Transposon insertion sites in <i>R</i>. <i>parkeri</i>.

<p>Transposon insertion sites in <i>R</i>. <i>parkeri</i>.</p

Transposon mutagenesis of <i>R</i>. <i>parkeri</i>.

<p>(A) Map of the pMW1650 plasmid used in this study for transposon mutagenesis (IR, inverted repeats). (B) Experimental scheme for transposon mutagenesis and isolation of individual mutants.</p

Mapping the transposon insertion sites.

<p>(A) Diagram showing the insertion of the transposon cassette into a chromosomal region (in grey). Primers specific to the transposon ends were paired with universal primers to amplify the chromosome- transposon junctions (red triangles), using semi-random nested PCR. Two nested PCR reactions were done to improve amplification of the chromosome-transposon junction directly from boiled bacteria. (B) <i>R</i>. <i>parkeri</i> chromosomal map showing all transposon insertion sites (see red lines) identified in this screen.</p

Expression of FLAG-RickA in <i>R. parkeri</i>.

<p>Immunoblots of <i>R. parkeri</i> strains that are not transformed (untransformed), transformed with pMW1650 to express GFP_UV (GFP-1, 2), and transformed with pMW1650-FLAG-RickA to express GFP_UV and FLAG-RickA (FLAG-RickA-1, 2, 3), probed with anti-RickA (top blot; higher molecular mass band is FLAG-RickA, lower is endogenous RickA), anti-FLAG (middle blot) or anti-GFP antibodies (bottom blot). Equal volumes were loaded into each lane, and the levels of endogenous RickA serve as a built in loading control.</p

Expression of FLAG-RickA does not affect actin-based motility.

<p><i>R. parkeri</i> strains (green) transformed with (A) pMW1650 to express GFP_UV, or (B) pMW1650-FLAG-RickA to express GFP_UV and FLAG-RickA, were imaged in live Cos7 cells expressing the actin marker mCherry-Lifeact (red). Scale bar 10 µm. (C) Scatter plots of the rates of actin-based motility for untransformed bacteria (control) or strains expressing GFP_UV or GFP_UV and FLAG-RickA.</p

Transposon insertion sites for <i>R. parkeri</i> strains.

<p>Transposon insertion sites for <i>R. parkeri</i> strains.</p

Physical maps for pMW1650 and pRIE.

<p>Both pMW1650 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037310#pone.0037310-Liu1" target="_blank">[9]</a> and pRIE contain a <i>Himar1</i> transposase gene under the control of the <i>Borrelia burgdorferi flgB</i> promoter, and a kanamycin resistance gene (KanR). Within the transposable element, bounded by inverted repeats (IR), are an <i>E. coli</i> ColE1 origin of replication, a GFP_UV gene under the control of the <i>R. rickettsii ompA</i> promoter, and a rifampicin resistance gene (RifR) under the control of the <i>rpsL</i> promoter. In addition, pRIE contains two multiple cloning sites: MCS1 for insertion of fluorescent protein genes under the control of the <i>R. rickettsii ompA</i> promoter, and MCS2 for insertion of other genes to be expressed under the control of their native promoter.</p

Expression of fluorescent proteins in <i>R. parkeri</i>.

<p><i>R. parkeri</i> strains expressing (A) GFP_UV, (B) EGFP, (C) mCherry, and (D) 3XmCherry are shown in infected Cos7 cells alone (left) and together with actin filaments (right). Colors are indicated in the image. Scale bar 10 µm.</p

Detection of FLAG-RickA in bacteria.

<p><i>R. parkeri</i> strains that were not transformed (<i>Rp</i>) or transformed with pMW1650-FLAG-RickA (<i>Rp</i> FLAG-RickA) were used to infect Vero cells and then (A) labeled by immunofluorescence with anti-RickA antibody and stained for DNA with DAPI, or (B) labeled by immunofluorescence with anti-FLAG antibody and stained for DNA with DAPI. In the merged images, RickA or FLAG are labeled in green, and DNA in red. Scale bar 10 µm. Higher magnification images of individual bacteria (highlighted in boxes in the lower magnification images) are shown on the right.</p

The GTPase-binding domain (GBD) of N-WASP is a dominant negative inhibitor of EHEC pedestal formation and binds with high efficiency to a single EspF_U repeat in yeast two-hybrid assays.

<p>(A) The modular structure of N-WASP is depicted, featuring WASP homology-1 (WH1), GTPase-binding (GBD), proline-rich (PRD), and WH2/verprolin-connector-acidic (VCA) domains. Several N-WASP-binding partners are shown above their interacting domains. (B) HeLa cells transfected with plasmids encoding Flag-N-WASP constructs were infected with EHECΔ<i>dam</i> (a mutant that binds to mammalian cells and generates pedestals with considerably higher efficiency than wild type EHEC <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000191#ppat.1000191-Campellone4" target="_blank">[44]</a>), fixed, and treated with DAPI to identify bacteria, a Flag antibody to visualize tagged N-WASP (top panels), and phalloidin to detect F-actin (bottom panels). Flag-N-WASP recruitment was only evaluated in cells expressing low levels of these tagged proteins (top panels), while effects on actin pedestal formation were only assessed in cells expressing high levels of Flag-N-WASP (bottom panels). Pedestal formation indices were determined by calculating the percentage of mock-transfected or Flag-N-WASP overexpressing cells harboring five or more actin pedestals. Data represent the mean+/−SD of three experiments. (C) HeLa cells expressing GFP alone, a GFP-tagged GBD, or a GFP-tagged GBD H208D point mutant were infected with EHECΔ<i>dam</i> or EPEC and treated with DAPI to identify bacteria and phalloidin to detect F-actin. Pedestal formation indices were determined as in (B). (D) Plasmids encoding the N-WASP GBD fused to the LexA DNA-binding domain and EspF_U fragments fused to the Gal4 transcriptional activation domain were co-transformed into a yeast two-hybrid reporter strain. Data represent the mean+/−SD of β-galactosidase activity for three co-transformants for each pairwise combination.</p