Complete DNA sequence and structural analysis of the enteropathogenic Escherichia coli adherence factor plasmid

Abstract

The complete nucleotide sequence and organization of the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid of EPEC strain B171 (O111:NM) were determined. The EAF plasmid encodes two known virulence-related operons, the bfp operon, which is composed of genes necessary for biosynthesis of bundle-forming pili, and the bfpTVW (perABC) operon, composed of regulatory genes required for bfp transcription and also for transcriptional activation of the eae gene in the LEE pathogenicity island on the EPEC chromosome. The 69-kb EAF plasmid, henceforth designated pB171, contains, besides the bfp and bfpTVW (perABC) operons, potential virulence-associated genes, plasmid replication and maintenance genes, and many insertion sequence elements. Of the newly identified open reading frames (ORFs), two which comprise a single operon had the potential to encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic E. coli. Another ORF, located between the bfp and bfpTVW operons, showed high similarity with trcA, a bfpT-regulated chaperone-like protein gene of EPEC. Two sites were found to be putative replication regions: one similar to RepFIIA of p307 or F, and the other similar to RepFIB of R100 (NR1). In addition, we identified a third region that contains plasmid maintenance genes. Insertion elements were scattered throughout the plasmid, indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained.

FIG. 1

Map of the entire pB171 plasmid. The outer circle shows ORFs, with their orientations denoted by their positions: outside the ring indicates clockwise, and inside the ring indicates counterclockwise. ORFs encoding previously documented virulence proteins are indicated by red boxes; ORFs encoding newly identified, putative virulence proteins are indicated by pink boxes. IS-associated ORFs are indicated by green boxes; ORFs encoding proteins related to replication and plasmid maintenance functions are indicated by yellow boxes. The inner circle shows IS elements (green) with the scale in kilobase pairs. Nomenclature of the ORFs is given in Table 1.

FIG. 2

Alignment of TrcP (Orf21) with TrcA and related proteins. The predicted amino acid sequence encoded by ORF21 (trcP) was aligned by using CLUSTAL V (18) with the predicted amino acid sequences of TrcA (50), Orf19 in LEE (12), and IpgB of S. flexneri (1, 31, 41). Identical residues in all proteins are indicated by asterisks, and conserved residues are indicated by dots. Identical residues in TrcA, Orf19, and IpgB that correspond to residues in TrcP are indicated with shaded boxes.

FIG. 3

Schematic representation of homologous sequences in the pB171 ORF35–ORF36 region and in the pO157 toxB region. First and third lines indicate DNA segments of pB171 and pO157, respectively. The scales (positions) are indicated above the lines according to the sequence of pB171 (this study) and pO157 (accession no. AB011549). Homologous sequences are indicated by closed boxes. These regions are surrounded by IS elements (hatched boxes). Deduced proteins encoded by these regions are indicated by arrows under the coding sequence. From the sequence of pB171 DNA, two separate protein products, Orf35 and Orf36, are predicted, while only a single, large product, 3,169 aa in length, is predicted to be encoded by the toxB region of pO157. The amino acid sequences of Orf35 and Orf36 correspond to different segments in the C-terminal part of ToxB (indicated by the closed box or arrows together with their positions in the amino acid sequence).

FIG. 4

Primary structures of RepFIIA and the ccdAB regions. (A) Comparison of the RepFIIA region of pB171 and the corresponding regions of the R100 (NR1), F, and pSU233 plasmids. ORFs are indicated by arrows under the DNA of the pB171 plasmid. Nucleotide positions are indicated on the line. Homologous sequences of NR1 (accession no. X12776), F (M12987), and pSU233 (X55893) are indicated by lines under arrows, showing homologous regions in nucleotide position in each sequence with percent identity. Note that only the sequence of pB171 corresponding to the copB region is replaced by pSU233. (B) Schematic representation of the primary structure of the plasmid maintenance region. ORFs are indicated by arrows under the DNA of pB171. The nucleotide sequence of the ccdAB region showed high homology with that of the F plasmid (M12987) and the pO157 plasmid of E. coli O157:H7 (AB011549). The impB region showed high homology with the PT110 plasmid of Salmonella typhimurium (X53528) and with the SA100 plasmid of S. flexneri (AF079316).

FIG. 5

Base composition of pB171. The plots showing G+C content were derived by using the DNASIS program, which also shows selected ORFs by hatched boxes to the correct scale. The scale on each boxes indicates its position in the plasmid in kilobase pairs.