Genomic organization of <i>cosR</i> flanking regions of <i>Campylobacter</i> species and other bacterial species in <i>ε-</i>Proteobacteria.

<p>Genomic organization of <i>cosR</i> homolog (black arrows) flanking regions shows: (A) the absence of <i>cosS</i> in thermotolerant <i>Campylobacter</i> spp.; <i>C. jejuni</i> NCTC11168 (GenBank accession number: AL111168.1), <i>C. coli</i> RM2228 (AAFL00000000.1), <i>C. lari</i> RM2100 (NC_012039.1), (B) the presence of <i>cosS</i> (gray arrows) in non-thermotolerant <i>Campylobacter</i> spp.; <i>C. fetus</i> 82-40 (CP000487.1), <i>C. concisus</i> 13826 (CP000792.1), <i>C. curvus</i> 525.92 (CP000767.1), <i>C. hominis</i> ATCC BAA-391 (CP000776.1), (C) different prevalence in the other bacterial species of ε-Proteobacteria; <i>W. succinogenes</i> DSM174 (NC_005090.1), <i>A. butzleri</i> RM4018 (CP000361.1), <i>S. deleyianum</i> DSM6946 (CP001816.1), <i>H. pylori</i> 26695 (NC_000915.1).</p

Autophosphorylation and phosphotransfer of <i>C. fetus</i> CosS.

<p>(A) Analysis of autophosphorylation of MBP-tagged cytoplasmic domain of the sensor histidine kinase <i>C. fetus</i> CosS (MBP-trCosS). The status of the MBP-trCosS autophosphorylation was analyzed over time after incubation with [γ-³²P] ATP by SDS-gel electrophoresis and autoradiography. The <i>C. fetus</i> rCosR (rCosR_F) and <i>C. jejuni</i> rCosR (rCosR_J) proteins were incubated for 30 min with [γ-³²P] ATP. (B) Phosphorylation of rCosR_F by MBP-trCosS. Autophosphorylation of MBP-trCosS (2 µM) was accomplished by incubation of the protein with [γ-³²P] ATP for 2 min. Time course of phosphotransfer from ³²P-labeled MBP- trCosS is indicated on top. (C) Non-phosphorylation of <i>C. jejuni</i> rCosR_J and CosR_J mutant (CosRJ_N51D) by MBT-trCosS.</p

Divergent Distribution of the Sensor Kinase CosS in Non-Thermotolerant <i>Campylobacter</i> Species and Its Functional Incompatibility with the Response Regulator CosR of <i>Campylobacter jejuni</i>

<div><p>Two-component signal transduction systems are commonly composed of a sensor histidine kinase and a cognate response regulator, modulating gene expression in response to environmental changes through a phosphorylation-dependent process. CosR is an OmpR-type response regulator essential for the viability of <i>Campylobacter jejuni</i>, a major foodborne pathogenic species causing human gastroenteritis. Although CosR is a response regulator, its cognate sensor kinase has not been identified in <i>C. jejuni</i>. In this study, DNA sequence analysis of the <i>cosR</i> flanking regions revealed that a gene encoding a putative sensor kinase, which we named <i>cosS</i>, is prevalent in non-thermotolerant <i>Campylobacter</i> spp., but not in thermotolerant campylobacters. Phosphorylation assays indicated that <i>C. fetus</i> CosS rapidly autophosphorylates and then phosphorylates <i>C. fetus</i> CosR, suggesting that the CosRS system constitutes a paired two-component signal transduction system in <i>C. fetus</i>. However, <i>C. fetus</i> CosS does not phosphorylate <i>C. jejuni</i> CosR, suggesting that CosR may have different regulatory cascades between thermotolerant and non-thermotolerant <i>Campylobacter</i> species. Comparison of CosR homolog amino acid sequences showed that the conserved phosphorylation residue (D51), which is present in all non-thermotolerant <i>Campylobacter</i> spp., is absent from the CosR homologs of thermotolerant <i>Campylobacter</i> species. However, <i>C. jejuni</i> CosR was not phosphorylated by <i>C. fetus</i> CosS even after site-directed mutagenesis of N51D, implying that <i>C. jejuni</i> CosR may possibly function phosphorylation-independently. In addition, the results of <i>cosS</i> mutational analysis indicated that CosS is not associated with the temperature dependence of the <i>Campylobacter</i> spp. despite its unique divergent distribution only in non-thermotolerant campylobacters. The findings in this study strongly suggest that thermotolerant and non-thermotolerant <i>Campylobacter</i> spp. have different signal sensing mechanisms associated with the CosR regulation.</p></div

Amino acid sequence analysis of CosR homologs.

