Identification of Genes From Mycobacterium smegmatis mc²155 Involved in the Decolorization of the Triarylmethane Dye Malachite Green

EPA Grant Number: MA916303
Title: Identification of Genes From Mycobacterium smegmatis mc²155 Involved in the Decolorization of the Triarylmethane Dye Malachite Green
Investigators: Lopez, Denise L.
Institution: California State University - Fresno
EPA Project Officer: Zambrana, Jose
Project Period: January 1, 2004 through December 31, 2006
Project Amount: $89,826
RFA: GRO Fellowships for Graduate Environmental Study (2004) RFA Text |  Recipients Lists
Research Category: Fellowship - Natural and Life Sciences , Academic Fellowships , Biology/Life Sciences


The objectives of this research project are to: (1) expand a transposon mutant library of mutant Mycobacterium smegmatis mc²155; (2) screen the transposon mutant library for mutants unable to decolorize malachite green, a toxic dye widely used in fisheries outside the United States; (3) identify the gene responsible for each mutant phenotype, confirm identity by complementation, and determine if the identified gene is induced by malachite green; and (4) investigate whether M. smegmatis is able to detoxify other compounds used in agriculture within the United States.


We have demonstrated that M. smegmatis is able to decolorize the toxic, triarylmethane dye malachite green. A library of M. smegmatis transposon mutants currently is being constructed. We have created a high-throughput method to screen mutants generated in this manner for their inability to decolorize malachite green. Sorted mutants are replica-plated into ELISA plates containing liquid media supplemented with malachite green. The ELISA plates are incubated at 37°C for 48 hours and then inspected for mutants that are unable to decolorize the dye. Three thousand mutants have been screened to date. One mutant does not decolorize malachite green, and the dye accumulates within the growing colonies on solid media. Three others have lower minimum inhibitory concentrations for malachite green than wild-type. The flanking genomic DNA will be sequenced, and the sequence will be searched against M. smegmatis genome database to identify the disrupted gene. To determine whether the gene is induced by the presence of malachite green, Northern blots will be performed on RNA extracted from wild-type cells grown in the presence of the dye as well as in normal media. To ensure that the gene identified is responsible for the phenotype, the mutant will be complemented with the wild-type gene. Restoration of the wild-type phenotype will confirm that the gene is indeed involved in the decolorization of malachite green. We have determined M. smegmatis also can grow on media supplemented with other various toxic compounds. Oxygen uptake studies will be performed to investigate whether M. smegmatis is able to detoxify these compounds, such as the herbicides simazine and diuron that are known groundwater contaminants in the Central Valley of California.

This research is expected to result in the identification of genes involved in the decolorization of malachite green by M. smegmatis . It also will be determined whether other compounds can be detoxified by this bacterium. These results may have important implications in the field of bioremediation as these compounds currently are used in the international marketplace and therefore have the potential to bioaccumulate.

Supplemental Keywords:

fellowship, bioremediation, bioaccumulation, mycobacteria, detoxification, decolorization, dye, malachite green, triarylmethane dye, herbicide, simazine, diuron, ground water, contaminant, transposon, phenotype, transposon mutants, gene, mutagenesis, disruption, genome, bacteria, malachite green,, Scientific Discipline, Waste, TREATMENT/CONTROL, Remediation, Environmental Chemistry, Treatment Technologies, Biochemistry, bacterial detoxification, transposon mutant research, genetic engineering, agriculture, mycobacterium smegmatis

Relevant Websites:

2004 STAR Graduate Fellowship Conference Poster (PDF, 1p., 112KB, about PDF)

Progress and Final Reports:

  • 2004
  • 2005
  • Final