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The Degradation Pathway for Propargyl Bromide in Soil Bacteria
EPA Grant Number: U916085Title: The Degradation Pathway for Propargyl Bromide in Soil Bacteria
Investigators: Ramage, Holly R.
Institution: California State University - Long Beach
EPA Project Officer: Just, Theodore J.
Project Period: January 1, 2002 through January 1, 2004
Project Amount: $24,303
RFA: GRO Graduate Fellowships (2002)
Research Category: Academic Fellowships , Biology/Life Sciences , Fellowship - Natural and Life Sciences
Description:
Objective:The overall goal of this research project is to elucidate information on the biotic degradation of propargyl bromide (3-bromopropyne). The specific objectives are to: (1) identify the species of the 10 degrader microorganisms; (2) establish rates of propargyl bromide degradation by the microorganisms; and (3) identify gene(s) that are involved in the degradation pathway. One of the primary pesticides used by farmers since the 1960s is methyl bromide, a soil fumigant. Methyl bromide is a broad-spectrum fumigant that is used during preplanting soil preparation to control disease caused by soil-dwelling weeds, fungi, nematodes, and bacteria. Concerns about the effects of methyl bromide on the ozone layer are great, and the fumigant is being phased out in the United States (and other countries) because it has high reactivity with ozone. Propargyl bromide is a promising alternative to methyl bromide. If propargyl bromide is selected as a feasible alternative to methyl bromide, enhancing biotic processes to expedite its removal from soil is desirable.
Approach:Through enrichment culture techniques, we have isolated 10 bacterial strains that can use propargyl bromide as a sole carbon source. The use of genome-based identification of bacterial species (by using the sequences of the small [16S] ribosomal subunit) will provide an accurate, efficient method for the identification of a large amount of these microorganisms. Primers used to amplify the 16S subunit have been designed to align with highly conserved sequence and should amplify the small ribosomal subunit of all eubacteria. Because the primers are designed for the conserved region, the amplification of the 16S subunits of diverse bacterial organisms is possible with one set of primers.
The strains will be further characterized using gas chromatography (GC). The main points of interest will be the rate of degradation of propargyl bromide and the accumulation of any compounds that may be intermediates, products, or byproducts of the degradation pathway. The rate of degradation of propargyl bromide can be measured by adding each of the strains to media containing a known amount of propargyl bromide and measuring the disappearance of propargyl bromide over time. All compounds that accumulate at detectable levels will be identified as new peaks in the GC analysis of the culture supernatant.
Transposon mutagenesis will be used to create a group of mutants characterized by the loss of ability to degrade propargyl bromide. A transposon delivery system will allow the delivery of the transposon into the target cell, resulting in the disruption of genes. If a gene in the propargyl bromide degradation pathway is disrupted, that organism can no longer use propargyl bromide as a sole carbon source. These mutants can be identified, and the site of transposon insertion in the genome will be recovered, allowing for the identification of the gene in the degradation pathway.
Supplemental Keywords:fellowship, propargyl bromide, soil bacteria, small ribosomal subunit, 16S, gas chromatography, GC.