<p>(A) Multiple alignment of CosR homologs (GenBank accession number indicated in parentheses) in <i>Campylobacter spp.</i>: <i>C. jejuni</i> CosR (Cj0355c: YP_002343793.1), <i>C. coli</i> CosR (CCO0443: WP_002778246.1), <i>C. lari</i> CosR (Cla_0175: YP_002574789.1), <i>C. fetus</i> CosR (CFF8240_0242: YP_891446.1), <i>C. concisus</i> CosR (CCC13826_0980: YP_001466303.1), <i>C. curvus</i> CosR (CCV52592_1693: YP_001408852.1), <i>C. hominis</i> CosR (CHAB381_0745: YP_001406322.1), <i>Helicobacter pylori</i> HP1043 (NP_223100.1), <i>Helicobacter pullorum</i> HPMG 439 (EEQ62982.1), and <i>Helicobacter canadensis</i> HCAN 1051 (EES89763.1). The highly conserved residues and a phosphate-accepting aspartate residue in the receiver domain are indicated by an arrowhead and a star, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089774#pone.0089774-Schr1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089774#pone.0089774-Itou1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089774#pone.0089774-Volz1" target="_blank">[33]</a>. (B) Phylogenetic tree of CosR homologs in campylobacters. The tree was generated by using the MegAlign program (DNASTAR) based on the Jotun-Hein alignment of amino acid sequences of CosR homologs. Other CosR homologs in ε-Proteobacteria include <i>Arcobacter butzleri</i> Abu0375 (YP_001489319.1), <i>Sulfurospirillum deleyianum</i> Sde1_0235 (YP_003303308.1), and <i>Wolinella succinogenes</i> WS0306 (NP_906557.1).</p

Effects of <i>cosS</i> on <i>Campylobacter</i> growth and survival under oxidative stress condition.

<p>(A) Growth of a <i>cosS</i> knockout mutant of <i>C. fetus</i> and a <i>C. jejuni</i> strain harboring <i>cosS</i> at 37°C and 42°C. (B) Aerotolerance and (C) sensitivity to oxidative stress reagents of the <i>cosS</i> mutant and its complementation strain (cosS-C) of <i>C. fetus</i>. After exposure to atmospheric condition for 12 h or 20 mM of paraquat and H₂O₂ for 2 h, changes in viability were determined by dotting 10 µl of bacterial cultures on agar plates. The results are representative of three independent experiments with similar results.</p

Binding of <i>C. fetus</i> CosR (rCosR_F), <i>C. jejuni</i> CosR (rCosR_J) and its mutant (CosRJ_N51D) to the promoter regions of genes of the <i>C. jejuni</i> CosR regulon.

<p>The binding efficiency of <i>C. fetus</i> CosR is significantly lower than that of <i>C. jejuni</i> CosR. The target genes are selected based on previous reports <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089774#pone.0089774-Hwang1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089774#pone.0089774-Hwang2" target="_blank">[19]</a>.</p

The regulatory function of LsrR was restored by exogenous autoinducer-2.

<p>(A) Schematic of the genomic context of the part of <i>lsr</i> operon, and the <i>lsrR</i>, and <i>lsrK</i> loci in the wild-type (WT) and P<i>cat-lsr</i> strains. The promoters of the <i>lsr</i> operon (<i>lsrA</i>) and <i>lsrK</i> have been replaced with the constitutively expressed promoter of the chloramphenicol resistant gene (P<i>cat</i>). (B and C) The P<i>cat</i>-<i>lsr</i> strains harboring pJH1 carrying a chromosomal <i>invF-lacZ</i> (B) or <i>fliC-lacZ</i> (C) transcriptional fusion were grown in LB containing 100 µM IPTG to induce LsrR expression, with shaking. When indicated, the signal molecule, AI-2 (DPD), was added at final concentrations of 48 and 144 µM. (D) The SDS-PAGE gel pattern of secreted flagella proteins was evaluated in the absence or presence of 100 µM IPTG and/or 144 µM DPD. The secreted proteins were recovered from cell-free spent culture media by TCA precipitation. (E) Western blot analysis was conducted on cell extracts prepared from P<i>cat</i>-<i>lsr</i> strains harboring pJH1 grown in LB or LB containing 100 µM IPTG and/or 144 µM DPD, with shaking. These strains express the SopB protein tagged with a HA-epitope (SopB-HA) from the normal chromosomal location. (F) Monolayer of HEp-2 epithelial cells were infected with wild-type (WT) <i>Salmonella</i>, WT <i>Salmonella</i> harboring pJH1, and P<i>cat</i>-<i>lsr Salmonella</i> harboring pJH1 in the presence or absence of 100 µM IPTG and/or 144 µM DPD. After the gentamicin treatment, the numbers of internalized bacteria were determined by plating the bacteria on LB agar following appropriate dilutions. Values represent the relative amount of the intracellular bacteria and have been standardized to the level of internalization of WT strain, which was set at 1.00. The values are the average and standard deviation from three independent experiments, each done in triplicate.</p

LsrR negatively controls the expression of flagella.

