Biochemistry and physiology of halorespiration by Desulfitobacterium dehalogenans

Halorespiration is a novel respiratory pathway, which has been discovered as a result of the search for microorganisms that can be used in bioremediation of chlorinated compounds. Halorespiring bacteria are able to use these compounds as terminal electron acceptor for growth in anaerobic environments. These bacteria have developed enzyme systems with high dechlorination rates and low threshold values. These characteristics are important for the application of dechlorinating bacteria in bioremediation.Figure 1. A 16S rRNA based phylogenetic tree reflecting the relationships of halorespiring bacteria (marked *) with other bacteria.The diversity of bacteria capable of using chlorinated compounds as terminal electron acceptor indicates that halorespiration is widespread throughout the bacterial domain (Fig 1). Insight in the physiology and biochemistry of these bacteria is currently lacking. This study aimed to get a better comprehension of the biochemistry of halorespiration. The research has focused on three topics:elucidation of the coupling of reductive dechlorination to ATP formation in Desulfitobacterium dehalogenans,isolation and characterization of dehalogenases from different Desulfitobacterium species, andisolation and characterization of a novel Desulfitobacterium strain from human feces.In Chapter 1 , an overview is given of microbial dehalogenation mechanisms with emphasis on halorespiration. The halorespiring bacteria that have been obtained in pure culture, the current models for 3-chlorobenzoate and tetrachloroethene (PCE) respiration, and the characteristics of reductive dehalogenases, are also reviewed.Desulfitobacterium dehalogenans is an anaerobic Gram-positive bacterium that uses ortho -chlorinated phenolic compounds as terminal electron acceptor for growth. In Chapter 2, the growth yields of D. dehalogenans grown with hydrogen, formate, pyruvate, or lactate as electron donor and Cl-OHPA as electron acceptor have been compared. In addition, the activities of the different electron donating and electron accepting enzymes were localized. These results indicate that the oxidation of lactate and pyruvate coupled to the reduction of Cl-OHPA yields 1 ATP per mole of acetate produced by substrate level phosphorylation. When formate or hydrogen is used as electron donor for reductive dechlorination, the growth yield is approximately 1/3 of the growth yield with pyruvate as electron donor. Under these growth conditions, energy cannot be conserved via substrate-level phosphorylation.However, a proton motive force (PMF) may be established, which can be used by a proton-driven ATPase for ATP-formation. A model has been postulated in which the localization of the electron-donating enzyme (e.g. hydrogenase, formate dehydrogenase, lactase dehydrogenase, or pyruvate ferredoxin oxidoreductase) determines whether a PMF is established. In contrast to the electron transport by the electron transport chain (ETC) and the reduction of the chlorinated compound by the reductive dehalogenase, which do not contribute to the PMF. We have investigated the composition of the ETC, which is involved in electron transport from formate to Cl-OHPA in cell suspensions and have compared it with the ETC involved in fumarate respiration with formate as electron donor ( Chapter 3 ). Menaquinone, cytochrome c, and b were components that were found to be present in cells grown with formate and either Cl-OHPA or fumarate. We have demonstrated that these components could be reduced by formate and oxidized upon addition of the induced electron acceptor. This suggests that (a part of) the halorespiratory chain is shared with fumarate respiration. However, the ETCs involved in halorespiration and fumarate respiration are not identical. The involvement of cytochrome b in fumarate respiration could be demonstrated while this was not possible for halorespiration. The results suggest that cytochrome b is the direct electron donor for fumarate reductase.The electron transport chain from formate to Cl-OHPA has been investigated in more detail by electron paramagnetic resonance spectroscopy. In these experiments, we have shown that molybdenum, iron-sulfur clusters, cobalamin, a high spin heme and an unknown iron-sulfur cluster are components that were reduced by formate and oxidized by Cl-OHPA. This may indicate that the formate dehydrogenase which is active in halorespiration is a molybdenum and