Screening of an E. coli O157:H7 Bacterial Artificial Chromosome Library by Comparative Genomic Hybridization to Identify Genomic Regions Contributing to Growth in Bovine Gastrointestinal Mucus and Epithelial Cell Colonization

Abstract

Enterohemorrhagic E. coli (EHEC) O157:H7 can cause serious gastrointestinal and systemic disease in humans following direct or indirect exposure to ruminant feces containing the bacterium. The main colonization site of EHEC O157:H7 in cattle is the terminal rectum where the bacteria intimately attach to the epithelium and multiply in the intestinal mucus. This study aimed to identify genomic regions of EHEC O157:H7 that contribute to colonization and multiplication at this site. A bacterial artificial chromosome (BAC) library was generated from a derivative of the sequenced E. coli O157:H7 Sakai strain. The library contains 1152 clones averaging 150 kbp. To verify the library, clones containing a complete locus of enterocyte effacement (LEE) were identified by DNA hybridization. In line with a previous report, these did not confer a type III secretion (T3S) capacity to the K-12 host strain. However, conjugation of one of the BAC clones into a strain containing a partial LEE deletion restored T3S. Three hundred eighty-four clones from the library were subjected to two different selective screens; one involved three rounds of adherence assays to bovine primary rectal epithelial cells while the other competed the clones over three rounds of growth in bovine rectal mucus. The input strain DNA was then compared with the selected strains using comparative genomic hybridization (CGH) on an E. coli microarray. The adherence assay enriched for pO157 DNA indicating the importance of this plasmid for colonization of rectal epithelial cells. The mucus assay enriched for multiple regions involved in carbohydrate utilization, including hexuronate uptake, indicating that these regions provide a competitive growth advantage in bovine mucus. This BAC-CGH approach provides a positive selection screen that complements negative selection transposon-based screens. As demonstrated, this may be of particular use for identifying genes with redundant functions such as adhesion and carbon metabolism.

Keywords: EHEC; bacterial artificial chromosome; comparative genomic hybridization; locus of enterocyte effacement; mucus; sugar utilization.

Figure 1

Complementation of T3S in TUV93-0 Δ148A. The LEE containing clone pBAC3/A2 was introduced into the TUV93-0 Δ148A containing a partial deletion of the LEE. A western blot of secreted proteins isolated from TUV93-0 (wt), TUV93-0 Δ148A, and the complemented strain TUV93-0 Δ148A pBAC3/A2 are shown.

Figure 2

Bacterial artificial chromosome-CGH of primary tissue culture. E. coli O157:H7 strain Sakai BAC clones with increased adherence were selected by multiple rounds of adherence to primary bovine terminal rectum cells. Total gDNA from output (adherent) and input (384 clones from the library) pools were subjected to CGH analysis. The ratio of output and input were normalized and a sliding average (window = 50 genes) applied. The black solid line indicates average ratio (output/input), the red line indicates the average p value and gray shading indicates an arbitrary two-fold cut-off for significance. Green lines and numbers above the plot indicate the location of O-islands. Solid arrowheads and designations indicate select genes implicated in adherence of E. coli.

Figure 3

Bacterial artificial chromosome-CGH of mucus-cultured clones. E. coli O157:H7 strain Sakai BAC clones were selected cultured were selected by multiple rounds of growth in bovine terminal rectal mucus. Total gDNA from output (mucus grown) and input (384 clones from the library) pools were subjected to CGH analysis. The ratio of output and input were normalized and a sliding average (window = 50 genes) applied. The black solid line indicates average average ratio (output/input), the red line indicates the average p value and gray shading indicates an arbitrary two-fold cut-off for significance. Green lines and numbers above the plot indicate the location of O-islands. Solid arrowheads and designations indicate select genes implicated in sugar utilization of E. coli. The red dashed box indicates the region selected for further confirmation (see Figure 4).

Figure 4

Growth of clone I12 in bovine terminal rectal mucus. Clone I12 encodes the positively selected region containing uxaC and agaWEFA (see Figure 3). DH10B control (pV41) and I12 were tagged with Nal^r and competed against reciprocal Nal^s strains in bovine rectal mucus. CFU/ml of total and Nal^r cells were recorded over 48 h.