Organohalide respiration in Firmicutes: energy metabolism and transcription regulation

Organohalides are a class of compounds often considered as persistent pollutants and harmful to environmental and human health. Some bacteria, among which are representatives from the Firmicutes phylum, are capable of using these compounds as terminal acceptors in a process called organohalide respiration (OHR). To do so, organohalide-respiring bacteria (OHRB) are equipped with enzymes called reductive dehalogenases which perform the terminal reduction in the OHR respiratory chain by replacing the halide by a hydrogen. OHR is not only interesting from a biological point of view but it also represents a powerful process for bioremediation purposes. The present work focuses on two different fundamental aspects of OHR which remain poorly understood.

The first part of the work focused on the transcriptional regulation of the reductive dehalogenase genes. OHRB can encode up to several dozens of different reductive dehalogenases in their genome which suggest the use of a broad number of organohalides, but this diversity remains largely unexplored. In Firmicutes OHRB, RdhK are transcriptional regulators specialised in the activation of reductive dehalogenase genes upon exposure of the cell to organohalides. Thus, the identification of RdhK binding partners (organohalide effector and DNA target sites) represents an interesting alternative to identify new OHR substrates while preventing the tedious and challenging characterisation of the complex membrane-associated and oxygen-sensitive reductive dehalogenases. Here, a strategy based on RdhK hybrid proteins was designed and optimised, and is proposed to serve to improve the efficiency of RdhK characterisation. The approach enabled a functional decoupling of the two domains of RdhK regulators targeting either the effector or the DNA target site. Therefore, it reduces the complexity of the screening procedure in the identification of RdhK binding partners. Further implementation of the hybrid strategy will increase the global comprehension of Firmicutes OHR regulatory networks and ultimately provide an indirect way to explore the reductive dehalogenase substrate diversity.

The second part of the thesis aimed to increase knowledge in the energy metabolism of Firmicutes OHRB. Quantitative comparative proteomics was applied to Desulfitobacterium hafniense strain DCB-2, which revealed the proteome adaptations to the growth on different substrates promoting either fermentation or respiration. In addition, possible roles of the complex I-like enzyme was investigated using physiological and biochemical approaches. Respiratory complex I is the entry point of electrons produced by cytoplasmic metabolic activity in the respiratory chain and is composed of three modules. Most OHRB express a version of the complex which lacks the N-module responsible for the electron uptake from NADH, suggesting the use of alternative electron donors. While a few candidate partners for the OHR complex I-like enzyme were proposed based on proteomics results, the study of the physiological role of the enzyme helped in developing different respiratory models for strain DCB-2, which mainly differ in their dependence on the complex I-like enzyme.

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

The genome of Dehalobacter restrictus - high gene redundancy for a restricted metabolism

Christof Holliger, Julien Maillard, Aamani Rupakula Boyanapalli, Hauke Smidt

Organohalide respiration (OHR) is a bacterial anaerobic respiratory metabolism dedicated to use halogenated compounds as terminal electron acceptors. OHR bacteria are strictly anaerobic microorganisms which use reductive dehalogenases (RdhA) as key enzymes in this respiration process (1,2). Dehalobacter restrictus, an obligate OHR bacterium has been shown to use exclusively tetra- (PCE) and trichloroethene (TCE) as electron acceptors. The PCE RdhA (so called PceA) has been characterized previously (3). D. restrictus genome is currently being sequenced (in collaboration with JGI) revealing an unexpected high number of rdhA genes. Aim: The overall aim of this study is to characterize the functional diversity of rdhA genes in D. restrictus. Specific attention will be given to the use made by this bacterium of such redundant genetic information, and to the possible evolution mechanisms by which these numerous gene copies have emerged. Methods: The rdh gene clusters will be analyzed using bioinformatics comparing their diversity and gene composition to the well-characterized cpr (2) and pce (4) gene clusters in OHR isolates. Although D. restrictus is characterized by a restricted metabolism, different culture conditions will be tested either by replacing PCE with other organohalides, or by spiking organohalides in a culture growing on PCE. The level of transcription of the rdhA genes in D. restrictus will be measured by a targeted approach using optimized RNA extraction, RT, PCR and qPCR protocols. A special focus will also be given to the operon structure of the active pceABCT gene cluster of D. restrictus. Results: So far the analysis of the 2.9 Mb draft genome of D. restrictus revealed the presence of 20 different rdh gene clusters. While most of them are scattered in the available genome contigs, an array of 4 consecutive rdhAB operons is interspersed with regulatory genes suggesting a tight transcription regulation. When compared to the vast family of RdhA sequences in databases, D. restrictus RdhAs reflect the diversity observed in Firmicutes and are fairly distant to Dehalococcoides-type RdhAs. Preliminary transcription analysis of the 20 rdhA genes in standard growth conditions suggests that only the known pceA gene is significantly used in PCE/TCE dechlorination. Proteomic data will be obtained to confirm this view. More transcriptional analysis from cells subjected to other organohalides will be also presented. Conclusion: Based on genome analysis, D. restrictus seems to adopt an intermediate position between the facultative OHR bacteria such as Desulfitobacteria, and the obligate ones like Dehalococcoides members. The high rdhA gene redundancy appears as an evolutionary strategy to grow on many organohalides. This hypothesis still needs to be proven experimentally. References: (1) Holliger et al., Eds. Häggblom & Bossert, Kluwer Academic, 2003, p115. (2) Smidt & de Vos, Annu. Rev. Microbiol., 2004, 58:43. (3) Maillard et al., Appl. Environ. Microbiol., 2003, 69:4628. (4) Maillard et al., Environ. Microbiol., 2005, 7:107.

2012