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Title: MUTATIONS IN THE CSGD PROMOTER ASSOCIATED WITH VARIATIONS IN CURLI EXPRESSION IN CERTAIN STRAINS OF ESCHERICHIA COLI O157:H7

Author
item Uhlich, Gaylen
item Keen, James
item ELDER, ROBERT - FORMER ARS EMPLOYEE

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/16/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: Cattle- and human-origin strains of the food-borne pathogen, Escherichia coli serotype O157:H7, were tested for the presence of curli fibers; hair- like surface structures that may enhance bacterial survival in the environment. Curli-expressing bacteria bind Congo red dye and form red colonies on solid culture media containing Congo red. Two human-origin strains, ATCC 43895 and ATCC 43894, produced a mixture of both curli- producing (red) and curli-deficient (white) cells. All other strains produced white colonies only. Daily passage of the red strains in liquid media resulted in a mixture of both red and white colonies by the third day. The white strains remained white under all growth conditions tested. Amplification and size-comparison of the genes involved in curli fiber production demonstrated that both red and white strains contained a complete set of these genes. The product of one of the genes, named csgD, regulates the expression of the other genes required for curli production. Nucleotide sequence comparison of the regulatory regions of the csgD genes revealed that a single nucleotide variation existed between the red and white strains. When a copy of the csgD gene from a red strain, including its single nucleotide variation, was introduced into the white strains, the white strains formed red colonies. This study suggests that certain strains of E. coli O157:H7 produce curli fibers and that their expression is regulated by a single nucleotide change in a regulatory gene. Comparing the red and white strains for their ability to metabolize biochemical substrates showed that the red strains could metabolize 1 or 2 additional substrates, suggesting that the csgD gene controls other functions besides curli formation.

Technical Abstract: Bovine and human isolates of Escherichia coli serotype O157:H7 were screened for the expression of curli fibers using their ability to bind Congo red dye. Human pathogenic strains ATCC (American Type Culture Collection) 43895 and ATCC 43894 produced two distinct colony morphologies on media containing Congo red dye: rough, dry, red colonies and smooth, moist, white colonies. Red strains reverted to a mixed population following serial passage at 37 C, while white strains remained stable under all growth conditions tested. Comparison of the csgDEFG and csgBA operons of white and red variants failed to detect operon insertions or deletions. The csgDEFG promoter sequence of red strains contained a single base-pair difference at one of two locations when compared to white variants. Assays of csgD::lacZ promoter fusions demonstrated significantly greater expression from the promoters of red variants compared to white variants. Polymerase chain reaction- (PCR-) amplified and plasmid-cloned product fro the csgDEFG operon of a strain 43894 red variant transformed strains 43895 and 43894 white variants, curli-deficient bovine, and curli-deficient human origin strains to a red phenotype. Red variants of 43895 and 43894 tested for substrate utilization showed an increased capacity to utilize either arginine or pyruvate, or both substrates when compared to the white variants, indicating functional differences other than curli expression. These results suggest that certain strains of E. coli O157:H7 may express curli fibers in a phase variant manner.