Figure 1.
HrrF is upregulated in an 86-028NPrpsLΔfur background.
Total RNA was isolated from strains 86-028NPrpsL, 86-028NPrpsLΔfur, and 86-028NPrpsLΔfur(pT-fur) grown in (DIS) to mid-logarithmic phase at 37°C with shaking at 50rpm. After rRNA removal, cDNA libraries were generated for RNA-Seq analysis on the Illumina HiSeq2000. An intergenic transcript was identified between NTHI1320 (pmbA) and NTHI1321 (hpt). A representative image using the Integrated Genomics Viewer is shown. The transcript was upregulated in the 86-028NPrpsLΔfur background compared to the parent strain. Upregulation of the intergenic transcript was reversed by complementation of the fur mutant strain. The vertical axes represent a read depth ranging from 0-1800 reads. The Fur binding site (green line), transcriptional start site (*), and predicted Rho-independent terminators (red lines) are indicated.
Figure 2.
HrrF is conserved among the Pasteurellaceae.
hrrF sequences from 12 Pasteurellaceae species were aligned using CLUSTALW. The purple bar indicates the coding sequence for pmbA. The red bar indicates the sequence for hrrF. The asterisk indicates the TSS. The black dotted line indicates the location of the Rho-independent terminator. Nucleotides that match 86-028NP sequence are shaded black. NTHi: nontypeable Haemophilus influenzae, HAE: H. influenzae biogroup aegyptius, H.i.: Haemophilus influenzae, H.p.: Haemophilus parainfluenzae, A.a. HK1651: Aggregatibacter actinomycetemcomitans, A.a. NJ8700: Aggregatibacter aphrophilus, A.s.: Actinobacillus succinogenes, M.s.: Mannheimia succiniciproducens
Figure 3.
HrrF expression is iron-responsive and Fur-dependent.
A. Total RNA was purified from strains 86-028NPrpsL, 86-028NPrpsLΔfur, 86-028NPrpsLΔfurΔhrrFL, and 86-028NPrpsLΔfurΔhfq grown in DIS to mid-logarithmic phase at 37°C with shaking at 50rpm. Northern blots were performed using an RNA probe specific for HrrF. The parent strain produced low levels of the ∼97nt HrrF transcript. Strain 86-028NPrpsLΔfur produced HrrF transcript at a greater level than the parent strain. The ∼260nt HrrFL transcript was only produced at detectable levels in the 86-028NPrpsLΔfur strain. Transcript levels of HrrF produced in the 86-028NPrpsLΔfurΔhfq strain were similar to that produced in the 86-028NPrpsLΔfur strain indicating that Hfq was not required for HrrF stability. B. Total RNA was collected from strains 86-028NPrpsL, 86-028NPrpsLΔhrrFL, and 86-028NPrpsLΔhfq grown as in (A). At mid-logarithmic phase cultures were split in two and 2,2′-bipyridine was added to one aliquot at a final concentration of 500 µM for 15min. Total RNA was then collected and used for northern blot analysis. HrrF transcript levels increased upon iron chelation. The ∼260nt HrrFL transcript was only detectable upon addition of 2,2′-bipyridine. HrrF transcript levels produced in the 86-028NPrpsLΔhfq strain were similar to parental HrrF levels after iron chelation indicating that Hfq was not required for HrrF stability. HrrF transcript was not detectable in RNA from the hrrFL mutant strain demonstrating the RNA probe's specificity. C. NTHi 86-028NPrpsL was chelated with 2,2′-bipyridine as in (B). After 15 minutes of chelation FeSO4 was added to the chelated culture for 20 minutes. Culture aliquots were collected for total RNA extraction before chelation, after 15 minutes of chelation, and 5, 10, and 20 minutes after the addition of FeSO4. HrrF transcript levels decreased over time in response to iron addition. All blots were stripped and probed again with an RNA probe specific for the 5S rRNA to serve as a loading control.
Figure 4.
Fur binds upstream of the hrrF promoter.
A. Location of the three predicted Fur boxes in the hrrF promoter region. Fur boxes 1, 2, 3, and 1-3 are indicated by the green, orange, purple, and black lines respectively. B. Soluble protein fractions from 86-028NPrpsLΔfur(pT-fur) (+) or 86-028NPrpsLΔfur (-) were prepared. Biotinylated oligonucleotides containing the promoter regions of sxy, tbp2, and hrrF were incubated for 30 minutes with the protein fractions followed by pull-down with streptavidin-agarose beads. Beads were washed, and the bound proteins resolved on a 4.0 to 20.0% SDS-polyacrylamide gradient gel, transferred to nitrocellulose, and probed with anti-Pseudomonas Fur antibody. Fur was enriched in the pulldown with the hrrF promoter oligo compared to negative controls which contained no oligo, the sxy promoter, or the scrambled Fur boxes 1–3 (FB 1–3). Fur binding to the hrrF promoter oligo was similar to Fur binding to the promoter of the Fur-regulated gene tbp2. The absence of Fur detection with oligonucleotides incubated with 86-028NPrpsLΔfur extracts demonstrated the specificity of the anti-Fur antibody. C. Strains were constructed in which the predicted hrrF Fur box sequences, shown in (A), were scrambled in the NTHi 86-028NPrpsL genome. Strains were grown in DIS medium to mid-logarithmic phase and split in two. 2,2′-bipyridine was added to one aliquot to a final concentration of 500 µM for 15 minutes. Total RNA was collected from both cultures. Northern blots were performed using an RNA probe specific to HrrF. Blots were stripped and probed with an RNA probe specific to the 5S rRNA as a loading control. Scrambling the Fur box 1 sequence disrupted Fur regulation, but transcription was maintained at parental levels. Scrambling the Fur box 2 or 3 sequence, nearly abolished hrrF transcription, suggesting that these putative Fur boxes overlap the hrrF promoter. Scrambling Fur boxes 1–3 simultaneously completely abolished hrrF transcription. FB = Fur box.
Figure 5.
The half-life of HrrF is not dependent on Hfq.
The 86-028NPrpsLΔfur(pT), 86-028NPrpsLΔfurΔhfq(pT), and 86-028NPrpsLΔfurΔhfq(pT-hfq) strains were grown to mid-logarithmic phase in DIS at 37°C with shaking at 50rpm. Rifampicin was then added to a final concentration of 250 µM to inhibit transcription. At 0, 2, 4, 6, 10, 20, and 30 minutes after rifampicin addition total RNA was collected. RNA was probed via northern blot with an RNA probe specific for the 5′ end of HrrF. Blots were stripped and probed again with an RNA probe specific for 5S rRNA to serve as a loading control (data not shown). The calculated half-life of HrrF was approximately seven minutes in the 86-028NPrpsLΔfur strain. There was no significant difference in the calculated HrrF half-life in any of the strains tested. This indicated that the stability of hrrF was not dependent on the RNA chaperone Hfq.
Table 1.
HrrF targets identified via RNA-Seq.