An unusual tandem‐domain rhodanese harbouring two active sites identified in <i>Desulfitobacterium hafniense</i>

Prat, Laure; Maillard, Julien; Rohrbach-Brandt, Emmanuelle; Holliger, Christof

doi:10.1111/j.1742-4658.2012.08660.x

Cited by 15 publications

(10 citation statements)

References 47 publications

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“…The pellet was quickly resuspended in 1 ml of LifeGuard solution (MoBio, Carlsbad, CA), flash-frozen in liquid N 2 , and stored at Ϫ80°C. An optimized TRIzol-based RNA extraction protocol was used for total RNA extraction as previously described (33). Reverse transcription (RT) was performed as follows.…”

Section: Methodsmentioning

confidence: 99%

Functional Genotyping of Sulfurospirillum spp. in Mixed Cultures Allowed the Identification of a New Tetrachloroethene Reductive Dehalogenase

Buttet

Holliger

Maillard

2013

Appl Environ Microbiol

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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 PceA TCE , 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 sequencesubstrate relationships within tetrachloroethene reductive dehalogenases are also discussed. Widespread use of chlorinated solvents in cleaning and metaldegreasing operations over the last century has resulted in extensive environmental contamination by chlorinated ethenes. Tetrachloroethene (PCE) is currently one of the main contaminants of aquifers. Different remediation strategies have been developed, including bioremediation, which uses microorganisms for the degradation of pollutants (1). Several genera of strictly anaerobic bacteria, including Desulfitobacterium (2), Dehalococcoides (3), Dehalobacter (4), and Sulfurospirillum (5), are able to conserve energy via the reductive dehalogenation of chloroethenes by a respiratory metabolism (6-9) recently coined organohalide respiration (OHR). The enzyme class involved in OHR for the reduction of halogenated compounds is known as the reductive dehalogenase (RdhA) family (10, 11). Most RdhA enzymes have been isolated from OHR bacteria (OHRB) as 48-to 65-kDa monomers consisting of a single polypeptide chain and containing one corrinoid cofactor and two iron-sulfur clusters. The majority of currently known OHRB carry multiple rdhA genes, i.e., 1 to 36 genes, depending on the strain (12). Several studies have demonstrated that the presence of multiple nonidentical rdhA genes is a typical feature of OHRB (13-15). This suggests that the substrate range of the OHRB may be far greater than previously believed (16). However, substrate specificity has been determined for only about 15 enzymes within the several hundreds of putative rdhA sequences reported in databases (12). Moreover, transcriptomic studies on strains displaying multiple rdhA genes often failed to clearly identify which reductive dehalogenase was responsible for the dechlorination of specific substrates (12,(17)(18)(19)(20).The genus Sulfurospirillum comprises microaerophilic or facultative anaerob...

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Section: Methodsmentioning

confidence: 99%

Functional Genotyping of Sulfurospirillum spp. in Mixed Cultures Allowed the Identification of a New Tetrachloroethene Reductive Dehalogenase

Buttet

Holliger

Maillard

2013

Appl Environ Microbiol

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“…A. arabaticum and D. dehalogenans cells were harvested in exponential phase by centrifugation at 4°C and 4,000 g for 10 min and RNA was extracted by using TRIzol as described in ref. 41. Reverse transcription was performed on 1 μg of RNA mixed with 200 ng of random hexamers, in the presence of the SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Carbon source-dependent expansion of the genetic code in bacteria

Prat

Heinemann

Aerni

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Despite the fact that the genetic code is known to vary between organisms in rare cases, it is believed that in the lifetime of a single cell the code is stable. We found Acetohalobium arabaticum cells grown on pyruvate genetically encode 20 amino acids, but in the presence of trimethylamine (TMA), A. arabaticum dynamically expands its genetic code to 21 amino acids including pyrrolysine (Pyl). A. arabaticum is the only known organism that modulates the size of its genetic code in response to its environment and energy source. The gene cassette pylTSBCD , required to biosynthesize and genetically encode UAG codons as Pyl, is present in the genomes of 24 anaerobic archaea and bacteria. Unlike archaeal Pyl-decoding organisms that constitutively encode Pyl, we observed that A. arabaticum controls Pyl encoding by down-regulating transcription of the entire Pyl operon under growth conditions lacking TMA, to the point where no detectable Pyl-tRNA Pyl is made in vivo. Pyl-decoding archaea adapted to an expanded genetic code by minimizing TAG codon frequency to typically ∼5% of ORFs, whereas Pyl-decoding bacteria (∼20% of ORFs contain in-frame TAGs) regulate Pyl-tRNA Pyl formation and translation of UAG by transcriptional deactivation of genes in the Pyl operon. We further demonstrate that Pyl encoding occurs in a bacterium that naturally encodes the Pyl operon, and identified Pyl residues by mass spectrometry in A. arabaticum proteins including two methylamine methyltransferases.

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“…Standards were prepared by serial dilution from 10 7 to 10 2 copy numbers/l. RNA extraction and reverse transcription (RT) were performed as described previously (48). Runs of qPCR consisted of the standard dilution series targeting a single gene and samples in triplicates.…”

Section: Methodsmentioning

confidence: 99%

Diversity of Cobalamin Riboswitches in the Corrinoid-Producing Organohalide Respirer Desulfitobacterium hafniense

et al. 2013

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The strategic adaptation of prokaryotes in polluted niches involves the efficient regulation of their metabolism. The obligate anaerobe and metabolically versatile Desulfitobacterium hafniense reductively dechlorinates halogenated organic compounds (socalled organohalides). Some D. hafniense strains carry out organohalide respiration (OHR), a process which requires the use of corrinoid as a cofactor in reductive dehalogenases, the key enzymes in OHR. We report here the diversity of the cobalamin riboswitches that possibly regulate the corrinoid metabolism for D. hafniense. The analysis of available D. hafniense genomes indicates the presence of 18 cobalamin riboswitches located upstream of genes whose products are mainly involved in corrinoid biosynthesis and transport. To obtain insight into their function, the secondary structures of three of these RNA elements were predicted by Mfold, as well as analyzed by in-line probing. These RNA elements both display diversity in their structural elements and exhibit various affinities toward adenosylcobalamin that possibly relates to their role in the regulation of corrinoid metabolism. Furthermore, adenosylcobalamin-induced in vivo repression of RNA synthesis of the downstream located genes indicates that the corrinoid transporters and biosynthetic enzymes in D. hafniense strain TCE1 are regulated at the transcriptional level. Taken together, the riboswitch-mediated regulation of the complex corrinoid metabolism in D. hafniense could be of crucial significance in environments polluted with organohalides both to monitor their intracellular corrinoid level and to coexist with corrinoid-auxotroph OHR bacteria.

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An unusual tandem‐domain rhodanese harbouring two active sites identified in Desulfitobacterium hafniense

Cited by 15 publications

References 47 publications

Functional Genotyping of Sulfurospirillum spp. in Mixed Cultures Allowed the Identification of a New Tetrachloroethene Reductive Dehalogenase

Functional Genotyping of Sulfurospirillum spp. in Mixed Cultures Allowed the Identification of a New Tetrachloroethene Reductive Dehalogenase

Carbon source-dependent expansion of the genetic code in bacteria

Diversity of Cobalamin Riboswitches in the Corrinoid-Producing Organohalide Respirer Desulfitobacterium hafniense

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