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United States Department of Agriculture

Agricultural Research Service

Research Project: Microbial Communities and Interactions and Their Impact on Food Safety

Location: Molecular Characterization of Foodborne Pathogens

Title: Phage insertion in mlrA and variations in rpoS limit curli expression and biofilm formation in Escherichia coli serotype O157:H7

Authors
item Uhlich, Gaylen
item Chen, Chin-Yi
item Cottrell, Bryan
item Hofmann, Christopher
item Dudley, Edward -
item Strobaugh, Terence
item Nguyen, Ly Huong

Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 3, 2013
Publication Date: June 6, 2013
Citation: Uhlich, G.A., Chen, C., Cottrell, B.J., Hofmann, C.S., Dudley, E., Strobaugh Jr, T.P., Nguyen, L.T. 2013. Phage insertion in mlrA and variations in rpoS limit curli expression and biofilm formation in Escherichia coli serotype O157:H7. Microbiology. 159:1586-1596.

Interpretive Summary: Under nutrient-deprived conditions, bacteria grow in a tightly packed community and encase themselves in a protective polysaccharide matrix, a process called biofilm formation. Such a state provides the bacteria with protection against physical stresses and allows slowed growth and metabolism, which extends bacterial survival. Although biofilm formation provides distinct advantages to food-borne bacteria, one such pathogen, Escherichia coli serotype O157:H7 forms little biofilm under laboratory conditions, although some formation is seen under severe stress conditions. This indicates that serotype O157:H7 uses a complex system for controlling biofilm expression. In this study, we show that biofilm formation in serotype O157:H7 is blocked by two distinct mechanisms: 1) insertion of DNA from a bacterial virus into the serotype O157:H7 biofilm-regulating gene mlrA, causing its inactivation; and 2) the high occurrence of spontaneous DNA sequence-altering mutations in a second bioflm-regulating gene, rpoS. Identifying these blocks in the common pathway for biofilm formation will allow us to find the alternative regulatory pathways being used to initiate serotype O157:H7 biofilm.

Technical Abstract: Biofilm formation in Escherichia coli is a tightly controlled process requiring the expression of adhesive curli fibers and certain polysaccharides such as cellulose. The transcriptional regulator CsgD is central to biofilm formation, controlling the expression of the curli structural and export proteins, and the diguanylate cyclase, adrA, which indirectly activates cellulose production. CsgD itself is highly regulated by two sigma factors (RpoS and RpoD), multiple DNA-binding proteins, silencing RNAs, and several GGDEF/EAL proteins acting through c-di-GMP. One such transcription factor, MlrA, binds the csgD promoter to enhance the RpoS-dependant transcription of csgD. Curli fibers and biofilm formation are poorly expressed in strains of E. coli serotype O157:H7 cultured under laboratory conditions in rich media and studies have shown that bacteriophage, often carrying the stx1 gene, utilize an insertion site in the proximal mlrA coding region. Using a bank of 55 serotype O157:H7 strains, we investigated the consequences of bacteriophage insertion and determined that mlrA trans-complementation restored curli and/or biofilm formation to nearly half of the prophage-bearing strains. Moreover, we discovered RpoS mutations in >70% of the strains that often attenuated or inactivated RpoS-dependent functions. We conclude that bacteriophage interruption of mlrA and RpoS mutations provide major obstacles limiting curli expression and biofilm formation in most serotype O157:H7 strains.

Last Modified: 10/31/2014
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