<p>(A) Wild-type (WT) and mutant strains carrying <i>fliC-lacZ</i> fusion on chromosome were diluted in LB medium grown with shaking, and β-galactosidase activity (Miller units) was determined at 4 h. Values are the means and standard deviation of three independent experiments. (B, left) Wild-type (WT) <i>Salmonella</i> carrying a chromosomal <i>invF-lacZ</i> fusion and either the control plasmid, pUHE21-2<i>lacI^q</i> or the <i>lasR</i>⁺ plasmid pJH1 were grown in LB with shaking for 4 h. Production of LsrR was induced by addition of 100 µM IPTG. (B, right) WT strain carrying pJH1 was grown in LB or LB with 100 µM IPTG, with shaking for 4 h. The mRNA levels of flagella genes were determined by qRT-PCR. Values are means and standard deviations of three independent experiments. (C) Representative SDS-PAGE gel of secreted proteins. Overnight cultures of the WT strain harboring either pUHE21-2<i>lacI^q</i> or pJH1 were diluted (1∶100) into fresh LB broth in the presence or absence of 100 µM IPTG and grown for 4 h with (aerobic) or without (anaerobic) shaking. Secreted proteins were recovered from cell-free spent culture media by TCA precipitation. (D) Electron microscopic observation of flagella using negative stain. Samples were prepared from WT cells harboring either pUHE21-2<i>lacI^q</i> or pJH1 grown in LB with 100 µM IPTG. The scale bar indicates 0.5 µm. (E) Phenotypic assay for motility was performed to confirm the down-regulation of flagella genes in LsrR-overexpressing <i>Salmonella</i> cells. A 1 µl aliquot of washed WT cells harboring either pUHE21-2<i>lacI^q</i> or pJH1 was stab inoculated into 0.3% LB agar with or without 100 µM IPTG. The images were taken following 8 h of growth at 37°C.</p

MOESM1 of The complete genome sequence of Cronobacter sakazakii ATCC 29544T, a food-borne pathogen, isolated from a child’s throat

Additional file 1: Figure S1. The morphology of C. sakazakii ATCC 29544 imaged by energy-filtering transmission electron microscopy (EF-TEM). EF-TEM photograph was obtained by 2% uranyl acetate on copper grids and examined with ET-TEM at a voltage of 120 kV (LIBRA 120, Zeiss, Oberkochen, Germany). Figure S2: Comparative analysis of two ATCC 29544-specific gene clusters: (A) lac operon and (B) arsenic resistance. The amino acid sequence identities between associated genes are indicated as percentages

Receptor Diversity and Host Interaction of Bacteriophages Infecting <em>Salmonella enterica</em> Serovar Typhimurium

<div><h3>Background</h3><p><em>Salmonella enterica</em> subspecies <em>enterica</em> serovar Typhimurium is a Gram-negative pathogen causing salmonellosis. <em>Salmonella</em> Typhimurium-targeting bacteriophages have been proposed as an alternative biocontrol agent to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated.</p> <h3>Methodology/Principal Findings</h3><p>Twenty-five <em>Salmonella</em> phages were isolated and their receptors were identified by screening a Tn5 random mutant library of S. Typhimurium SL1344. Among them, three types of receptors were identified flagella (11 phages), vitamin B₁₂ uptake outer membrane protein, BtuB (7 phages) and lipopolysaccharide-related O-antigen (7 phages). TEM observation revealed that the phages using flagella (group F) or BtuB (group B) as a receptor belong to <em>Siphoviridae</em> family, and the phages using O-antigen of LPS as a receptor (group L) belong to <em>Podoviridae</em> family. Interestingly, while some of group F phages (F-I) target FliC host receptor, others (F-II) target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain.</p> <h3>Conclusions/Significance</h3><p>In this report, three receptor groups of 25 newly isolated <em>S.</em> Typhimurium-targeting phages were determined. Among them, two subgroups of group F phages interact with their host receptors in different manner. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for <em>Salmonella</em> phages and will inform development of optimal phage therapy for protection against <em>Salmonella</em>.</p> </div