Hfq, SmpB Targets in Salmonella Table 2. Strains and plasmids used in this study. Strain or plasmid Description S. enterica serovar Typhimurium 14028s HY2688 HY4261 HYW-pduE HYW-pduA HYW-htrA HYW-STM1513 HYW-osmY HYW-fliC HYW-yciF HYS-pduE HYS-pduA HYS-fliC HYS-yciF HYH-pduE HYH-pduA HYH-htrA HYH-STM1513 HYH-osmY Plasmid pKD13 pKD13-2HA pKD46 pCP20 Wild-type smpB Hfq pduE-2HA pduA-2HA htrA-2HA STM1513-2HA osmY-2HA fliC-2HA yciF-2HA smpB pduE-2HA smpB pduA-2HA smpB fliC-2HA smpB yciF-2HA hfq pduE-2HA hfq pduA-2HA hfq htrA-2HA hfq STM1513-2HA hfq osmY-2HA Materials and Methods Reagents The following reagents were used in sample preparation: Nanopure or Milli-Q quality water (,18 megohm?cm or better); ammonium bicarbonate (NH4HCO3); bicinchoninic acid (BCA) or coomassie protein assay reagents (Pierce, Rockford, IL); urea; thiourea; dithiothreitol (DTT); 3-((3-cholamidopropyl)dimethy- lammonio)-1-propanesulfonate (CHAPS); calcium chloride; se- quencing-grade modified trypsin (Promega); HPLC-grade meth- anol (MeOH); trifluoroacetic acid (TFA); acetonitrile (ACN); ammonium formate; formic acid; and ammonium hydroxide (NH4OH). All reagents were obtained from Sigma Aldrich (St. Louis, MO) unless otherwise specified. Bacterial strains and culture conditions Bacterial strains and plasmids used in this study are listed in Table 2. Salmonella enterica serovars Typhimurium strain 14028 wildtype and mutant strains were grown and harvested using standard batch culture procedures. Briefly, cells from a single colony were inoculated into 5 mL of Luria–Bertani (LB) medium and then grown for 16 h with shaking at 37uC. This starter culture was then diluted 1:100 into 300 mL of fresh LB medium in a 2 L Erlenmeyer flask and grown for 16 h with shaking (200 rpm) at 37uC. Once the logarithmic and stationary phase cultures reached an OD 600 of 0.6 and 2.0, respectively, the cells were harvested via centrifugation at 40006g. The acidic minimal media 1 (AMM- 1) cultures used cells pre-cultured in magnesium minimal medium (MgM) pH5.0 overnight at inoculation. Bacteria under stationary phase in MgM media were diluted 1:100 into fresh pH5.0 MgM and then incubated at 37uC with shaking (200 rpm) for 4 h. For the acidic minimal media 2 (AMM-2) culture, cells were first grown in LB medium to stationary phase as described above, rinsed twice with magnesium minimal medium (MgM) pH 5.0, resuspended in an equal volume of MgM pH 5.0, and then incubated at 37uC with shaking (200 rpm) for 4 h. The cells were harvested as described above. All experiments were performed three times for every bacterial strain, on three different days. After the cells were harvested for each culture condition, a portion of the pellets was lysed for RNA isolation and the remainder cell pellets were washed twice with Cellgro Dulbecco’s phosphate-buffered saline (PBS; Mediatech) and finally pelleted in microcentrifuge tubes to an approximate wet weight of 0.1 g/tube. The cells were frozen and stored at 280uC until needed. Genetic manipulation of chromosomal genes of Salmonella Typhimurium The primers used for deleting or tagging chromosomal genes of Salmonella Typhimurium are listed in Table S5. The procedures of gene deleting and tagging were the same as those described previously [28,49]. Non-polar in-frame gene deletion was carried out to eliminate the entire coding region except the initiation codon and last 7 amino acids of smpB and hfq. For HA tagging, pKD13 was modified to encode in-frame double HA tag after the kan cassette removal. Two oligonucleotide sequences (2HA-F1 and 2HA-R1) were annealed together as indicated. Using the modified plasmid, pKD13-2HA, as a template, PCR-based chromosomal gene recombination was performed and the sequences encoding 2HA epitopes were inserted in-frame after the last codon (see Table S6 for the scar sequence). All resulting Salmonella Typhimurium strains made in this study are listed in Table 2. The deleted mutations were tested in mice for attenuation in virulence and for growth in macrophages. The strains tagged with PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4809 Ref. or source M.J. Worley This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study repR6Kc ApR FRT KmR FRT K.A.Datsenko pKD13 derivative harboring 2HA This study reppSC101ts ApR PBADgam bet exo K.A.Datsenko reppSC101ts ApR CmR cI857 lPRflp K.A.Datsenko doi:10.1371/journal.pone.0004809.t002 the HA epitope were used to validate the LC-MS results by Western hybridization using DnaK as a control [50]. In these experiments wells were loaded with equivalent numbers of bacteria. Animal experiments For experimental continuity, 5 six-week-old female BALB/c mice were intraperitoneally (i.p.) infected with 14028s, DsmpB, and Dhfq strains respectively. Bacteria cells were grown in LB overnight, washed with PBS and diluted to 2 colony-forming units (cfu)/ml in PBS. Each mouse was administered 200 cfu i.p. and the dose was enumerated by plating on LB agar media. Mouse mortality was monitored for 21 days and all animal experiments were in accordance with the protocol of Oregon Health & Science University Animal Care and Use Committee. Macrophage infection assays RAW 264.7 macrophage-like cell lines were plated in 24-well plates at 2.56105 cells/well and incubated overnight at 37uC with 5% CO2. The next day, stationary phase bacteria were added at an input multiplicity of infection (m.o.i.) of 50 (resulting in approximately 1 bacteria per cell). Infections were initiated by centrifuging the bacteria onto the cell monolayers at 2506g for 5 min and then incubated at 37uC with 5% CO2 for 30 min. To remove extracellular bacteria after the infection, the monolayers were washed twice with PBS and supplemented with DMEM plus gentamicin (100 ug/ml final concentration) for 1 h. The presence