PMC

Identification of ecs1581 as a positive regulator of T3S. A. SDS-PAGE gel showing complementation of T3S in an EHEC ΔOI-51 strain expressing a cloned OI-51 fragment containing genes z1835-z1843 (pZ1835-pZ1843). The empty vector alone acted as a control (pWControl). B. SDS-PAGE gel showing the T3S profiles for the parent strain TUV93-0 and ΔOI-51 mutant expressing pECs1581 or pWControl. C. SDS-PAGE gel showing the T3S profiles for parent strain TUV93-0, ΔOI-51 and a Δecs1581 mutant expressing pZ1835–Z1843, pECs1581 or pWControl. Protein samples were prepared and analysed as described in Experimental procedures.

Organization of OI-51 from E. coli O157 and nucleotide sequence homology with related regions from E. coli ED1a and E. coli CFT073. The E. coli O157 sequence used for the representation and analysis was from the Sakai strain and the region shown is between the chromosomal co-ordinates: 1594585–1610169 which lie between ycdF and phoQ of E. coli K-12 MG1655. The partially homologous region from E. coli CFT073 (chromosomal co-ordinates: 1377764–1393349) lies in the same chromosomal location. The related prophage in E. coli ED1a lies between ydgD and ydgG (chromosomal co-ordinates: 1740689–1756274). The selected regions were compared by blastn; regions and level of homology are indicated by the grey shading with genes encoding RdgR (ECs1581 in Sakai) and homologues (ECED1_1787 in ED1a and C1493 in CFT073) shown in orange. Other characterized genes are annotated as shown with ssDNA indicating a conserved gene in the three regions that is predicted to encode a single-stranded DNA binding protein.

Effects of natural and engineered variants of ECs1581 on T3S and motility. A. SDS-PAGE gel showing T3S levels in EHEC strain TUV93-0 and Δecs1581 mutant expressing wild-type and engineered variants of commensal protein ECED1_1787 from a low copy number plasmid (pECED1_1787, pECED1_1787C20R and pECED1_1787N35K). pWControl in the ecs1581 mutant strain was used as a negative control and wild-type variants pECs1581 and pC1493 were included as a reference for high T3S levels. B. Analysis of motility in strain TUV93-0 and Δecs1581 mutant expressing pECED1_1787, pECED1_1787C20R, pECED1_1787N35K or pWControl. C. SDS-PAGE gel showing T3S levels in strain TUV93-0 and Δecs1581 mutant expressing wild-type and engineered variants of ECs1581 from a low copy number plasmid (pC1493, pECs1581, pECs1581R20C, pECs1581R20C20N, pECs1581K35N, pECs1581R20C + K35N, pECs1581R20C + D10A, pECs1581R20C + H45R, pECs1581R20C + R61D, pECs1581R20C + Q27K and pWControl). D. Analysis of motility in the same strain set. Analysis of culture supernatants and motility was carried out as described in Experimental procedures. The motility figures (B and D) are composite images from photographs of motility agar plates inoculated with the strain of interest. The same images are used for the wild types and controls in B and D.

Screening of EHEC O157:H7 O-island (OI) mutants for altered levels of T3S. SDS-PAGE gels showing culture supernatant secretion profiles for EHEC strain TUV93-0 and a selection of 24 isogenic deletion strains. Parent strain TUV93-0 also acted a positive control for T3S and a T3S system mutant (ΔLEE1–3) provided a negative control for secretion. OI deletions are as defined with reference to the original designations in ). The translocon protein bands are indicated, EspB/D and EspA as well as BSA which was added as a loading control to rule out the precipitation process as a source of variation and to act as a co-precipitant. Strains were cultured in MEM-HEPES to an OD₆₀₀ of 1.0 and culture supernatants were TCA-precipitated, separated by SDS-PAGE and stained with Colloidal blue as described in Experimental procedures.

