Pathogenesis of strains in murine and Wax Moth models of infection.

<p>(A) CFUs of the Δ<i>frvA</i> and Δ<i>frvA</i> complemented strain enumerated from livers and spleens three days post infection. Error bars represent standard error of the mean and asterisks represent P<0.001 by the Student's t-test when compared to the wild-type and complement strains. (B) Pathogenesis of strains in the <i>Galleria mellonella</i> model of infection. Dotted line and cross indicates LT-50 (time in which 50% of insects had perished). (C) Pathogenesis of the Δ<i>lmo0642</i> mutant in the murine model of infection. Strains were inoculated into mice by the ip route and numbers were enumerated in the spleens at day three post-infection. Student t-test did not detect a significant difference between the wild-type and mutant strain.</p

Quantitative real-time PCR.

<p>Induction of <i>lmo0641</i> (<i>frvA</i>) transcription in Δ<i>fur</i> compared to the wild-type (black bar) and induction of gene transcriptions in Δ<i>frvA</i> compared to the wild-type (gray bars) in BHI. Up-regulated genes are represented by bars above the x-axis and the down-regulated gene (<i>fur</i>) is represented by the bar below the axis. Asterisks represent Fur-regulated genes. Error bars represent the mean ± SD of the relative change in gene expression of independent duplicate samples.</p

Bacterial growth.

<p>The rates and extent of bacterial growth (A: EGD-e; C: Δ<i>lmo0641</i>; E:Δ<i>lmo0641</i>/pPL2<i>lmo0641</i>) were determined in iron-restricted MOPS-L media supplemented with Hb (panels <b>A</b>–<b>C</b>; open, gray and black symbols represent addition of 0.0, 0.02 and 2 uM Hb, respectively) or Hn (<b>D</b>, <b>E</b>; open, gray and black symbols represent addition of 0.0, 0.2 and 2 uM Hn, respectively), and in BHI broth (<b>F</b>). The bacteria were cultured in BHI broth overnight. In <b>A–E</b> they were then subcultured in MOPS-L to stationary phase, and at t = 0 subcultured again at 1% into MOPS-L containing different concentrations of Hb or Hn. In <b>F</b>, at t = 0 they were subcultured into BHI broth. The flasks were shaken at 37°C and absorbance at 600 nm (initially close to zero for all cultures) was monitored for 12–26 h (note different scales). Because of the slow growth of <i>L. monocytogenes</i> in iron-restricted minimal media, this graphic representation focuses on the comparison of the mutant strains at later times in the growth cycle.</p

⁵⁹Fe binding and uptake assays.

<p>Uptake affinity (K_m in nM) and velocity (V_max in pMol per 10⁹ cells per minute) by which the wild-type (open circles) and Δ<i>0641</i> (Δ<i>frvA</i>) (closed circles) strains transport [⁵⁹Fe]-citrate (A) and [⁵⁹Fe]-Hn (B) were assessed. Overall K_m and V_max of [⁵⁹Fe] transport are listed in the tables on right-hand side. Data was plotted using the Enzyme Kinetics algorithm of Grafit 7 (Erithacus Ltd, West Sussex, UK) and represent the mean of independent experiments done in triplicate.</p

Identification and role in virulence of Fur-regulated gene systems.

<p>(A) The classical Fur box is represented as a 19 bp sequence. Recent studies have suggested that a more accurate representation of the Fur box is that of a 7-1-7 motif. The 19 bp sequence was used to search the <i>Listeria monocytogenes</i> EGDe genome sequence (Listilist). (B) Identified sequences were aligned and a graphical display of the results was generated using the web based programme sequence logo (17). (C) Genetic organisation of 29 putative Fur regulated genes (black/gray) at 12 chromosomal loci. All genes are drawn approximately to scale using the <i>L. monocytogenes</i> EGDe genome sequence data. Lmo numbers refer to the National Centre for Biotechnology Information annotation scheme. Fur boxes are represented by black circles. Gray genes indicate those disrupted in EGDe in the course of this study. Lollipops are used to illustrate putative stem loop terminator regions. (D) RT-PCR analysis was used to confirm Fur regulation of all identified genes and to give an indication of the increase in expression levels. Control primers were used to ensure that template cDNAs were of equal concentration. Samples were removed at various cycles of PCR (cycle number in brackets) and visualised on agarose gels. A repeat experiment demonstrated similar results. Results were also verified through real-time PCR analysis. (E) In vivo survival of disruption mutants in Fur-regulated loci in the murine infection model. Mice were injected i.p. with either the wild-type or mutants and the number of bacteria recovered from the spleen was determined three days post-inoculation. Error bars represent the standard deviations from the mean (n = 4). * indicates means are significantly different to the wild-type (P<0.05). ND, not detected.</p

Nutrition tests.

<p>Tests demonstrate the halo of growth surrounding a paper disc embedded with 10 µl aliquots of the test iron compound. Concentrations of compounds are indicated as µM. Fc (ferrichrome) and FcA (ferrichrome A), FxB (ferrioxamine B), Hb (haem/haemoglobin) and Hn (haemin) were tested on BHI agar containing 0.1 mM BP. The experiment was repeated several times with similar results. No differences were seen between mutant strains and the wild-type in these iron nutrition assays.</p