Primers used in this study.

<p>Primers used in this study.</p

Detection of HemS expression level in <i>B. henselae</i> pNS2Trc and <i>B. henselae</i> pNS2Trc::hemS_AS by immunoblotting.

<p>20 µg samples of <i>B. henselae</i> pNS2Trc (1) and <i>B. henselae</i> pNS2Trc::<i>hemS</i>_AS (2), 20 ng sample of purified his-tagged HemS (3) were loaded on SDS-PAGE. After electrophoresis, one gel was stained with comassie brilliant blue R (A). Another gel was transferred to a nitrocellulose filter to do immune blotting as decribed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037630#s2" target="_blank">Materials and Methods</a>” (B). Measurement of HemS band intensity using Image J software gave the following results: <i>B. henselae</i> pNS2Trc: mean gray value: 24, integrated density: 3218; <i>B. henselae</i> pNS2Trc<i>::hemS</i> AS: mean gray value: 14, integrated density: 2075.</p

HemS knockdown decreases <i>B. henselae</i> ability to face exposure to H₂O₂.

<p><i>B. henselae</i> pNS2Trc and <i>B. henselae</i> pNS2Trc::<i>hemS</i>_AS were challenged with 10 mM H₂O₂ as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037630#s2" target="_blank">Materials and Methods</a>”. Experiments were performed in triplicate and a single representative experiment is presented.</p

Binding of heme to HemS.

<p>(A): Increasing amounts of heme (1 µM–20 µM final concentration) were added to HemS (10 µM) as decribed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037630#s2" target="_blank">Materials and Methods</a>” and the spectrum (300 nm–700 nm) was recorded after 5 min for each addition. The Soret band at 411 nm increases with each addition of heme as demonstrated by absorbance peak increases at 411 nm. (B): Absorbance at 411 nm was measured for each sample and plotted versus heme concentration. Experiments were performed in triplicate and a single representative experiment is presented.</p

HemS and heme degrading or trafficking enzymes sequence alignment.

<p>ClustalW alignment of HemS from <i>B. henselae</i>, PhuS <i>from Pseudomonas aeruginosa </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037630#pone.0037630-Kaur1" target="_blank">[52]</a>, ChuS from <i>E. coli </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037630#pone.0037630-Suits1" target="_blank">[46]</a>, ShuS from <i>Shigella dysenteriae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037630#pone.0037630-Wyckoff2" target="_blank">[43]</a>, HemS from <i>Yersinia enterocolitica </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037630#pone.0037630-Stojiljkovic1" target="_blank">[21]</a>, HmuS from <i>Yersinia pestis </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037630#pone.0037630-Thompson1" target="_blank">[47]</a>. This alignment was generated by Clustal W. Amino acids conserved in five or more polypeptides are highlighted in grey. Amino acids conserved in all protein are indicated with a star.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

HemS dependent degradation of heme.

<p>(A): 10 mM final concentration of ascorbate was added to the HemS-heme complex (10 µM). The spectral changes from 300–700 nm were recorded every 1 min. (B): Cytochrome P450 reductase was added to 10 µM HemS-heme complex with a 0.3∶1 molar ratio and heme degradation was initiated by adding NADPH 10 µM increments to a final concentration of 100 µM. The spectra were recorded from 300–700 nm after each addition. All Experiments were performed in triplicate and a single representative experiment is presented.</p

Functional complementation of the <i>E. coli</i> mutants impaired in iron release from heme.

<p><i>E. coli</i> strains FB8.27 pAM<i>::hasR</i> (pBAD24) (<b>A</b>), FB8.27 <i>efeB::Kan yfeX::Cmp</i> (pAM<i>::hasR</i>) (pBAD24) (B) and FB8.27 <i>efeB::Kan yfeX::Cmp</i> (pAM<i>::hasR</i> ), (pBAD24::<i>hemS</i>_his) (C) were tested for the use of heme as an iron source on iron depleted medium M63 (Gly 0.4%, Ara 0.2%, Dip 70 µM, Spc, Amp). Growth around the wells containing 1 µM, 5 µM, 10 µM, or 50 µM Hb were performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037630#s2" target="_blank">Materials and Methods</a>”. Growth around the wells was assessed by visible turbidity in the agar. These pictures were taken after 48 hours of growth at 37°C. Experiment was repeated three times. A representative result is presented.</p

Geographical distribution of <i>B. henselae</i> STs in different continents.

<p>The lower panel shows the ST distribution in European countries that were represented by at least 10 isolates.</p

Frequency of feline and human <i>B. henselae</i> isolates within each ST in correlation with the geographic origin of the isolates.

<p>Frequency of feline and human <i>B. henselae</i> isolates within each ST in correlation with the geographic origin of the isolates.</p