Complete DNA sequence analysis of enterohemorrhagic Escherichia coli plasmid pO157_2 in β-glucuronidase-positive E. coli O157:H7 reveals a novel evolutionary path

Abstract

Strains of enterohemorragic Escherichia coli (EHEC) O157:H7 that are non-sorbitol fermenting (NSF) and β-glucuronidase negative (GUD(-)) carry a large virulence plasmid, pO157 (>90,000 bp), whereas closely related sorbitol-fermenting (SF) E. coli O157:H(-) strains carry plasmid pSFO157 (>120,000 bp). GUD(+) NSF O157:H7 strains are presumed to be precursors of GUD(-) NSF O157:H7 strains that also carry pO157. In this study, we report the complete sequence of a novel virulence plasmid, pO157-2 (89,762 bp), isolated from GUD(+) NSF O157:H7 strain G5101. PCR analysis confirmed the presence of pO157-2 in six other strains of GUD(+) NSF O157:H7. pO157-2 carries genes associated with virulence (e.g., hemolysin genes) and conjugation (tra and trb genes) but lacks katP and espP present in pO157. Comparative analysis of the three EHEC plasmids shows that pO157-2 is highly related to pO157 and pSFO157 but not ancestral to pO157. These results indicated that GUD(+) NSF O157:H7 strains might not be direct precursors to GUD(-) NSF O157:H7 as previously proposed but rather have evolved independently from a common ancestor.

Fig 1

EHEC plasmid pO157-2 map and comparison with two other EHEC plasmids. (A) Genetic map of the novel virulence EHEC plasmid pO157-2 generated with CGView (25). Blue block arrows in outer circle denote coding regions in plasmid indicating ORF transcription direction. G+C content is shown in the middle circle, and deviation from the average G+C content (47.71%) is shown in the innermost circle. BLAST comparisons with other two EHEC plasmids are shown in red (pO157) and green (pSFO157). (B) Comparison of whole sequences of plasmid pO157-2 and plasmids pO157 and pSFO157 with MAUVE. Each plasmid genome is laid out in a horizontal track, and homologous segments are indicated in the same color and connected across genomes. Respective scales show the sequence coordinates in base pairs. A colored similarity plot is shown for each genome, the height of which is proportional to the level of sequence identity in that region.

Fig 2

Schematic comparison of plasmids pO157-2, pO157, and pSFO157. Genes are shown in blocks; shaded blocks share less similarity with other genes. (A to E) Comparative analysis of gene contents for all three plasmids (pO157, pO157-2, and pSFO157) divided by regions according to pSFO157 (1). Block coloring indicates type of region: insertion sequences are represented by red blocks, transposons are dark orange, hly genes are dark green, etp genes are blue, tra genes are light green, and toxB is light orange. Areas not present in the plasmids are shown with dotted lines. tr, truncated; hp, genes coding for a hypothetical protein.

Fig 2

Schematic comparison of plasmids pO157-2, pO157, and pSFO157. Genes are shown in blocks; shaded blocks share less similarity with other genes. (A to E) Comparative analysis of gene contents for all three plasmids (pO157, pO157-2, and pSFO157) divided by regions according to pSFO157 (1). Block coloring indicates type of region: insertion sequences are represented by red blocks, transposons are dark orange, hly genes are dark green, etp genes are blue, tra genes are light green, and toxB is light orange. Areas not present in the plasmids are shown with dotted lines. tr, truncated; hp, genes coding for a hypothetical protein.

Fig 3

Proposed evolution of plasmid pO157-2 based on the stepwise evolution model for EHEC O157:H7. Possible acquisitions and/or losses of genes are indicated in branches. Clonal complexes (CCs) A3 to A7 are shown; changes predicted to have occurred: blue, gain of genes or regions; red, loss of genes or regions; gray, particular gene was truncated; green arrow, introduction of insertion sequence excision enhancer (IEE) in strains. Red circle, newly introduced CC A7 with plasmid pO157-2. Ancestral strain and plasmids A3 and A5 (shaded circles) have not been reported.