Understanding the metabolism of tetrachloroethene-respiring Dehalobacter restrictus:from genome analysis, corrinoid cofactor biosynthesis to regulation of reductive dehalogenases

Tetra- and trichloroethene (PCE, TCE) are organohalides polluting the environment as a result of inappropriate use, storage, and disposal by various industries. Anthropogenic pollution by organohalides is a major source of concern because of their undesirable effects on human health. Remediation of contaminated sites by the use of microorganisms is a promising approach, especially under anaerobic conditions. Dehalobacter restrictus represents the paradigmatic member of the genus Dehalobacter, which in recent years has proven to be a major player in the biodegradation of a growing number of organohalides, both in situ and in the laboratory. D. restrictus grows only through anaerobic respiration of PCE and TCE with hydrogen as electron donor by a process known as organohalide respiration (OHR). To this day, only a single reductive dehalogenase (PceA/RdhA), the key enzyme in the OHR process, has been characterized on genetic and biochemical levels. However, recent genome analysis of D. restrictus has revealed the presence of 25 rdhA genes. Chapter 2 of this thesis describes a functional genomics and proteomics approach on D. restrictus with a focus on the diversity, composition and expression of rdh gene clusters. Genome analysis also revealed a complete corrinoid biosynthetic pathway, WL pathway for CO2 fixation and hydrogenases. Some of these were identified in proteomic analysis along with main PceABCT, RdhA14 and a few RdhK. OHR bacteria (OHRB) have developed different strategies to satisfy their need of corrinoid (Cobalamin/Vitamin B12 derivatives), as it is an essential cofactor of RdhAs forming the basis for Chapter 3. Obligate OHRB such as Dehalococcoides spp. and D. restrictus cannot de novo synthesize corrinoid. However. genome analysis revealed that in contrast to Dehalococcoides mccartyi, the genome of D. restrictus surprisingly has the complete series of genes for biosynthesis of corrinoid, however a single non-functional gene could account for the corrinoid auxotrophy. Comparative genomics within Dehalobacter spp. revealed that one of the five operons associated with the biosynthesis of corrinoid is unique to D. restrictus, which encoded enzymes corrinoid- salvaging and transport proteins. Omics during corrinoid starvation highlighted the importance of operon-2 in corrinoid homeostasis in D. restrictus along with indicating its augmented corrinoid salvaging strategy. Chapter 4 finally analyses the diversity of RdhK proteins in D. restrictus belonging to the CRP-FNR family of transcriptional regulators. Earlier studies in Desulfitobacterium spp. have allowed the identification and characterization of a transcriptional regulator, CprK known to be involved in the regulation of cpr gene cluster involved in OHR. Moreover recent genome analysis in D. restrictus, revealed the presence of 25 cprK-like rdhK genes found to be located in the direct neighbourhood of the rdh gene clusters strongly suggesting they could be implicated in regulating OHR in D. restrictus. A combination of in silico, in vivo and in vitro analyses have been attempted to characterize the role of a few RdhK proteins and understand the tri-partite interaction of the RdhK with the putative organohalide along with the putative-DNA binding regions (dehaloboxes). However, further efforts are still needed to elucidate the network regulating the OHR metabolism in D. restrictus

Corrinoid auxotrophy in the obligate organohalide-respiring Dehalobacter restrictus

Corrinoids (derivatives of vitamin B12) are an essential cofactor of reductive dehalogenases, the key enzymes involved in the environmental-friendly process of organohalide respiration (OHR). However, genomic and physiological analyses of obligate OHR bacteria (such as Dehalobacter and Dehalococcoides) have delivered contrasting results on the ability of de novo corrinoid biosynthesis. This raised the question of the source of corrinoids for obligate OHR bacteria in the environment and their biosynthesis/scavenging mechanism of corrinoids. Dehalobacter is an important bacterial genus for the bioremediation of organohalides such as chloroethenes, chloroethanes and chloroform. Genomic analysis of D. restrictus revealed the presence of all the genes required for the production of corrinoids (1), however the strain is incapable of de novo biosynthesis. The general aim of the present study is to better understand the corrinoid metabolism of the genus Dehalobacter at the level of biosynthesis, regulation and transport. A detailed analysis revealed that the cbiH gene whose product is involved in the corrin ring contraction displays a frame-shift mutation which was confirmed experimentally, suggesting that it might represent a possible checkpoint behind the corrinoid auxotrophy. Moreover, in proteomic data of a D. restrictus culture growing in presence of vitamin B12 in the medium, several corrinoid biosynthesis proteins were not detected arguing for specific regulation mechanisms. The transport and scavenging metabolism of corrinoid by D. restrictus is now under scrutiny

