Strains and plasmids used in this study.

<p>BCCM = Belgian Co-ordinated Collections of Micro-organisms, NCPPB = National Collection Plant Pathogenic Bacteria, SCRI = Scottish Crop Research Institute.</p

Domain structure, homology and molecular phylogeny of pectocins M1 and M2.

<p>a) Domain structure of pectocin M1 and relationship to colicin M and plant ferredoxin. b) Sequence alignment of pectocin M1, M2 and pectocin P (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033033#s3" target="_blank">discussion</a>) with [2Fe-2S] ferredoxin type proteins and colicin M. For clarity of presentation prior to alignment pectocin P was truncated to amino acids 1–101 (N-terminal domain) and colicin M was truncated to amino acids 128–271 (C-terminal domain). Genbank/PDBaccession numbers are as follows: Ferredoxin I [<i>S. oleracea</i>] 1704156A, plant-like ferredoxin [<i>Pcc</i> PC1] YP_003017870, pectocin M1 [<i>Pcc</i> PC1] YP_003017875, pectocin M2 [<i>Pcb</i> BPR1692] ZP_03825528, colicin M [<i>E. coli</i> SMS-3-5] YP_001739994, pectocin P [<i>Pcc</i> WPP14] ZP_03830397. Invariant residues are highlighted in black, residues with similar properties in gray b) Nearest neighbour joining molecular phylogenetic tree of [2Fe-2S] ferredoxins and pectocin ferredoxin domains. Bootstrap values (%) at major nodes are indicated. Species names represent independent ferredoxin proteins from listed species, typifying the class of ferredoxin. Proteins discussed in the study are named with species designation in brackets. Plant ferredoxins and adrenodoxin were aligned with signal peptides removed, pectocin sequences were trimmed to minimum region on homology with plant-like ferredoxin from <i>Pcc</i> PC1. Ellipses designate the following: blue = plant-type ferredoxins, red = ferredoxins found predominately in γ-proteobacteria, yellow = ferredoxins involved in electron transport to cytochrome P450. Scale represents substitutions per amino acid site.</p

Genomic context of the pectocin M1 gene.

<p>a) Position of genomic regions on the chromosome of <i>Pcc</i> PC1 containing the pectocin M1 gene and a related plant-like ferredoxin gene. b) Alignment of genomic regions from above, containing the pectocin M1 gene and the related plant-like ferredoxin gene showing annotated open reading frames and nucleotide homology shared between the two regions.</p

Purification and characterisation of pectocin M proteins.

<p>a) SDS PAGE of purified pectocin M2, M1 and M1 D222A b) Absorbance spectrum of pectocin M1 at a concentration 1.2 mg ml⁻¹. Maxima at 330, 423 and 466 nm, identical to those observed in plant ferredoxins, indicate the presence of a [2Fe-2S] cluster in pectocin M1. Spectra with identical absorbance peaks were obtained for the pectocin D222A mutant and pectocin M2. c) Agar overlay spot tests of a 3-fold serial dilution (68 µM-0.385 nM) of pectocin M1 spotted onto overlay of <i>P. atrosepticum</i> LMG 2386 cells grown in the presence (top) and absence (bottom) of the iron chelator 2,2′-bipryidine (200 µM). d) Liquid growth inhibition assay, test strain LMG 2386, grown in LB broth with 200 µM 2,2′-bipyridine. Purified PM1 was added when indicated.</p

Susceptibility of <i>Pectobacterium</i> strains to pectocin M1 and M2.

<p>Susceptibility of <i>Pectobacterium</i> strains to pectocin M1 and M2.</p

Genomic and phylogenomic context of the <i>yhaOMKJ</i> locus.

<p>(A) Genomic context of the D-serine tolerance locus (blue) in three distinct <i>E</i>. <i>coli</i> isolates–CFT073 (UPEC), EDL933 (EHEC) and MG1655 (K-12). The system encodes DsdC (a LysR type transcriptional regulator), DsdX (a D-serine outer membrane transporter) and DsdA (a D-serine deaminase). In EDL933 the D-serine tolerance locus is truncated and replaced with the sucrose utilization locus (<i>cscRAKB</i> highlighted in green). (B) Genomic context of the second putative D-serine sensory locus (red) in CFT073, EDL933 and MG1655. The system encodes YhaJ (a putative LysR type transcriptional regulator), YhaK (a redox-sensitive bicupin), YhaM (a putative deaminase) and YhaO (a putative inner membrane D-serine transporter). (B) The <i>yhaOMKJ</i> locus is highly conserved across the <i>E</i>. <i>coli</i> phylogeny. Circularized phylogenomic tree of 1591 <i>E</i>. <i>coli</i> and <i>Shigella</i> isolates overlaid with gene carriage for the <i>dsdCXA</i> locus and the <i>yhaOMKJ</i> locus. The <i>yhaOMKJ</i> genes are indicated by red blocks and the <i>dsdCXA</i> locus by blue blocks. Ordering of the genes is numbered and corresponds to the gene in the legend labeled *. Presence of a gene is determined by > 80% identity over > 80% of the coding sequence. Pseudogenes are indicated as yellow blocks. <i>E</i>. <i>coli</i> phylogroups are subdivided by color with the branch point labeled on the tree. Phylogroup A = Blue; Phylogroup B1 = Green; Phylogroup B2 = Red; Phylogroup C = Magenta; Phylogroup D = Purple; Phylogroup E = Cyan; Phylogroup F = Brown; <i>Shigella</i> = Gold. The position of prototypical strains is indicated on the outside of the figure.</p

YhaO and YhaJ are required for attaching and effacing lesion formation on host cells.

