Functional genomics of corrinoid starvation in the organohalide-respiring bacterium Dehalobacter restrictus strain PER-K23

De novo comnoid biosynthesis represents one of the most complicated metabolic pathways in nature. 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, of which cbiH appears to be truncated and therefore non-functional, possibly explaining the corrinoid auxotrophy of this obligate OHRB. Comparative genomics within Dehalobacter spp. revealed that one (operon-2) of the five distinct comnoid biosynthesis associated operons present in the genome of D. restnctus appeared to be present only in that particular strain, 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 comnoid homeostasis in D. restnctus and the augmented salvaging strategy this bacterium adopted to cope with the need for this essential cofactor.

Understanding the metabolism of tetrachloroethene-respiring Dehalobacter restrictus

Aamani Rupakula Boyanapalli

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.

EPFL2015

Corrinoid auxotrophy in the obligate organohalide respiring Dehalobacter restrictus

Christof Holliger, Julien Maillard, Aamani Rupakula Boyanapalli, Hauke Smidt

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.

2013

Corrinoid auxotrophy in the obligate organohalide-respiring Dehalobacter restrictus

Christof Holliger, Aamani Rupakula Boyanapalli

2013

Understanding the metabolism of tetrachloroethene-respiring Dehalobacter restrictus

Aamani Rupakula Boyanapalli

EPFL2015