GrlA of enterohemorrhagic Escherichia coli O157:H7 activates LEE1 by binding to the promoter region.
OBJECTIVE
The locus of enterocyte effacement (LEE) of enterohemorrhagic Escherichia coli (EHEC) O157:H7 encodes virulence factors that lead cooperatively to an attaching and effacing lesion on host large intestine cells. Global regulator of LEE activator (GrlA), encoded by the open reading frame 3 in the EHEC LEE, is known to serve as a positive regulator of LEE expression. However, how it functions to orchestrate gene expression remains unclear.


METHODS
A grlA-deleted mutant strain was created, and the determinants needed for the LEE activation were addressed by complementation experiments. A DNA electrophoresis mobility-shifting assay was used to test a hypothesis that the activation occurs via a direct binding on the putative promoter region.


RESULTS
Activation of the major LEE operons could be rescued by an over-expression of LEE-encoded regulator (Ler), except for the LEE1 operon, expression of which absolutely required GrlA. Consistent with the latter observation, GrlA bound specifically to the putative LEE1 promoter region. Furthermore, determinants critical for this activity have been mapped to the N-terminal region of GrlA.


CONCLUSIONS
GrlA upregulates the expression of LEE through binding of the LEE1 promoter, which in turn increases the level of Ler allowing it to function as a downstream activator. The opposing effect of global regulator of LEE repressor (GrlR) is explainable by in vitro findings that GrlR interacts with GrlA, suppressing the specific binding of GrlA on the LEE1 promoter.
Huang
Ling-Hui
LH
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.
Syu
Wan-Jr
WJ
D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019453:Escherichia coli O157 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D009876:Operon; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C513430:GrlA protein, E coli; C457340:LEE protein, E coli; D010750:Phosphoproteins; D015534:Trans-Activators