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United States Department of Agriculture

Agricultural Research Service

Title: Identification and Evaluation of Regulatory Factors Modulating the Expression of Ler in Enterohemorrhagic Escherichia Coli O157:h7

Authors
item Fenton, Karla - IOWA STATE UNIVERSITY
item Cornick, N - IOWA STATE UNIVERSITY
item Morgan, Robert
item SHARMA, VIJAY

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: February 21, 2003
Publication Date: May 22, 2003
Citation: FENTON, K., CORNICK, N.A., MORGAN, R.W., SHARMA, V.K. IDENTIFICATION AND EVALUATION OF REGULATORY FACTORS MODULATING THE EXPRESSION OF LER IN ENTEROHEMORRHAGIC ESCHERICHIA COLI O157:H7. AMERICAN SOCIETY FOR MICROBIOLOGY. 2003. ABSTRACT P. 96.

Technical Abstract: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 encodes multiple virulence factors that enable it to colonize host intestine and produce characteristic attaching and effacing (A/E) lesions. Many of the virulence factors implicated in the formation of the A/E phenotype are contained within a horizontally acquired segment of DNA, called locus of enterocyte effacement (LEE). A total of 41-ORFs, most of which are organized into five polycistronic operons (LEE1 - LEE5), constitute LEE. The Ler protein, encoded by the gene ler of the LEE1 operon, induces transcription of many of the LEE operons. The expression of ler is positively regulated by the host integration factor (IHF) and an autoinducer encoded by the gene luxS (quorum sensing pathway). However, the genes implicated in the negative regulation of ler and the mechanism by which these regulatory factors reduce ler transcription in environments unfavorable for host colonization are not fully understood. We have previously reported that the gene hha down regulates the expression of ler by binding to a DNA sequence containing the ler promoter region. In this study, we present experimental evidence that additional, yet unidentified, genes may also be implicated in the negative regulation of ler expression. A temperature-sensitive plasmid containing a transcriptional fusion between the ler promoter and a lacZ reporter cassette was constructed (pSM139) and introduced into a deltahha derivative of E. coli O157:H7 strain 86-24. The strain 86-24/pSM139 was then cultured under conditions that facilitated transfer of ler::lac fusion from pSM139 to the host chromosome by allelic exchange events. The determination of b-galactosidase activity of strain 86-24 deltahha ler::lac (parent strain) revealed a maximal expression after 4 h of growth. Transpositional mutagenesis of this strain resulted in isolation of clones (mutant strains) expressing 2- to 3-fold higher levels of beta-galactosidase as compared to the parent strain. These results showed that the transposon insertion inactivated a gene in the mutant strain causing an increased expression of ler. Identification of the gene inactivated by the transposon insertion, its' ability to complement the mutant strain, and the mechanism by which this gene affects ler transcription are currently under investigation.

Last Modified: 9/29/2014