The ETT2 gene cluster, encoding a second type III secretion system from Escherichia coli, is present in the majority of strains but has undergone widespread mutational attrition

Abstract

ETT2 is a second cryptic type III secretion system in Escherichia coli which was first discovered through the analysis of genome sequences of enterohemorrhagic E. coli O157:H7. Comparative analyses of Escherichia and Shigella genome sequences revealed that the ETT2 gene cluster is larger than was previously thought, encompassing homologues of genes from the Spi-1, Spi-2, and Spi-3 Salmonella pathogenicity islands. ETT2-associated genes, including regulators and chaperones, were found at the same chromosomal location in the majority of genome-sequenced strains, including the laboratory strain K-12. Using a PCR-based approach, we constructed a complete tiling path through the ETT2 gene cluster for 79 strains, including the well-characterized E. coli reference collection supplemented with additional pathotypes. The ETT2 gene cluster was found to be present in whole or in part in the majority of E. coli strains, whether pathogenic or commensal, with patterns of distribution and deletion mirroring the known phylogenetic structure of the species. In almost all strains, including enterohemorrhagic E. coli O157:H7, ETT2 has been subjected to varying degrees of mutational attrition that render it unable to encode a functioning secretion system. A second type III secretion system-associated locus that likely encodes the ETT2 translocation apparatus was found in some E. coli strains. Intact versions of both ETT2-related clusters are apparently present in enteroaggregative E. coli strain O42.

FIG. 1.

TP-PCR.

FIG. 2.

ETT2 and eip structures. (a) Structure of the ETT2 pathogenicity island in a number of E. coli and Shigella strains and comparisons with regions of Spi-1 and Spi-3 from S. enterica serovar Typhimurium. Homologous genes are vertically aligned. Insertions relative to the complete ETT2 sequence (as seen in strains Sakai and EAEC 042) are indicated with dashed lines. Dotted lines indicate deletions. (b) Structure of the eip island in EAEC 042 and comparison with the backbone sequence seen in Sakai (and other sequenced E. coli strains). Numbered brackets in both sections indicate the positions of primer pairs used for long PCR (see the text for details).

FIG. 3.

Phylogenetic trees showing relationship of ETT2 to other TTSSs based on neighbor-joining analysis of EivC and its homologues (a) and of EicA and YgeG TPR chaperones to each other and to other chaperones (b). The numbers on the branches indicate the percentages of bootstrap support based on 1,000 replicates. Numbers in parentheses are GI numbers of published sequences.

FIG. 4.

Illustrative PCR results for ETT2 gene cluster. Strains, from top to bottom: O157 Sakai strain (complete ETT2 gene set), ECOR1 (a B171-8-like strain with an 8.7-kb deletion), K-12 (14.6-kb deletion), and CFT073 (UPEC, with no ETT2). Lanes, from left to right: molecular weight markers, ∼5-kb amplicons obtained with ETT2 TP-PCR primer pairs 1 to 10 (see text for details), negative control (DNA, no primers), and molecular weight markers (HyperLadder I; Bioline).

FIG. 5.

TP-PCR results superimposed on phylogenetic structure of E. coli. The tree was obtained by neighbor-joining analysis of the ECOR MLEE data (available at http://foodsafe.msu.edu/whittam/ecor) using the program Neighbor, part of the PHYLIP package (J. Felsenstein; available from http://evolution.genetics.washington.edu/phylip.html). Branches containing one of the three most common genotypes are indicated by bold, dashed, or gray lines. Filled circles indicate strains with eip clusters.

FIG. 6.

Indel-specific short PCRs. The first two rows show the results for ECOR strains from 200-bp PCRs across the deletion seen in strain B171-8. The second two rows show the results for ECOR strains from 600-bp PCRs across the ETT2 insertion site (see Table 1 for details). Positive results are labeled with ECOR strain numbers or pathotypes.