Chromosomal instability in enterohaemorrhagic Escherichia coli O157:H7: impact on adherence, tellurite resistance and colony phenotype

Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (TelR) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five EHEC O157:H7 strains that differentiate into large (L) colonies and small (S) colonies with high and low Tel minimal inhibitory concentrations (MICs) respectively. S colonies (Tel-MICs ≤ 4 µg ml−1) sustained large internal deletions within the TelR OIs via homologous recombination between IS elements and lost ter and iha. Moreover, complete excision of the islands occurred by site-specific recombination between flanking direct repeats. Complete excision of OI 43 and OI 48 occurred in 1.81 × 10−3 and 1.97 × 10−4 cells in culture, respectively; internal deletion of OI 48 was more frequent (9.7 × 10−1 cells). Under iron limitation that promotes iha transcription, iha-negative derivatives adhered less well to human intestinal epithelial cells and grew slower than did their iha-positive counterparts. Experiments utilizing iha deletion and complementation mutants identified Iha as the major factor responsible for these phenotypic differences. Spontaneous deletions affecting TelR OIs contribute to EHEC O157 genome plasticity and might impair virulence and/or fitness.

. PCR strategy to characterize tellurite resistance (Tel R )-encoding islands and their integration sites in L and S colonies of E. coli O157:H7 strains.
A. Physical map of O island (OI) 48 and its flanks in E. coli O157:H7 strain EDL933 and positions of primers used to map Tel R -encoding islands.
Large arrows indicate ORFs of OI 48 (designations above arrows). Striped arrows denote putative transposases. Dotted arrows indicate insertion sequences (IS) and IS-associated genes. ter genes are grey. The duplicated arrows at the 5´ and 3´ends of the island indicate respective ORFs at these positions in OI 48 (Z1559 [putative P4-family integrase of OI 48] and Z1664, respectively; lower arrows) and in OI 43 (Z1120 [putative P4-family integrase of OI 43] and Z1226, respectively; upper arrows). Moreover, genes upstream of the first ORF of OI 48 (ycdU) and OI 43 (clpA) and downstream of the last ORF of OI 48 (serX, ycdW) and OI 43 (serW, infA) are indicated by double arrows. The scale (in bp) is above the graph. Small arrows indicate positions of PCR primers; the numbers below the small arrows indicate PCR numbers in Table S1, which describes PCR conditions. B. Strategy to investigate occupancy of serW and serX, the respective integration sites for OI 43 and OI 48 in EDL933.

Fig. S2.
Number of copies of Tel R -encoding island in L strains. The terC DNA was quantified using light-cycler PCR and normalized to gyrB DNA. The terC/gyrB DNA ratio for each strain represents a mean ± standard deviation from three independent experiments. Strains with two copies or one copy of terC are depicted by black or grey bars, respectively. Strains EDL933 (GenBank accession no. AE005174; 2 copies of the ter cluster), Sakai RIMD 0509952 (GenBank accession no. NC_002695; 1 copy of the ter cluster), and 154S (lacking the ter cluster) were used as controls.

Fig. S3
. PFGE of XbaI-digested genomic DNA from L and S strains. Chromosomal DNA from strains 81L, 95L, 134L, 135L, and 154 L and from three independent S colonies derived from each L strain was digested with XbaI and subjected to PFGE as described in Experimental procedures.
The cluster analysis was performed using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) in BioNumerics software 5.1. Strain numbers are shown to the right from the gel (three independent S colonies derived from each L strain are designated by strain number and the end numbers 1 to 3). Molecular weight scale (based on XbaI-digested DNA of Salmonella enterica serovar Braenderup strain H9812) is given above the gel.  L strains were cultured in DMEM (iron content < 0.05 µg ml -1 ), DMEM with 10 µM FeCl 2 (iron content 0.50 µg ml -1 ) or LB broth (iron content 0.59 µg ml -1 ) and iha transcription was determined using quantitative real-time reverse transcription PCR and normalized to gapA transcription. The ratio of iha/gapA transcription was 8.4-24.9, ≤ 1.0, and ≤ 0.04, respectively. Data are presented as means ± standard deviations from three experiments performed with three independently isolated RNA preparations. Differences between relative transcription of iha in DMEM and in each of the other two media are indicated * (p < 0.05) and ** (p < 0.001) (unpaired Student´s t test).

Fig. S6
. Transcription analysis of iha and non-iha adhesin genes (eae, lpfA1, lpfA2, ehaA) in L and S strains grown under iron-limited conditions. The strains were cultured in DMEM (iron content < 0.05 µg ml -1 ) and transcription of each gene was determined using quantitative real-time reverse transcription PCR and normalized to gapA transcription. Data are presented as means ± standard deviations from three experiments performed with three independently isolated RNA preparations.
e. Position of the primer within ycdU (Z1558), which is located directly upstream of the integrase gene of OI 48 in strain EDL933 (Fig.   S1A).
f. Last genes of OI 43 and OI 48, respectively.
g. Positions of the primer within serW and serX genes (which are 100% identical).
h. Position of the primer within infA (Z1228), which is located downstream of OI 43 (Fig. S1A).
i. Position of the primer within ycdW (Z1666) which is located downstream of OI 48 (Fig. S1A).
j. n.a., not applicable k. Position of the primer within recA(Z4002).
l. Full sequencing of the PCR product was not possible due to the presence of an additional IS629 besides Z1638/Z1339 in the PCR fragment. However, the existence of two IS elements has been verified by restriction digestion with HindIII, a single cutting enzyme in IS629, which resulted in three restriction fragments of the expected size (data not shown). According to sequencing data additional HindIII restriction sites in flanking regions were excluded. All primers were designed in this study except for LP43 and LP44 (Friedrich et al., 2002) and wrbA1 and wrbA2 (Bielaszewska et al., 2006b).