The RNA chaperone Hfq impacts growth, metabolism and production of virulence factors in Yersinia enterocolitica.

To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs) which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin

Analysis of Yop proteins secreted by <i>Y</i>. <i>enterocolitica</i>.

<p>Proteins secreted into the supernatant (SN, lanes 1-4, 9–10) and proteins from total bacterial cell extracts (Cells, lanes 5–8, 11–12) were analyzed by Coomassie blue staining (upper panel) and by immunoblotting using antibodies specific for YopB, YopD, LcrV, YopE and YopP. Loading was as follows: molecular weight markers (in kDa); 1 and 5, parental strain WA-314; 2 and 6, <i>hfq</i> mutant SOR3; 3 and 7, <i>hfq</i> mutant SOR4; 4 and 8, TTSS-defective <i>lcrD</i> mutant strain WA-314(pYV-515); 9 and 11, parental strain JB580v; 10 and 12, <i>hfq-</i>negative strain SOR17.</p

Pesticin sensitivity assay^a.

<p>^a , all strains were tested in duplicate at least twice and a representative experiment is shown; ^b, size of growth inhibition obtained with undiluted pesticin preparate; ^c, MID: minimum inhibitory dilution factor for pesticin preparate to inhibit bacterial growth.</p

Primers used in this study.

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

Influence of <i>hfq</i> on indole production and ornithine decarboxylase activity.

<p>(A and B) The concentration of indole present in culture supernatants was determined after growth in LB at 27°C. (A) Bacteria were grown for four hours at 27°C. Data represent mean and standard deviation of at least three independent experiments each performed with triplicate independent cultures. (B) Complementation analysis. Bacterial cultures were grown for 16 h at 27°C, since strains carrying plasmids were delayed in their indole production. Because of the variability of indole concentration produced by parental strains carrying plasmids (between 0.07 and 1.5 mM in four independent experiments), results were expressed relative to the indole produced by the parental strain JB580v carrying the control vector(which was set at 100%). Data represent mean and standard deviation of four independent experiments each performed with at least triplicate independent cultures. Significance was calculated with Student‘s unpaired <i>t</i>-test (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001). (C) Ornithine decarboxylase activity detected using the API-20E strip. All wells are positive (negative wells remain yellow), but wells inoculated with <i>hfq</i>-negative strains turn red, whereas those inoculated with parental strains are more orange.</p

Influence of <i>hfq</i> on bacterial survival to acidic and oxidative stress.

<p>(A) Bacterial survival to exposure to pH 4.0 for 90 min. (B) Bacterial survival to exposure to 1 mM H₂O₂ for 90 min. Results are expressed as % survival relative to bacteria incubated in PBS pH 7.5 and are the mean and standard deviation of at least three experiments performed with three separate cultures. Complementation assays correspond to two independent experiments performed with at least three separate cultures. Significance was calculated with Student‘s unpaired <i>t</i>-test (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001). Bacterial strains are WA-314 and its <i>hfq</i>-negative derivative SOR4, JB580v and its <i>hfq</i>-negative derivative SOR17.</p

2-DE analysis of total soluble (A) and total membrane (B) proteins stained with Coomassie blue.

<p>Bacteria were grown in triplicate at 37°C for 5 h. One representative gel per strain is shown. Proteins were separated in 2-DE gels (for all gels: pH range 3–10, molecular weight (MW) range 15–150 kDa). Highlighted spots were identified by mass spectrometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086113#pone-0086113-t003" target="_blank">Table 3</a>). MW marker size is indicated in kDa.</p

Role of <i>hfq</i> in production of yersiniabactin and its receptor FyuA.

<p>(A) Immunodetection of FyuA in strains grown for 24 h in LB supplemented with DIP (LBD). Loading was as follows: 1, WA fyuA; 2, WA-314; 3, SOR4; 4, JB580v; 5, SOR17; 6, WA-314(pACYC184ts); 7, WA-314(pAhfq); 8, SOR4(pACYC184ts); and 9, SOR4(pAhfq). Upper panel shows the immunoblot. The relative signal for each band compared to wild type (which was set to 100%) is indicated. Bottom panel shows part of Coomassie blue-stained gel used as loading control. (B) Reporter assay measuring yersiniabactin production. Following growth for 24 h in LBD at 37°C, bacterial culture supernatants were harvested. They were applied to a reporter strain which expresses luciferase in response to yersiniabactin. Luciferase activity was determined after incubation of the reporter strain for 24 h at 37°C. Results are the mean and standard deviation of duplicate cultures each assessed in triplicate. Significance was calculated with Student‘s unpaired <i>t</i>-test (**<i>P</i><0.01; ***<i>P</i><0.001). Similar results were obtained in three independent experiments.</p

Growth of <i>Y</i>. <i>enterocolitica</i> strains in BHI (A, B) and LB (C,D).

<p>(A and B) Bacteria were grown in BHI at 27°C (A) and 37°C (B): parental strains WA-314 (black diamonds) and JB580v (black squares), <i>hfq</i>-negative strains SOR3 (white diamonds), SOR4 (white triangles) and SOR17 (white squares). (C and D) Growth in LB of complemented strains at 27°C (C) and 37°C (D): JB580v(pACYC184) (black squares and straight line, parental strain harbouring the control plasmid), JB580v(pAhfq) (white square and dotted line, parental strain with complementing plasmid), SOR17(pACYC184) (black triangle and black line, <i>hfq</i>-negative strain with control plasmid), and SOR17(pAhfq) (white triangle and dotted line, complemented <i>hfq</i> strain). Full complementation of the growth defect of strains SOR3 and SOR4 was also observed after introduction of plasmid pAhfq (data not shown). Results are the mean and standard deviation (error bars) of two cultures and are representative of at least two independent experiments.</p

Immunodetection of the 19-kDa urease beta subunit in total protein extracts of <i>Y</i>. <i>enterocolitica</i>.

<p>The relative signal for each band compared to wild type (which was set to 100%) is indicated. Upper panel shows the immunoblot, bottom panel shows part of the Coomassie blue-stained gel used as loading control. (A) Loading was as follows: 1, WA-314; 2, SOR4; 3, SOR3; 4, urease-negative control strain 8081-U-GB; 5, JB580v, and 6, SOR17. (B) Complementation analysis. Loading was as follows: 1, SOR4(pACYC184ts); 2, SOR4(pAhfq); 3, WA-314(pAhfq); 4, WA-314(pACYC184ts); 5, WA-314; and 6, 8081-U-GB. In another experiment, we also observed restoration of the production of UreB in the <i>hfq</i>-negative strain SOR17 carrying pAhfq (data not shown).</p