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.

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