Purification & characterization of PceC protein involved in dehalorespiration by desulfitobacterium & dehalobacter

Dehalorespiration is a process during which microbes derive energy by the reductive dehalogenation of chlorinated organic pollutants, by using them as electron acceptors. Characterization of the full dehalorespiration pathway in dehalogenating organisms is of great importance, as their physiological properties and substrate range will determine the extent of the bioremediation process and the conditions that should be used. Various Desulfitobacterium strains and Dehalobacter restrictus harbor the pceABCT gene cluster responsible for tetrachloroethene (PCE) anaerobic respiration. Our lab has previously focused on isolation & characterization of the proteins PceA and PceT, the reductive dehalogenase and its specific maturation factor, respectively. PceB is expected to play the role of the membrane anchor for PceA. We are now currently investigating the role of PceC in dehalorespiration. Sequence analysis revealed that PceC is homologous to CprC encoded in the chlorophenol reductive dehalogenase operon which has been postulated to be a membrane-bound FMN-binding transcription regulator of the NosR /NirI family. The goal of the work presented here is the heterologous production and purification of the predicted soluble FMN-binding domain in E. coli in order to raise PceC antibodies and allow an accurate detection and localization of PceC in membranes of Desulfitobacterium and Dehalobacter cells. This should also help in providing stoichiometric analysis of PceC in relation to PceA. For further biochemical characterization reconstitution of the active FMN-binding domain will be attempted

Corrinoid auxotrophy in the obligate organohalide respiring Dehalobacter restrictus

Corrinoids (derivatives of vitamin B12) are an essential cofactor of reductive dehalogenases, the key enzymes involved in the environmental-friendly process of organohalide respiration (OHR). However, genomic and physiological analyses of obligate OHR bacteria (such as Dehalobacter and Dehalococcoides) have delivered contrasting results on the ability of de novo corrinoid biosynthesis. This raised the question of the source of corrinoids for obligate OHR bacteria in the environment and their biosynthesis/scavenging mechanism of corrinoids. Dehalobacter is an important bacterial genus for the bioremediation of organohalides such as chloroethenes, chloroethanes and chloroform. Genomic analysis of D. restrictus revealed the presence of all the genes required for the production of corrinoids (1), however the strain is incapable of de novo biosynthesis. The general aim of the present study is to better understand the corrinoid metabolism of the genus Dehalobacter at the level of biosynthesis, regulation and transport. A detailed analysis revealed that the cbiH gene whose product is involved in the corrin ring contraction displays a frame-shift mutation which was confirmed experimentally, suggesting that it might represent a possible checkpoint behind the corrinoid auxotrophy. Moreover, in proteomic data of a D. restrictus culture growing in presence of vitamin B12 in the medium, several corrinoid biosynthesis proteins were not detected arguing for specific regulation mechanisms. The transport and scavenging metabolism of corrinoid by D. restrictus is now under scrutiny

Corrinoid auxotrophy in the obligate organohalide respiring Dehalobacter restrictus

Background Corrinoids are an essential cofactor of reductive dehalogenases, the key enzymes of the environmental-friendly process of organohalide respiration (OHR). Dehalobacter restrictus strain PER-K23 is an obligate OHR bacterium (OHRB) able to conserve energy with tetrachloroethene, but is unable to de novo synthesize corrinoids (1). Genome analysis of D. restrictus however revealed the presence of the complete corrinoid biosynthesis pathway (2,3). Objectives The aim of the present study is to understand the corrinoid metabolism of D. restrictus at the level of biosynthesis, regulation and transport and to compare it to contrasting situations in other OHR bacteria. Methods Genome analysis was performed with standard bioinformatic tools. Both transcriptomic and proteomic approaches were applied on D. restrictus cells cultivated in media with alternative corrinoid conditions. Gene expression was further addressed using targeted reverse transcription and quantitative PCR. Conclusions Annotation and analysis of genes involved in corrinoid metabolism revealed a 101-bp deletion in the cbiH gene resulting in a shift of the reading frame and leading to a non-functional enzyme. This mutation, which is not present in the genome of other Dehalobacter spp., indicates that cbiH represents a possible checkpoint behind corrinoid auxotrophy. The expression of most corrinoid biosynthetic genes is likely to be controlled by cobalamin riboswitches. Experimental evidence of this latter mechanism is under scrutiny. Comparative ’omics’ analyses of corrinoid-starved cells revealed an increased production of corrinoid transporters and proteins involved in corrinoid salvaging. Taken together, these data suggest that D. restrictus has recently lost its capacity of de novo corrinoid synthesis. References (1) Holliger et al. (1998), Arch Microbiol 169, 313. (2) Kruse et al. (2013), Stand Genomic Sci, submitted. (3) Rupakula et al. (2013), R Soc Phil Trans B, in press

