyrene-Induced Proteins in Mycobacterium sp. Strain 6PY1: Evidence for Two Ring-Hydroxylating Dioxygenases

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [(14)C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M(r)s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.

Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a Sphingomonas Strain That Degrades Various Polycyclic Aromatic Hydrocarbons

ABSTRACT In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [14C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.

Betaproteobacteria dominance and diversity shifts in the bacterial community of a PAH-contaminated soil exposed to phenanthrene.

In this study, the PAH-degrading bacteria of a constructed wetland collecting road runoff has been studied through DNA stable isotope probing. Microcosms were spiked with (13)C-phenanthrene at 34 or 337 ppm, and bacterial diversity was monitored over a 14-day period. At 337 ppm, PAH degraders became dominated after 5 days by Betaproteobacteria, including novel Acidovorax, Rhodoferax and Hydrogenophaga members, and unknown bacteria related to Rhodocyclaceae. The prevalence of Betaproteobacteria was further demonstrated by phylum-specific quantitative PCR, and was correlated with a burst of phenanthrene mineralization. Striking shifts in the population of degraders were observed after most of the phenanthrene had been removed. Soil exposed to 34 ppm phenanthrene showed a similar population of degraders, albeit only after 14 days. Results demonstrate that specific Betaproteobacteria are involved in the main response to soil PAH contamination, and illustrate the potential of SIP approaches to investigate PAH biodegradation in soil.

Enhanced Nitrogenase Activity in Strains of Rhodobacter capsulatus That Overexpress the rnf Genes

In the photosynthetic bacterium Rhodobacter capsulatus, a putative membrane-bound complex encoded by the rnfABCDGEH operon is thought to be dedicated to electron transport to nitrogenase. In this study, the whole rnf operon was cloned under the control of the nifH promoter in plasmid pNR117 and expressed in several rnf mutants. Complementation analysis demonstrated that transconjugants which integrated plasmid pNR117 directed effective biosynthesis of a functionally competent complex in R. capsulatus. Moreover, it was found that strains carrying pNR117 displayed nitrogenase activities 50 to 100% higher than the wild-type level. The results of radioactive labeling experiments indicated that the intracellular content of nitrogenase polypeptides was marginally altered in strains containing pNR117, whereas the levels of the RnfB and RnfC proteins present in the membrane were four- and twofold, respectively, higher than the wild-type level. Hence, the enhancement of in vivo nitrogenase activity was correlated with a commensurate overproduction of the Rnf polypeptides. In vitro nitrogenase assays performed in the presence of an artificial electron donor indicated that the catalytic activity of the enzyme was not increased in strains overproducing the Rnf polypeptides. It is proposed that the supply of reductants through the Rnf complex might be rate limiting for nitrogenase activity in vivo. Immunoprecipitation experiments performed on solubilized membrane proteins revealed that RnfB and RnfC are associated with each other and with additional polypeptides which may be components of the membrane-bound complex.

Characterization of an fdxN mutant of Rhodobacter capsulatus indicates that ferredoxin I serves as electron donor to nitrogenase.

