Regulation of substrate dependent expression and activity from dehalogenating enzymes in Dehalococcoides mccartyi strain CBDB1

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The biochemistry of organohalide respiration is still elusive because some of the species capable of it do not provide sufficient biomass for classical biochemistry on purified enzymes. The improvement of sensitivity in mass spectrometry of peptides together with available genomic information will enhance the study of the biochemical principles of organohalide respirers. The species Sulfurospirillum multivorans, Dehalococcoides sp. CBDB1, DCMB5 and BTF08 will be provided by the subproject partners 1, 2, 3 and 4 within the framework of this Research Unit and cultivated with different electron donors, electron acceptors and carbon sources. The limited biomass of the Dehalococcoides spp. requires optimisation of both protein extraction protocols and measurement by high-resolution mass spectrometers. Shotgun proteomic analyses will provide a deeper insight into the proteins involved and thus the corresponding biochemical pathways. Among the different organohalide respiring species a wide variety of reductive dehalogenase genes are found and neither their substrate specificity nor the regulation of their synthesis in response to the substrate is understood. An accurate detection and quantification of the different gene products of the reductive dehalogenases will be performed by selected reaction monitoring – mass spectrometry (srm-MS). This targeted proteomics approach uses standard peptides labelled with heavy isotopes for quantification of peptides of interest in digested protein extracts by qtrap-MS. The specific quantification will yield insights into the correlation between substrate, culture conditions and the biochemical pathways involved as well as the expression of orthologous reductive dehalogenases.