Table 1.
Bacteria and plasmids used in this study.
Table 2.
Primers for fis and vapBC-1 cloning.a
Table 3.
Gel shift substrates.a
Figure 1.
Expression of NTHi fis following nutrient upshift.
Quantitative real-time PCR was performed for fis mRNA following nutrient upshift of NTHi wild type strain R2866. The zero time point is fis expression in the stationary phase culture prior to dilution into fresh media, while samples at 10, 30, 60, and 120 minutes indicate fis mRNA levels after nutrient upshift. Each time point included three biological replicates and three technical replicates. Error bars are standard deviations.
Figure 2.
Expression from the NTHi vapBC-1 promoter.
β-galactosidase activity indicating lacZ expression under vapBC-1 promoter control in wild type R2866 (diamonds), and R2866 mutants, Δfis (squares), ΔvapBC-1 (crosses), and Δfis ΔvapBC-1 (triangles), was measured every 30 minutes for 2 hours after nutrient upshift. The zero time point indicates expression in the stationary phase culture prior to dilution into fresh media. Each data point is the average of three independent assays performed in triplicate. Error bars are standard deviations.
Figure 3.
DNase I protection of the vapBC-1 locus control region by Fis and Vap proteins.
(A) The 32P-labeled sense strand of 153 bp DNA substrate containing vapB-1 TIR and upstream sequence in the vapBC-1 locus control region, is shown with numbers indicating the distance from the 5′-labeled end. The putative Fis site (underline), inverted repeat regions (arrows), vapB-1 translation start ATG (italics), and G cleavage products (*) seen in (D, lane G) are noted. On each gel shown, a 10 bp DNA ladder (lane M), 153 bp substrate without protein (lane 1), and DNase I digest of the substrate (lane 2) are indicated. Gels show DNase I cleavage products from samples containing: (B) a Fis∶DNA molar ratio of 2∶1, 7.5∶1, 15∶1, or 30∶1 (lanes 3–6), (C) a VapBC-1∶DNA molar ratio of 2∶1, 7.5∶1, 15∶1, or 30∶1 (lanes 3–6) or 40∶1 VapB-1∶DNA (lane 7), and (D) VapC-1∶DNA molar ratio of 40∶1 or 80∶1 (lanes 3 and 4). Vertical bars indicate the DNase I footprint from protein binding. Arrows (<) in panel D indicate DNase I hypersensitive sites. The gels each represent one of two independent experiments.
Figure 4.
Fis, VapC-1 and VapBC-1 bind the vapB-1 TIR.
(A) The sequence of the 50TIR indicating the putative Fis binding site (underline) and the inverted repeat regions (arrows). (B) Gel shift products from samples containing 50TIR alone (lane 1) and 10, 30, 150 or 600 molar ratios of Fis to DNA. (C) Products from samples containing 50TIR alone (lane 1), VapC-1 (lane 2), VapC-1 followed by VapB-1 (lane 3), VapB-1 (lane 4), or the reconstituted VapBC-1 complex (lane 5). VapB-1 and VapC-1 are present in a 150∶1 molar ratio to 50TIR. In VapBC-1 samples, VapB-1 and VapC-1 are at a 3∶1 molar ratio, with a VapC-1∶DNA molar ratio of 150∶1. This ratio is reported since VapC-1 is the DNA binding protein and the actual amount of VapBC-1 complexes cannot be determined. The identity of each band is noted at the right of each gel. Each gel represents one of two independent experiments.
Figure 5.
Fis interacts non-specifically with the vapB-1 TIR.
Gel shift products from a titration of Fis, lacking a polyhistidine tag, with (A) 50TIR, (B) GC50TIR or (C) 50US are shown. The gels show the products of the following samples: DNA only (lane 1) and the addition of Fis at 10, 30, 90 or 300 molar ratios with the DNA substrate (lanes 2–5). The identity of each band is noted at the right of each gel. Each gel represents one of two independent experiments.
Figure 6.
VapBC-1 specifically interacts with the vapB-1 TIR.
(A) The sequence of the 50TIR is shown with arrows indicating the inverted repeat regions and bases above and below the sequence indicating the position of the G to C and T to G substitutions in the TG50TIR and 2M50TIR substrates (see Table 3). Gel shift products from a titration of VapBC-1 with (B) 50TIR or (C) TG50TIR are shown. The lanes in panels B and C contain the following samples: DNA only (lane 1), the addition of VapBC-1 at a VapC-1∶DNA ratio of 10, 25, 50, 100, 200, 400 or 800 molar ratio with the DNA substrate (lanes 2–8), and VapC-1 only at an 800∶1 protein to DNA ratio (lane 9). (D) A comparison of VapBC-1 binding at a 400∶1 molar ratio with each DNA substrate (lanes 2, 4, and 6). Lanes 1, 3, and 5 contain only DNA. The identity of each band is noted at the right of each gel. Each gel represents one of three independent experiments.
Figure 7.
Competition binding of 50TIR and 50US by Vap proteins.
Gel shift products from addition of 50US substrate into samples containing 50TIR substrate and either (A) VapBC-1 or (B) VapC-1 at a 150∶1 molar ratio of VapC-1 protein to DNA. VapB-1 and VapC-1 are at a 3∶1 molar ratio in the VapBC-1 samples, but VapC-1∶DNA molar ratios are reported since VapC-1 is the DNA binding protein and the actual amount of VapBC-1 complexes cannot be determined. The gels show the products of the following samples: 50TIR without protein (lane 1), protein with only 50TIR (lane 2), the addition of a 1∶1, 5∶1, 10∶1 or 50∶1 molar ratio of cold 50US:50TIR (lanes 3–6), and DNA only at 50∶1 molar ratio of 50US:50TIR (lane 6). The identity of each band is noted at the right of each gel. Each gel represents one of two independent experiments.
Figure 8.
VapB-1 targets VapC-1 to the vapB-1 translation initiation region.
Gel shift products from a titration of VapB-1 into samples following VapC-1 binding to (A) 50TIR or (B) 50US at a 100∶1 molar ratio of protein to DNA. The gels show the products of the following samples: VapC-1 and DNA without VapB-1 (lane 1), the addition of Vap B-1 at a 50, 100, 200 or 300 molar ratio with the DNA substrate (lanes 2–5), and VapB-1 only at a 300∶1 protein to DNA ratio (lane 6). The identity of each band is noted at the right of each gel. Each gel represents one of two independent experiments.
Figure 9.
Model for the regulation of the vapBC-1 locus.
(A) During colonization under favourable conditions, the VapBC-1 complex binds to and autorepresses TA operon transcription. (B) Stress induces Lon and Clp proteases that degrade VapB-1, releasing active VapC-1 toxin. (C) The ribonuclease activity of VapC-1 facilitates a state of bacteriostasis, resulting in nonspecific antibiotic tolerance. (D) Upon improved conditions, Fis activates vapBC-1 operon transcription, displacing any bound VapC-1. Fis levels decrease in early exponential growth, allowing the VapBC-1 complex to bind and restore transcriptional equilibrium.