Molecular characterization of key enzymes involved in dehalorespiration with tetrachloroethene

Chloroethenes, and most particularly tetra- (PCE) and trichloroethene (TCE) are major groundwater pollutants due to their extensive industrial use as solvents since the 1920s. The strong electronegativity of the chlorines renders them very stable under aerobic conditions. However, biodegradation of chloroethenes under anaerobic conditions has been shown to be a promising strategy for remediation of chloroethene-contaminated sites. To date around fifteen bacterial strains have been isolated with the property of using chloroethenes as terminal electron acceptor in a process called dehalorespiration. Anaerobic dehalorespiring bacteria show an unequal chloroethene substrate range and an unequal extent of dechlorination. While most of the dehalorespiring bacteria dechlorinate PCE and TCE to cis-1,2-dichloroethene cis-1,2-DCE) and belong to the phyla Firmicutes, δ- and ε-Proteobacteria, a few strains of the genus Dehalococcoides affiliated with the phylum Chloroflexi and are able to dechlorinate cis-1,2-DCE and vinyl chloride (VC) to the non-toxic ethene. Dehalobacter restrictus and Dehalococcoides isolates were found to be completely restricted to dehalorespiration which gave rise to some basic evolutionary questions. Identification of the key enzyme in the dechlorination reaction, the reductive dehalogenase, has revealed a new class of enzymes containing a corrinoid and two ironsulfur clusters as cofactors. At the beginning of this thesis, nine chloroethene reductive dehalogenases have been characterized on biochemical level, while only little information was available on molecular level. Therefore, the overall goal of this thesis was to characterize on a molecular level the reductive dehalogenases involved in tetrachloroethene dehalorespiration and to get some indications on the evolution of this novel anaerobic respiration process. Starting from the N-terminal sequence of the PCE reductive dehalogenase (PceA) of Dehalobacter restrictus and from a conserved amino acid stretch found in two already sequenced reductive dehalogenases, a degenerate PCR approach allowed the isolation of the gene encoding PceA. Comparison with unpublished data from Desulfitobacterium sp. strain PCE-S showed 100% sequence identity. The full sequence of the pceAB gene of strain PCE-S helped to isolate the corresponding gene cluster from D. restrictus and Desulfitobacterium hafniense strain TCE1, which has also been shown to contain an identical N-terminal sequence. Sequence analysis confirmed the presence of a Twin-Arginine Translocation (Tat) signal peptide, which is involved in the incorporation of the reductive dehalogense into the cytoplasmic membrane. Detailed analysis of the iron-sulfur cluster binding motifs present in PceA of D. restrictus and the chlorophenol reductive dehalogenase (CprA) of Desulfitobacterium dehalogenans revealed differences in the second motif, which may explain results obtained by EPR spectroscopy, namely the presence of two [4Fe-4S] clusters in the former enzyme and the presence of one [3Fe-4S] and one [4Fe-4S] cluster in the latter one. Structure breaking residues such as glycine and proline are present at the two extremities of the ten amino acid stretch separating the first and second ironbinding cysteine residues of the second motif in PceA, but not in CprA. This primary structure probably allows the formation of a loop in the tertiary structure and the participation of the first cyteine as a ligand in a [4Fe-4S] cluster. In both new sequences, the presence of a short gene (pceB) encoding a hydrophobic protein with three conserved trans-membrane α-helices was confirmed, indicating a possible role in anchoring the catalytic unit of the reductive dehalogenase into the membrane. The complete sequence identity observed in the newly isolated reductive dehalogenases raised the question of a possible horizontal gene transfer between Dehalobacter restrictus and Desulfitobacterium hafniense strain TCE1. Therefore, the flanking regions of the reductive dehalogenase genes (pceAB) in Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus were investigated. This study revealed the presence of a composite transposon (named Tn-Dha1) in strain TCE1 bordered with two identical insertion sequences (ISDha1, including the transposase gene tnpA1) and containing six open reading frames: the already characterized pceAB, two genes (pceCT) related to members of the o-chlorophenol reductive dehalogenase gene cluster of Desulfitobacterium dehalogenans, and two possibly truncated genes with homology to another transposase (tnpA2) and to a subunit of the Tat machinery (tatA), respectively. In contrast, only the pceABCT gene cluster (i.e. without the transposon structure and the other two genes) was present in Dehalobacter restrictus, indicating that the genes encoding the key enzymes for the dechlorination activity are stably integrated into the genome. A detailed investigation of Tn-Dha1 by PCR and Southern blot analysis indicated that Tn-Dha1 may form various circular molecules, an indication for an active mobile genetic element. A model for the transposition of Tn-Dha1 was proposed, in which the transposon may excise from the chromosome and circularize, forming an unstable structure with two abutted ISDha1. The strong promoter formed by the junction of both IS would lead to high expression of the transposase, which in turn reacts with the circular element by either re-integrating it in the chromosome or excising one or both ISDha1 from that element. The resulting structures would be single IS, IS tandems and circular molecules with one or no remaining IS, both latter structures being dead-end products of the transposition event. The hypothesis of mobile reductive dehalogenase genes was also investigated using a genomic approach in preliminary sequence data (released by The Institute for Genome Research, TIGR) of the genome of Dehalococcoides ethenogenes, a dehalorespiring bacterium capable to completely dechlorinate PCE to ethene. The genome was shown to contain the extraordinary number of eighteen different copies of reductive dehalogenase genes, including the well characterized tceA. A genomic signature of D. ethenogenes was obtained by calculating the frequency of 4-letter DNA words along the genome and was graphically represented. Local disruptions of the genomic signature in certain segments of the genome were highlighted, corresponding to DNA, which may have been acquired by horizontal gene transfer, so-called original regions. It revealed that from the eighteen putative reductive dehalogenase genes present in the genome of D. ethenogenes, fifteen were located in original regions. Moreover, several genes encoding for recombinases (transposase, integrase) were found within these original regions, strongly indicating that these may have been acquired horizontally. The complete electron transport chain leading the electrons to the reductive dehalogenase has not yet been characterized for any of dehalorespiring bacteria and the direct electron-donor has not yet been elucidated for any of reductive dehalogenases. Therefore, the presence of cytochromes in cells of Desulfitobacterium hafniense strain TCE1 was investigated with regard to the presence or absence of PCE in the growth medium. Detection of cytochromes using a sensitive detection method based on chemiluminescence revealed a strongly enhanced signal in the membrane fractions of strain TCE1 cells grown on PCE instead of fumarate as terminal electron acceptor. Western blot analysis revealed the presence of a 45 kDa protein in membrane fraction, corresponding most probably to a c-type cytochrome. UV-visible spectroscopy confirmed the presence of c-type cytochromes in membrane fractions. This study, although further investigations are needed, indicated that a c-type cytochrome may be involved in the direct electron transfer to the PCE reductive dehalogenase of D. hafniense strain TCE1. At present, numerous sequences of reductive dehalogenase genes have been reported and deposited on sequence databases, revealing the great interest shown for this new anaerobic respiration pathway. While several degenerate PCR approaches have led to the isolation of 22 mostly partial genes, analysis of preliminary genome sequence data from Dehalococcoides ethenogenes and Desulfitobacterium hafniense strain DCB-2 has revealed 18 and 6 sequences, respectively. Sequence alignment and homology analysis of the 66 reductive dehalogenases genes available in August 2004 revealed four main clusters, two corresponding to chlorophenol and chloroethene reductive dehalogenases found in the phylum Firmicutes, one with sequences mostly isolated from ε-Proteobacteria, and one containing most of the genes isolated from the genus Dehalococcoides. Hence, the reductive dehalogenases appear to be rather conserved wihtin phylogenetic groups, indicating a relatively ancient enzyme class. Reductive dehalogenases show some features such as the presence of a Tat signal peptide and iron-sulfur clusters that are common to most of terminal reductases. However, the presence of a corrinoid at the catalytic center and of several specific conserved amino acid stretches makes them a new class of anaerobic reductases. Finally, the strong variation in the topology of the dehalorespiration chain and the variable presence and involvement of different electron transferring components such as quinones and cytochromes in dehalorespiring bacteria indicate that reductive dehalogenases may have been integrated into existing respiration chains rather than that dehalorespiration has evolved as a whole

