BMC Genomics 2009, 10:165 http://www.biomedcentral.com/1471-2164/10/165 ulators of the GadA and GadB glutamate decarboxylases [6,7]. In recent years, an increasing number of sncRNAs have been identified, but the functions of the vast major- ity of these are unknown. This prompted us to investigate whether sncRNAs in addition to DsrA and GadY, were involved in AR; and if so, to establish how they exerted their regulatory actions. In the present study, seventy-nine snRNAs identified prior to 2006 [8-10] were selected for the construction of a sin- gle-gene knockout library within E. coli K12-MG1655. Comparison of each mutant with the wild type strain ena- bled us to identify the sncRNA GcvB as a previously unknown regulator of AR in E. coli. We demonstrate that GcvB positively regulates AR predominantly by upregulat- ing the levels of the RpoS transcription factor (Sigma S). Our findings provide further insight into the regulatory roles played by sncRNAs in AR, which contributes to viru- lence of E. coli. Results Construction of single-sncRNA gene knockout mutant collection in E. coli K-12 MG1655 To systematically characterize sncRNAs, we established a single-sncRNA gene knockout library within E. coli K12- MG1655. Recombineering techniques were used to replace the target gene with a selectable chloramphenicol- resistance cassette, which was generated by polymerase chain reaction (PCR) using primers containing homology (45 nt) to the gene-flanking regions. This homologous recombination-based approach enabled precise gene deletion with minimal interference to adjacent genes (polar effects) [11,12]. In total, seventy-nine genes encod- ing sncRNAs identified prior to 2006 [8-10] were deleted individually from the MG1655 chromosome (see addi- tional file 1). The sncRNA GcvB is a novel regulator of AR in E. coli We investigated the involvement of sncRNAs in the AR of E. coli by challenging the sncRNA mutants as well as the wild type strain by incubation in LB medium at pH 2.0 (for 15 or 30 minutes), and comparing their percent sur- vival and/or rates of recovery after pH neutralization (termed acid recovery). Unless otherwise stated, bacterial cells were grown for 5 hrs to the early stationary phase before the acid challenge. As expected, cells lacking DsrA, which is a known AR regulator, were sensitive to the acid challenge, confirming previous findings (Fig. 1A) [4,5]. However, deleting gadY did not affect acid survival (Fig. 1A), suggesting that GadY is not required for AR in spite of its positive control of GadX, a transcriptional activator of the GadA and GadB glutamate decarboxylases [6,7]. Interestingly, the absence of the sncRNA GcvB resulted in a significant reduction in acid survival (Fig. 1A, B). The same AR phenotypes in the sncRNA mutants were also rRFeeisggisutrlaentoc1reyinroEleschoef rtichheias mcoalill noncoding RNA GcvB in acid Regulatory role of the small noncoding RNA GcvB in acid resistance in Escherichia coli. (A) MG1655-K12 wild type strain (wt) and various deletion mutants were grown in Luria Broth broth (pH 7.0) for 5 hr at 37°C. Cells were acid shocked at pH 2.0 for 30 min, and then neutralized by adding alkalinized LB (pH 9.3). Cells were allowed to grow for an additional 3 hrs before measurement of optical density at 600 nm (OD600). Two-tailed paired t tests were employed to compare each mutant with the wt in terms of acid resistance. (B) Because the gcvB mutant displayed a lower growth rate than the wt (data not shown), percent acid survival was determined to confirm the data of acid recovery measured as OD600. Cell viability was determined by plate counts and percent acid survival was calculated as the number of cfu remaining after the 30 min-acid treatment divided by the ini- tial cfu at time zero (quoted as a percentage). (C) A plasmid expressing GcvB under the control of its native promoter was transformed into a gcvB mutant of E. coli MG1655. The mutant carrying an empty vector was used as a control. Cells were incubated for 5 hrs before being acid challenged for 15 min. Error bars represent standard deviation (* P-value < 0.05). Page 2 of 7 (page number not for citation purposes)