The biphenyl dioxygenase (BPDO) catalyses a stereospecific dioxygenation of biphenyl and analogs of it. Aside from being involved in the destruction and detoxification of toxic pollutants in soil, in the context of the green chemistry concept, this enzyme is a promising biocatalyst to design new more selective and more environmentally friendly approaches to manufacture fine chemicals. At this time, most of our knowledge about the variability of key residues determining the substrate specificity and regiospecificity of the enzyme oxygenase component (BphAE) toward biphenyl analogs and about the effect of altering these residues on catalytic properties is based on investigations made with BphAEs from cultured organisms and engineered enzymes derived from them. The purpose of this work was to examine the diversity of the amino acid sequence patterns of the α subunit (BphA) C-terminal domain deduced from PCR products amplified from DNA extracted from cultured bacteria of various phylogenetic lines and from the soil microflora of PCB-contaminated soils. Of special interest were segments of the C-terminal portion called regions I, III and IV. Altogether, the phylogenetic tree obtained from aligning the deduced amino acid sequences of BphAs C-terminal domain from cultured bacteria belonging to various ecological niches and from uncultured soil bacteria reveals that most of the BphAs were linked to the three clusters of BphAs previously reported. However, few belong to new branches that diverge from the previously known branches showing a high diversity of BphAs in natural environment. Furthermore, data show a wide distribution of BphAs with family linkages that not only crosses bacterial taxonomic frontiers but also ecological niches. Nevertheless, in spite of this divergence, the sequence patterns of regions III and IV amino acids that are known to influence substrate specificity and regiospecificity are rather conserved among BphAs and the pattern was independent of the family cluster to which they belong. In most cases, regions III and IV amino acid patterns are closer to those of Pseudomonas pseudoalcaligenes KF707 BphA1 than to the most versatile Burkholderia xenovorans LB400 BphA. This might suggest that the PCB-degrading potency of soil bacteria is closer to the one observed for KF707 BphAE than from LB400 BphAE. However, the fact that among less than 20 PCR products amplified from soil DNA that we have sequenced, one of them was very homologous to that of LB400 BphA and in addition, residues 335 and 336 of LB400 were replaced by residues that previous enzyme engineering had shown to extend the range of PCB substrate used by the enzyme strongly suggest that PCB-degrading bacteria are evolving in soil to optimize their PCB-degrading capacity.
Previous work has shown that the C-terminal portion of BphA, especially two amino acid segments designated region III and region IV, influence the regiospecificity of the biphenyl dioxygenase (BPDO) toward 2,2-dichlorobiphenyl (2,2-CB). In this work, we evolved BPDO by shuffling bphA genes amplified from polychlorinated biphenyl-contaminated soil DNA. Sets of approximately 1-kb DNA fragments were amplified with degenerate primers designed to amplify the C-terminal portion of bphA. These fragments were shuffled, and the resulting library was used to replace the corresponding fragment of Burkholderia xenovorans LB400 bphA. Variants were screened for their ability to oxygenate 2,2-CB onto carbons 5 and 6, which are positions that LB400 BPDO is unable to attack. Variants S100, S149, and S151 were obtained and exhibited this feature. Variant S100 BPDO produced exclusively cis-5,6-dihydro-5,6-dihydroxy-2,2-dichlorobiphenyl from 2,2-CB. Moreover, unlike LB400 BPDO, S100 BphA catalyzed the oxygenation of 2,2,3,3-tetrachlorobiphenyl onto carbons 5 and 6 exclusively and it was unable to oxygenate 2,2,5,5-tetrachlorobiphenyl. Based on oxygen consumption measurements, variant S100 oxygenated 2,2-CB at a rate of 16 ؎ 1 nmol min ؊1 per nmol enzyme, which was similar to the value observed for LB400 BPDO. cis-5,6-Dihydro-5,6-dihydroxy-2,2-dichlorobiphenyl was further oxidized by 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) and 2,3-dihydroxybiphenyl dioxygenase (BphC). Variant S100 was, in addition, able to oxygenate benzene, toluene, and ethyl benzene. Sequence analysis identified amino acid residues M 237 S 238 and S 283 outside regions III and IV that influence the activity toward doubly ortho-substituted chlorobiphenyls.
La recomposition sociodémographique des campagnes entraîne des répercussions sur les interactions locales et les rapports de pouvoir. Les appréhender demeure un défi, car les analyses se limitent souvent aux seuls conflits entre néoruraux et ruraux de longue date autour d’enjeux partiels, sans inclure les décideurs. Pour dépasser cette vision conflictuelle et fragmentaire, notre objectif est de dégager un portrait global des relations tant de coopération que d’opposition de quatre groupes, soit les néoruraux, les ruraux de longue date, les dirigeants d’organismes et les élus municipaux, à propos de l’ensemble des enjeux les concernant. Cet article s’appuie sur des données recueillies auprès de ces différents acteurs dans deux territoires ruraux contrastés du Québec (Canada). Après un bilan mitigé des liens sociaux entretenus lors de la participation locale des néoruraux, nous nous concentrons sur les zones de collaborations et/ou de conflits de tous ces protagonistes quant aux enjeux démographiques, économiques, socioculturels, politiques, environnementaux et agricoles. Trois tendances émergent, révélant des modalités inattendues d’interactions, des rapports de force complexes ainsi que des conceptions antagoniques de l’espace rural et de son développement futur.
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