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

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.

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

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

Identification and characterization of proteins supporting dehalorespiration in Desulfitobacterium hafniense strain TCE1

Laure Prat

Respiration is a fundamental catalytic process in the aerobic and anaerobic energy metabolism of many prokaryotic and most eukaryotic organisms. The major difference between these organisms is that various organic and inorganic substrates can be used to donate or accept electrons in the bacterial and archaeal kingdoms, in various ranges of redox potentials in order to drive aerobic (with oxygen as electron acceptor) and anaerobic respiratory pathways. During the last few decades, several bacterial strains have been reported to use chlorinated compounds as terminal electron acceptor under anaerobic conditions, in a process called dehalorespiration. Among more than thirty dehalorespiring bacterial strains isolated to date, the Dehalococcoides group and the Gram-positive genus Desulfitobacterium comprise the largest number of reductively dehalogenating pure cultures. While most Dehalococcoides isolates appeared as highly specialized bacteria that depend strictly on dehalorespiration for growth, members of the genus Desulfitobacterium are very versatile microorganisms with regards to the electron donors and acceptors utilized. Globally, this remarkable prokaryotic respiratory flexibility is guaranteed by an elaborate reservoir of complex redox enzymes. Regarding the specific electron transport chain involved in dehalorespiration, rather little knowledge has been obtained so far with the exception of the key enzyme, the reductive dehalogenase, well described in several dehalorespiring bacteria, and of little information on b-type cytochromes and menaquinones. The overall objective of this thesis was to identify new possible components of the electron transport chain by comparative proteomic analysis, to investigate the presence/absence of a regulatory pathway leading to the biosynthesis of the reductive dehalogenase, and finally to characterize newly identified proteins that might support the dehalorespiration process. A comparative 2D proteomic analysis was performed on Desulfitobacterium hafniense strain TCE1, a bacterium capable of using tetrachloroethene (PCE) as electron acceptor. Soluble and membrane-associated proteins were analyzed from cells cultivated on different couples of electron donors and acceptors, lactate – fumarate (LF), lactate – PCE (LP), hydrogen – fumarate (HF), and hydrogen – PCE (HP). Cells growing on LP or HP revealed 72 and 93 protein spots in membrane fraction, and 49 and 12 spots in soluble fraction that correspond to increasingly expressed proteins compared to their fumarate-grown counterparts. More than 80 proteins were identified by mass spectrometry analysis, among which the key enzyme in the dehalorespiration process, the PCE reductive dehalogenase (PceA), as well as interesting candidates which could support dehalorespiration. The possible role of these proteins was analyzed in further details. Involved in energy and carbon metabolisms, electron transfer flavoproteins (ETF) and proteins related to the Wood-Ljungdahl pathway of CO2 fixation were identified in HP growth conditions. Proteins associated to stress response such as the phage shock protein PspA, and to regulatory pathways, the transcriptional repressor CodY, were also observed and discussed. Although only slightly induced by PCE, an additional protein displaying homology to sulfur-transferases was found in a large abundance in the fraction of membrane-associated proteins. This protein was named PhsE, as the corresponding gene belongs to a gene operon with homology to the molybdoenzyme thiosulfate/polysulfide reductase operons (phs/psr) found in Salmonella typhimurium or Wolinella succinogenes. PhsE protein architecture resembles to the class of tandem-domain rhodaneses, with the particularity to contain two active-site cysteines. It is also characterized by the presence of a typical lipoprotein signal peptide (LSP), which anchors PhsE on the outside of the cytoplasmic membrane. In the genome sequence of D. hafniense strain Y51, phsE (DSY3892) is part of an apparent operon encoding one out of about sixty different members of the complex iron-sulfur molybdoenzyme (CISM) family. PhsA, the catalytic molybdoenzyme, displays significant sequence homology with the thiosulfate/polysulfide reductase (PhsA/PsrA) subfamily. So far no other CISM operon has been described with a sulfur-transferase encoding gene. Despite the numerous functions proposed for rhodanese in cyanide detoxification, Fe/S cluster formation, oxidative stress protection, sulfur mobilization and involvement in general sulfur metabolism, the question of the physiological role of PhsE remains open. The expression of the Phs complex appeared to be influenced by the addition of sulfide in the culture medium, generally used as reducing agent in anaerobic cultures. Sulfide is known to affect the intracellular redox status. Therefore, it is hypothesized that PhsE might be involved in the control of the redox balance of the cell, notably by scavenging potentially toxic reduced sulfur species. To support data obtained at the protein level during proteomic analysis, genes and the corresponding messenger RNA from D. hafniense strain TCE1 were studied. While in silico DNA sequence analysis did not reveal any obvious features regulating the transcription of pceA, puzzling results were obtained in proteomic analysis showing an almost complete absence of PceA in cells grown on fumarate. To characterize this phenomenon, starting from a routinely PCE-grown inoculum, strain TCE1 was repeatedly transferred on medium containing fumarate as electron acceptor. The pool of pceA gene in the total culture DNA decreased significantly already after fifteen successive sub-cultures on fumarate, suggesting a shift in strain TCE1 population upon relief of the PCE selection pressure. Consequently the level of pceA mRNA and PceA protein also followed the same trend. Secondly, a PCE pulse experiment was performed in an anaerobic continuous culture of strain TCE1 on fumarate. It revealed that PCE itself has no power of induction on genetic level, except for the positive effect observed on pspA transcripts which encodes a protein involved in cell membrane integrity. Finally the expression study of the phsFABCDE gene cluster revealed that all phs genes are co-transcribed, and most likely build an operon. Thus phsE seemed to be strictly coregulated with the phs operon, and not under the influence of another promoter. In conclusion, proteomic analysis enabled to investigate the general physiological status of Desulfitobacterium hafniense strain TCE1 during dehalorespiration. A tentative model of the dehalorespiration pathway is proposed in summarizing recent data obtained on the topology of the PCE reductive dehalogenase and on sequence similarity with proteins involved in other anaerobic respiratory pathways. Regarding the possible support of PhsE towards the dehalorespiration, the complicated chemistry of sulfur does not allow to clearly attribute a function to the Phs complex, but there are indications that it might build a 'sulfur' oxidoreductase which could catalyze the conversion of sulfide to polysulfide with concomitant electron transfer to the quinone pool. The sulfide oxidation activity might be useful to D. hafniense to scavenge toxic sulfur species, avoiding that they disturb the redox balance of the cell, and redox-sensitive enzymatic complexes.

EPFL2009