A spermidine riboswitch class in bacteria exploits a close variant of an aptamer for the enzyme cofactor S-adenosylmethionine.

Natural polyamines such as spermidine and spermine cations have characteristics that make them highly likely to be sensed by riboswitches, such as their general affinity to polyanionic RNA and their broad contributions to cell physiology. Despite previous claims that polyamine riboswitches exist, evidence of their biological functions has remained unconvincing. Here, we report that rare variants of bacterial S-adenosylmethionine-I (SAM-I) riboswitches reject SAM and have adapted to selectively sense spermidine. These spermidine-sensing riboswitch variants are associated with genes whose protein products are directly involved in the production of spermidine and other polyamines. Biochemical and genetic assays demonstrate that representatives of this riboswitch class robustly function as genetic "off" switches, wherein spermidine binding causes premature transcription termination to suppress the expression of polyamine biosynthetic genes. These findings confirm the existence of natural spermidine-sensing riboswitches in bacteria and expand the list of variant riboswitch classes that have adapted to bind different ligands.

Riboswitches.

Riboswitches are structured noncoding RNA domains that are typically found embedded in messenger RNAs, where they sense specific target molecules or elemental ions and regulate gene expression. These RNAs thus serve as genetic switches that can activate or repress gene expression in response to changing levels of their target ligand. To many observers, riboswitches might seem like rare oddities that are not as sophisticated as, or competitive with, the various protein factors that perform these same roles. However, as the number of experimentally validated riboswitch classes increases, and their true biochemical sophistication is recognized, it is becoming clearer that many species from all three domains of life entrust RNAs to make important chemical sensing and gene control decisions without the necessary participation of protein factors.

Differential synthesis of novel small protein times Salmonella virulence program.

Gene organization in operons enables concerted transcription of functionally related genes and efficient control of cellular processes. Typically, an operon is transcribed as a polycistronic mRNA that is translated into corresponding proteins. Here, we identify a bicistronic operon transcribed as two mRNAs, yet only one allows translation of both genes. We establish that the novel gene ugtS forms an operon with virulence gene ugtL, an activator of the master virulence regulatory system PhoP/PhoQ in Salmonella enterica serovar Typhimurium. Only the longer ugtSugtL mRNA carries the ugtS ribosome binding site and therefore allows ugtS translation. Inside macrophages, the ugtSugtL mRNA species allowing translation of both genes is produced hours before that allowing translation solely of ugtL. The small protein UgtS controls the kinetics of PhoP phosphorylation by antagonizing UgtL activity, preventing premature activation of a critical virulence program. Moreover, S. enterica serovars that infect cold-blooded animals lack ugtS. Our results establish how foreign gene control of ancestral regulators enables pathogens to time their virulence programs.

RNA chaperone activates Salmonella virulence program during infection.

Organisms often harbor seemingly redundant proteins. In the bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium), the RNA chaperones CspC and CspE appear to play redundant virulence roles because a mutant lacking both chaperones is attenuated, whereas mutants lacking only one exhibit wild-type virulence. We now report that CspC-but not CspE-is necessary to activate the master virulence regulator PhoP when S. Typhimurium experiences mildly acidic pH, such as inside macrophages. This CspC-dependent PhoP activation is specific to mildly acidic pH because a cspC mutant behaves like wild-type S. Typhimurium under other PhoP-activating conditions. Moreover, it is mediated by ugtL, a virulence gene required for PhoP activation inside macrophages. Purified CspC promotes ugtL translation by disrupting a secondary structure in the ugtL mRNA that occludes ugtL's ribosome binding site. Our findings demonstrate that proteins that are seemingly redundant actually confer distinct and critical functions to the lifestyle of an organism.

Biochemical Validation of a Fourth Guanidine Riboswitch Class in Bacteria.

