Characterization of a small PlcR-regulated gene co-expressed with cereolysin O-4

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a small PlcR-regulated gene co-expressed with cereolysin O"</p><p>http://www.biomedcentral.com/1471-2180/7/52</p><p>BMC Microbiology 2007;7():52-52.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1913518.</p><p></p>spreading on LB agar. We spotted 15 μl of Pep13, at dilutions of 7.26 mM to 0.93 mM, on indicator strains. Antibacterial activity was assessed after overnight incubation at 37°C

Characterization of a small PlcR-regulated gene co-expressed with cereolysin O-0

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a small PlcR-regulated gene co-expressed with cereolysin O"</p><p>http://www.biomedcentral.com/1471-2180/7/52</p><p>BMC Microbiology 2007;7():52-52.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1913518.</p><p></p> and the putative RBS binding site (bold italic) are indicated. The transcription initiation site (+1) is indicated by the arrow. (B) Total RNA (20 μg) extracted from at the onset of stationary phase (T0), two hours (T2) and four hours (T4) after T0 was subjected to primer extension analysis, using an oligonucleotide binding 65 nucleotides downstream from the Clo start codon. The same oligonucleotide was used to prime dideoxy sequencing reactions from the corresponding region obtained by PCR amplification (lanes C, T, A, G)

Characterization of a small PlcR-regulated gene co-expressed with cereolysin O-2

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a small PlcR-regulated gene co-expressed with cereolysin O"</p><p>http://www.biomedcentral.com/1471-2180/7/52</p><p>BMC Microbiology 2007;7():52-52.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1913518.</p><p></p>underlined), -10 and -35 boxes (underlined) and the putative RBS-binding site (bold italic) are indicated. The transcription initiation site of is shown in bold

Characterization of a small PlcR-regulated gene co-expressed with cereolysin O-3

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a small PlcR-regulated gene co-expressed with cereolysin O"</p><p>http://www.biomedcentral.com/1471-2180/7/52</p><p>BMC Microbiology 2007;7():52-52.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1913518.</p><p></p>wild-type (closed symbols) and Δ (open symbols) strains. Grey symbols, ODof cultures of bacteria. Cells were grown at 37°C in LB medium. Time zero indicates entry into stationary phase. Standard deviations of triplicate measurements are shown for β-galactosidase activity

Characterization of a small PlcR-regulated gene co-expressed with cereolysin O-1

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a small PlcR-regulated gene co-expressed with cereolysin O"</p><p>http://www.biomedcentral.com/1471-2180/7/52</p><p>BMC Microbiology 2007;7():52-52.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1913518.</p><p></p>he Multalin version 5.4.1 program [40]. The numbers indicate copy number (1, 2 or 3) in the available genome sequences from the group. Each number 1 corresponds to a Pep orthologue encoded by an ORF positioned upstream from a cholesterol-dependent cytolysin-encoding gene. Bc14579: ATCC14579; Bc10987: ATCC10987; BcZK: EL33; BcG9241: G9241; Btk: serovar strain 97-27; Bt407: strain 407Cry-; Bti: serovar ATCC 35646. For , the finished and unfinished genome sequences of the various strains gave the same Pep sequences, which are indicated only once. The strains tested were: strain Ames Ancestor, strain Ames, strain Sterne, strain Kruger B, strain A1055, strain CNEVA-9066, strain Western North America USA6153, strain Vollum, and strain Australia 94. In the strain A2012 unfinished genome sequence, only orthologue number 1 was identified. *: Pep orthologue identified in an unfinished genome sequence

Characterization of a small PlcR-regulated gene co-expressed with cereolysin O-5

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a small PlcR-regulated gene co-expressed with cereolysin O"</p><p>http://www.biomedcentral.com/1471-2180/7/52</p><p>BMC Microbiology 2007;7():52-52.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1913518.</p><p></p> and the putative RBS binding site (bold italic) are indicated. The transcription initiation site (+1) is indicated by the arrow. (B) Total RNA (20 μg) extracted from at the onset of stationary phase (T0), two hours (T2) and four hours (T4) after T0 was subjected to primer extension analysis, using an oligonucleotide binding 65 nucleotides downstream from the Clo start codon. The same oligonucleotide was used to prime dideoxy sequencing reactions from the corresponding region obtained by PCR amplification (lanes C, T, A, G)

Predicted binding mode common to PLANTS and GOLD docking of glucose-6P (cyan sticks) to the ligand-binding cavity of HlyIIR (yellow ribbons, yellow sticks).

<p>Main receptor-interacting residues are labelled at their C-αatoms. Intermolecular hydrogen bonds are displayed as green broken lines. Nitrogen and oxygen atoms are coloured in blue and red, respectively.</p

HlyIIR negatively regulates <i>hlyII</i> expression in <i>B. cereus</i>.

<p>(A) Expression levels of the target <i>hlyII</i> gene relative to the endogenous standard 16S RNA were measured by RT-qPCR throughout bacterial growth in the wild type Bt407 (circle) and the Bt407Δ<i>hlyIIR</i> (square) strains. Data are expressed as the ratio of <i>hlyII</i> mRNA normalized to 16S RNA. Values are means of two independent experiments. (B) The specific β-galactosidase activity (Miller unit) of strains Bt407 and Bt407 Δ<i>hlyIIR</i> harboring the transcriptional pHT-P<i>hlyII</i>’<i>Z</i> fusion were measured from bacteria grown in LB medium at 37°C from 2 h before the culture entry into stationary phase (t−2) to 4 h after (t4). Results represent mean values of at least three independent experiments. (C) The specific β-galactosidase activity (Miller unit) of strain Bt407 harboring the pHT-P<i>hlyIIR</i>’<i>Z</i> fusion was measured from bacteria grown in LB medium at 37°C from 2 h before the culture entered into stationary phase (t−2) to 4 h after (t4). Results represent mean values of at least three independent experiments.</p

Model of the role and expression of <i>hlyII</i> during infection.

<p>A) As long as iron and glucose are abundant in the bacterial environment, the bacteria will be able to use these resources for growth. Glucose will enter the bacteria as glucose 6P (blue rectangles) and will bind HlyIIR (orange plain cross). Iron (purple circles) will bind Fur (red ovals). These bindings will promote HlyIIR and Fur repressor activities, leading to HlyIIR- and Fur-based transcriptional repression on <i>hlyII</i> gene expression. B) During host infection, bacteria find themselves in an environment, which is low in glucose and iron. Levels of these nutrients are further lowered during bacterial proliferation. The decrease in the concentration of glucose during bacterial proliferation will lead to an inhibition of HlyIIR activity, thus allowing <i>hlyII</i> expression. The decrease in the concentration of iron, partially due to its sequestration by immune cells, will lead to an inhibition of Fur activity, thus allowing <i>hlyII</i> expression. Thus, when glucose and iron are getting scarce, <i>hlyII</i> expression is activated. HlyII will then be released in the environment and induce macrophage and erythrocyte lysis. The dead cells will release their intracellular content, allowing access to metabolites that are essential for bacterial growth.</p

Glucose 6P binds to HlyIIR to enhance its DNA binding capacity.

<p>(A) Isothermal titration calorimetry (ITC) assays were performed to measure heat variations generated upon binding between glucose 6P (G6P) and HlyIIR (red line) or arabinose and HlyIIR (blue squares). (B) ITC assays were performed to measure binding between a DNA fragment containing the HlyIIR binding site and HlyIIR supplemented either with glucose 6P (red line) or with arabinose (blue line).</p