Characterization of the surfaceome of the metal-reducing bacterium Desulfotomaculum reducens

Desulfotomaculum reducens strain MI-1 is a Gram-positive, sulfate-reducing bacterium also capable of reducing Fe(III). Metal reduction in Gram-positive bacteria is poorly understood. Here, we investigated Fe(III) reduction with lactate, a non-fermentable substrate, as the electron donor. Lactate consumption is concomitant to Fe(III) reduction, but does not support significant growth, suggesting that little energy can be conserved from this process and that it may occur fortuitously. D. reducens can reduce both soluble [Fe(III)-citrate] and insoluble (hydrous ferric oxide, HFO) Fe(III). Because physically inaccessible HFO was not reduced, we concluded that reduction requires direct contact under these experimental conditions. This implies the presence of a surface exposed reductase capable of transferring electrons from the cell to the extracellular electron acceptor. With the goal of characterizing the role of surface proteins in D. reducens and of identifying candidate Fe(III) reductases, we carried out an investigation of the surface proteome (surfaceome) of D. reducens. Cell surface exposed proteins were extracted by trypsin cell shaving or by lysozyme treatment, and analyzed by liquid chromatography-tandem mass spectrometry. This investigation revealed that the surfaceome fulfills many functions, including solute transport, protein export, maturation and hydrolysis, peptidoglycan synthesis and modification, and chemotaxis. Furthermore, a few redox-active proteins were identified. Among these, three are putatively involved in Fe(III) reduction, i.e., a membrane-bound hydrogenase 4Fe-4S cluster subunit (Dred_0462), a heterodisulfide reductase subunit A (Dred_0143) and a protein annotated as alkyl hydroperoxide reductase but likely functioning as a thiol-disulfide oxidoreductase (Dred_1533).

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

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

Assessing the Bacterial Ecology of Organohalide Respiration for the Design of Bioremediation Strategies

Noam Shani

Groundwater is essential for human activities and is sometimes referred to as a non-renewable resource in the same way as oil and gas. During the past century, precious groundwater reserves worldwide have been threatened by anthropogenic release of chemical compounds. Among these, chlorinated ethenes (CEs) such as tetrachloroethene (PCE) and trichloroethene (TCE) belong to the most common class of groundwater contaminants. In oxic conditions, PCE and TCE are recalcitrant to any form of degradation and constitute a long-term source of groundwater contamination. It has been reported that CEs can be degraded biologically in anoxic conditions, serving as electron acceptors of an anaerobic respiratory process called organohalide respiration (OHR). In this process, PCE is sequentially reduced to TCE, cis-dichloroethene (cDCE), vinyl chloride (VC), and finally to harmless ethene (Eth). Engineering strategies based on reductive dechlorination, such as monitored natural attenuation (MNA), have been designed for the bioremediation of aquifers contaminated with CEs. However, stalling of the sequence of dechlorination has often been observed, resulting from incomplete or impeded biodegradation of the highly toxic daughter molecules (cDCE and VC) and leading to their accumulation in situ. It was shown in laboratory experiments that OHR of CEs is more efficient in mixed cultures and that organohalide-respiring bacteria (OHRB) live in association with other microorganisms in microbial consortia. In the aquifer ecosystem, OHRB rely on complex interactions between members of bacterial communities for their electron donors and carbon supplies. On the other hand, they are in competition for these resources with other terminal electron-accepting processes (TEAPs). These complex interactions together with the intrinsic structural heterogeneity of aquifers make it difficult to understand the reasons for lower CEs accumulation, to predict the fate of OHR, and to design bioremediation strategies. This thesis aimed at elucidating the reasons for lower CEs accumulation in situ. An ecological approach considering the aquifer ecosystem in its whole complexity was developed and applied to the specific cases of two contaminated sites showing accumulation of lower CEs. The proposed methodology relies on the precise depiction of both aquifer habitat and microbial communities interacting therein. However, the potential impact of the first technical step, namely the impact of the pumping parameters on groundwater samples used for the description of the bacterial communities, was not known at the start of this thesis work. Results of investigations addressing this topic showed that parameters related to the tubing characteristics did not impact the apparent bacterial community structures (BCS) in a laboratory experiment. However, the study revealed a significant impact of the pumping flow rate on apparent BCS extracted from the aquifer. Terminal-restriction fragment length polymorphism (T-RFLP), coupled with an appropriate groundwater sampling strategy, enabled an accurate profiling of the BCS. However no indication was provided concerning the identities of the community members. A bioinformatics tool called PyroTRF-ID was developed to overcome this obstacle. The software enabled affiliating terminal-restriction fragments (T-RFs) to precise phylotypes by coupling T-RFLP and pyrosequencing data. An additional function enables in silico assessment of restriction enzymes for designing appropriate T-RFLP procedures. The developed methodology was applied all along the thesis work. Optimized tools and procedures were used to investigate the reasons for the accumulation of VC, and to a lesser extent cDCE, in the first aquifer showing relatively homogeneous lithological composition and slow groundwater fluxes. Statistical findings indicated that VC reduction was outcompeted by Fe(III) reduction in some sections of the aquifer. T-RFs showing significant correlations with VC reduction variables were identified by sequencing and with PyroTRF-ID. Results showed sequences closely affiliated to uncultured bacteria of the "Lahn Cluster" (LC) within the class Dehalococcoidetes, previously reported as PCE-to-cDCE dechlorinating microorganisms, and to the genus "Dehalococcoides". According to present knowledge, only members of this genus are able to reduce cDCE and VC. A major T-RF negatively correlated with the LC T-RF affiliated to the genus Rhodoferax, containing iron-reducing bacteria (IRB). Furthermore, indications were obtained of a mutualistic interaction between IRB and sulfate-reducing bacteria, potentially playing an important role by reducing the Fe(III) contents locally. The importance of the aquifer hydrogeological structure and functioning was exemplified in the study of the second aquifer ecosystem, where discrepancies in the fate of OHR were observed. In the immediate vicinity of the source zone, complete OHR was occurring, with production of Eth, whereas cDCE was accumulating further downstream, in apparent similar redox conditions. The profiling and metagenomic techniques revealed drastically different bacterial communities in the zones displaying different OHR fate. The source zone displayed bacterial populations typically found in highly reduced environments and involved in OHR, such as Dehalococcoides sp. In contrast, the downstream zone showing an accumulation of cDCE displayed bacterial populations typically found in more oxidized environments, such as aerobic bacteria, nitrifiers, and denitrifiers. In this zone, the conditions were probably slightly oxidized periodically by the combination of aquifer recharge, a specific lithological configuration, and lower organic matter content. Finally, and based on these considerations, a methodology for the investigation of the reasons for lower CEs accumulation in contaminated aquifers is proposed and discussed. This methodology follows on a multidisciplinary ecological approach for the description of the functioning of the aquifer ecosystem, and enables formulating scenarii of the reasons for lower CEs accumulation.

EPFL2012