An intriguing consequence of ongoing riboswitch discovery efforts is the occasional identification of metabolic or toxicity response pathways for unusual ligands. Recently, we reported the experimental validation of three distinct bacterial riboswitch classes that regulate gene expression in response to the selective binding of a guanidinium ion. These riboswitch classes, called guanidine-I, -II, and -III, regulate numerous genes whose protein products include previously misannotated guanidine exporters and enzymes that degrade guanidine via an initial carboxylation reaction. Guanidine is now recognized as the primal substrate of many multidrug efflux pumps that are important for bacterial resistance to certain antibiotics. Guanidine carboxylase enzymes had long been annotated as urea carboxylase enzymes but are now understood to participate in guanidine degradation. Herein, we report the existence of a fourth riboswitch class for this ligand, called guanidine-IV. Members of this class use a novel aptamer to selectively bind guanidine and use an unusual expression platform arrangement that is predicted to activate gene expression when ligand is present. The wide distribution of this abundant riboswitch class, coupled with the striking diversity of other guanidine-sensing RNAs, demonstrates that many bacterial species maintain sophisticated sensory and genetic mechanisms to avoid guanidine toxicity. This finding further highlights the mystery regarding the natural source of this nitrogen-rich chemical moiety.

The phosphorelay BarA/SirA activates the non-cognate regulator RcsB in Salmonella enterica.

To survive an environmental stress, organisms must detect the stress and mount an appropriate response. One way that bacteria do so is by phosphorelay systems that respond to a stress by activating a regulator that modifies gene expression. To ensure an appropriate response, a given regulator is typically activated solely by its cognate phosphorelay protein(s). However, we now report that the regulator RcsB is activated by both cognate and non-cognate phosphorelay proteins, depending on the condition experienced by the bacterium Salmonella enterica serovar Typhimurium. The RcsC and RcsD proteins form a phosphorelay that activates their cognate regulator RcsB in response to outer membrane stress and cell wall perturbations, conditions Salmonella experiences during infection. Surprisingly, the non-cognate phosphorelay protein BarA activates RcsB during logarithmic growth in Luria-Bertani medium in three ways. That is, BarA's cognate regulator SirA promotes transcription of the rcsDB operon; the SirA-dependent regulatory RNAs CsrB and CsrC further increase RcsB-activated gene transcription; and BarA activates RcsB independently of the RcsC, RcsD, and SirA proteins. Activation of a regulator by multiple sensors broadens the spectrum of environments in which a set of genes is expressed without evolving binding sites for different regulators at each of these genes.

Antagonistic functions between the RNA chaperone Hfq and an sRNA regulate sensitivity to the antibiotic colicin.

The RNA chaperone Hfq is a key regulator of the function of small RNAs (sRNAs). Hfq has been shown to facilitate sRNAs binding to target mRNAs and to directly regulate translation through the action of sRNAs. Here, we present evidence that Hfq acts as the repressor of cirA mRNA translation in the absence of sRNA. Hfq binding to cirA prevents translation initiation, which correlates with cirA mRNA instability. In contrast, RyhB pairing to cirA mRNA promotes changes in RNA structure that displace Hfq, thereby allowing efficient translation as well as mRNA stabilization. Because CirA is a receptor for the antibiotic colicin Ia, in addition to acting as an Fur (Ferric Uptake Regulator)-regulated siderophore transporter, translational activation of cirA mRNA by RyhB promotes colicin sensitivity under conditions of iron starvation. Altogether, these results indicate that Fur and RyhB modulate an unexpected feed-forward loop mechanism related to iron physiology and colicin sensitivity.

Regulating iron storage and metabolism with RNA: an overview of posttranscriptional controls of intracellular iron homeostasis.

Iron (Fe) is a double-edged sword for most living organisms. Although it is essential for the catalytic activity of a large number of enzymes, ferrous iron (Fe(2+) ) becomes cytotoxic in the presence of normal respiratory by-products such as H(2) O(2) . Because of this toxicity, intracellular iron concentrations ought to be regulated by elaborated homeostasis systems that, despite decades of extensive studies, have not yet revealed all of their surprising arrays of mechanistic details. Within the last few years, our understanding of iron metabolism has revealed that posttranscriptional regulation represents a major contribution to iron homeostasis in a host of organisms. While the small RNA RyhB regulates iron homeostasis in bacteria, its functional homolog protein Cth2 performs a similar task in yeasts. Recent advances in the elucidation of the mechanism of action and functions of RyhB have been made in Escherichia coli. In addition, other RyhB-like small RNAs have been identified in several bacterial species, such as Pseudomonas aeruginosa, Salmonella enterica, Vibrio cholerae, Neisseria meningitidis, and Shigella spp. These recent findings have shed light on the complexity of iron homeostasis.

