Properties of Enzymes Involved in Tetrachloroethene Respiration: From Physiology to Protein Function

Tetrachloroethene (PCE) pollution threatens nature and human health due to its toxic and carcinogenic potential. Due to industrial activities, large amounts of PCE were discharged into the environment over the last decades and represent one of major groundwater pollutants. Organohalide respiration (OHR) is a bacterial anaerobic respiration in which the halogenated compounds, such as chloroethenes, are used as terminal electron acceptors. This process requires the presence of an electron transport chain located in the cytoplasmic membrane which allows proton translocation and establishes a proton-motive force across the membrane. Redox proteins and other non-protein electron shuttles are usually combined in the membrane to accomplish that task. In members of the genera Desulfitobacterium and Dehalobacter, which represent a paradigmatic group of organohalide-respiring bacteria (OHRB), the pceABCT gene cluster has been identified as responsible for PCE respiration, and can be considered as a model system for studying OHR. While the function of PceA, the key enzyme of this process, and PceT, the dedicated chaperon, are well established, it is very likely that PceB plays the role of membrane anchor for PceA. The remaining gene, pceC, codes for a predicted membrane-bound flavoprotein harboring conserved cysteine motifs in the transmembrane domain. Despite the fact that it has been considered as a putative transcriptional regulator, PceC presents all the features that could potentially fulfill the role of electron shuttle between reduced menaquinones and PceA. In the Laboratory for Environmental Biotechnology, a bacterial consortium (SL2-PCEb) and derived subcultures thereof has been used as a model OHRB community. SL2-PCEb has been previously characterized for its stepwise dechlorination of PCE to cis-DCE via a significant accumulation of TCE. Two derived subcultures, named SL2-PCEc and SL2-TCE, harbor a distinct population of Sulfurospirillum sp. (SL2-1 and SL2-2, respectively). Both subcultures showed a different pattern of dechlorination, as strain SL2-1 was only able to dechlorinate PCE to trichloroethene (TCE), while strain SL2-2 kept the potential of the parental consortium, namely PCE to cis-DCE, however, without TCE accumulation. The key enzyme in the consortium SL2-PCEc is known as PceATCE and displays 92% amino acid sequence identity with the well-characterized PceA enzyme from S. multivorans. The overall goal of this thesis was to provide new insights into the physiology and biochemistry of tetrachloroethene-respiring bacteria, to characterize at the biochemical level a new reductive dehalogenase identified from a bacterial consortium, and to assess the kinetic parameters of strains present in this consortium and competing for tetrachloroethene. Finally, the characterization of PceC, a predicted membrane-bound flavoprotein was undertaken.


Related material