Frequent concomitant presence of Desulfitobacterium spp. and " Dehalococcoides ” spp. in chloroethene-dechlorinating microbial communities

The presence of chloroethene dechlorination activity as well as several bacterial genera containing mainly organohalide-respiring members was investigated in 34 environmental samples from 18 different sites. Cultures inoculated with these environmental samples on tetrachloroethene and amended weekly with a seven organic electron donor mixture resulted in 11 enrichments with cis-DCE, ten with VC, and 11 with ethene as dechlorination end product, and only two where no dechlorination was observed. "Dehalococcoides” spp. and Desulfitobacterium spp. were detected in the majority of the environmental samples independently of the dechlorination end product formed. The concomitant presence of Dehalococcoides spp. and Desulfitobacterium spp. in the majority of the enrichments suggested that chloroethene dechlorination was probably the result of catalysis by at least two organohalide-respiring genera either in parallel or by stepwise catalysis. A more detailed study of one enrichment on cis-DCE suggested that in this culture Desulfitobacterium spp. as well as Dehalococcoides spp. dechlorinated cis-DCE whereas dechlorination of VC was only catalyzed by the latte

Multiple Dual C−Cl Isotope Patterns Associated with Reductive Dechlorination of Tetrachloroethene

Dual isotope slopes are increasingly used to identify transformation pathways of contaminants. We investigated if reductive dechlorination of tetrachloroethene (PCE) by consortia containing bacteria with different reductive dehalogenases (rdhA) genes can lead to variable dual C−Cl isotope slopes and if different slopes also occur in the field. Two bacterial enrichments harboring Sulfurospirillum spp. but different rdhA genes yielded two distinct δ13C to δ37Cl slopes of 2.7 ± 0.3 and 0.7 ± 0.2 despite a high similarity in gene sequences. This suggests that PCE reductive dechlorination could be catalyzed according to at least two distinct reaction mechanisms or that rate-limiting steps might vary. At two field sites, two distinct dual isotope slopes of 0.7 ± 0.3 and 3.5 ± 1.6 were obtained, each of which fits one of the laboratory slopes within the range of uncertainty. This study hence provides additional insight into multiple reaction mechanisms underlying PCE reductive dechlorination. It also demonstrates that caution is necessary if a dual isotope approach is used to differentiate between transformation pathways of chlorinated ethenes