iron-sulfur containing formate dehydrogenase. This enzyme donates its electrons either to cytochrome c, or the electrons are transferred to cytochrome b. The electrons may then be transferred to menaquinone which takes 2 protons from the cytoplasm and, depending on the localization of the reductive dehalogenase, the protons are released at the outside or inside of the cell, as is shown in figure 2 model A and B, respectively. In addition, oxidation of cobalamin, a cofactor of chlorophenol reductive dehalogenase, was observed in cell suspensions upon addition of Cl-OHPA. This observation strongly suggests that the dehalogenase, which we have characterized, is involved in in vivo halorespiration.Figure 2: The electron transport system of D. dehalogenans catalyzing the oxidation of formate coupled to reductive dechlorination of 3-chloro-4-hydroxyphenyl acetate. It shows two tentative models for the generation of a proton gradient based on the localization of the ortho -chlorophenol reductive dehalogenase at the outer (model A) or the inner aspect (model B) of the cytoplasmic membrane.The isolation and characterization of a chlorophenol reductive dehalogenase is described in Chapter 4 . This enzyme was purified anaerobically from a Triton X-100 extract of the membrane fraction. The purified enzyme catalyzed the dechlorination of Cl-OHPA with a V max of 28 units/mg protein and a K m of 20 mM. In addition, the purified dehalogenase catalyzed the reductive dehalogenation of several ortho -chlorinated phenols and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (E m = -440 mV), one [3Fe-4S] cluster (E m = 170 mV), and one cobalamin per 48-kDa monomer. The Co + /Co 2+ transition had an E m of -370 mV. The corresponding gene has been isolated, cloned, and sequenced, and revealed the presence of two closely linked genes: (i) cpr A, encoding the o-chlorophenol reductive dehalogenase, (ii) cpr B, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. Moreover, cprA contains a twin-arginine type signal sequence that is processed in the purified enzyme.Besides ortho -chlorinated phenols, D. dehalogenans is able to use other electron acceptors. In Chapter 5 , the influence of other electron acceptors on the induction of dechlorinating activity and on the dechlorinating activity in cell suspensions and cell extracts is described. The results indicate that D. dehalogenans does not have a preferred electron acceptor in batch cultures, but it utilizes several electron acceptors simultaneously. This could be relevant for in situ bioremediation techniques because the presence of multiple electron acceptors in polluted sediments is not unusual.While D. dehalogenans is able use ortho -chlorinated phenols as terminal electron acceptors for growth, Desulfitobacterium sp. strain PCE1 is able to use both chlorophenols and PCE and Desulfitobacterium frappieri strain TCE1 can use PCE and TCE. We compared the substrate spectrum of the enzymes in cell extracts of these strains grown with Cl-OHPA or PCE as electron acceptors ( Chapter 6 ). The results indicate that strain PCE1 contains separate enzymes for PCE and chlorophenol dechlorination. This was studied in more detail by the isolation of the chlorophenol reductive dehalogenase and the PCE reductive dehalogenase of strain PCE1 and the PCE/TCE reductive dehalogenase from strain TCE1. Based on the N-terminal sequence, size and substrate spectrum, the chlorophenol reductive dehalogenase of strain PCE1 was found to be very similar to the dehalogenase of D. dehalogenans. The PCE/TCE reductive dehalogenase of strain TCE1 has similar characteristics as have been described for PCE reductive dehalogenase of strain PCE-S. The PCE reductive dehalogenase from strain PCE1 was found to be a novel type of reductive dehalogenase. The enzyme catalyzed the reduction of PCE, and had a low activity with TCE. The purified enzyme had a subunit size of 45 kDa on SDS-PAGE. The activity of this enzyme as well as of the chlorophenol reductive dehalogenase of strain PCE1 was found to be inhibited upon addition of the cobalamin inhibitors 1-iodopropane and NO to cell extracts.In Chapter 7 , the isolation and characterization of a new strain of Desulfitobacterium frappieri is described. This isolate is the first Desulfitobacterium strain described that is not able to use chlorinated ethenes or phenols as terminal electron acceptor.Keywords :Halorespiration, Desulfitobacterium dehalogenans , anaerobic dechlorination, bioremediation, PCE, chlorophenol.