Michel Sylvestre

Cloning and expression of genes involved in 4-chlorobiphenyl transformation by Pseudomonas testosteroni: homology to polychlorobiphenyl-degrading genes in other bacteria

The genes of Pseudomonas testosteroni strain B-356, specifying the transformation of 4-chlorobiphenyl (4-CB) into 4-chlorobenzoic acid (4-CBA) were cloned into Pseudomonas putida KT2440 using a broad-host-range cosmid, pPSA842. Of 10,000 clones tested, four were able to transform 4-CB. Gas chromatographic and mass spectrometric analysis of the catabolic products from two of the 4-CB-transforming clones carrying the hybrid plasmids, pDA1 and pDA2, demonstrated that pDA1 carried a complete set of structural genes involved in the transformation of 4-CB into 4-CBA, while pDA2 contained part of the pathway genes leading up to the meta-cleavage compound. Restriction endonuclease mapping and subcloning of pDA1 and pDA2 showed that the clones contained a common stretch of DNA of about 9.1 kb and that pDA2 carried gene(s) involved in regulation. Probing blots of genomic DNA from 13 different polychlorinated biphenyl(PCB)-degrading bacteria with radio-labelled pDA1 and pDA2, suggested that many PCB-degrading pathways have a common phylogenetic origin.

Total biodegradation of 4-chlorobiphenyl (4 CB) by a two-membered bacterial culture

SummarySeveral bacterial strains that can oxidize mono- and dichlorinated biphenyls with one unsubstituted ring have already been described. The major route for this biodegradation leads ultimately to the corresponding chlorobenzoic acid, but several other minor chlorinated metabolites that might possibly be of concern for the environment have also been described previously. Since none of the bacterial strains that are able to oxidize these chlorinated biphenyls in pure culture are known to degrade chlorobenzoic acid, the oxidation of these substrates by axenic cultures always generates chlorobenzoates plus several other metabolites. In the present study, we have estimated the biodegradation of 4-chlorobiphenyl (4CB) by a two-membered bacterial culture containing one strain able to grow on 4CB and to transform it into 4-chlorobenzoate (4CBA) and one strain able to degrade 4CBA. The results were encouraging, since it was shown that the degradation of 4CB was more rapid and complete with the double bacterial culture.

Biphenyl/Chlorobiphenyls catabolic pathway of Comamonas testosteroni B-356: Prospect for use in bioremediation

This review summarizes our present knowledge concerning bacterial PCB oxidative catabolic pathways. Using, as an example, some of the results that were obtained mainly with Comamonas testosteroni B-356, some of the major features of PCB catabolic pathways will be depicted. Based on our present knowledge, some of the factors that should be considered in priority when looking for strategies to obtain new strains with enhanced capabilities to degrade those persistent pollutants will be discussed. The major features that will be discussed include the bioavailability of PCB as substrate, the use of co-substrate, the significance of the substrate reactivity pattern of the biphenyl oxygenase (the first enzyme of the pathway) and the importance of the stringent control of metabolite production.

Factors affecting the enhancement of PCB degradative ability of soil microbial populations

Abstract In this study, we compared the capacity of freshly isolated microbial populations obtained from soils with a history of contamination and evaluated the effect of analog enrichment on the potential of PCB degradation attained by the enhanced populations. It has previously been suggested that PCB degrading ability should improve through mutations of the oxygenase that can increase reactivity of the enzyme towards various individual congeners. Our results show that the chlorine content of the PCB mixture and duration of soil contamination (number of years) have no effect on the natural evolution of the ability of soil microbial populations to degrade higher chlorinated PCBs. Moreover, although our results provided evidence that analog enrichment can favor the selection of oxygenases with increased activities, we failed to select for populations that are able to degrade higher chlorinated PCBs more efficiently. Our data also clearly showed that chlorobenzoates, the end-products of the chlorobiphenyl degradation pathway (or their metabolites) interfere with PCB degradation. Therefore, the efficiency of PCB degradation is not only impaired by the substrate selectivity pattern of the chlorobiphenyl oxygenases, but also by the stringent control of metabolite production and it is likely that population adjustment to prevent production of metabolites that could interfere with PCB degradation would be difficult to achieve through natural selection.

Substrate selectivity pattern of Comamonas testosteroni strain B-356 towards dichlorobiphenyls

Abstract In this work, we have investigated the substrate selectivity pattern of strain B-356 resting cell suspensions and cell lysates towards selected chlorobiphenyl congeners. The strain showed a preference for the double meta-substituted congener 3,3′-dichlorobiphenyl over the double ortho-substituted congener 2,2′-dichlorobiphenyl and the double para-substituted congener 4,4′-dichlorobiphenyl. The results are discussed with reference to the substrate selectivity pattern reported for Pseudomonas sp. strain LB400.

