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United States Department of Agriculture

Agricultural Research Service

Research Project: PREVENTION OF LOSSES FROM COLIBACILLOSIS AND O157:H7 AND OTHER SHIGA TOXIN-PRODUCING E. COLI (STEC) IN CATTLE AND SWINE Title: Identification of Hha As a Potential Substrate for the Clpxp Protease, a Modulator of Lee Expression in Enterohemorrhagic Escherichia Coli 0157:h7

item Mesterhazy, Karla
item Sharma, Vijay

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: February 17, 2006
Publication Date: May 22, 2006
Citation: Mesterhazy, K.A., Sharma, V.K. 2006. Identification of Hha as a potential substrate for the ClpXP protease, a modulator of LEE expression in enterohemorrhagic Escherichia coli 0157:h7 [abstract]. American Society for Microbiology. p.264.

Technical Abstract: Locus of enterocyte (LEE) encodes factors for attachment of Enterohemorrhagic Escherichia coli (EHEC) O157:H7 to host intestinal cells and production of attaching and effacing (A/E) lesions. The five LEE operons (LEE1 - LEE5) encode functions for producing A/E phenotype. The expression of LEE operons is positively regulated by LEE1-encoded regulator ler. Several positive and negative regulators modulate ler expression. Recent studies have demomstrated that ClpXP protease regulates LEE expression by controlling intracellular levels of RpoS and GrlR. We have reported that Hha represses the expression of LEE4::lac transcriptional fusion by reducing ler transcription. In the present study, we demonstrate that Hha may serve as a potential target of ClpXP. Methods: A clpXP deletion was introduced into the chromosome of EHEC O157:H7 strain 86-24 'hha carrying a LEE4::lac transcriptional fusion. '-galactosidase activity of the mutant strain was compared to 86-24 'hha LEE4::lac and 86-24 LEE4::lac strains. Cell-free extracts of the parent strain 86-24 and its 'clpXP, 'hha, and 'hha 'clpXP derivatives were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blotting using anti-GST-Hha antibodies. The blots were developed by using horseradish peroxidase conjugated antibodies. Results: 86-24 'hha 'clpXP LEE4::lac was highly repressed in the expression of LEE4::lac fusion as compared to 86-24 'hha LEE4::lac. The level of LEE4::lac expression in the double mutant strain was similar to that of 86-24 LEE4::lac strain. Western blotting showed Hha accumulation in 86-24 'clpXP, while no Hha was detected in 86-24 and its 'hha, and 'hha 'clpXP mutants . Inability to detect Hha in 86-24 could be attributed to ClpXP activity, which presumably keeps levels of Hha below the detection limits of the assay. Conclusions: Our results suggest that the reduced expression of LEE4::lac fusion in 86-24 'hha 'clpXP may be caused by accumulation of an unknown repressor of LEE4. Accumulation of Hha in strains lacking ClpXP suggests that this protease plays an important role in LEE expression by controlling levels of Hha.

Last Modified: 4/18/2015
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