Silent but deadly: IS200 promotes pathogenicity in Salmonella Typhimurium.

Bacterial transposons were long thought of as selfish mobile genetic elements that propagate at the expense of 'host' bacterium fitness. However, limited transposition can benefit the host organism by promoting DNA rearrangements and facilitating horizontal gene transfer. Here we discuss and provide context for our recently published work which reported the surprising finding that an otherwise dormant transposon, IS200, encodes a regulatory RNA in Salmonella Typhimurium. This previous work identified a trans-acting sRNA that is encoded in the 5'UTR of IS200 transposase mRNA (tnpA). This sRNA represses expression of genes encoded within Salmonella Pathogenicity Island 1 (SPI-1), and accordingly limits invasion into non-phagocytic cells in vitro. We present new data here that shows IS200 elements are important for colonization of the mouse gastrointestinal tract. We discuss our previous and current findings in the context of transposon biology and suggest that otherwise 'silent' transposons may in fact play an important role in controlling host gene expression.

Evolving Mistranslating tRNAs Through a Phenotypically Ambivalent Intermediate in Saccharomyces cerevisiae.

The genetic code converts information from nucleic acid into protein. The genetic code was thought to be immutable, yet many examples in nature indicate that variations to the code provide a selective advantage. We used a sensitive selection system involving suppression of a deleterious allele (tti2-L187P) in Saccharomyces cerevisiae to detect mistranslation and identify mechanisms that allow genetic code evolution. Though tRNASer containing a proline anticodon (UGG) is toxic, using our selection system we identified four tRNASerUGG variants, each with a single mutation, that mistranslate at a tolerable level. Mistranslating tRNALeuUGG variants were also obtained, demonstrating the generality of the approach. We characterized two of the tRNASerUGG variants. One contained a G26A mutation, which reduced cell growth to 70% of the wild-type rate, induced a heat shock response, and was lost in the absence of selection. The reduced toxicity of tRNASerUGG-G26A is likely through increased turnover of the tRNA, as lack of methylation at G26 leads to degradation via the rapid tRNA decay pathway. The second tRNASerUGG variant, with a G9A mutation, had minimal effect on cell growth, was relatively stable in cells, and gave rise to less of a heat shock response. In vitro, the G9A mutation decreases aminoacylation and affects folding of the tRNA. Notably, the G26A and G9A mutations were phenotypically neutral in the context of an otherwise wild-type tRNASer These experiments reveal a model for genetic code evolution in which tRNA anticodon mutations and mistranslation evolve through phenotypically ambivalent intermediates that reduce tRNA function.

A transposon-derived small RNA regulates gene expression in Salmonella Typhimurium.

Bacterial sRNAs play an important role in regulating many cellular processes including metabolism, outer membrane homeostasis and virulence. Although sRNAs were initially found in intergenic regions, there is emerging evidence that protein coding regions of the genome are a rich reservoir of sRNAs. Here we report that the 5΄UTR of IS200 transposase mRNA (tnpA) is processed to produce regulatory RNAs that affect expression of over 70 genes in Salmonella Typhimurium. We provide evidence that the tnpA derived sRNA base-pairs with invF mRNA to repress expression. As InvF is a transcriptional activator of SPI-1 encoded and other effector proteins, tnpA indirectly represses these genes. We show that deletion of IS200 elements in S. Typhimurium increases invasion in vitro and reduces growth rate, while over-expression of tnpA suppresses invasion. Our work indicates that tnpA acts as an sRNA 'sponge' that sets a threshold for activation of Salmonella pathogenicity island (SPI)-1 effector proteins and identifies a new class of 'passenger gene' for bacterial transposons, providing the first example of a bacterial transposon producing a regulatory RNA that controls host gene expression.

A cis-encoded sRNA, Hfq and mRNA secondary structure act independently to suppress IS200 transposition.