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

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

The Membrane-Bound C Subunit of Reductive Dehalogenases: Topology Analysis and Reconstitution of the FMN-Binding Domain of PceC

Organohalide respiration (OHR) is the energy metabolism of anaerobic bacteria able to use halogenated organic compounds as terminal electron acceptors. While the terminal enzymes in OHR, so-called reductive dehalogenases, are well-characterized, the identity of proteins potentially involved in electron transfer to the terminal enzymes remains elusive. Among the accessory genes identified in OHR gene clusters, the C subunit (rdhC) could well code for the missing redox protein between the quinol pool and the reductive dehalogenase, although it was initially proposed to act as transcriptional regulator. RdhC sequences are characterized by the presence of multiple transmembrane segments, a flavin mononucleotide (FMN) binding motif and two conserved CX3CP motifs. Based on these features, we propose a curated selection of RdhC proteins identified in general sequence databases. Beside the Firmicutes from which RdhC sequences were initially identified, the identified sequences belong to three additional phyla, the Chloroflexi, the Proteobacteria, and the Bacteriodetes. The diversity of RdhC sequences mostly respects the phylogenetic distribution, suggesting that rdhC genes emerged relatively early in the evolution of the OHR metabolism. PceC, the C subunit of the tetrachloroethene (PCE) reductive dehalogenase is encoded by the conserved pceABCT gene cluster identified in Dehalobacter restrictus PER-K23 and in several strains of Desulfitobacterium hafniense. Surfaceome analysis of D. restrictus cells confirmed the predicted topology of the FMN-binding domain (FBD) of PceC that is the exocytoplasmic face of the membrane. Starting from inclusion bodies of a recombinant FBD protein, strategies for successful assembly of the FMN cofactor and refolding were achieved with the use of the flavin-trafficking protein from D. hafniense TCE1. Mass spectrometry analysis and site-directed mutagenesis of rFBD revealed that threonine-168 of PceC is binding FMN covalently. Our results suggest that PceC, and more generally RdhC proteins, may play a role in electron transfer in the metabolism of OHR

Corrinoid Auxotrophy in the Obligate Organohalide-Respiring Dehalobacter restrictus strain PER-K23

Introduction: Corrinoids are an essential cofactor of reductive dehalogenases, the key enzymes of organohalide respiration (OHR). Dehalobacter restrictus strain PER-K23 is an obligate OHR bacterium able to conserve energy with tetrachloroethene, but is unable to de novo synthesize corrinoids. In contrast, genome analysis of D. restrictus revealed the presence of the complete corrinoid biosynthesis pathway. Objectives: To understand the corrinoid metabolism of D. restrictus at the level of biosynthesis, regulation and transport and compare it to other emerging Dehalobacter genomes Methods: Genomes of Dehalobacter spp. was obtained from the JGI. Proteomics were applied on D. restrictus cells cultivated in media supplemented with corrinoids spanning a range from 0 to 250 µg/l. Gene expression analysed using targeted reverse transcription and quantitative PCR. Results and Discussion: A non-functional cbiH gene All the genes required for corrinoid biosynthesis & transport were identified in D. restrictus. However, there was a 101-bp deletion in the precorrin-3B C17-methyltransferase encoding cbiH. This mutation is not present in the genome of other Dehalobacter strains indicating that a non-functional cbiH could be the possible cause of corrinoid auxotrophy in D. restrictus. Active regulation of corrinoid metabolism The expression of corrinoid biosynthetic genes in D. restrictus was verified experimentally to be regulated by cobalamin riboswitches. Analysis of corrinoid-starved D. restrictus cells revealed an increased transcription of the genes encoded directly downstream of the riboswitches. After corrinoid addition all operons were repressed, with most pronounced reduction for two operons, termed operon 1 and 2, encoding enzymes involved in corrinoid transport and salvaging only found in the genome of D. restrictus (73- and 346-fold, respectively). Overproduction of corrinoid-salvaging proteins (CbiZ) and transporters Proteomics of corrinoid-starved cells revealed on average a 45-fold over-production of operon 2 thereby reinforcing the trend from transcriptomics. Taken together, these data suggest that D. restrictus has lost its ability of de novo corrinoid synthesis and instead evolved a strategy for augmented corrinoid uptake and modification to fulfill its corrinoid requirement