<p>(A) Wide-field fluorescence microscopy images of HeLa cells incubated with TUV93-0, <i>ΔyhaO</i>, <i>ΔyhaO</i> + p<i>yhaO</i>, <i>ΔyhaJ</i> and <i>ΔyhaJ</i> + p<i>yhaJ</i> in MEM-HEPES (LEE-inducing conditions). Host cells were stained with FITC-Phalloidin to fluorescently label actin green (488) and bacterial cells were either transformed with a plasmid constitutively expressing RFP (<i>ΔyhaO</i> and <i>ΔyhaJ</i>) or stained with Alexafluor 555 (p<i>yhaO</i> and p<i>yhaJ</i>) to label them red. Merged channels clearly show the areas of localized actin condensation beneath colonized bacterial cells, which corresponds to A/E lesion and pedestal formation as indicated by a white arrow. (B) Quantification of the average percentage of colonized host cells in the <i>ΔyhaO</i> and <i>ΔyhaJ</i> mutants and corresponding complementation backgrounds relative to TUV93-0. (C) Quantification of the average percentage of attached bacteria forming A/E lesions on bound host cells. Data was calculated from three biological replicates with at least twenty-five random fields of view taken per replicate. ***, ** and * denote P ≤ 0.001, P ≤ 0.01 and P ≤ 0.05 respectively.</p

Identification of YhaO and YhaJ as potential virulence determinants.

<p>(A) Screening of the <i>yhaOMKJ</i> locus for a role in virulence. SDS-PAGE profile of secreted proteins from TUV93-0, <i>yhaO</i>, <i>yhaM</i>, <i>yhaK</i> and <i>yhaJ</i> cultured in MEM-HEPES. Arrows indicate the location of the major LEE-encoded secreted effectors Tir, EspD and EspA as identified by mass-spectrometry. Samples were normalized according to cellular OD⁶⁰⁰ to normalize loading into each well. Immunoblot analysis of EspD levels from secreted (Sec) and whole cell lysate (WCL) fractions confirmed the SDS-PAGE results. Anti-GroEL was used to verify equal concentrations of WCL, which corresponded to OD⁶⁰⁰ normalized culture samples, loaded into each well (B) SDS-PAGE analysis highlighting complementation of the <i>ΔyhaO</i> and <i>ΔyhaJ</i> phenotypes by plasmids p<i>yhaO</i> and p<i>yhaJ</i>. SDS PAGE and immunoblot analysis of secreted protein profiles and EspD cytoplasmic expression confirmed the results. Protein secretion experiments were performed on multiple occasions.</p

YhaJ directly regulates the LEE in EHEC.

<p>(A) Schematic representation of the LEE pathogenicity island. The master regulator <i>ler</i> upstream regulatory region is expanded to illustrate the rationale behind the design of the nested deletion series to monitor <i>LEE1</i> promoter activity as described by Islam <i>et al</i>. Promoters P1 and P2 as well as corresponding -10 and -35 elements are indicated. (B) Monitoring the impact of YhaJ on <i>LEE1</i> expression in TUV93-0. LEE10 and LEE20 plasmids were transformed into TUV93-0 (grey) and <i>ΔyhaJ</i> (orange) and LacZ activity was measured in Miller units at an OD⁶⁰⁰ of approximately 0.7 during growth in MEM-HEPES. The presence of promoters P1 and P2 in each assay is indicated above the graph. * and NS denote P ≤ 0.05 and no significance respectively and the data was calculated from three biological replicates. (C) Purified YhaJ was tested for its ability to bind the <i>LEE1</i> P1 and P2 promoter regions by EMSA. DIG-labeled LEE1 P1 and P2 specific DNA probes were incubated with increasing concentrations of YhaJ. A shift in free-DNA that corresponds to a YhaJ-DNA complex was only observed for <i>LEE1</i> P1 and this was in agreement with the data presented in panel B. Specificity of the binding reaction was tested by the addition of a 100-fold excess (+) of unlabeled P1 or P2 probe to the binding reaction to outcompete binding of the DIG-labeled probe to YhaJ. A 100-fold excess of unlabeled <i>kan</i> probe was also used as a non-specific competitor for YhaJ binding to the P1 region (<i>LEE1</i> P1 vs <i>kan</i>) to ensure specify of the band shift pattern. EMSA experiments were performed in triplicate to confirm the results.</p

Schematic model of LEE regulation by the YhaO/YhaJ D-serine sensory system.

<p>Summary of small molecule signals that are encountered by EHEC in the intestinal (red) and extraintestinal (blue) environments [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005359#ppat.1005359.ref009" target="_blank">9</a>]. In the intestinal environment LEE expression is affected by signals such as fucose, ethanolamine and quorum sensing molecules (epinephrine, norepinephrine and AI-3). YhaJ constitutively regulates <i>yhaO</i> as well as stimulating the LEE (+) helping to promote A/E lesion formation and colonization of host tissue. In the extraintestinal environment D-serine can be encountered in high concentrations leading to repression (-) of the LEE by an unknown (?) direct mechanism [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005359#ppat.1005359.ref021" target="_blank">21</a>]. Expression of <i>yhaO</i> is also increased resulting in further uptake of D-serine and thus a greater transcriptional response to this signal (+++) promoting inhibition of colonization in unfavorable environments. The outer membrane (OM), peptidoglycan layer (PG) and inner membrane (IM) of EHEC are indicated.</p