In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 13 ( 2012-03-27)
Abstract:
Our current search for Hfq-dependent sRNAs in Salmonella has identified ∼130 validated candidates, most of which are likely to perform regulatory functions. Because we are just beginning to understand the complex interplay between core genomic elements and HGT-acquired genes ( 5 ), we believe that additional studies will reveal a greater role than previously expected for sRNA-mediated regulation in the acquisition and control of virulence determinants. This role should be the case in both Salmonella and other bacterial pathogens. The binding energies (i.e., the strength of molecular interactions) of the SgrS– sopD and SgrS– sopD2 duplexes were calculated; these energies indicated that G-U conferred a lower stability interaction compared with G-C that was decreased by ∼1.2 kcal/mol. How can such a marginal difference in RNA duplex strength confer selective target discrimination? By generating a number of SgrS mutant alleles, we found that the positioning of G-C vs. G-U base pairing is critical for successful target discrimination. The proximal end of the sRNA that is responsible for RNA duplex formation, the so-called seed-sequence, identifies genuine target mRNAs by the strength and accuracy of base pairing. A characteristic of HGT genes is that they are more likely to undergo duplication than so-called core genes. Gene duplication is a well-studied phenomenon accelerating evolutionary change in bacterial pathogens ( 4 ). For example, the sopD gene has been duplicated to generate sopD2 throughout the S. enterica species, except in the ancestral S. bongori ( 1 ). A bioinformatic comparison of the sopD and sopD2 sequences showed that the SgrS targeting region is well-conserved between both genes; in other words, the component sequences do not change greatly. However, our experiments showed that SgrS negatively regulated the expression of sopD but not sopD2 . That is, SgrS discriminates between these two potential target mRNAs. This finding led us to discover that the SgrS– sopD2 interaction differed slightly from the original SgrS– sopD RNA duplex because of the exchange of just one of the component base pairs: a G-U for G-C exchange is responsible for this short interaction ( Fig. P1 ). G-C base pairs engage three hydrogen bonds instead of the two formed by G-U base pairs, and therefore, they confer a higher degree of stability on the RNA duplex. Indeed, the replacement of the G-U base pair with G-C in the SgrS– sopD2 interaction produced a fully functional SgrS target gene that displayed a regulatory pattern similar to sopD . Such differential stability allows the sRNA to discriminate between its targets. We reproduced target discrimination between sopD vs. sopD2 in vitro using both the full-length SgrS molecule and a 14-nt RNA corresponding to the targeting region of SgrS. Importantly, we discovered that, in addition to preventing the accumulation of phosphorylated sugars by blocking sugar import, SgrS reduces the expression of the horizontally acquired gene that encodes the virulence factor SopD ( Fig. P1 ). SgrS does this reduction by binding to the mRNA molecule associated with the sopD gene. The suppressive activity of SgrS requires the Hfq protein, which acts as an RNA chaperone, maintains sRNA 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. Furthermore, our genetic and biochemical analyses of the resultant RNA duplex formation revealed that SgrS binding sequesters the sopD start codon to block the initiation of the translation process. To our knowledge, it has not previously been shown that Hfq-binding sRNAs can control the expression of horizontally acquired virulence factors. Salmonella and E. coli not only share many regulatory proteins but also share several highly conserved sRNAs. These so-called core sRNAs often serve a central function in bacterial metabolism or the response to environmental stress, and they control target genes that cluster into distinct functional groups. A good example is the SgrS sRNA that prevents the accumulation of phosphorylated sugars in E. coli ( 2 ). In this study, we showed that SgrS performs a similar function in Salmonella . Small noncoding RNAs (sRNAs) constitute a vital group of so-called posttranscriptional regulators that shape the gene expression of eukaryotic and prokaryotic organisms. In bacteria, sRNAs generally act through base pairing to reduce or increase the translation of target mRNAs into protein. Most of our current knowledge of sRNA numbers and functions stems from two species, Escherichia coli and Salmonella enterica serovar Typhimurium. Both organisms display a high degree of sequence conservation across about three-quarters of the chromosome that constitutes their core genome. An additional ∼25% of Salmonella genes were acquired by horizontal gene transfer (HGT)—a mechanism by which genes move between organisms—and are required for the virulent lifestyle of this pathogen ( 1 ). Little is known about interactions between the core genome and these horizontally acquired genomic islands at the regulatory level. We have found that posttranscriptional control by sRNA regulators plays an important role.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1119414109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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