Dataset Information

Functional genotyping of Sulfurospirillum spp. in mixed cultures allowed the identification of a new tetrachloroethene reductive dehalogenase.

ABSTRACT: Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceATCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed.

SUBMITTER: Buttet GF

PROVIDER: S-EPMC3811552 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Functional genotyping of Sulfurospirillum spp. in mixed cultures allowed the identification of a new tetrachloroethene reductive dehalogenase.

Buttet Géraldine F GF Holliger Christof C Maillard Julien J

Applied and environmental microbiology 20130830 22

Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the ...[more]

PMID: 23995945

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Project description:The organohalide-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated, and their use as cofactor by PceA was analyzed. Exogenously applied 5-methylbenzimidazole (5-MeBza), 5-hydroxybenzimidazole (5-OHBza), and 5-methoxybenzimidazole (5-OMeBza) were found to be efficiently incorporated as lower bases into norcobamides (NCbas). Structural analysis of the NCbas by nuclear magnetic resonance spectroscopy uncovered a regioselectivity in the utilization of these precursors for NCba biosynthesis. When 5-MeBza was added, a mixture of 5-MeBza-norcobamide and 6-MeBza-norcobamide was formed, and the PceA enzyme activity was affected. In the presence of 5-OHBza, almost exclusively 6-OHBza-norcobamide was produced, while in the presence of 5-OMeBza, predominantly 5-OMeBza-norcobamide was detected. Both NCbas were incorporated into PceA, and no negative effect on the PceA activity was observed. In crystal structures of PceA, both NCbas were bound in the base-off mode with the 6-OHBza and 5-OMeBza lower bases accommodated by the same solvent-exposed hydrophilic pocket that harbors the adenine as the lower base of authentic norpseudo-B12 In this study, a selective production of different norcobamide isomers containing singly substituted benzimidazoles as lower bases is shown, and unique structural insights into their utilization as cofactors by a cobamide-containing enzyme are provided.IMPORTANCE Guided biosynthesis of norcobamides containing singly substituted benzimidazoles as lower bases by the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans is reported. An unprecedented specificity in the formation of norcobamide isomers containing hydroxylated or methoxylated benzimidazoles was observed that implicated a strict regioselectivity of the norcobamide biosynthesis in the organism. In contrast to 5,6-dimethylbenzimidazolyl-norcobamide, the incorporation of singly substituted benzimidazolyl-norcobamides as a cofactor into the tetrachloroethene reductive dehalogenase was not impaired. The enzyme was found to be functional with different isomers and not limited to the use of adeninyl-norcobamide. Structural analysis of the enzyme equipped with either adeninyl- or benzimidazolyl-norcobamide cofactors visualized for the first time structurally different cobamides bound in base-off conformation to the cofactor-binding site of a cobamide-containing enzyme.

Project description:The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 +/- 1 kDa, whereas the native molecular mass was 71 +/- 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-beta-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 +/- 0.1 mol of cobalamin, 0.6 +/- 0.02 mol of cobalt, 7.1 +/- 0.6 mol of iron, and 5.8 +/- 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 +/- 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K(m) values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 +/- 3.2, 23.7 +/- 5.2, and 47 +/- 10 micro M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.

Project description:Organohalide respiration (OHR) is a bacterial anaerobic process that uses halogenated compounds, e.g., tetrachloroethene (PCE), as terminal electron acceptors. Our model organisms are Dehalobacter restrictus strain PER-K23, an obligate OHR bacterium (OHRB), and Desulfitobacterium hafniense strain TCE1, a bacterium with a versatile metabolism. The key enzyme is the PCE reductive dehalogenase (PceA) that is encoded in the highly conserved gene cluster (pceABCT) in both above-mentioned strains, and in other Firmicutes OHRB. To date, the functions of PceA and PceT, a dedicated molecular chaperone for the maturation of PceA, are well defined. However, the role of PceB and PceC are still not elucidated. We present a multilevel study aiming at deciphering the stoichiometry of pceABCT individual gene products. The investigation was assessed at RNA level by reverse transcription and (quantitative) polymerase chain reaction, while at protein level, proteomic analyses based on parallel reaction monitoring were performed to quantify the Pce proteins in cell-free extracts as well as in soluble and membrane fractions of both strains using heavy-labeled reference peptides. At RNA level, our results confirmed the co-transcription of all pce genes, while the quantitative analysis revealed a relative stoichiometry of the gene transcripts of pceA, pceB, pceC, and pceT at ~ 1.0:3.0:0.1:0.1 in D. restrictus. This trend was not observed in D. hafniense strain TCE1, where no substantial difference was measured for the four genes. At proteomic level, an apparent 2:1 stoichiometry of PceA and PceB was obtained in the membrane fraction, and a low abundance of PceC in comparison to the other two proteins. In the soluble fraction, a 1:1 stoichiometry of PceA and PceT was identified. In summary, we show that the pce gene cluster is transcribed as an operon with, however, a level of transcription that differs for individual genes, an observation that could be explained by post-transcriptional events. Despite challenges in the quantification of integral membrane proteins such as PceB and PceC, the similar abundance of PceA and PceB invites to consider them as forming a membrane-bound PceA2B protein complex, which, in contrast to the proposed model, seems to be devoid of PceC.

Project description:Organohalide respiration (OHR) is a bacterial anaerobic process in which halogenated compounds, e.g. tetrachloroethene (PCE), are used as terminal electron acceptors. Our model organisms are Dehalobacter restrictus strain PER-K23, an obligate OHR bacterium, and Desulfitobacterium hafniense strain TCE1, a bacterium with a versatile energy metabolism. The key catalytic enzyme is the tetrachloroethene reductive dehalogenase (PceA) that is, at genomic level, encoded in a highly conserved gene cluster (pceABCT) in both D. restrictus and D. hafniense strain TCE1. To date, the functions of PceA and PceT, a dedicated molecular chaperone for the maturation of PceA, are well defined. However, the role of PceB and PceC are still not elucidated. Here, we present a multilevel study aiming at deciphering the stoichiometry of the pceABCT individual gene products. The investigation was assessed at RNA level by reverse transcription and (quantitative) polymerase chain reaction, while at protein level, proteomic analyses based on parallel reaction monitoring were performed to quantify the PceA, B, C and T proteins in cell-free extracts as well as in soluble and membrane fractions of both strains using heavy-labelled reference peptides. At RNA level, our results confirmed the co-transcription of all pce gene products, while the quantitative analysis revealed a relative stoichiometry of the gene transcripts of pceA, pceB, pceC and pceT at approximately 1.0:3.0:0.1:0.1 in D. restrictus. This trend was not observed in D. hafniense strain TCE1, where no substantial difference was measured for the four genes. At proteomic level, a 2:1 stoichiometry of PceA and PceB was observed in the membrane fraction, and a low abundance of PceC in comparison to the other two proteins. In the soluble fraction, a 1:1 stoichiometry of PceA and PceT was identified. In summary, we show that the pce gene cluster is transcribed as an operon with, however, a level of transcription that differs for individual genes, an observation that could be explained by post-transcriptional events, such as RNA processing and differential RNA stability. Results at protein level suggest that PceA and PceB form a membrane-bound PceA2B protein complex, which, in contrast to the proposed model, seems to be devoid of PceC.

Dataset Information

Functional genotyping of Sulfurospirillum spp. in mixed cultures allowed the identification of a new tetrachloroethene reductive dehalogenase.

Publications

Functional genotyping of Sulfurospirillum spp. in mixed cultures allowed the identification of a new tetrachloroethene reductive dehalogenase.

Similar Datasets

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