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A Transposon-Derived Small RNA Regulates Gene Expression in Salmonella Typhimurium Michael J 5470–5486 Nucleic Acids Research, 2017, Vol. 45, No. 9 Published online 21 February 2017 doi: 10.1093/nar/gkx094 A transposon-derived small RNA regulates gene expression in Salmonella Typhimurium Michael J. Ellis, Ryan S. Trussler, Onella Charles and David B. Haniford* Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada Received September 21, 2016; Revised January 30, 2017; Editorial Decision January 31, 2017; Accepted February 06, 2017 ABSTRACT would maintain sequence identity of paralogs; however, Downloaded from https://academic.oup.com/nar/article/45/9/5470/3038232 by guest on 06 October 2021 IS200 is an essentially dormant transposon (1,2). Conser- Bacterial sRNAs play an important role in regulating vation and copy number might therefore reflect a selective many cellular processes including metabolism, outer pressure on the host bacterium to maintain IS200. Trans- membrane homeostasis and virulence. Although sR- posons can contribute to host fitness in several ways includ- NAs were initially found in intergenic regions, there ing: (i) by mediating DNA rearrangements that influence is emerging evidence that protein coding regions of host gene expression and gene structure (4); (ii) contribut- the genome are a rich reservoir of sRNAs. Here we ing passenger genes such as antibiotic resistance determi- report that the 5UTR of IS200 transposase mRNA nants (5); (iii) providing a rich source of DNA regulatory (tnpA) is processed to produce regulatory RNAs that sequence (6); (iv) providing proteins and/or protein motifs affect expression of over 70 genes in Salmonella Ty- from transposase proteins that can be domesticated by the phimurium. We provide evidence that the tnpA de- host (7); and (v) providing regulatory RNAs that affect host gene expression (8,9). As a simple insertion sequence, IS200 rived sRNA base-pairs with invF mRNA to repress does not encode any passenger genes, and the dormancy expression. As InvF is a transcriptional activator of of IS200 suggests that this element would not contribute SPI-1 encoded and other effector proteins, tnpA in- transposition-dependent functions to the host. directly represses these genes. We show that dele- Non-coding RNAs (ncRNA) play a crucial role in reg- tion of IS200 elements in S. Typhimurium increases ulating many critical processes in bacteria including outer invasion in vitro and reduces growth rate, while membrane homeostasis, metabolism and virulence (10,11). over-expression of tnpA suppresses invasion. Our The largest class of bacterial ncRNA are small RNAs work indicates that tnpA acts as an sRNA ‘sponge’ (sRNA) that base-pair with target mRNAs and affect trans- that sets a threshold for activation of Salmonella lation and/or transcript stability. sRNAs are typically ex- pathogenicity island (SPI)-1 effector proteins and pressed from intergenic regions and therefore have limited identifies a new class of ‘passenger gene’ for bacte- sequence complementarity with their trans-encoded tar- gets. A related class of ncRNA are antisense RNAs (as- rial transposons, providing the first example of a bac- RNA) which are encoded on the opposite strand of DNA terial transposon producing a regulatory RNA that to their target mRNA. Accordingly, asRNAs have much controls host gene expression. more extensive complementarity with their cis-encoded tar- gets. Note that both sRNAs and asRNAs act by an anti- INTRODUCTION sense mechanism, but are classified based on their genomic IS200 is the smallest prokaryotic transposon and is widely context relative to target mRNAs. The third and smallest conserved in Enterobacteriaceae and found throughout Eu- class of bacterial ncRNAs act by binding to and regulat- / bacteria and Archaea. One unusual feature of IS200 ele- ing protein activity (e.g. 6S RNA, CsrB C). The classic dis- ments is the high copy number achieved in Yersinia and tinction between these three classes of ncRNA has been Salmonella spp. (1–3). Many strains of Yersinia pestis con- challenged with continually emerging examples of dual- tain more than 50 copies of the IS200 ortholog IS1541, function ncRNA, including sRNAs derived from mRNAs while strains of Salmonella enterica subsp. enterica serovar (12–15), base-pairing sRNAs acting to modulate protein Typhimurium (S. Typhimurium) typically contain 5–12 activity (16–19) and asRNAs acting in trans to regulate copies and S. Typhi contains 26 copies of IS200 per genome. genes expressed from different loci (20,21). One common In the above cases, all IS200 