Absolute quantification of pceABCT gene products and characterization of the membrane protein complex responsible for the reduction of tetrachloroethene in Dehalobacter restrictus

The pceABCT gene cluster from Dehalobacter restrictus is one model system for tetrachloroethene (PCE) respiration. In the present study we aim to decipher the stoichiometry of the pceABCT gene products and the composition of PceA-containing membrane-bound protein complexes in D. restrictus. Quantitative proteomics (qProt), membrane extraction, CN-PAGE, and an in-gel enzymatic assay were developed. A qProt analysis performed on the membrane fraction revealed a 1.5:1 stoichiometric relationship between PceA and PceB, while PceC was about 60-times less abundant than PceA. Data from CN-PAGE and a newly developed in-gel PceA enzymatic assay revealed several forms of active PceA-containing complexes with masses of approximately 140, 180, and 250 kDa, depending on the physiological state of D. restrictus cells.

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

Lorenzo Cimmino, Christof Holliger, Julien Maillard, Adrian Schmid

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.

2021

ORGANOHALIDE RESPIRATION: STUDY OF THE MEMBRANE PROTEIN COMPLEX RESPONSIBLE FOR THE REDUCTION OF HALOGENATED COMPOUNDS

Lorenzo Cimmino, Christof Holliger, Julien Maillard, Adrian Schmid

Organohalide respiration (OHR) is a bacterial anaerobic respiratory process in which halogenated compounds are used as terminal electron acceptors. Desulfitobacterium and Dehalobacter, paradigmatic organohalide-respiring bacteria (OHRB), harbour the pceABCT gene cluster, representing one model system in OHR. While both Dehalobacter restrictus and Desulfitobacterium hafniense strain TCE1 dechlorinate tetrachloroethene (PCE), 3,5-dichlorophenol (DCP) is dechlorinated by D. hafniense strain DCB-2. The relatively high DNA sequence identity (70%) and similar gene composition and regulation between the pceABCT and dcpABCT gene clusters triggered our interest in studying the possible co-transcription of the genes, in establishing stoichiometric relationships between the corresponding proteins, and in trying to decipher the reductive dehalogenase (RDH) protein complex from the cytoplasmic membrane of those bacteria. Gene co-transcription was investigated by a combination of RNA extraction and RT-qPCR, while the stoichiometry of the proteins is analyzed via quantitative proteomics. Clear-Native PAGE technology combined with an in-gel reductive dehalogenase activity assay are established to identify proteins from the RDH complex. The transcriptional analysis conducted on D. hafniense strain DCB-2 showed that dcp gene cluster is actively up-regulated by 3,5-DCP. At protein level, reference peptides were defined for PceA, B, C and T from D. restrictus and D. hafniense strain TCE1 and they will be applied for measuring the relative stoichiometry of the targeted proteins. Furthermore, preliminary results from CN-PAGE analysis suggest that the PCE reductive dehalogenase (PceA) catalytic subunit is part of a ~150 kDa protein complex in D. restrictus. At transcriptional level the results suggest that dcp gene cluster shows an operon structure. However, the possibility of additional promoters regulating the transcription of individual genes within the operon are under investigation. At protein level, the identification of the RDH complex allows us to address the question of the proteins that are associated with PceA.

2019

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

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

Lorenzo Cimmino, Christof Holliger, Julien Maillard, Adrian Schmid

2021

ORGANOHALIDE RESPIRATION: STUDY OF THE MEMBRANE PROTEIN COMPLEX RESPONSIBLE FOR THE REDUCTION OF HALOGENATED COMPOUNDS

Lorenzo Cimmino, Christof Holliger, Julien Maillard, Adrian Schmid

2019