The membrane-bound C subunit of reductive dehalogenases: topology analysis and reconstitution of the FMN-binding domain of PceC

Organohalide respiration (OHR) is the energy metabolism of anaerobic bacteria able to use halogenated organic compounds as terminal electron acceptors. While the terminal enzymes in OHR, so-called reductive dehalogenases, are well-characterized, the identity of proteins potentially involved in electron transfer to the terminal enzymes remains elusive. Among the accessory genes identified in OHR gene clusters, the C subunit (rdhC) could well code for the missing redox protein between the quinol pool and the reductive dehalogenase, although it was initially proposed to act as transcriptional regulator. RdhC sequences are characterised by the presence of multiple transmembrane segments, a flavin mononucleotide (FMN) binding motif and two conserved CX3CP motifs. Based on these features, we propose a curated selection of RdhC proteins identified in general sequence databases. Beside the Firmicutes from which RdhC sequences were initially identified, sequences belong to three additional phyla, the Chloroflexi, the Proteobacteria and the Bacteriodetes. The diversity of RdhC sequences mostly respects the phylogenetic distribution, suggesting that rdhC genes emerged relatively early in the evolution of the OHR metabolism. PceC, the C subunit of the tetrachloroethene (PCE) reductive dehalogenase is encoded by the conserved pceABCT gene cluster identified in Dehalobacter restrictus PER-K23 and in several strains of Desulfitobacterium hafniense. Surfaceome analysis of D. restrictus cells confirmed the predicted topology of the FMN-binding domain (FBD) of PceC that is the exocytoplasmic face of the membrane. Starting from inclusion bodies of a recombinant FBD protein, strategies for successful assembly of the FMN cofactor and refolding were achieved with the use of the flavin-trafficking protein from D. hafniense TCE1. Mass spectrometry analysis and site-directed mutagenesis of rFBD revealed that threonine-168 of PceC is binding FMN covalently. Our results suggest that PceC, and more generally RdhC proteins, may play a role in electron transfer in the metabolism of organohalide respiration

The Membrane-Bound C Subunit of Reductive Dehalogenases: Topology Analysis and Reconstitution of the FMN-Binding Domain of PceC

Organohalide respiration (OHR) is the energy metabolism of anaerobic bacteria able to use halogenated organic compounds as terminal electron acceptors. While the terminal enzymes in OHR, so-called reductive dehalogenases, are well-characterized, the identity of proteins potentially involved in electron transfer to the terminal enzymes remains elusive. Among the accessory genes identified in OHR gene clusters, the C subunit (rdhC) could well code for the missing redox protein between the quinol pool and the reductive dehalogenase, although it was initially proposed to act as transcriptional regulator. RdhC sequences are characterized by the presence of multiple transmembrane segments, a flavin mononucleotide (FMN) binding motif and two conserved CX3CP motifs. Based on these features, we propose a curated selection of RdhC proteins identified in general sequence databases. Beside the Firmicutes from which RdhC sequences were initially identified, the identified sequences belong to three additional phyla, the Chloroflexi, the Proteobacteria, and the Bacteriodetes. The diversity of RdhC sequences mostly respects the phylogenetic distribution, suggesting that rdhC genes emerged relatively early in the evolution of the OHR metabolism. PceC, the C subunit of the tetrachloroethene (PCE) reductive dehalogenase is encoded by the conserved pceABCT gene cluster identified in Dehalobacter restrictus PER-K23 and in several strains of Desulfitobacterium hafniense. Surfaceome analysis of D. restrictus cells confirmed the predicted topology of the FMN-binding domain (FBD) of PceC that is the exocytoplasmic face of the membrane. Starting from inclusion bodies of a recombinant FBD protein, strategies for successful assembly of the FMN cofactor and refolding were achieved with the use of the flavin-trafficking protein from D. hafniense TCE1. Mass spectrometry analysis and site-directed mutagenesis of rFBD revealed that threonine-168 of PceC is binding FMN covalently. Our results suggest that PceC, and more generally RdhC proteins, may play a role in electron transfer in the metabolism of OHR