paralogs appear to be 100% feature for base-pairing ncRNAs is that the RNA-binding conserved and in general IS200 orthologs share >90% protein Hfq is typically required to facilitate pairing when identity. A highly active transposon might be expected to there is limited complementarity between an sRNA and achieve this high copy number and repeated transposition mRNA (22). In general, an interaction between Hfq and an *To whom correspondence should be addressed. Tel: +1 519 661 4013; Fax: +1 519 661 3175; Email: [email protected] C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2017, Vol. 45, No. 9 5471 This regulatory element ensures that impinging transcrip- A tion does not activate tnpA expression and terminates ∼85% tnpAtrunc-255 UUUUUU of upstream transcripts (23). Second, translation of tnpA +1 255 is strongly repressed by mRNA secondary structure that (100) ~660 AUG includes the Shine-Dalgarno sequence (SD) (Figure 1B). tnpA This stem-loop element represses tnpA expression 20-fold by preventing 30S ribosome binding. Third, art200 base- IS200 (707bp) pairs with tnpA to inhibit ribosome binding, and reduces translation 15-fold. Lastly, tnpA translation is inhibited di- art200 rectly by the RNA-binding protein Hfq, which recognizes a sequence immediately upstream of the SD and accord- 5’ B 3’ ingly stericly occludes ribosome binding. The three post- transcriptional mechanisms act independently and together Downloaded from https://academic.oup.com/nar/article/45/9/5470/3038232 by guest on 06 October 2021 supress translation of tnpA by at least 750-fold, ensuring 5’ art200 3’ almost no TnpA protein is produced (1). While these reg- ulatory mechanisms appear to be redundant, tnpA expres- M1 sion is reasonably high in S. Typhimurium for a transpo- son (∼10% the expression of hfq in mid-exponential phase (25)). It therefore appears that IS200 elements have evolved 50 AUG to maintain moderate transcription of tnpA from an IS200 5’ SD ...3’ encoded promoter, but close to no synthesis of TnpA. An- 100 other noteworthy feature of IS200-encoded RNAs is that AUG art200 expression appears to be growth phase regulated, 5’ SD ...3’ Degradation with increased expression when S. Typhimurium transitions tnpA trunc to stationary phase in rich media, as well as in growth media Transcript that stimulate Salmonella pathogenicity island (SPI) expres- C trunc sion (Supplementary Figure S1; (24)). Additionally, art200 tnpA art200 tnpA interacts with Hfq in vivo, although Hfq is dispensable for antisense regulation of tnpA expression. Intriguingly, while M1 Relative pTet-tnpAtrunc art200 expression is increased in stationary-phase, tnpA ex- Expression pression decreases ∼5-fold (25), which may indicate that high art200 expression is altered to control tnpA RNA levels. pTet-tnpA WT trunc One explanation for the unusual characteristics of IS200- Plasmid encoded RNAs is that a moderately expressed but never translated tnpA provides a way in which IS200 transposition Vector low could be rapidly activated under certain conditions. How- ever, previous work found that IS200 transposition is re- markably rare, even when post-transcriptional regulation is Figure 1. IS200 and experimental approach. (A) IS200 encodes a trans- completely eliminated (1). With respect to art200’s expres- posase mRNA (tnpA, red) and an antisense RNA (art200, blue). The sion patterns and Hfq-binding properties, this could simply tnpAtrunc-255 transcript encodes the first 255 nt of tnpA fused to the last 108 nt of SgrS (black, includes an intrinsic terminator) and is expressed reflect stochastic evolution of the promoter and sequence from the Tet promoter. (B) Approach used to deplete art200. Pairing be- of a regulatory RNA. A more intriguing explanation for tween tnpA (red) and art200 (blue) results in degradation of art200. The the peculiar properties of tnpA and art200 is that one or M1 mutation alters three critical nucleotides in the terminal loop of tnpA and prevents pairing with art200. The sequestered Shine-Dalgarno se- both IS200-encoded RNAs serves a regulatory role inde- quence (SD) of tnpA is indicated with a box and the translation start pendent of controlling transposition. In this scenario, an codon (AUG) is shown. (C) Heat map showing expected expression of IS200 encoded RNA might provide a selective advantage IS200 RNAs in Salmonella Typhimurium LT2 containing plasmids over- to Salmonella spp. and accordingly explain the conservation expressing either wild-type (WT) or M1 forms of tnpAtrunc-255. Note that and high copy number of this transposon. ‘tnpA’ signifies endogenous
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