The ancestral SgrS RNA discriminates horizontally acquired Salmonella mRNAs through a single G-U wobble pair

Kai Papenforta, Dimitri Podkaminskia, Jay C. D. Hintonb, and Jörg Vogela,1 aRNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, D-97080 Würzburg, Germany; and bDepartment of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland AUTHOR SUMMARY

Small noncoding A characteristic of HGT (sRNAs) constitute a vital group is that they are more likely of so-called posttranscriptional to undergo duplication than so- SgrS RNA regulators that shape the Core genome Horizontally acquired called core genes. Gene dupli- expression of eukaryotic and elements pathogenicity genes cation is a well-studied phe- prokaryotic organisms. In bac- nomenon accelerating evolu- teria, sRNAs generally act duplication tionary change in bacterial through base pairing to reduce pathogens (4). For example, the Hfq or increase the translation of ptsG/manXYZ mRNAs sopD mRNA sopD2 mRNA sopD gene has been duplicated target mRNAs into protein. target (G-C base-pair) pseudotarget (G-U base-pair) to generate sopD2 throughout Most of our current knowledge the S. enterica species, except in of sRNA numbers and functions the ancestral S. bongori (1). A Fig. P1. Posttranscriptional interaction between the core and stems from two species, Escher- horizontally acquired genome through Hfq and sRNAs. The SgrS sRNA bioinformatic comparison of the ichia coli and is encoded by the Salmonella core genome and conserved in many sopD and sopD2 sequences serovar Typhimurium. Both bacterial species, including E. coli and Salmonella. Aided by the RNA showed that the SgrS targeting organisms display a high degree chaperone Hfq, SgrS reduces the expression of mRNAs encoding for region is well-conserved be- of sequence conservation across sugar transport proteins (ptsG and manXYZ), both of which are core tween both genes; in other about three-quarters of the genomic elements. SgrS also represses the sopD mRNA, which encodes words, the component sequen- chromosome that constitutes a Salmonella virulence factor and was acquired by HGT (i.e., transfer ces do not change greatly. their core genome. An addi- from another organism). In contrast, SgrS does not repress the However, our experiments tional ∼25% of Salmonella duplicated sopD2 mRNA, a process that is mediated by discrimination showed that SgrS negatively between G-C (sopD) and G-U (sopD2) base-pairing. genes were acquired by hori- regulated the expression of sopD zontal gene transfer (HGT)—a but not sopD2. That is, SgrS mechanism by which genes move between organisms—and discriminates between these two potential target mRNAs. This are required for the virulent lifestyle of this pathogen (1). Little finding led us to discover that the SgrS–sopD2 interaction is known about interactions between the core genome and these differed slightly from the original SgrS–sopD RNA duplex horizontally acquired genomic islands at the regulatory level. because of the exchange of just one of the component base pairs: We have found that posttranscriptional control by sRNA regu- a G-U for G-C exchange is responsible for this short inter- lators plays an important role. action (Fig. P1). G-C base pairs engage three hydrogen bonds Salmonella and E. coli not only share many regulatory proteins instead of the two formed by G-U base pairs, and therefore, they but also share several highly conserved sRNAs. These so-called confer a higher degree of stability on the RNA duplex. Indeed, core sRNAs often serve a central function in bacterial metabo- the replacement of the G-U base pair with G-C in the SgrS– lism or the response to environmental stress, and they control sopD2 interaction produced a fully functional SgrS target target genes that cluster into distinct functional groups. A good gene that displayed a regulatory pattern similar to sopD. Such example is the SgrS sRNA that prevents the accumulation of differential stability allows the sRNA to discriminate between phosphorylated sugars in E. coli (2). In this study, we showed that its targets. We reproduced target discrimination between sopD SgrS performs a similar function in Salmonella. vs. sopD2 in vitro using both the full-length SgrS molecule Importantly, we discovered that, in addition to preventing the and a 14-nt RNA corresponding to the targeting region of SgrS. accumulation of phosphorylated sugars by blocking sugar import, The binding energies (i.e., the strength of molecular inter- SgrS reduces the expression of the horizontally acquired gene actions) of the SgrS–sopD and SgrS–sopD2 duplexes were cal- that encodes the virulence factor SopD (Fig. P1). SgrS does this culated; these energies indicated that G-U conferred a lower reduction by binding to the mRNA molecule associated with the stability interaction compared with G-C that was decreased by sopD gene. The suppressive activity of SgrS requires the Hfq ∼1.2 kcal/mol. How can such a marginal difference in RNA protein, which acts as an RNA chaperone, maintains sRNA duplex strength confer selective target discrimination? By gen- stability, and facilitates the joining or annealing of the sRNA to its target mRNA (3). SgrS reduces SopD expression under both infection-relevant and standard laboratory conditions. Further- Author contributions: K.P. and J.V. designed research; K.P. and D.P. performed research; more, our genetic and biochemical analyses of the resultant K.P., D.P., J.C.D.H., and J.V. analyzed data; and K.P., J.C.D.H., and J.V. wrote the paper. RNA duplex formation revealed that SgrS binding sequesters the The authors declare no conflict of interest. sopD start codon to block the initiation of the translation This Direct Submission article had a prearranged editor. process. To our knowledge, it has not previously been shown that 1To whom correspondence should be addressed. E-mail: [email protected]. Hfq-binding sRNAs can control the expression of horizontally See full research article on page E757 of www.pnas.org. acquired virulence factors. Cite this Author Summary as: PNAS 10.1073/pnas.1119414109.

4726–4727 | PNAS | March 27, 2012 | vol. 109 | no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1119414109 Downloaded by guest on September 23, 2021 erating a number of SgrS mutant alleles, we found that the po- virulence determinants. This role should be the case in both PNAS PLUS sitioning of G-C vs. G-U base pairing is critical for successful Salmonella and other bacterial pathogens. target discrimination. The proximal end of the sRNA that is responsible for RNA duplex formation, the so-called seed- 1. McClelland M, et al. (2001) Complete genome sequence of Salmonella enterica serovar sequence, identifies genuine target mRNAs by the strength Typhimurium LT2. Nature 413:852–856. and accuracy of base pairing. 2. Vanderpool CK (2007) Physiological consequences of small RNA-mediated regulation Our current search for Hfq-dependent sRNAs in Salmonella of glucose-phosphate stress. Curr Opin Microbiol 10:146–151. has identified ∼130 validated candidates, most of which are likely 3. Vogel J, Luisi BF (2011) Hfq and its constellation of RNA. Nat Rev Microbiol 9: 578–589. to perform regulatory functions. Because we are just beginning 4. Hooper SD, Berg OG (2003) Duplication is more common among laterally transferred to understand the complex interplay between core genomic genes than among indigenous genes. Genome Biol 4:R48. elements and HGT-acquired genes (5), we believe that addi- 5. Navarre WW, McClelland M, Libby SJ, Fang FC (2007) Silencing of xenogeneic DNA by tional studies will reveal a greater role than previously expected H-NS-facilitation of lateral gene transfer in by a defense system that for sRNA-mediated regulation in the acquisition and control of recognizes foreign DNA. Genes Dev 21:1456–1471. GENETICS

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