A mutant of Rhodobacter capsulatus, carrying an insertion into the fdxN gene encoding ferredoxin I (FdI), has been studied by biochemical analysis and genetic complementation experiments. When compared to the wild-type strain, the fdxN mutant exhibited altered nitrogen fixing ability and 20-fold lower levels of nitrogenase activity as assayed in vivo. When assayed in vitro with an artificial reductant, nitrogenase activity was only 3- to 4-fold lower than in the wild type. These results suggested that the FdI-deleted mutant had impaired electron transport to nitrogenase. Immunochemical assay of both nitrogenase components showed that the fdxN mutant contained about 4-fold less enzyme than wild-type cells. Results of pulse-chase labeling experiments using [35S]methionine indicated that nitrogenase was significantly less stable in the FdI-deleted mutant. When a copy of fdxN was introduced in the mutant in trans, the resulting strain appeared to be fully complemented with respect to both diazotrophic growth and nitrogenase activity. Depending on whether fdxN expression was driven by a nif promoter or a fructose-inducible promoter, FdI was synthesized either at wild-type level or in 10-fold lower amounts. The strain producing 10-fold less FdI did, however, display normal N2-fixing ability. Analysis of cytosolic proteins by bidimensional electrophoresis revealed that the fdxN mutant produced a 14 kDa polypeptide in amounts about 3-fold greater than wild-type cells. This protein was identified by N-terminal microsequencing as a recently purified [2Fe-2S] ferredoxin, called FdV, which cannot reduce nitrogenase. It is concluded that FdI serves as the main electron donor to nitrogenase in R. capsulatus and that an ancillary electron carrier, distinct of FdV, is responsible for the residual nitrogenase activity observed in the FdI-deleted mutant.

Characterization of a ring-hydroxylating dioxygenase from phenanthrene-degrading Sphingomonas sp. strain LH128 able to oxidize benz[a]anthracene

Sphingomonas sp. strain LH128 was isolated from a polycyclic aromatic hydrocarbon (PAH)-contaminated soil using phenanthrene as the sole source of carbon and energy. A dioxygenase complex, phnA1fA2f, encoding the α and β subunit of a terminal dioxygenase responsible for the initial attack on PAHs, was identified and isolated from this strain. PhnA1f showed 98%, 78%, and 78% identity to the α subunit of PAH dioxygenase from Novosphingobium aromaticivorans strain F199, Sphingomonas sp. strain CHY-1, and Sphingobium yanoikuyae strain B1, respectively. When overexpressed in Escherichia coli, PhnA1fA2f was able to oxidize low-molecular-weight PAHs, chlorinated biphenyls, dibenzo-p-dioxin, and the high-molecular-weight PAHs benz[a]anthracene, chrysene, and pyrene. The action of PhnA1fA2f on benz[a]anthracene produced two benz[a]anthracene dihydrodiols.

Purification and characterization of an arene cis-dihydrodiol dehydrogenase endowed with broad substrate specificity toward polycyclic aromatic hydrocarbon dihydrodiols

ABSTRACT Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent Mr of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with Km values in the range of 1.4 to 7.1 μM, and exhibited a much higher Michaelis constant for NAD+ (Km of 160 μM). At pH 7.0, the specificity constant ranged from (1.3 ± 0.1) × 106 M−1 s−1 with benz[a]anthracene 1,2-dihydrodiol to (20.0 ± 0.8) × 106 M−1 s−1 with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.

Characterization of Three XylT-Like [2Fe-2S] Ferredoxins Associated with Catabolism of Cresols or Naphthalene: Evidence for Their Involvement in Catechol Dioxygenase Reactivation

The xylT gene product, a component of the xylene catabolic pathway of Pseudomonas putida mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in Escherichia coli, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were -286 mV and -323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an M(r) of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction. In complementation experiments, plasmids expressing xylT or its analogues were introduced into an XylT-null mutant of P. putida which is unable to grow on p-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.

The crystal structure of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1

The ring‐hydroxylating dioxygenase (RHD) from Sphingomonas CHY‐1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four‐ and five‐fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY‐1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 Å by molecular replacement revealed the enzyme to share the same global α3β3 structural pattern. The catalytic domain distinguishes itself from other bacterial non‐heme Rieske iron oxygenases by a substantially larger hydrophobic substrate binding pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 Å. An 11 Å long water channel extends from the interface between the α and β subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY‐1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.

ApoFnr binds as a monomer to promoters regulating the expression of enterotoxin genes of Bacillus cereus.

Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-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 (Fnr(His)) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) ((Strep)Fnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnr(His) was mainly monomeric, while apo(Strep)Fnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apo(Strep)Fnr was dithiothreitol sensitive, underlining the importance of a disulfide 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.