Signal Quality Monitoring for New GNSS Signals

International audienceIn the context of GNSS signals and associated augmentation systems modernization, new modulations are envisaged. More precisely Galileo E1C, the pilot component of the E1 Open Service signal (CBOC(6,1,1/11) modulation), Galileo E5a and GPS L5 (BPSK(10) modulation) are signals that will be used by civil aviation airborne receivers for pseudorange computation. To meet stringent requirements defined for civil aviation GNSS receivers, the characterization of distortions which could affect a GNSS signal in a hazardous way is required. In particular, expected signal distortions generated at payload level are described by Threat Models (TM). Distortions incorporate in the TM are also called Evil Waveform (EWF). These TMs, and their associated parameter ranges, referred to as Threat Space (TS) are powerful and necessary tools to design and test the performance of Signal Quality Monitor (SQM). The SQM is a mean to detect the presence of dangerous signal distortions and is necessary to protect users with high requirements in terms of integrity, accuracy, availability, and continuity (for example civil aviation users). Nowadays, this monitoring task is performed by GBAS and SBAS reference station for GPS L1 C/A to warn the user in a timely manner. In this paper SQMs for Galileo E1C and Galileo E5a will be designed and compared by mean of an innovative representation inspired from [1]. From this representation, SQM performance is assessed based on the highest differential tracking error entailed by a signal distortion included in the TM and not detected by the SQM within allocated Pfa and Pmd.. It is noteworthy that performance of SQM is dependent on several parameters and in particular on the C/N0 at which the reference station is operating. One of the advantage of the proposed representation is that performances of the SQM can be assessed for different equivalent C/N0 from one figure. Using this representation, different SQMs are compared and an optimized SQM is proposed to monitor signal distortions on Galileo E5a and Galileo E1C signals

Explore RdhK based regulatory network of organohalide respiration using a hybrid proteins strategy

Reductive dehalogenase (rdh) gene clusters are encoding proteins that enable organohalide respiring bacteria (OHRB) to couple the degradation of halogenated molecules to energy conservation. The transcription of rdh gene clusters is regulated by RdhK regulators belonging to the CRP/FNR-family. RdhK6 (previously called CprK1) in Desulfitobacterium hafniense was shown to activate the transcription of the chlorophenol rdh genes in presence of 3-hydroxy-4-chlorophenylacetate1,2. RdhK effector-binding domain binds to organohalides which triggers protein conformational change and allows the interaction with specific DNA motifs (dehalobox, DB) upstream of rdh gene clusters3,4. The genome of Dehalobacter restrictus PER-K23 encodes 24 rdh gene clusters, suggesting a great OHR potential. Each cluster has a rdhK paralogue in close proximity5. The elucidation of the regulation network represents an indirect way to reveal yet unknown substrates for D. restrictus. However, the challenge resides in the fact that for each new RdhK, there are a large number of potential organohalides and possible DB sequences, resulting in a high amount of combinations to be tested. This project aims to develop a strategy involving RdhK hybrid proteins to screen for DB and organohalides individually. The hybrids are composed by one domain (i.e. DNA- or effector-binding domain) of D. hafniense RdhK6 and the complementary domain from any RdhK of interest. The proof of concept as well as strategy limitations and alternatives will be discussed. References 1. Kim et al. (2012). BMC Microbiol. 12:21. 2. Gábor et al. (2006). J Bacteriol. 188:2604. 3. Joyce et al. (2006). J Biol Chem. 281:28318. 4. Levy et al. (2008). Mol Microbiol. 70:151. 5. Rupakula et al. (2013). Philos Trans R Soc Lond B Biol Sci. 368(20120325):1

Frequent concomitant presence of Desulfitobacterium spp. and “Dehalococcoides” spp. in chloroethene-dechlorinating microbial communities

The presence of chloroethene dechlorination activity as well as several bacterial genera containing mainly organohalide respiring members was investigated in 34 environmental samples from 18 different sites. Cultures inoculated with these environmental samples on tetrachloroethene and amended weekly with a seven organic electron donor mixture resulted in eleven enrichments with cis-DCE, ten with VC, and eleven with ethene as dechlorination end product, and only two where no dechlorination was observed. “Dehalococcoides” spp. and Desulfitobacterium spp. were detected in the majority of the environmental samples independently of the dechlorination end product formed. The concomitant presence of “Dehalococcoides” spp. and Desulfitobacterium spp. in the majority of the enrichments suggested that chloroethene dechlorination was probably the result of catalysis by at least two organohalide respiring genera either in parallel or by stepwise catalysis. A more detailed study of one enrichment on cis-DCE suggested that in this culture Desulfitobacterium spp. as well as “Dehalococcoides” spp. dechlorinated cis-DCE whereas dechlorination of VC was only catalyzed by the latter