Comparison between GI-T4SS clusters in <i>Legionella</i> and SPI-7 family ICEs.

<p>LpcGI-2 from <i>L. pneumophila</i> strain Corby is representative of the conserved LGI cluster in <i>Legionella</i> spp. and compared to the well-characterised SPI-7 family ICEs from <i>Haemophilus influenzae</i> (ICE<i>Hin</i>1056), <i>Pseudomonas aeruginosa</i> (PAPI-1) and <i>Salmonella bongori</i> (ICE<i>Sb</i>1). Similarity between the different GI-T4SS clusters is represented by grey bars, shaded according to the percentage of amino acid identity as shown in the key. Genes are coloured according to category as described in the key.</p

Phylogeny of GI-T4SS across representative Proteobacterial genomes.

<p>Maximum likelihood tree of LgiN/VirB4/TraC homologues showing the divergent <i>Legionella</i> GI-T4SS clade (red). Representatives from the SPI-7 family of ICE are highlighted in blue, with representatives of the T4ASS (<i>A. tumefaciens</i> plasmid Ti, <i>E. coli</i> F plasmid and IncP-alpha plasmid) and T4BSS (Plasmid R64) in black. Asterisks indicate branches with percentage support from 1000 bootstrap replicates above 80% (** &gt;90%, * &gt;80%).</p

Phylogenetic relationship between LGI-T4SS clusters.

<p>A consensus network tree showing the relationship between the 14 <i>Legionella</i> GI-T4SS clusters, constructed from the individual maximum likelihood trees of 15 conserved <i>lgi</i> genes. Branch labels indicate the number of trees that support each branch. Branch lengths show the number of substitutions per site, as indicated by the scale bar, and are averaged across the trees that contain that branch.</p

Genetic organisation of the 14 LGI-T4SS clusters in <i>Legionella</i>.

<p>Each of the 14 <i>Legionella</i> GI-T4SS (LGI-T4SS) clusters found in the genomes of sequenced <i>L. pneumophila</i>, <i>L. drancourtii</i> and <i>L. longbeachae</i> strains are presented, and labeled according to the LGI on which they are located. Common to all LGI-T4SSs are a highly conserved cluster of 20 <i>lgi</i> genes (<i>lgiA-T</i>), and regulatory <i>lvr</i> genes. CDSs are represented as arrows, coloured according to their function as described in the key. The translated nucleotide sequence identity (tBLASTx) between each cluster is represented by grey bars, shaded according to the percentage of amino acid identity as shown in the key.</p

Distribution of GI-T4SSs in <i>Legionella</i>.

<p><i>attL</i> to last <i>attR.</i> Size from </p><p><i>attL</i>: <i>attR1</i> (no. mismatches), <i>attR2</i> (no. mismatches). Number of base pairs in direct repeat of </p

ICE core genes in ICE<i>Hin</i>1056, ICE<i>Sb</i>1 and PAPI-1 and corresponding LGI homologues.

<p><b>α</b><i>ssb</i> is only present near LGI-1 clusters in <i>L. pneumophila</i> strains and LdrGI-c.</p><p><b>β</b><i>topB</i> is only present near LdrGI-c and LdrGI-d.</p><p><b>χ</b> all LGIs have integrases unrelated to those in ICE<i>Hin</i>1056, ICE<i>Sb</i>1 and PAPI-1.</p>**<p>Essential for conjugation.</p>*<p>Non-essential but deficiency significantly reduces conjugation.</p>∧<p>Not required for conjugation.</p

Effect of <i>fis</i> deletion on <i>sslE</i> expression in strains IHE3034, 536 and EC958 determined via western blot and qRT-PCR analysis.

<p>Western blot analysis of SslE using preparations from (A) IHE3034, (B) 536 and (C) EC958; each with their respective <i>fis</i> mutant and complemented strains. In each analysis, (i) whole-cell lysates and (iii) supernatant fractions were examined. In addition, (ii) a control for whole cell lysate samples was performed using an OmpA antibody. (D) Relative fold-difference of <i>sslE</i> transcript levels of IHE3034, 536 and EC958, and their respective <i>fis</i> mutant and complemented strains. All mRNA levels were calculated relative to the level of IHE3034 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation. Statistical analysis was performed using an unpaired, two-tailed t-test.</p

Effect of <i>fis</i> and <i>hns</i> double deletion on <i>sslE</i> expression in UTI89 determined via western blot and qRT-PCR analysis.

<p>(A) Western blot analysis of SslE using (i) whole-cell lysates and (iii) supernatant fractions prepared from UTI89, UTI89<i>hns</i>, UTI89<i>fis</i> and UTI89<i>fis hns</i>. (ii) Western blot loading control for whole cell lysate samples using an OmpA antibody. The overall level of SslE was reduced in UTI89<i>fis hns</i> compared to wild-type UTI89. (B) Relative fold-difference of <i>sslE</i> transcript levels of UTI89, UTI89<i>hns</i>, UTI89<i>fis</i> and UTI89<i>fis hns</i>. All mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation.</p

Effect of <i>fis</i> and <i>typA</i> deletion in UTI89 on <i>sslE</i> expression determined via western blot and qRT-PCR analysis

<p>(A) Western blot analysis of SslE using (i) whole-cell lysates and (iii) supernatant fractions prepared from UTI89, UTI89<i>fis</i> and UTI89<i>fis</i>(pFis). (ii) Western blot loading control for whole cell lysate samples using an OmpA antibody. (B) Western blot analysis of SslE using whole-cell lysates and supernatant fractions prepared from UTI89, UTI89<i>typA</i> and UTI89<i>typA</i>(pTypA). (ii) Western blot loading control for whole cell lysate samples using an OmpA antibody. (C) Relative fold-difference of <i>sslE</i> transcript levels of UTI89, UTI89<i>fis</i> and UTI89<i>fis</i>(pFis); mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. (D) Relative fold-difference of <i>sslE</i> transcript levels of UTI89, UTI89<i>typA</i> and UTI89<i>typA</i>(pTypA); mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation. Statistical analysis was performed using an unpaired, two-tailed t-test.</p