Abstract
Recently, we reported the global regulatory roles of the two small RNAs in Salmonella typhimurium, ryhB-1 and ryhB-2. However, the genetic basis of the sRNA-target interactions remains unknown. To identify the nucleotides of RyhB-1 that are functionally important for its regulatory actions, we introduced random single-point mutations into ryhB-1 gene on the chromosome of Salmonella typhimurium carrying a sodB–lacZ translational fusion by an error-prone PCR method. We reasoned that mutants expressing variant RyhB-1 with weakened interaction with sodB transcript would produce a higher level of SodB when compared to wild type, leading to darker blue colonies on X-gal agar plates. Five mutants displaying a significant increase in β-galactosidase activity under the condition inducing RyhB-1 expression were isolated. Quantitative real-time PCR analysis showed that the expression levels of eight target mRNAs in these five mutants were significantly changed when compared to the parent strain. Interestingly, two mutations affected growth and cell survival under H2O2-stressed conditions. The results suggest that there are strong selective constraints against mutational changes in ryhB-1 gene sequence, leading to high levels of nucleotide conservation in ryhB-1 gene sequences among the genus of Salmonella.
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References
Bossi L, Figueroa-Bossi N (2007) A small RNA downregulates LamB maltoporin in Salmonella. Mol Microbiol 65(3):799–810. doi:10.1111/j.1365-2958.2007.05829.x
Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2(1):28–33
Cherepanov PP, Wackernagel W (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158(1):9–14
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12):6640–6645. doi:10.1073/pnas.120163297
Ellermeier JR, Slauch JM (2008) Fur regulates expression of the Salmonella pathogenicity island 1 type III secretion system through HilD. J Bacteriol 190(2):476–486. doi:10.1128/JB.00926-07
Figueroa-Bossi N, Lemire S, Maloriol D, Balbontin R, Casadesus J, Bossi L (2006) Loss of Hfq activates the sigmaE-dependent envelope stress response in Salmonella enterica. Mol Microbiol 62(3):838–852. doi:10.1111/j.1365-2958.2006.05413.x
Geissmann TA, Touati D (2004) Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator. EMBO J 23(2):396–405. doi:10.1038/sj.emboj.7600058
Hao Y, Zhang ZJ, Erickson DW, Huang M, Huang Y, Li J, Hwa T, Shi H (2011) Quantifying the sequence–function relation in gene silencing by bacterial small RNAs. Proc Natl Acad Sci USA 108(30):12473–12478. doi:10.1073/pnas.1100432108
Horton RM, Cai ZL, Ho SN, Pease LR (1990) Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8(5):528–535
Jacques JF, Jang S, Prevost K, Desnoyers G, Desmarais M, Imlay J, Masse E (2006) RyhB small RNA modulates the free intracellular iron pool and is essential for normal growth during iron limitation in Escherichia coli. Mol Microbiol 62(4):1181–1190. doi:10.1111/j.1365-2958.2006.05439.x
Kawamoto H, Koide Y, Morita T, Aiba H (2006) Base-pairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq. Mol Microbiol 61(4):1013–1022. doi:10.1111/j.1365-2958.2006.05288.x
Kim JN, Kwon YM (2013) Genetic and phenotypic characterization of the RyhB regulon in Salmonella Typhimurium. Microbiol Res 168(1):41–49. doi:10.1016/j.micres.2012.06.007
Kim JN, Kwon YM (2013) Identification of target transcripts regulated by small RNA RyhB homologs in Salmonella: RyhB-2 regulates motility phenotype. Microbiol Res 168(10):621–629. doi:10.1016/j.micres.2013.06.002
Maloy SR, Stewart VJ, Talyor RK (1996) Genetic analysis of pathogenic bacteria. Cold Spring Harbor Laboratory, Plainview
Masse E, Escorcia FE, Gottesman S (2003) Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev 17(19):2374–2383. doi:10.1101/gad.1127103
Masse E, Gottesman S (2002) A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci USA 99(7):4620–4625. doi:10.1073/pnas.032066599
Masse E, Vanderpool CK, Gottesman S (2005) Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 187(20):6962–6971. doi:10.1128/JB.187.20.6962-6971.2005
Morita T, Maki K, Aiba H (2005) RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs. Genes Dev 19(18):2176–2186. doi:10.1101/gad.1330405
Oglesby AG, Murphy ER, Iyer VR, Payne SM (2005) Fur regulates acid resistance in Shigella flexneri via RyhB and ydeP. Mol Microbiol 58(5):1354–1367. doi:10.1111/j.1365-2958.2005.04920.x
Tjaden B, Goodwin SS, Opdyke JA, Guillier M, Fu DX, Gottesman S, Storz G (2006) Target prediction for small, noncoding RNAs in bacteria. Nucleic Acids Res 34(9):2791–2802. doi:10.1093/nar/gkl356
Velayudhan J, Castor M, Richardson A, Main-Hester KL, Fang FC (2007) The role of ferritins in the physiology of Salmonella enterica sv. Typhimurium: a unique role for ferritin B in iron–sulphur cluster repair and virulence. Mol Microbiol 63(5):1495–1507. doi:10.1111/j.1365-2958.2007.05600.x
Vogel J, Papenfort K (2006) Small non-coding RNAs and the bacterial outer membrane. Curr Opin Microbiol 9(6):605–611. doi:10.1016/j.mib.2006.10.006
Acknowledgments
This work was supported by Arkansas Biosciences Institute (ABI). We thank Dr. James M. Slauch (University of Illinois at Urbana-Champaign) for providing the Salmonella typhimurium sodB–lacZ translational fusion strain.
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Kim, J.N., Kwon, Y.M. Phenotypic Characterization of Salmonella RyhB-1 Mutations that Modulate Target Regulation. Curr Microbiol 69, 212–217 (2014). https://doi.org/10.1007/s00284-014-0572-1
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DOI: https://doi.org/10.1007/s00284-014-0572-1