with DNaseI (Qiagen) treatment. Salmonella microarray slide preparation and general microarray analysis followed the procedures described by Porwollik et al. [63]. Total RNA from 14028s, smpB, and hfq was reverse transcribed with Cy3-linked dUTP and genomic DNA from 14028s was labeled with Cy5- linked dUTP as reference for hybridization. Hybridization mixtures containing same amounts of Cy3-labled probes and Cy5-labeled probes were applied on the Salmonella array slide and incubated in a hybridization chamber (Corning) at 42uC overnight. For each strain and condition, three biological repeats were hybridized on separate slides. Slides contained identical triplicate arrays. Slides hybridized with Cy3/Cy5-labled probes were scanned using ScanArray Express software (Packard Bioscience, BioChip Technologies), and the fluorescent signal intensities were quantified using QuantArray (Packard Bioscience, BioChip Technologies). The raw data was processed in WebArray [64], using print-tip Loess normalization within slides, and Scale normalization procedures between slides. The Salmonella data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE11486. Immunoblot analysis Chromosomal haemaglutanin (HA)-tagged Salmonella Typhi- murium 14028 wildtype and mutant strains were grown as described above, and the final optical density of each culture was recorded. Cells corresponding to an OD600 of 0.05 were harvested by centrifugation at 10000 g in a microfuge at 4uC, washed twice with an equal volume of ice-cold DPBS, resuspended in 50 mL of Laemmli 16sample buffer, and boiled for 5 min. A 1/ 10th volume of the total cell lysate was loaded into the wells of 15% Tris-Cl sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) gels (BIO-RAD). After the proteins were separated on the gel, the proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked in Tris-buffered saline (TBS) plus 5% powdered nonfat dry milk for 1 h, probed with an anti-HA monoclonal antibody (Covance, 1:1,000 dilution in block solution) and an anti-DnaK monoclonal antibody (Stressgen, 1:4,000 dilution in block solution) for 1 h, washed 3 times for 5 min with TBS, probed with a goat-antimouse 2u antibody (Sigma A4416, 1:5000 dilution in block solution) for 30 min, and finally washed 3 times for 5 min with TBS. The immune complexes were detected via chemiluminescence using Perkin- Elmer’s ‘‘Western Lightning’’ reagents and then exposed to XAR Biofilm (Kodak). Quantitative PCR analysis Wild-type strain and mutant strain cells were grown as described above and harvested for RNA extraction. Total RNA was isolated using RNAprotectTM (Qiagen), RNeasy kit (Qiagen), and DNase set (Qiagen) according to the manufacturer’s recommendations. cDNA was synthesized using iScript cDNA systhesis kit (Bio Rad) and subjected to real-time PCR (ABI 7700, Applied Biosystems) with SYBR green PCR master mix (Applied Biosystems) and primer sets. Primers used in RT-PCR are listed in Table S5. The expression ratio of each gene is the average from three independent RNA samples and was normalized to the level of gyrB. gyrB is assumed to be a steadily transcribed housekeeping gene. Hfq, SmpB Targets in Salmonella Supporting Information Table S1 List of gene products (proteins) regulated by Hfq and gene products (proteins) regulated by SmpB under the growth conditions examined. Found at: doi:10.1371/journal.pone.0004809.s001 (1.17 MB XLS) Table S2 List of proteins that overlap between Hfq-regulated proteins and Hfq-associated mRNAs. Found at: doi:10.1371/journal.pone.0004809.s002 (0.07 MB XLS) Table S3 Validation of proteomics dataset by cross-comparison with the literature. Found at: doi:10.1371/journal.pone.0004809.s003 (0.05 MB XLS) Table S4 List of gene transcripts (RNA) regulated by Hfq and gene transcripts (RNA) regulated by SmpB under the growth conditions examined. Found at: doi:10.1371/journal.pone.0004809.s004 (0.23 MB XLS) Table S5 List of primers used in this study. Found at: doi:10.1371/journal.pone.0004809.s005 (0.05 DOC) Table S6 Scar sequences. Found at: doi:10.1371/journal.pone.0004809.s006 (0.03 DOC) MB MB Figure S1 Growth curves of strains in LB and minimal acidic media: Growth phenotypes of wild-type (open corcle), DsmpB (closed square), and Dhfq (closed triangle) were compared in LB (A) and minimal acidic (B) media. For the growth in minimal acidic media, cells were pre-cultured in MgM (pH5.0) overnight and diluted 1:100 into fresh MgM (pH5.0) media. Found at: doi:10.1371/journal.pone.0004809.s007 (0.77 MB TIF) Figure S2 Immunoblot analysis of the protein levels of selected proteins from varying Salmonella wildtype and mutant strain cultures: Salmonella wildtype and Dhfq mutant strains harboring HA-tag at the HtrA, STM1513 and OsmY genes (Panel A) and Salmonella wildtype and DsmpB mutant strains harboring HA-tag at the FliC and YciF genes (Panel B) were grown under the indicated conditions as described. Same amount of cell lysates was loaded in each lane and probed by Western blot analysis for the indicated proteins and a control protein DnaK. AMM = Acidic minimal media. The level of YciF-encoded protein expressed in each strain was normalized to the DnaK level. Intracellular DnaK level in each lane was set to 100% (Panel C). Found at: doi:10.1371/journal.pone.0004809.s008 (2.35 MB TIF) Acknowledgments The authors gratefully acknowledge the help of Penny Colton for assistance in preparing this manuscript. Mass spectrometry data and results can be found at http://omics.pnl.gov/; http://www.proteomicsresource.org/ Author Contributions Conceived and designed the experiments: CA HY JNA FH RS. Performed the experiments: CA HY SP HMB BP. Analyzed the data: CA HY SP JNA FH. Contributed reagents/materials/analysis tools: NJ MM. Wrote the paper: CA HY JNA FH. PI for grants that provided partial support: RS FH. Contributed to initial drafts of the manuscript: SP. PLoS ONE | www.plosone.org 11 March 2009 | Volume 4 | Issue 3 | e4809