Regulation der Organohalidrespiration in Dehalococcoides mccartyi-Stämmen
Zusammenfassung der Projektergebnisse
Wichtigste Ergebnisse: The genome sequences of the D. mccartyi strains DCMB5 and BTF08 were analyzed and revealed the presence of 23 and 20, respectively, rdhAB genes. The annotated RdhAs confirmed, and correlated with, the different substrate spectra of both strains. - The strain DCMB5 was shown to dechlorinate diverse chlorinated aromatic compounds including chlorinated dibenzo-p-dioxins, phenols and benzenes. It encodes an ortholog of the chlorobenzene reductive dehalogenase CbrA in the genome, which was also the most abundant RdhA in its proteome. The importance of CbrA for organohalide respiration with chlorinated benzenes and likely also dibenzo-p-dioxins was confirmed by quantitative transcription analyses of cbrA in D. mccartyi strain CBDB1. - Two RdhR proteins (MarR-type regulators) were shown to act as repressors of rdhA gene transcription in D. mccartyi strain CBDB1 by in vitro and heterologous in vivo studies. - Rdh1R (cbdbA1625) negatively regulates the transcription of its own gene and the corresponding rdhA gene (cbdbA1624). - Rdh2R (cbdbA1456) regulates transcription of two rdhA genes (cbdbA1453 and cbdbA1455) located in the immediate vicinity of its encoding gene. The determined conserved binding sites comprise the transcriptional start sites of the rdhA genes and consist of a direct repeat, which contains in its half sites short palindromes. Rdh2R is a dimer in solution, but, interestingly, binds as tetramer to the direct repeat, probably effectively interfering with RNA polymerase binding. The moderate affinity of binding (Kd 63 nM), however, might enable relatively fast dissociation of the complex, when electron acceptors for organohalide respiration are available. - Transcriptome analyses of strain CBDB1 grown with 1,2,3- or 1,2,4-TCB confirmed the high expression of cbrA and cbdbA80 with both electron acceptors. Eight further rdhA genes were significantly transcribed. Among those, cbdbA1588, an ortholog of pceA of D. mccartyi 195, was most differentially up-regulated in the presence of 1,2,4-TCB. - Proteome analyses of strain CBDB1 grown with 1,2,3- or 1,2,4-TCB confirmed the abundant synthesis of CbrA and CbdbA80 as well as the 1,2,4-TCB-specific synthesis of CbdbA1588, suggesting that rdhA gene expression is mainly regulated on the transcriptional level. - Indirect evidence for the catalytic activity of CbdbA1588 (PceA) was obtained by the two- to ten-fold increase of specific activities towards 1,2,4-TCB and 2,3-dichlorophenol (reported to be a substrate of PceA in strain 195) in crude extracts of 1,2,4-TCB-grown cells compared to 1,2,3-TCB-grown cells and suggests a role of CbdbA1588 in the dechlorination of 1,2,4-TCB. - According to the genomic context, the interaction of the response regulator RdhP (cbdbA78) of a two-component system (TCS) with the promoters of cbrA and cbdbA80 was assumed. Heterologously produced RdhP precipitated in inclusion bodies. Solubilised and re-folded RdhP did not interact with the promoters of both rdhA genes, which was in accord with results of extended heterologous in vivo interaction studies with promoter-lacZ reporter strains. However, preliminary results suggest an interaction of RdhP with the intergenic regions upstream of cbiZ (cbdbA81), encoding an amidohydrolase involved in cobinamide salvaging, and rdhA (cbdbA88), pointing to a more global regulatory function of this TCS. - Nε-lysine acetylation plays a role in the metabolic regulation of D. mccartyi strain CBDB1. As expected, the highest amount of acetylated proteins was observed in the stationary growth phase. Interestingly, the two most abundant RdhA´s, CbrA and CbdbA80, were also acetylated, indicating their partial inactivation and storage in the cytoplasm.
Projektbezogene Publikationen (Auswahl)
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2013. Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi. Phil Trans R Soc B 368: 20120317
Wagner A, Segler L, Kleinsteuber S, Sawers G, Smidt H, Lechner U
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2015. Dehalococcoides mccartyi strain DCMB5 respires a broad spectrum of halogenated aromatic compounds. Appl Environ Microbiol 81: 587-596
Pöritz M, Schiffmann C, Hause G, Heinemann U, Seifert J, Jehmlich N, Von Bergen M, Nijenhuis I, Lechner U
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2016. Comparative genomics and transcriptomics of organohalide respiring bacteria and regulation of rdh gene transcription, p 345-376. In Adrian L, Löffler F. (eds) Organohalide-respiring bacteria. Springer, Berlin
Kruse T, Smidt. H, Lechner, U
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2016. The MarR-type regulator Rdh2R regulates rdh gene transcription in Dehalococcoides mccartyi CBDB1. J Bacteriol 198: 3130-3141
Krasper L, Lilie H, Kublik A, Adrian L, Golbik R, Lechner U
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2018. Anaerobic microbial dehalogenation and its key players in the contaminated Bitterfeld-Wolfen megasite. FEMS Microbiol Ecol 94
Nijenhuis I, Stollberg R, Lechner U
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2018. Desulfitobacterium contributes to the microbial transformation of 2,4,5-T by methanogenic enrichment cultures from a Vietnamese active landfill. Microb Biotechnol 11: 1137–1156
Lechner U, Türkowsky D, Dinh TTH, Al-Fathi H, Schwoch S, Franke S, Gerlach M-S, Koch M, von Bergen M, Jehmlich N, Dang TCH
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2019. Anaerobic degradation of 2,4,5-trichlorophenoxyacetic acid by enrichment cultures from freshwater sediments. Env Sci Pollut Res
Al-Fathi H, Koch M, Lorenz WG, Lechner U
