Gene expression profiling in enterohemorrhagic escherichia coli
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AbstractThe pathogen Enterohemorrhagic Escherichia coli (EHEC) is a continuing problem in outbreaks associated with contaminated food and water. This study describes the development of a novel high throughput method for monitoring global gene expression in EHEC. The random promoter library is based on a promoter trap method and three reporters lux, gfp and lacZ, were compared for efficacy. The lux reporter system was found to be the best candidate. Random promoter library construction methods were optimized using a partial E. coli MG 1655 library. Subsequently, an EHEC random promoter library was constructed and characterized by assaying gene expression from 3,840 clones under many conditions. Unique expression profiles were observed, the distribution of cloned fragments was random and over-coverage of the genome was indicated by redundancy in the sequencing results. Hierarchical clustering was used to analyze the gene expression data. Co-clustering clones with response profiles unique to certain groups of assay conditions were characterized and sequenced although the results illustrated the difficulties associated with cluster analysis. In contrast, clusters associated with the LEE promoters were well defined and sequence results were classified into groups based on putative function. Gene products associated with response to different cell stresses were represented. Cell wall modifying enzymes were also identified as was miaA, required for tRNA modification and virulence in Shigella. This dataset, including expression profiles in many conditions and sequence information, is a valuable tool and resource for understanding EHEC gene regulation.
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