# Role of Hibernation Promoting Factor
## 4 Materials and Methods
### Strains
Aliquots of cells containing approximately \(10^{10}\) CFU were removed, washed twice with PBS pH 7.2, resuspended in PBS, and added to 60 mL of PBS to achieve \(2\times 10^{8}\) cells/mL. The resulting starvation cultures were incubated at 37 \({}^{\circ}\)C with shaking at 200 rpm for eight days, and sampled daily for use in cell viability, protein, and RNA assays. Starvation cultures following growth in MOPS-minimal media were prepared similarly, with the following modifications. Overnight cultures were washed twice in MOPS buffer then used to inoculate flasks containing 100 mL MOPS minimal media supplemented with either 40 mM fructose or 40 mM glucose, to O.D.\({}_{600}\) = 0.05. Cells were incubated in 100 mL MOPS-fructose or MOPS-glucose until the OD\({}_{600}\) reached 0.3 or 1.0. At this point, approximately \(10^{10}\) CFU were removed by centrifugation, washed twice with PBS pH 7.2, resuspended in PBS to approximately \(2\times 10^{8}\) CFU/mL. Starvation cultures were incubated with shaking at 200 rpm at 37 \({}^{\circ}\)C and sampled daily for use in cell viability, protein, and RNA assays. **Determination of cell viability. ** Over the course of growth and starvation, the drop plate method was used to quantify colony forming units (CFUs). Briefly, cells were serially diluted in 0.85% NaCl and replicate 10 \(\upmu\) volumes were plated on TSA. Colonies were counted after 18 h incubation at 37 \({}^{\circ}\)C. For fold-reduction over time, CFUs were normalized by dividing by the number of cells at zero days of starvation. The mean and standard deviations from at least three biological replicates per experiment were plotted in GraphPad Prism v8.3. Two-way ANOVA with [PERSON]'s correction for multiple comparisons was performed to determine statistically significant differences at \(\alpha\) = 0.01. **Protein analysis during starvation conditions. ** On days 0, 2, 4, 6, and 8 of starvation, volumes ranging from 4.5 to 9 mL were removed from starvation cultures. The cells were collected by centrifugation at 7690\(\times\)\(g\) for three min at 4 \({}^{\circ}\)C, the supernatants were removed, and cell pellets were immediately frozen at \(-80\)\({}^{\circ}\)C. Cells were then lysed in 100 \(\upmu\)L RIPA buffer and total protein was quantified as described previously for growth studies. For comparison of strains cultured in different media, the reduction of cellular protein over starvation time was plotted as the change from the initial time point. For immunoblot analysis, 15 \(\upmu\)g of total cellular protein from each sample at each timepoint was loaded per lane for SDS-PAGE analysis. Immunoblotting and densitometry of the ribosomal protein bands was performed as described above. The mean and standard error from at least three independent biological replicates were plotted in GraphPad Prism v 8.3. 2-way ANOVA was performed with [PERSON]'s or [PERSON]'s multiple comparison test, as appropriate. Multiplicity adjusted \(p\)-values were reported at \(\alpha\) of 0.01. _P. aeruginosa hpf_ **expression from P\({}_{\textit{hpf}}\). ** To assay for _hpf_ expression over the course of growth, overnight cultures of _P. aeruginosa_ PAO1::P\({}_{\textit{hpf}}\)-HPF-YFP were centrifuged at 9500\(\times\)\(g\) for one min. The cell pellets were washed twice with MOPS buffer pH 7.2, then used to inoculate flasks containing 50 mL MOPS minimal media supplemented with either 40 mM fructose or 40 mM glucose to an initial OD\({}_{600}\) of 0.05. Cultures were incubated at 37 \({}^{\circ}\)C with shaking at 200 rpm. Samples were collected over 48 h to assay for growth as optical density at 600 nm on the Ultrospec 2100 Pro (Amersham Biosciences, Little Chalfont, UK) and for HPF production as YFP fluorescence on the Cytation 5 plate reader (BioTek Instruments, Winooski, VT, USA). This experiment was performed on three independent biological replicates. **RNA extraction and analysis.