IS200 is found throughout Enterobacteriaceae and transposes at a notoriously low frequency. In addition to the transposase protein (TnpA), IS200 encodes an uncharacterized Hfq-binding sRNA that is encoded opposite to the tnpA 5'UTR. In the current work we asked if this sRNA represses tnpA expression. We show here that the IS200 sRNA (named art200 for antisense regulator of transposase IS200) basepairs with tnpA to inhibit translation initiation. Unexpectedly, art200-tnpA pairing is limited to 40 bp, despite 90 nt of perfect complementarity. Additionally, we show that Hfq and RNA secondary structure in the tnpA 5'UTR each repress tnpA expression in an art200-independent manner. Finally, we show that disrupting translational control of tnpA expression leads to increased IS200 transposition in E. coli. The current work provides new mechanistic insight into why IS200 transposition is so strongly suppressed. The possibility of art200 acting in trans to regulate a yet-unidentified target is discussed as well as potential applications of the IS200 system for designing novel riboregulators.

Hfq binds directly to the ribosome-binding site of IS10 transposase mRNA to inhibit translation.

Hfq is a critical component of post-transcriptional regulatory networks in most bacteria. It usually functions as a chaperone for base-pairing small RNAs, although non-canonical regulatory roles are continually emerging. We have previously shown that Hfq represses IS10/Tn10 transposase expression through both antisense RNA-dependent and independent mechanisms. In the current work, we set out to define the regulatory role of Hfq in the absence of the IS10 antisense RNA. We show here that an interaction between the distal surface of Hfq and the ribosome-binding site of transposase mRNA (RNA-IN) is required for repressing translation initiation. Additionally, this interaction was critical for the in vivo association of Hfq and RNA-IN. Finally, we present evidence that the small RNA ChiX activates transposase expression by titrating Hfq away from RNA-IN. The current results are considered in the broader context of Hfq biology and implications for Hfq titration by ChiX are discussed.

Probing Hfq:RNA interactions with hydroxyl radical and RNase footprinting.

RNA footprinting and structure probing techniques are used to characterize the interaction between RNA-binding proteins and RNAs in vitro. Hydroxyl radical footprinting results in the identification of protein binding site(s) in an RNA. Ribonuclease (RNase) structure probing is a complementary technique that also provides information about protein binding sites, as well as RNA structure and possible protein-directed RNA remodeling. Here we provide a comprehensive protocol for studying the interaction between Hfq and an mRNA or sRNA of interest using a combination of RNase A, T1, and V1 as well as hydroxyl radical footprinting techniques. Detailed protocols for in vitro synthesis of (32)P-labeled RNA; formation of Hfq:RNA binary complex(es), RNase, and hydroxyl radical footprinting; preparation and running of sequencing gels; and data analysis are provided.

Tn5 transposition in Escherichia coli is repressed by Hfq and activated by over-expression of the small non-coding RNA SgrS.

BACKGROUND: Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base pairing of mRNAs with trans-encoded sRNAs. It was previously shown that Hfq down-regulates Tn10 transposition by inhibiting IS10 transposase expression at the post-transcriptional level. This provided the first example of Hfq playing a role in DNA transposition and led us to ask if a related transposon, Tn5, is similarly regulated. RESULTS: We show that Hfq strongly suppresses Tn5 transposition in Escherichia coli by inhibiting IS50 transposase expression. However, in contrast to the situation for Tn10, Hfq primarily inhibits IS50 transposase transcription. As Hfq does not typically function directly in transcription, we searched for a transcription factor that also down-regulated IS50 transposase transcription and is itself under Hfq control. We show that Crp (cyclic AMP receptor protein) fits these criteria as: (1) disruption of the crp gene led to an increase in IS50 transposase expression and the magnitude of this increase was comparable to that observed for an hfq disruption; and (2) Crp expression decreased in hfq (-) . We also demonstrate that IS50 transposase expression and Tn5 transposition are induced by over-expression of the sRNA SgrS and link this response to glucose limitation. CONCLUSIONS: Tn5 transposition is negatively regulated by Hfq primarily through inhibition of IS50 transposase transcription. Preliminary results support the possibility that this regulation is mediated through Crp. We also provide evidence that glucose limitation activates IS50 transposase transcription and transposition.