Hybrid Transcriptional Regulators for the Screening of Target DNA Motifs in Organohalide-Respiring Bacteria

The bioremediation of persistent organohalide molecules under anoxic conditions mostly relies on the bacterial process called organohalide respiration (OHR). Organohalide-respiring bacteria (OHRB) are phylogenetically diverse anaerobic bacteria that share the capacity to use organohalides as terminal electron acceptors in an energy-conserving process. The reductive dehalogenase (rdh) gene clusters encode for proteins specialized in the respiration of one or a limited number of organohalides. One particular OHRB may harbor up to several dozens of rdh gene clusters suggesting a wide potential for bioremediation. To avoid wasting energy in producing unnecessary proteins, rdh gene clusters often include a transcriptional regulator. In organohalide-respiring Firmicutes, RdhK is a dedicated transcriptional regulator of OHR and represents a subfamily of proteins among the CRP/FNR superfamily of regulators. RdhK proteins are composed of an effector-binding domain (EBD) which recognizes a given organohalide and subsequently controls the interaction of its C-terminal DNA-binding domain (DBD) with a DNA motif (referred to as dehalobox, or DB) located in the promoter region of the target rdh genes. The two binding partners (i.e. an organohalide molecule and a DB sequence) of RdhK proteins are interdependent which impairs the exploration of OHR regulatory networks. Here, we propose a strategy relying on hybrid proteins to efficiently screen the DNA target of a single RdhK protein without prior knowledge on its effector. To demonstrate the potential of the method, two hybrids with alternative fusion points were designed based on RdhK6 EBD and RdhK1 DBD from Desulfitobacterium hafniense. Electrophoretic mobility shift assay was performed with purified hybrids along with the parental proteins and their binding properties were further tested in vivo through a beta-galactosidase reporter assay. Along with revealing new RdhK6 features, we show that both hybrids resulted in active regulatory proteins with distinct binding patterns. While Hybrid A was less specific for the DNA motif, Hybrid B successfully mimicked the binding behavior of the parental proteins and thus represents a promising template for the design of new RdhK hybrids to screen yet uncharacterized RdhK proteins and also possibly other members of the CRP/FNR superfamily.

Metal reduction and sporulation in Desulfotomaculum reducens

Elena Cristina Dalla Vecchia

The genera Desulfotomaculum and Clostridium, belonging to the phylum Firmicutes, comprise Gram-positive, low G+C genomic content, anaerobic, spore- forming bacteria. Desulfotomaculum is a metabolically and environmentally versatile genus capable of growing fermentatively as well as by respiring sulfate. D. reducens is also capable of metal reduction and has been reported to conserve energy from this process. In this thesis, iron (Fe(III)) reduction by D. reducens in the presence of pyruvate or lactate as electron donors was investigated. When pyruvate is present, D. reducens grows fermentatively. If Fe(III) is present, it can act as an electron sink alternative to protons and consume excess reducing equivalents accumulated during pyruvate oxidation. Electron transfer to Fe(III) by D. reducens in the presence of pyruvate is mediated by a soluble electron carrier, likely riboflavin, released by cells during fermentation. This mechanism allows the microorganism to reduce extracellular, solid phase Fe(III) even if it is not directly accessible by physical contact. In the presence of lactate, D. reducens is unable to conserve significant energy from electron donor oxidation and Fe(III) reduction. In contrast to pyruvate, during lactate oxidation, no redox-active compound is released by the cells, and extracellular, solid phase Fe(III) can only be reduced by direct contact. This implies that a surface exposed reductase must be present in D. reducens cells. The investigation of the surface proteome of this organism led to the identification of three proteins, most likely localized to the cell surface, potentially involved in Fe(III) reduction. One is a heterodisulfide reductase subunit A, a putative intermediate in the Fe(III) reduction chain. The second is a membrane-associated hydrogenase. The third protein is annotated as alkyl hydroperoxide reductase although its actual function may be thiol-disulfide oxidoreduction; several lines of evidence suggest the involvement of the latter in metal reduction by D. reducens. Clostridium species are widespread in the environment and are capable of utilizing a great variety of organic substrates for fermentative growth, while they are unable to conserve energy through respiration. However, they can reduce terminal electron acceptors to eliminate excess reducing equivalents from fermentation. Among the electron acceptors used by Clostridium spp., are metals, such as iron and uranium (U(VI)). One species whose vegetative cells are capable of U(VI) reduction is C. acetobutylicum, although little information is available on the mechanism underlying this process. In this thesis, the potential involvement of the spores of C. acetobutylicum in U(VI) reduction is probed. It was found that the spores of this species can reduce the radionuclide, and that to do so they require the presence of an unidentified soluble compound released by growing cells and H2 as the electron donor. The involvement of spores in metal reduction is very interesting because generally spores are considered not to interact directly with the surrounding environment. In fact, spores are dormant, thus metabolically inactive, cells. Spore formation is a cell differentiation process activated in response to environmental stress and conditions that are hostile to vegetative growth. Sporulation has been extensively studied and characterized in Bacillus subtilis, for which a model has been developed. Some information is also available on sporulation in the clostridia. In this thesis, a genomic investigation of sporulation in the Desulfotomaculum genus was undertaken. The B. subtilis model was used as a reference and the information on Clostridium as a comparison. Significant similarities were found in the core regulatory process across genera, while some differences were identified in the initiation of sporulation. Substantial differences were highlighted in the spore structural proteins.