Persistence and fate of PCBs in sediments of the Saint Lawrence River

Abstract Polychlorinated biphenyl (PCB) congener concentrations and composition were determined in sediments from three sections of the St. Lawrence River that have been exposed to high PCB contamination. In at least one sampling station in each river section (upper Lac St-Francois (LSF) and two sections in metropolitan Montreal, Canada), high PCB concentrations were found in the top 10 cm of sediments (respectively 16.2, 9.8 and 3.8 mg/kg d.w. of Aroclor 1248 equivalents). In the case of sediments along the south shore of LSF, both PCB concentrations (regression analysis) and composition (correspondence analysis) were related to the distance of sampling sites downstream from the local source area. In the most contaminated station of the lake, located near an industrial facility, analysis of sediment core profile dated by 137 Cs revealed a relatively constant input of PCBs since ∼ 1982. The di- and tri-chlorinated congeners accounted for more than 70% of the quantified congeners in surface sediments of this station, whereas these congeners made up less than 30% of the PCBs found in less contaminated St. Lawrence River sediments. The PCB concentrations and compositions found in the river sediments suggest that local point sources, river hydrology, physicochemical processes and microbial dechlorination were major factors explaining the fate of PCB congeners in St. Lawrence River sediments.

Catalytic activity of Pseudomonas putida strain G7 naphthalene 1,2-dioxygenase on biphenyl

Naphthalene dioxygenase (NAP dox) catalyzes the first reaction of the naphthalene catabolic pathway encoded by the NAH plasmid. Similar to other aryl hydroxylating dioxygenases, NAP dox introduces molecular oxygen on two vicinal carbons of the aromatic ring. In this work it is shown that Pseudomonas putida G7 NAP dox (NAP dox G7 ) can catalyze the oxygenation of biphenyl quite efficiently. Similar to biphenyl dioxygenase which is the homologous reaction of the biphenyl catabolic pathway, the enzyme attack biphenyl molecule onto the ortho-meta carbons preferentially. However, unlike biphenyl dioxygenase of Pseudomonas sp. LB400, NAP dox G7 ) dioxygenates a non negligible portion of the substrate onto the meta-para carbons.

Functionality of biphenyl 2,3-dioxygenase components in naphthalene 1,2-dioxygenase

Abstract Naphthalene 1,2-dioxygenase (Nap dox) and biphenyl 2,3-dioxygenase (Bph dox) are related enzymes that have differentiated during evolution as their specificity has changed. Although their component arrangement is similar, the structure of each component has been modified quite extensively. The purpose of this work was to determine the catalytic capacity of purified Nap dox toward chlorobiphenyls and to investigate the functionality of Bph dox components in the Nap dox system. Both enzyme systems were purified by affinity chromatography as histidine-tagged fused proteins. Data show for the first time that Nap dox can catalyze the oxygenation of all three monochlorobiphenyl isomers, but it is unable to hydroxylate 2,5-, 2,2′-, 3,3′-, 4,4′-di- and 2,2′,5,5′-tetrachlorobiphenyl. The rates of cytochrome c reduction by the ferredoxin components of the two enzymes were identical when the Bph dox reductase component was used in the assay, showing an efficient electron transfer between the Bph dox reductase component and the Nap dox ferredoxin. However, when the Bph dox ferredoxin was used to reconstitute a hybrid Nap dox, the enzyme was only 22% as active as the parental enzyme. These data are discussed in terms of the potential use of Nap dox for the development of enhanced chlorobiphenyl-degrading dioxygenases.

Factors involved in bactericidal activities of formaldehyde and formaldehyde and formaldehyde condensate/isothiazolone mixtures

Enhanced antimicrobial activities of formaldehyde (FA)_and 5-choloro-2-methyl-4-isothiazolin-3-one (IT) mixtures were determined in tryptic soy broth (TSB), minimal salt-based medium, saline, and soluble oil emulsion using a Pseudomonas aeruginosa strain as a test organism. The apparent higher activity of mixture was the result of simultaneous and combined action of the biocides. The involvement of sulfhydryl groups as targets in the mode of action of both biocides was investigated using S-nitroso thioglycollic acid as a covalent modifier of exterior sulfhydryl groups of the cells. The results suggest their involvement in the mode of action of IT. These studies also indicate a considerable independent action by both biocides when the mixture is used with absence of cross-resistance in resistant isolates and independent induction of resistance in Pseudomonas aeruginosa to these biocides. Replacement of FA in the mixture with 1, 3,5-tris (2-hydroxyethyl)-hexahydrotriazine at equimolar concentrations of FA in TSB showed essentially the same levels of increased activity with no apparent adverse effects on the antimicrobial activity of IT from the amine portion of the molecule. Furthermore, in-vitro studies also indicated direct protection of IT by FA and FA condensate biocides in the presence of nucleophiles.

Microbial transformation of a dihydroxybiphenyl

The X-ray crystal structure of (3S,4R)-3,4-dihydroxy-5-(3-hydroxyphenyl)-5-cyclohexen-1-one, C 12 H 12 O 4 , was determined and showed that the molecule consists of a conjugated cyclohexenone ring substituted by two hydroxyl groups and a 3-hydroxyphenyl group. There are three O-H...O intermolecular hydrogen bonds