Project description:Sulfurospirillum multivorans is one of the few bacteria, which can anaerobically respire organohalides such as tetrachloroethene. The regulation of this organohalide respiration is in most parts unknown. Sulfurospirillum multivorans was shown to downregulate the expression of organohalide respiration-specific genes slowly when no substrate is present, over the time of approximately 100 generations. To unravel the molecular details of this peculiar regulation and the involved factors, we sequenced the primary transcriptome of the organism.
Project description:Organohalide respiration is an environmentally relevant type of anaerobic respiration. We show that Sulfurospirillum halorespirans undergoes the same type of downregulation of the organohalide respiratory genes as had been overserved before in S. multivorans when cultivated without chlorinated ethenes for a long period of time. We compared the proteomes and acetylomes of S. halorespirans cells cultivated in the presence of PCE with those of cells long- and short-term cultivated with nitrate as sole electron acceptor.
Project description:The organohalide-respiring Sulfurospirillum multivorans uses chlorinated ethenes as electron acceptors for growth under anoxic conditions. However, little is known about the interaction of these substrates with proteins. Here, we apply thermal proteome profiling (TPP) to analyze enzyme-trichloroethene interactions. TPP is commonly used to investigate protein-ligand binding through protein melting curve shifts. Several modifications in the protocol, e.g. performing the incubation under anaerobic conditions and increasing the temperature range up to 97°C, improved the detection range and allowed the investigation of oxygen-sensitive proteins. Enzymatic reductive dehalogenation was prevented by omitting the electron donor during incubations. This enabled detecting the interaction of the tetrachloroethene reductive dehalogenase PceA with trichloroethene and confirms the enzyme’s specificity for this substrate. Another 19 proteins showed significant melting curve shifts with trichloroethene, pointing to other proteins directly or indirectly interacting with trichloroethene. Interestingly, a putative response regulator reacted similarly towards trichloroethene, which is potentially in line with its proposed role in regulating trichloroethene respiration. The TPP approach is here proven to facilitate the identification of substrate-enzyme interactions of strictly anaerobic reductive dehalogenases and probably their regulators. This strategy can be used to identify yet unknown substrate specificities and potential signal-sensing proteins in other difficult to study bacteria.
Project description:The strictly anaerobic bacterium Dehalococcoides mccartyi is obligatory dependent on organohalide respiration for energy conservation and growth. Due to its capability to reductively dehalogenate a multitude of toxic halogenated electron acceptors, it plays an important role in the attenuation of these compounds at respective contaminated sites. Here, D. mccartyi strain CBDB1, specialized on the dehalogenation of chloroaromatic compounds, was grown in a two-liquid phase system with 1,2,3-trichlorobenzene as electron acceptor, acetate plus CO2 as carbon source and hydrogen as electron donor. The proteome and Nε-lysine acetylome were analyzed in the lag, exponential and stationary phases. The high and almost invariable abundance of the membrane-localized organohalide respiration complex consisting of the reductive dehalogenases CbrA and CbdbA80, the uptake hydrogenase HupLS and the organohalide respiration molybdoenzyme OmeAB was shown throughout growth and also after a prolonged stationary phase. Quantification of transcripts of reductive dehalogenase genes revealed their major synthesis starting in the lag phase, which might be a prerequisite for balanced growth in the exponential phase. The analyses of the coverage of functional pathways as well as indicator analysis revealed the growth-phase specificity of the proteome, with regulatory proteins identified as important indicators for the stationary phase. The number of acetylated proteins increased from the lag to the stationary phase. We detected pronounced acetylation of key proteins of the acetate metabolism, i.e. the synthesis of acetyl-CoA and its processing via gluconeogenesis and the incomplete Wood-Ljungdahl pathway, as well as of proteins central for the biosynthesis of amino acids, co-factors and terpenoids. In addition, the partial acetylation of the reductive dehalogenases as well as of TatA, a component of the twin-arginine translocation machinery, suggests that acetylation might be directly involved in the maintenance of the organohalide respiration capacity of D. mccartyi over periods without access to halogenated electron acceptors.
Project description:We compared the global transcriptomic analysis of Desulfoluna spongiiphila strain AA1, an organohalide-respiring Desulfobacterota isolated from a marine sponge, with 2,6-dibromophenol or with sulfate as electron acceptor. The most significant difference of the transcriptomic analysis was the expression of one reductive dehalogenase gene cluster (rdh16), which was significantly upregulated with 2,6-dibromophenol.