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United States Department of Agriculture

Agricultural Research Service

Title: Differential Regulation of Sepl and Esp Genes of Enterohemorrhagic Escherichia Coli O157:h7

Authors
item Sharma, Vijay
item Casey, Thomas
item Morgan, Robert

Submitted to: International Union of Microbiological Societies Proceedings/Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: April 14, 2005
Publication Date: July 23, 2005
Citation: Sharma, V.K., Casey, T., Morgan, R.W. 2005. Differential regulation of sepL and esp genes of enterohemorrhagic Escherichia coli O157:H7 [abstract]. Joint Meeting of the 3 Divisions of the International Union of Microbiological Societies. p. 159.

Technical Abstract: Background: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) genes required for the formation of attaching and effacing lesions on host epithelial cells are located in a horizontally acquired DNA fragment, termed locus of enterocyte effacement (LEE). The LEE1 through LEE5 operons encode factors essential for the formation of characteristic attaching and effacing lesions on epithelial cells. The expression of these operons is under the positive regulation of ler, the first gene of the LEE1 operon. Both in vivo and in vitro studies have shown that the interactions of Ler with the promoter elements of LEE operons are critical prerequisites for the expression of LEE. We have recently reported that the increased expression of ler in an hha mutant strain of EHEC O157:H7 causes enhanced expression of LEE operons and enable this strain to exhibit hyperadherence phenotype on Hep-2 cells. In the present study, we report that the EHEC O157:H7 monocistronic sepL operon is differentially regulated and transcribed independently of the downstream LEE4 operon unlike transcription of the sepL in EPEC strain E2348/69. Methods: The transcriptional levels of sepL were determined by RT-PCR using total RNA isolated from EHEC O157:H7 expressing or lacking hha. The sepL transcription was compared to the transcription of gapA (housekeeping gene used as a control) and espA encoded in the LEE4 operon. Relative binding of purified Ler to the DNA fragment containing sepL promoter was compared to the binding of Ler to LEE4 promoter fragment using gel shift assays. Results: The levels of sepL transcription were not affected in the presence or absence of hha. On the other hand, levels of espA increased by 100-fold in hha mutant strain. The in vitro gel shift assays showed that the purified Ler bound to the DNA fragment containing the sepL promoter with a reduced affinity relative to its binding to the LEE4 promoter. Conclusions: The results suggest two possible explanations for further investigations: either sepL is constitutively transcribed at low levels irrespective of the ler expression or the expression of sepL is governed by some additional regulatory factors.

Last Modified: 9/2/2014
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