Metal reduction and sporulation in Desulfotomaculum reducens

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.

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

Mechanism(s) of electron transfer and metabolic changes during iron reduction by Clostridium acetobutylicum

Cornelia List

Microorganisms catalyze many of the redox transformations driving the iron cycle in the geosphere. For instance, iron cycling includes the reduction of solid-phase and soluble ferric iron (Fe(III)) to ferrous iron (Fe(II)). However, in order to reduce solid-phase Fe(III), the reduction requires the delivery of electrons extracellularly, requiring a specialized electron transfer system. The mechanism of iron reduction by Gram-negative bacteria has been de-scribed in some detail, but very little is known for Gram-positive bacteria. These microorgan-isms are likely to contribute to iron reduction significantly, particularly in environments where Gram-negative bacteria fare poorly, such as metal contaminated sites, or hydrother-mal environments. Some Gram-positive bacteria use metal reduction as a form of enhanced fermentation, where the metal is a sink for excess reducing equivalents. In these cases, metal reduction is facultative and is not a mechanism for direct energy conservation. Here, we use Clostridium acetobutylicum, a Gram-positive fermenter, as a model organism for its ability to reduce iron and contaminant heavy metals. A major advantage of this model organism is that genetic tools are available to study genes involved in the metal reduction process. We investigated the reduction of solid-phase and soluble Fe(III) by C. acetobutyli-cum and found that it reduces both forms of iron. The bacteria do not require direct contact with solid-phase Fe(III) (as hydrous ferric oxide, HFO) for the reduction to occur. In fact, flavins were identified as likely to be involved in the extracellular electron transfer (EET) in this organism. We found that HFO reduction is increased by utilizing exogenous (resazurin, resorufin, anthraquinone-2,6-disulfonate) or endogenous (riboflavin, RF) electron shuttles. Inactivation of a putative flavin transporter (CAC2841) decreased HFO and Fe(III)-citrate reduction. We proposed that a complex CAC2841/ CAC3100/ CAC3101/ CAC3102 imports RF and CAC2841/ CAC3100 exports flavin adenine dinucleotide (FAD). FAD might be hy-drolyzed by CAC2761 and CAC2766, which interact with membrane-anchored flavoproteins potentially involved in EET (CAC2762 and CAC2767). Additionally, we performed a transcriptomic analysis of Fe(III)-citrate- and HFO-reducing cultures and compared them to fermentation alone. We found two Fe-S oxidoreductases (CA1254 and CAC1021) upregulated with Fe(III)-citrate, which have an unknown function and could be putative Fe(III)-citrate reductases. Additional upregulated redox active pro-teins with potential involvement in solid-phase Fe(III) reduction were identified as CA1044 and CAC3371. These proteins should be considered for further studies. Furthermore, iron reduction impacts bacterial metabolism by buffering the pH and shifting the carbon and electron flow to less reduced metabolites as compared to fermentation. In addition to catabolic pathways, metabolic modeling showed that iron reduction influences anabolic pathways. Finally, metabolic modeling suggests that NADH is the physiological elec-tron donor for iron reduction in this system. This study contributes to a better understanding of the mechanism of iron reduction by Gram-positive bacteria and provides implications for further studies to understand the EET by fermenters in natural environments.

EPFL2019

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