Sequence comparison of ECs1581 variants in E. coli. A. Multiple alignment of the predicted amino acid sequences of ECs1581 in different lineages of O157:H7. Residue divergence specific to lineage I EHEC O157:H7 isolates is marked by a pink shaded box. B. Multiple alignment of EHEC variant ECs1581 (EDL933/Sakai) with commensal variant ECED1_1787 (ED1a) and UPEC variant C1493 (CFT073). The RGD motif is at amino acid position 20–22. Residue divergence between the strains is indicated. C. SDS-PAGE gel showing stimulation of T3S in an EHEC Δecs1581 mutant expressing EHEC ECs1581 (pECs1581) or UPEC C1493 (pC1493) from a low copy number plasmid. pWControl alone in the ecs1581 mutant was used as a negative control strain. TCA-precipitated culture supernatants were analysed as described previously in Experimental procedures. D. Analysis of motility repression in an EHEC Δecs1581 and UPEC Δc1493 deletion background expressing pECs1581, pC1493 or pWControl. Motility was assessed as described in Experimental procedures. E. SDS-PAGE gel showing T3S profile for EHEC strain ZAP193 and isogenic ΔgrlA::Tn mutant expressing pECs1581 or pWControl. Strains were cultured in DMEM to an OD₆₀₀ of 1.0 and culture supernatant proteins were analysed exactly as described for strains grown in MEM-HEPES.

ECs1581 stimulates T3S via LEE1. A. Measurement of LEE1 promoter activity in EHEC strain TUV93-0 and isogenic Δecs1581 mutant. A 428 bp LEE1 promoter fragment (−444 to −16 upstream from the ler ATG start codon) was cloned into the promoter-less green fluorescence protein (GFP) plasmid pKC26 to create transcriptional fusion plasmid pLEE1–GFP. Strains transformed with this reporter were cultured in DMEM and the fluorescence produced by each bacterial population measured every 60 min as described in the Experimental procedures. Corresponding OD₆₀₀ measurements were taken at each time point and plotted against the mean fluorescence intensity values for each strain. The promoter-less plasmid pKC26 in each strain background acted as a control. B. Measurement of the pLEE1–GFP reporter and control plasmids in EHEC strain ZAP198 () with and without induced expression of ecs1581 (pECs1581) from low copy number plasmid pWSK29. C. Measurement of the pLEE1–GFP reporter and control plasmids in strain ZAP198Δler (ZAP1004, ) with and without induced expression of pECs1581. D. Measurement of the pLEE1–GFP reporter and control plasmids in strain ZAP198 with a constitutive LEE1 promoter (ZAP1327, ) with and without induced expression of pECs1581. E. RT-PCR measurement of ler expression levels in EHEC strains ZAP198, ZAP1004 and ZAP1327. F. Western blot of EspD secretion by EHEC Δecs1581 and the replaced LEE1 promoter mutant (ZAP1327) trans expressing Ler (pLer) or ECs1581 (pECs1581) from low copy number plasmids.

OI-51 contributes to ruminant colonization. Six animals were orally dosed with both wild-type (WT) (TUV93-0) and ΔOI-51 EHEC O157:H7 strains as described in Experimental procedures. A. Levels of both strains were determined daily from faecal samples. ‘PosE’ are samples that were positive for the strain following broth enrichment; ‘NegE’ samples were negative following broth enrichment. B. The cumulative shedding levels for each animal and strain were estimated for colonized animals from day 5 onwards to avoid inoculum effects. One animal was excluded from the analysis that was not properly colonized by either strain (< 20 cfu per gram of faeces). The different symbols represent individual animals and allow direct comparison of the two strains in each animal. The right-hand panel of the graph shows the percentage mean decrease of the mutant strain by comparison with the WT. The difference in cumulative shedding levels in the five colonized animals was assessed by a one-sample t-test compared to a null hypothesis of 0 change. The data were log10 transformed data to normalize the values, and the difference was statistically significant (P = 0.006).