Full PhD thesis available for download: http://infoscience.epfl.ch/record/210608/files/EPFL_TH6709.pdf
PhD abstract available at: http://infoscience.epfl.ch/record/210608
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.
Dehalobacter restrictus ; organohalide respiration metabolism ; biodegradation ; genome ; reductive dehalogenase ;corrinoid biosynthesis ; cobalamin riboswitches ; corrinoid auxotrophy ;functional genomics ; CRP-FNR transcriptional regulator proteins
Prof. Alexis Berne (président) ; Prof. Christof Holliger, Dr Julien Maillard (directeurs) ; Prof. Melanie Blokesch, Prof. Gabriele Diekert, Dr David Johnson (rapporteurs)
Public defense: 2015-8-27
Dehalobacter restrictus strain PER-K23 is an obligate organohalide respiring bacterium, which displays extremely narrow metabolic capabilities. It only grows via coupling energy conservation to anaerobic respiration of tetra- and trichloroethene with hydrogen as sole electron donor. D. restrictus represents the paradigmatic member of the genus Dehalobacter, which in recent years turned out to be a major player in the bioremediation of an increasing number of organohalides, both in situ and in laboratory studies. The recent elucidation of D. restrictus genome revealed a rather elaborate genome with predicted pathways that were not suspected from its restricted metabolism, such as a complete corrinoid biosynthetic pathway, the Wood-Ljungdahl pathway for CO2 fixation, abundant transcriptional regulators and several types of hydrogenases. However, one important feature of the genome is the presence of 25 reductive dehalogenase genes, from which so far only one, pceA, was characterized on genetic and biochemical levels. My first PhD project describes a multi-level functional genomics approach on D. restrictus across three different growth phases. A global proteomic analysis allowed to consider general metabolic pathways relevant to organohalide respiration, while the dedicated genomic and transcriptomic analysis focused on the diversity, composition and expression of genes associated with reductive dehalogenases.
Functional genomics of corrinoid starvation in the organohalide-respiring bacterium Dehalobacter restrictus strain PER-K23
Organohalide respiration (OHR) is an anaerobic bacterial respiration process of environmental interest as many anthropogenic halogenated organic compounds can be used as terminal electron acceptors by organohalide-respiring bacteria (OHRB), which remove the halogens and therefore contribute to bioremediation of environments polluted with these compounds. The key enzyme in OHR is the reductive dehalogenase (RDase), which strictly depends on corrinoid cofactors for the dehalogenation reaction. De novo corrinoid biosynthesis represents one of the most complicated metabolic pathways in nature, which only 40% of bacteria are capable of. Organohalide-respiring bacteria (OHRB) have developed different strategies to deal with their need of corrinoid, as it is an essential cofactor of reductive dehalogenases, the key enzymes in OHR metabolism.
In contrast to Dehalococcoides mccartyi, the genome of Dehalobacter restrictus strain PER-K23 contains a complete set of corrinoid biosynthetic genes with the exception of cbiH, which appears to be truncated and therefore non-functional, explaining the corrinoid auxotrophy of this obligate OHRB. Comparative genomics within Dehalobacter spp. revealed that one (operon-2) of the five distinct corrinoid biosynthesis associated operons present in the genome of D. restrictus appeared to be a unique operon, which encodes multiple members of corrinoid transporters and salvaging enzymes. Operon-2 was highly up-regulated upon corrinoid starvation both at the transcriptional (346-fold) and proteomic level (46-fold on average), in line with the presence of an upstream cobalamin riboswitch. Together, these data highlight the importance of this operon in corrinoid homeostasis in D. restrictus and the augmented salvaging strategy this bacterium adopted to cope with the need for this essential cofactor.