BMC Genomics 2009, 10:165 http://www.biomedcentral.com/1471-2164/10/165 by an unknown mechanism. Computational sequence analyses using the RNAhybrid server [31] have indicated that GcvB does not contain any extensive regions of sequence complementary to the 5' leader of rpoS mRNA (data not shown). As such, the mechanism by which GcvB regulates rpoS expression remains a subject of further investigation. Conclusion Here we reveal that the sncRNA GcvB positively regulates the ability of E. coli to survive low pH conditions by upreg- ulating the expression of RpoS. Our findings provide insight into the control of AR by GcvB. However, further detailed studies will be required to investigate the precise nature of the GcvB/rpoS interaction at the molecular level. Methods Bacterial strains, plasmids, and growth conditions E. coli K12-MG1655 was used for all gene deletion and overexpression experiments. The E. coli expression vector pET32a (+) (Novagen, Madison, WI) was used for the overexpression of sncRNAs. All bacterial strains were grown at 37°C, with shaking at 230 rpm, in Luria-Bertani (LB) medium supplemented with antibiotics when required. Bacterial growth was monitored by measuring optical density at 600 nm (OD600). The antibiotics amp- icillin (50 ug/ml), kanamycin (50 ug/ml), and chloram- phenicol (12.5 ug/ml) were used for selection. Gene deletions Gene deletion mutations were constructed using the λ- Red recombination system. E. coli MG1655 was trans- formed with plasmid pSim6 (a gift from Dr. Donald Court) [32] on which the expression of the λ recombina- tion proteins is induced at 42°C. PCR fragments encom- passing a loxP-cm-loxP ('floxed') chloramphenicol resistance cassette with homology (45 nt) to the regions immediately flanking each deletion locus were trans- formed into MG1655 harboring pSim6. After induction of λred, recombinants were selected for chloramphenicol (cm) resistance, and were further verified by colony PCR. Gene cloning The selectable loxP-cm-loxP cassette was first inserted immediately after the stop codon of the gene of interest on the chromosome (as described above). We then used recombineering to 'clone' this gene of interest preceded by its native promoter and 'floxed' cm cassette downstream of the T7 promoter in a pET32a expression vector. Specif- ically, we PCR amplified the gene of interest (including its native promoter) and adjacent 'floxed' cm cassette using primers that contained homology (45 nt) to the plasmid insertion site. The PCR product and expression vector were co-transformed into DY330[12] after induction of λred. Recombinants were selected for chloramphenicol resistance and verified by PCR and sequencing. The native promoter drove the gene overexpression in MG1655 with- out induction. Construction of chromosomal lacZ translational fusions and beta-galactosidase assays First, the loxP-cm-loxP selectable cassette was inserted immediately after the stop codon of the lacZ gene on the MG1655 chromosome using λred recombination (as described above). Next, the lacZ-loxP-cm-loxP cassette was PCR amplified and inserted (in frame) immediately prior to the stop codon of the target gene on the chromo- some. The inserted lacZ fragment started from the 8th codon of lacZ gene and co-transcribed and -translated with the fused genes. The expression of gene-lacZ fusions was quantified using a beta-galactosidase assay kit from Thermo Fisher Scientific (Rockford, Illinois, USA). Acid resistance assay Overnight cultures were diluted 1:50 and incubated at 37°C for 5 hrs in LB medium. 3 volumes of acidified LB medium (pH 1.9) was then added to 1 volume of the cell culture so that the final pH value was approximately 2.0. The acid challenge was maintained for 15 min for cells that were acid sensitive, and 30 min for acid-resistant cells. The acid challenge was stopped by adding 3 volumes of alkalinized LB medium (pH9.3), making the final pH value approximately 7.0. For samples that displayed sim- ilar growth rates, the neutralized cell cultures were allowed to grow for 3 hrs following the acid challenge, and acid survival levels were determined by measurement of optical density at 600 nm (OD600, termed acid recov- ery). When samples with different growth rates were examined, cell viability was determined by plate counts and percentage acid survival was calculated as the number of number of colony forming units (cfu) remaining after the acid treatment divided by the initial cfu at time zero. Statistical analysis Two-tailed paired t-tests were used for the comparison of means obtained from the beta-galactosidase and acid sur- vival assays. P values of < 0.05 were considered statisti- cally significant. Abbreviations AR: acid resistance; sncRNA: small noncoding RNA; UTR: unstranslated region. Authors' contributions YJ planned and designed the experiments, performed the experiments and the data analysis, wrote the main draft of the paper, and generated the figures. RW conceived of the study and helped to draft the manuscript. AD aided in the experimental design and in editing the manuscript. JH provided guidance over the entire project, and aided in Page 6 of 7 (page number not for citation purposes)