Bacteriophage P1

In all experiments, bacterial cells were cultured in 25mL Luria-Bertani broth (LB) for 16 hours at 37°C, 300RPM, and 80% humidity in 250mL flasks. Unless otherwise noted, the following concentrations were used: 10 μg/mL gentamicin, 100 μg/mL ampicillin, 5 μg/mL ofloxacin, 20 μM CCCP, 1 mM KCN. The concentration of all carbon sources added to potentiate aminoglycosides was normalized to deliver 60 mM carbon (e.g., 10 mM glucose, 20 mM pyruvate, etc.). E. coli (K12 EMG2) and S. aureus (ATCC 25923) were the two parent strains used in this study. Knockouts (Supplementary Table 1 and 2) were constructed by P1-phage transduction from the Keio knockout collection. In E. coli, non-persister stationary phase cells were killed by treatment with 5 μg/mL ofloxacin for 4 hours^{25 (link), 26 (link)}. Samples were then washed with phosphate buffered saline (PBS) and suspended in M9 salts with carbon source and antibiotic to determine metabolite-enabled killing of persisters. At specified time points, 10 μL aliquots of samples were removed, serially diluted, and spot-plated onto LB agar plates to determine colony forming units/mL (CFU/mL) and survival. Gent-TR was made as previously described^{27 (link)}. Aminoglycoside uptake was measured by incubating stationary phase samples with 10 μg/mL Gent-TR for 5 minutes at 37°C, 300RPM, and 80% humidity. 100 μL of each sample was then washed and resuspended in PBS and analyzed on a BD FACS Aria II flow cytometer. Biofilm survival assays were performed as previously described^{28 (link)}. Raw microarray data for S. aureus were downloaded from the Gene Expression Omnibus (GEO) series GSE20973^{29 (link)} and processed with RMA express using background adjustment, quantile normalization, and median polish summarization to compute RMA expression values^{30 (link)}. Mouse experiments were performed with female Charles River Balb/C mice in collaboration with ViviSource Laboratories and conformed to the ViviSource IACUC policies and Procedural Guidelines.

All E. coli strains used in this study are derivatives of E. coli K-12 MG1655 and are listed in Table 2. Plasmids were generally introduced into strains by TSS transformation (Chung and Miller, 1988 (link)). The ΔrybC∷kan, ΔgadX∷kan, ΔgadW∷kan and ΔgadXYW∷kan strains were generated by PCR amplification of the kan cassette of strain CRB316 (Ranquet and Gottesman, 2007 (link)) with the kan-for and kan-rev oligos listed in Table 3 followed by lambda Red recombinase-mediated gene replacement. Marked mutations were obtained and moved into the desired strain background using bacteriophage P1 transduction (Silhavy et al., 1984 ). Oligonucleotides used as probes and for polymerase chain reaction are described in Table 3. For cloning procedures, PCR amplification was carried out using the Expand High Fidelity PCR system (Roche) and DH5a was used as the recipient strain.
Strain PM1004 was created by first amplifying the cat-sacB cassette from strain NC397 (Svenningsen et al., 2005 (link)) using primers PBAD-cat-For and lacZ-sacB-rev. The PCR product was then recombined in strain PM1002 using mini λ mediated recombination as previously described (Court et al., 2003 (link)). The resulting strain, PM1004, was lacI’∷kan-P_BAD-cat-sacB-lacZ. Strain PM1205 was constructed by amplifying the P_BAD-cat-sacB-lacZ cassette using oligonucleotides lacI-PBAD-for and lacZ-sacB-rev and strain PM1004 genomic DNA as a template. The resulting PCR DNA fragment was recombined as previously described (Court et al., 2003 (link)) in the chromosome of strain PM1203, giving rise to strain PM1205.
The rybC (-245)-lacZ and P_BAD-(-89) dpiB-lacZ translational fusion were constructed as follows. A DNA fragment corresponding to nts -245 to +10 respective to the transcription start site of rybC was amplified using primers EcoRI-rybC(-245)-for and BamHI-rybC(-245)-rev. The PCR product was subsequently cloned in between the EcoRI and BamHI sites of the pRS415 plasmid (Simons et al., 1987 (link)), giving rise to pRSRybC. The P_BAD-(-89) dpiB-lacZ translational fusion (strain PM1011) was obtained by first amplifying the P_BAD promoter and a sequence corresponding to nts −89 to +10 respective to the ATG translation start codon of dpiB, using primers 5’PBAD and PBAD-dpiB(-89)-rev, and dpiB(-89)-for and SmaI-dpiB(-89)-rev, respectively. The two PCR fragments were then joined by an overlap extension PCR and the resulting PCR product was subsequently cloned in frame with lacZ between the EcoRI and SmaI sites of the pRS414 plasmid (Simons et al., 1987 (link)), resulting in pRSdpiB. The pRSRybC and pRSdpiB plasmids were then crossed with λRS468 bacteriophage and monolysogens were constructed in strain PM1001 as previously described (Simons et al., 1987 (link)), giving rise to strains PM1106 and PM1011, respectively.
The rybC (-101)-lacZ (PM1151) and rybC (-45)-lacZ (PM1152) lacZ fusions were constructed as follows. DNA fragments corresponding to nts -101 to +10 and to nts -45 to +10 respective to the transcription start site of rybC were amplified by PCR using oligonucleotides rybC(-101)-for or rybC(-45)-for and deeplac, and strain PM1106 as template. The PCR products were subsequently recombined in strain PM1004 as previously described (Court et al., 2003 (link)), resulting in strain PM1151 and PM1152, respectively.
The pRybC and pGadY plasmids were constructed by first PCR amplifying rybC and gadY from strain MG1655 using AatII-rybC-for and EcoRI-rybC-rev primers and AatII-gadY-for and EcoRI-gadY-rev primers, respectively. The PCR product was subsequently cloned into the pBR-plac vector digested with AatII and EcoRI. The C55G G56C G57C site directed mutants of pRybC was constructed using the Quickchange II site directed mutagenesis kit (Stratagene) following the manufacturers instructions, with pRybC as a template and primers QC-rybc-mut1-for and QC-rybc-mut1-rev.