Development of a Method to Efficiently Determine Prokaryotic Gene Sequences Relevant to Pollutant Biodegradation

Résumé du livre

Vast quantities of chemicals are generated in the U.S. each year for industrial and consumer purposes, and many of these chemicals ultimately become environmental pollutants. Environmental engineers have developed numerous biological treatment systems to degrade pollutants. To aid in the optimization of these technologies, molecular tools are being developed to study microbial degradation processes. In many cases, the use of such molecular tools has been hindered by a lack of available sequences for environmentally relevant genes. Therefore, the objective of this work was to develop a rapid and efficient molecular method to determine gene sequences for biodegradation enzymes. Suppression subtractive hybridization (SSH) Polymerase Chain Reaction (PCR) cDNA subtraction is a method that can amplify and identify genes that are expressed under specific conditions, such as growth on a given pollutant. Cells are grown either in the presence or absence of a pollutant; mRNA is isolated from both cultures and used to synthesize cDNA. Then the genes that are expressed in both cultures are "subtracted out" and only those genes that are uniquely expressed for the degradation of the pollutant are amplified during SSH PCR amplification. While excellent kits are commercially available for eukaryotic SSH PCR cDNA subtraction, no such kits exist for prokaryotes. This work developed methodology for the application of SSH PCR cDNA subtraction to prokaryotic systems using commercially available reagents from several sources. Pseudomonas putida mt-2, a fully sequenced bacterium, grown on toluene was selected as a model system; acetate was selected as the alternative carbon source because toluene-degradation genes are not highly expressed during growth on acetate. A protocol was developed for purification of up to 12 [Mu]g of mRNA (high quality, and DNA-free) from prokaryotes. A novel prokaryotic cDNA Subtraction (PCS) cDNA synthesis primer was designed, and a method for generating approximately 2 [Mu]g of prokaryotic cDNA using this primer was developed. cDNA from P. putida grown on toluene or acetate was subjected to the prokaryotic SSH PCR cDNA subtraction protocol developed here. 27 clones were isolated and sequenced, and 95% of these contained fragments of genes known to be related to toluene degradation. The sequences represented 10 distinct genes within two key operons. Based on the results presented here, prokaryotic SSH PCR cDNA subtraction shows promise as a focused gene sequencing tool for environmentally relevant bacteria