EPFL2014

Identifying the role of complex I in organohalide respiration of Firmicutes

Romain Hamelin, Christof Holliger, Julien Maillard, Mathilde Stéphanie Willemin

Organohalide-respiring bacteria (OHRB) are capable to link energy conservation with the use of organohalide compounds as terminal electron acceptors. While obligate OHRB relies only on this process for growing, facultative OHRB are able to use alternative electron acceptors. The enzymes catalyzing the final step of organohalide reduction are known as reductive dehalogenases and have been well characterized. However, the complete picture of the respiratory chain remains to be elucidated. Recent genomic and proteomic data suggested new potential players in the respiratory process, such as the respiratory complex I. Complex I, also called NADH:ubiquinone oxidoreductase (Nuo) is a well-recognized player in the classical respiratory electron transfer processes both in eukaryotes and prokaryotes. Its main role is to allow the coupling of proton pumping through the membrane with the transfer of two electrons from NADH to quinones. In bacteria, the complex is usually composed by fourteen subunits forming three modules: the NADH oxidase module, the quinone reductase module and the proton-pumping module. The presence of an 11-subunits version in Firmicutes OHRB, lacking the three subunits forming the NADH module suggests the use of alternative electron donor for the complex. Moreover, it is still unclear how to link the function of complex I with the enzymes involved in OHR. The goal of this study is therefore to investigate the role of complex I in OHRB, focusing on the Firmicutes phylum. Since OHRB are not genetically tractable, their study involves the use of alternative methodology. Therefore, blue-native PAGE will be use to investigate the complex I. The gel band containing members of the complex I will be cut out and send for MS analysis in order to identify possible partners of the complex. Moreover, pull down assays followed by MS analysis will be carry out with the subunits thought to play the role of entry site for the electrons into complex I. In parallel, comparative proteomics are envisaged to reveal the relative occurrence of complex I in the facultative OHRB Desulfitobacterium hafniense cultivated in different conditions (e.g. fermentation, organohalide or other respiration). The overall goal is to address the question whether the presence of the complex correlates with specific growth conditions as a first indication for its role. Preliminary results on complex I identification and characterization will be presented and discussed.

2018

Involvement of respiratory complex I in organohalide respiration?

Christof Holliger, Julien Maillard, Mathilde Stéphanie Willemin

Abstract Background The NADH:ubiquinone oxidoreductase (Nuo) is an assembly of proteins also known as complex I. It is the entry point for highly energetic electrons in the classical aerobic respiration process as it couples the transfer of two electrons from NADH to ubiquinone with proton pumping resulting in the establishment of a proton mortice force. It was assumed that to fulfil its function, the complex must contain at least 14 subunits. Yet the presence of an 11-subunit version of complex I as detected in genomic and proteomic analyses of strict anaerobes such as sulfate-reducing bacteria (SRB) or organohalide-respiring bacteria (OHRB) raised for the latter the question of its potential implication in the still poorly understood respiratory processes. Moreover, the 11-subunits complex I lacks the NADH oxidizing module suggesting the use of an alternative electron donor. Methods The goal of this work is to elucidate the role of complex I in the specific context of OHRB with a focus on Firmicutes. During organohalide respiration (OHR), organohalide compounds serve as final electron acceptors in an energy-conserving process. The complete picture of the electron transfer chain remains elusive and a good understanding of the proteins involved in this metabolism is of great interest for bioremediation. The development of targeted quantitative proteomics will serve to compare the abundance of the complex in Desulfitobacterium hafniense strain DCB-2 when cultivated in different growth conditions. This facultative OHRB is capable of using different combinations of electron donors/acceptors and offers the possibility to evaluate the prevalence of the complex in one or the other growth condition, reflecting its importance. Results Progress in the method development and preliminary proteomic data will be discussed. Conclusion In parallel, the biochemical characterization of the complex in Dehalobacter restrictus is currently under investigation by Blue Native PAGE to define its protein composition and identify possible electron donor candidates

2019