Phytoremediation of triphenylmethane dyes by overexpressing a Citrobacter sp. triphenylmethane reductase in transgenic Arabidopsis

ABSTRACT We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.

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Structural Insight into Bioremediation of Triphenylmethane Dyes byCitrobactersp. Triphenylmethane Reductase

Journal of Biological Chemistry ◽

10.1074/jbc.m804092200 ◽

2008 ◽

Vol 283 (46) ◽

pp. 31981-31990 ◽

Cited By ~ 26

Author(s):

Myung Hee Kim ◽

Yoonjeong Kim ◽

Hyo-Jung Park ◽

Jong Suk Lee ◽

Su-Nam Kwak ◽

...

Keyword(s):

Triphenylmethane Dyes ◽

Structural Insight ◽

Insight Into ◽

Triphenylmethane Reductase

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IncP-1β Plasmid pGNB1 Isolated from a Bacterial Community from a Wastewater Treatment Plant Mediates Decolorization of Triphenylmethane Dyes

Applied and Environmental Microbiology ◽

10.1128/aem.01177-07 ◽

2007 ◽

Vol 73 (20) ◽

pp. 6345-6350 ◽

Cited By ~ 22

Author(s):

Andreas Schlüter ◽

Irene Krahn ◽

Florian Kollin ◽

Gabriele Bönemann ◽

Michael Stiens ◽

...

Keyword(s):

Wastewater Treatment ◽

Bacterial Community ◽

Crystal Violet ◽

Wastewater Treatment Plant ◽

Treatment Plant ◽

Replication Initiation ◽

Host Bacterium ◽

Triphenylmethane Dyes ◽

Reductase Gene ◽

Triphenylmethane Reductase

ABSTRACT Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1β plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the α-proteobacterium Sinorhizobium meliloti and the γ-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1β plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.

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Genetic Characterization of Plasmid-Associated Triphenylmethane Reductase in Listeria monocytogenes

Applied and Environmental Microbiology ◽

10.1128/aem.01398-14 ◽

2014 ◽

Vol 80 (17) ◽

pp. 5379-5385 ◽

Cited By ~ 12

Author(s):

Vikrant Dutta ◽

Driss Elhanafi ◽

Jason Osborne ◽

Mira Rakic Martinez ◽

Sophia Kathariou

Keyword(s):

Listeria Monocytogenes ◽

Gene Cassette ◽

Triphenylmethane Dyes ◽

Environmental Persistence ◽

Content Type ◽

Hot Dog ◽

Encoding Genes ◽

Triphenylmethane Reductase

ABSTRACTThe enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome ofListeria monocytogenesH7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette (bcrABC) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene (tmr) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associatedtmrwas located two genes downstream ofbcrABCas part of a putative IS1216composite transposon. To confirm the role oftmrin triphenylmethane dye detoxification, we introduced varioustmr-harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanningbcrABCandtmrwere CV resistant, and in such constructstmrtranscription was induced by sublethal levels of either BC or CV. However, constructs harboring onlytmrand its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistantL. monocytogenesstrains revealed that all those harboringbcrABCand adjacent pLM80 sequences, includingtmr, were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence ofL. monocytogenes.

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