Figure 1: Hfq inhibits ompX and ail expression at the post-transcriptional level.
(a,b) Immunodetection of OmpX (a) and Ail (b) in Y. enterocolitica parental strain JB580v and hfq mutant SOR17 grown for 4 h in LB. Upper panels show immunoblots, and lower panels show part of the gels stained for total proteins. (v), vector pACYC184ts; (phfq), plasmid phfq. Percentages indicate the relative signal intensities: the signal was first normalized relative to total proteins loaded and then compared to that of the normalized signal for the parental strain. In (b) the monoclonal antibody reacts with three bands that are absent in the ail mutant (lane 1) and therefore correspond to different forms of Ail. In strains carrying plasmids (lanes 4–7), the larger Ail forms are less abundant and increase their apparent molecular weight. For our semi-quantitative analysis, we pooled the normalized signal for all bands. (c,d,f,g) Fluorescence of strain JB580v and derivatives carrying translational fusions with gfp was measured by flow cytometry upon growth in LB for 4 h (log.) and 22 h (stat.). Results are the mean fluorescence intensity (MFI) and standard deviation of at least three independent experiments, each with three independent cultures per strain. Significance was calculated with Student’s unpaired t-test (***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns, not significant P > 0.05). In these experiments, the MFI of strains carrying a promoter-less gfp gene was below 25 units. MFI of strains carrying: (c) ompX‘-’gfp fusion under control of PompX promoter, (d) ail‘-’gfp fusion under control of Pail promoter, (f) plasmids pFX-1 (gfp under control of Plac promoter) and pFX-Plac-ompX (ompX 5′UTR and ompX‘-’gfp fusion under control of Plac) and (g) pFX-2 (gfp under control of PLtetO-1 promoter) and pFX-PtetO-ail (ail 5′UTR and ail‘-’gfp fusion under control of PLtetO-1). (e) Northern blotting with DIG-labelled ompX- or ail-specific RNA probe. Bacteria were grown on four separate occasions in LB at 37 °C for 17 h (S1–S4). Upper two panels: Northern blots and bottom panel: ethidium-bromide stained total RNA. Due to an intervening sequence, the 23S rRNA is processed into two species that flank the 16S rRNA.
