Organohalide respiration: breakthrough towards the elucidation of the electron-accepting moiety of the respiratory chain

Background Organohalide respiration (OHR) is a bacterial anaerobic process in which chlorinated compound, e.g. tetrachloroethene (PCE), is used as terminal electron acceptors. Dehalobacter restrictus PER-K23, a paradigmatic organohalide-respiring bacterium (OHRB), harbours the pceABCT gene cluster, representing one model system for the study of PCE respiration. To date, the function of PceA, the key enzyme in the process, and PceT, the molecular chaperone for PceA maturation, are well defined. PceB and PceC are predicted integral membrane proteins, however no evidence has been obtained for their physiological function. The present work aims to investigate the stoichiometric relationships between the proteins encoded in the pceABCT gene cluster and to elucidate the membrane-bound reductive dehalogenase (RDH) protein complex. Methods The stoichiometry of the pceABCT gene products was assessed at RNA and protein level by RT-qPCR and quantitative proteomics, respectively. Clear-Native PAGE combined with an in-gel RDH activity assay was applied to identify the RDH complex from membrane extracts of D. restrictus. Results At RNA level, pceA, pceB, pceC and pceT genes appeared with relative stoichiometry (normalized for pceA) of approximately 1.0:3.0:0.1:0.1. At proteomic level, the cell-free extract of D. restrictus displayed a stoichiometry of 1.0:0.5:0.02:0.2 for the proteins PceA, PceB, PceC and PceT, respectively. CN-PAGE and in-gel enzymatic assay revealed an active RDH complex with a molecular mass of ~180 kDa identified in the membrane extract, while no in-gel activity could be detected in the soluble fraction. Conclusions Taken together, the results suggest that PceA and PceB form a complex in the membrane, while PceC might act as an accessory protein. Furthermore, the in-gel RDH activity assay allowed for the first time to visualise an active ~180 kDa PceA-containing complex from the membrane. The protein composition of the active RDH complex is currently underway and shall be presented at the conference.

Anaerobic Biodegradation of organohalide pollutants: a crucial step towards the elucidation of proteins involved

Lorenzo Cimmino, Christof Holliger, Julien Maillard, Adrian Schmid

Halogenated organic compounds (so-called organohalides) represent one of the major class of groundwater pollutants. The exploration of how organohalides are used as energy source is important in terms of ecosystem remediation but is also essential for the complete understanding of microbial metabolic interactions in the environment. Organohalide respiration (OHR) is a bacterial anaerobic process in which chlorinated compound, e.g. tetrachloroethene (PCE), is used as terminal electron acceptor. In the present work, Desulfitobacterium hafniense TCE1 and Dehalobacter restrictus, our model organohalide-respiring bacteria (OHRB) harbouring the pceABCT gene cluster, will be considered for the study of PCE respiration. To date, the function of PceA, the key catalytic enzyme in the process, and PceT, the dedicated molecular chaperone for PceA maturation, are well defined. However, the roles of PceB and PceC are not yet elucidated and the biochemistry of OHR electron transfer is still relatively elusive. Based on the genetic composition of the pce gene cluster, the hypothesis of a possible PceABC respiratory complex is tempting but the question remains largely unanswered. The present work represents an evaluation of the stoichiometry of PceA, PceB and PceC proteins via quantitative proteomics applied to the membranes fractions of our model organisms. In a second phase, the use of Blue-Native electrophoresis technology will be considered to investigate whether PceC participates in a membrane-bound protein complex together with PceA and PceB. The complementary results of both techniques will lead to identify the three proteins of interest in the membrane and is expected to shed light on the presence and composition of a PCE respiratory complex.

2019

ANAEROBIC BIODEGRADATION OF ORGANOHALIDE POLLUTANTS: A CRUCIAL STEP TOWARDS THE ELUCIDATION OF PROTEINS INVOLVED

Lorenzo Cimmino, Christof Holliger, Julien Maillard, Adrian Schmid

2019

Characterization of the electron-accepting part of the organohalide respiration chain of Dehalobacter and Desulfitobacterium

Lorenzo Cimmino

Organohalide respiration (OHR) is a bacterial anaerobic process that use halogenated compounds, e.g. tetrachloroethene (PCE), as terminal electron acceptors. D. restrictus strain PER-K23, an obligate OHR bacterium (OHRB), and D. hafniense strain TCE1, a bacterium with a versatile metabolism, harbour the pceABCT gene cluster, which represents our model system for the study of PCE respiration. To date, the function of PceA, the key enzyme in the process, and PceT, the molecular chaperone for PceA maturation, are well defined. However, the role of PceB and PceC are still not elucidated and the biochemistry of OHR electron transfer is still relatively elusive. Based on the genetic composition of the pce gene cluster, the hypothesis that PceB and PceC may play a role in electron transfer in the metabolism of organohalide respiration is tempting but the question remains largely unanswered. In the present Ph.D. thesis, a multilevel study aiming at characterizing the electron-accepting part of the respiratory chain of OHRB will be presented. The investigation was assessed at molecular level by the deciphering of the stoichiometry of pceABCT individual gene products, and at physiological and biochemical levels, where the characterization of the membrane PceA-containing protein complex of both obligate and facultative OHRB was the main focus. The stoichiometry analysis at RNA level revealed that the pce gene cluster is an operon with, however, a level of transcription that differs for individual genes, an observation that could be explained by post-transcriptional events. At proteomic level, an apparent 2:1 stoichiometry of PceA and PceB was obtained in the membrane fraction, while low abundance of PceC in comparison to the other two proteins was observed. In the soluble fraction, a 1:1 stoichiometry of PceA and PceT was identified. Furthermore, a combination of Clear-Native PAGE gel and a in-gel PceA enzymatic activity assay were applied to identify the RDH complex from the membrane of D. restrictus and D. hafniense strain TCE1. The results revealed an active RDH complex in the membrane extract of both organisms with an estimated molecular mass of 180 kDa, while no RDH activity could be detected in the soluble fraction. Furthermore, immunoblotting for PceA on CN-PAGE gel revealed the presence of a second, however inactive, PceA-containing complex with a molecular mass of 670 kDa, which was confirmed by MS analysis to be largely dominated by the molecular chaperone GroEL alongside with PceA and likely representing the GroEL maturation complex. The RDH complex was purified from the membrane extract by chromatography. The obtained fractions were analysed by LC-MS/MS showing unambiguously the presence of PceA and PceB. Furthermore, preliminary cryo-EM analysis led to propose a 3D reconstruction of the RDH complex, revealing the likely presence of a Pce(AB)2 complex. In summary, this study represents a road-map for the identification of RDH complexes from OHRB. The identification of PceA associated with the GroEL maturation complex raised new questions about the biosynthetic pathway of PceA and invites to consider the involvement of GroEL into the maturation of the key enzyme in organohalide respiration. Finally the preliminary results obtained via cryo-EM analysis set the basis for further investigation on the structure of the RDH complex and open new horizons towards the elucidation of the electron transfer chain involved in the reduction of PCE.

EPFL2022

Characterization of the electron-accepting part of the organohalide respiration chain of Dehalobacter and Desulfitobacterium

Lorenzo Cimmino

EPFL2022