Progress 10/01/02 to 09/30/05
Outputs
The goal of this project was to better understand the mechanisms of how E. coliO157:H7 persists in ruminent hosts. Specifically, we have sought to identify genes whose products are important for E. coli to colonize and survive in the digestive tract of ruminants. The interaction of E. coli with its host is likely to be complex with a variety of metabolic pathways potentially involved. Initially, a genetic footprinting strategy was used to identify E. coli O157:H7 genes that are required for survival in the rumen. We first performed an extensive mutagenesis of E. coli O157:H7 using a mariner transposable element. Insertion of this element shows specificity only for AT base pairs and is highly efficient in transposition. To test the genetic footprinting strategy we grew a mutagenized population of E. coli in both rich nutrient broth and defined, minimal medium supplemented with glucose. After growth for ~8 generations, the bacteria were subcultured and grown for an
additional 8-10 generations in each type of media. PCR primers were designed to amplify genes essential for amino acid biosynthesis (leuA and trpA). Total genomic DNA was prepared from each culture and PCR was performed. While specific PCR products were obtained from the culture grown in nutrient broth, products were greatly reduced from the culture grown under nutrient limiting conditions. These results are consistent with loss of mutants from the population where the leuA and trpA genes were disrupted by the transposable element. These mutant populations were subsequently tested for growth under anaerobic conditions supplemented with rumen fluid. However, conditions that permitted sufficient growth of the cultures in vitro were not found. Without sufficient growth the genetic footprinting strategy will not work in vivo, since loss of mutants require dilution from the population of mutants that are unable to grow under selective conditions. A separate approach was taken to identify
genes whose expression is differentially regulated in the rumen environment. DNA microarrays for E. coli O157:H7 became commercially available so we initiated experiments to perform transcriptional profiling of E. coli gene expression. We initially performed experiments with heat shocked E. coli to optimize conditions for performing microarrays. While initial experiments were performed with Cy-2 and Cy-5 fluorescent dyes, with improved results obtained by switching to more sensitive aminoallyl nucleotide labeling. Pilot experiments confirmed that we could detect increased expression of heat shock genes from cultures grown at 42oC. Additional improvements to the methodology were also incorporated, including use of hybridization chambers specifically designed for microarray hybridization, new fractionation method to purify and enrich for bacterial mRNA by removing the vast majority ribosomal RNA species. This study has brought us to a point where we can pursue transcriptional profiling
on bacteria grown under in vitro conditions that closely mimic the rumen environment, and eventually to characterize gene expression in an in vivo growth model.
Impacts
Identifying genes that are important for survival in the ecological niche exploited by E. coliO157:H7 represents a novel strategy to better understand the physiology and genetic responses of this microorganism in its natural environment. The products of the genes important for growth under these conditions will represent new targets for antimicrobial agents that could be added to feed and water, or for construction of new vaccines.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Establish conditions for genetic footprinting. Numerous functional genomic techniques have recently been developed to facilitate gene function analysis. Genetic footprinting in bacteria has been reported to be an effective technique for the identification of genes that are essential for growth under specific environmental conditions. We have initiated experiments to identify E. coli O157:H7 genes required for growth in defined medium. E. coli O157:H7 strain 933 was transformed with pGT-G69, a plasmid capable of delivering a mini-Tn10cam element at high frequency. Transformants were grown under conditions that promote expression of the transposase resulting in saturation mutagenesis of the bacterial genome with the mini-Tn10cam element. Pooled transposon mutants were then grown for greater than fifty generations in minimal salts medium in the absence of leucine. Cells were harvested, total cellular DNA isolated, and oligonucleotide primers specific to the mini-Tn10cam
element were used in combination with primers flanking the leu operon. Following PCR amplification, products were resolved by electrophoresis and analyzed by densitometric scanning. Since growth in minimal salts medium did not permit growth of transposon-induced leu auxotrophs, the PCR signal of the Tn-leu operon primer pair diminishes as the cells divide in the selective environment. Our results revealed that indeed PCR products were not obtained in the absence of leucine indicating that these mutants were not maintained under these growth conditions. These results indicate that a similar strategy should be effective at identifying specific E. coli O157:H7 genes that are required for growth in vivo. In preparation for performing genetic footprinting in vivo, work was also initiated on an ex vivo technique for analysis of E. coli O157:H7 gene function. Media consisting of minimal salts supplemented with rumen fluid was developed and used to propagate E. coli anaerobically. Growth
conditions have been established for genetic footprinting to be performed using controlled conditions that mimic the rumen environment.
Impacts
Understand how bacteria survive in the rumen environment is important to identify new strategies for control of microorganisms that are transmitted from cattle to humans. Identifying genes using genomic approaches represents a potentially powerful approach to discovering new targets for antimicrobial agents or treatments.
Publications
- No publications reported this period
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