A small RNA promotes siderophore production through transcriptional and metabolic remodeling.

Siderophores are essential factors for iron (Fe) acquisition in bacteria during colonization and infection of eukaryotic hosts, which restrain iron access through iron-binding protein, such as lactoferrin and transferrin. The synthesis of siderophores by Escherichia coli is considered to be fully regulated at the transcriptional level by the Fe-responsive transcriptional repressor Fur. Here we characterized two different pathways that promote the production of the siderophore enterobactin via the action of the small RNA RyhB. First, RyhB is required for normal expression of an important enterobactin biosynthesis polycistron, entCEBAH. Second, RyhB directly represses the translation of cysE, which encodes a serine acetyltransferase that uses serine as a substrate for cysteine biosynthesis. Reduction of CysE activity by RyhB allows serine to be used as building blocks for enterobactin synthesis through the nonribosomal peptide synthesis pathway. Thus, RyhB plays an essential role in siderophore production and may modulate bacterial virulence through optimization of siderophore production.

Dealing with oxidative stress and iron starvation in microorganisms: an overview.

Iron starvation and oxidative stress are 2 hurdles that bacteria must overcome to establish an infection. Pathogenic bacteria have developed many strategies to efficiently infect a broad range of hosts, including humans. The best characterized systems make use of regulatory proteins to sense the environment and adapt accordingly. For example, iron-sulfur clusters are critical for sensing the level and redox state of intracellular iron. The regulatory small RNA (sRNA) RyhB has recently been shown to play a central role in adaptation to iron starvation, while the sRNA OxyS coordinates cellular response to oxidative stress. These regulatory sRNAs are well conserved in many bacteria and have been shown to be essential for establishing a successful infection. An overview of the different strategies used by bacteria to cope with iron starvation and oxidative stress is presented here.

The small RNA RyhB activates the translation of shiA mRNA encoding a permease of shikimate, a compound involved in siderophore synthesis.

RyhB is a small RNA (sRNA) that downregulates about 20 genes involved in iron metabolism. It is expressed under low iron conditions and pairs with specific mRNAs to trigger their rapid degradation by the RNA degradosome. In contrast to this, another study has suggested that RyhB also activates several genes by increasing their mRNA level. Among these activated genes is shiA, which encodes a permease of shikimate, an aromatic compound participating in the biosynthesis of siderophores. Here, we demonstrate in vivo and in vitro that RyhB directly pairs at the 5'-untranslated region (5'-UTR) of the shiA mRNA to disrupt an intrinsic inhibitory structure that sequesters the ribosome-binding site (Shine-Dalgarno) and the first translation codon. This is the first demonstration of direct gene activation by RyhB, which has been exclusively described in degradation of mRNAs. Our physiological results indicate that the transported compound of the ShiA permease, shikimate, is important under conditions of RyhB expression, that is, iron starvation. This is demonstrated by growth assays in which shikimate or the siderophore enterochelin correct the growth defect observed for a ryhB mutant in iron-limited media.

Small RNAs controlling iron metabolism.

Iron is one of the most important metals in the metabolism of many organisms, including bacteria, in which it serves as a cofactor in multiple enzymatic reactions. Most of the earlier research on iron regulation in bacteria has focused on the transcriptional regulator Fur and its effect on the many genes involved in iron uptake. More recent work demonstrates the essential role of a small regulatory RNA, RyhB, in iron metabolism. RyhB downregulates a large number of transcripts encoding iron-using proteins, resulting in redistribution of the intracellular iron. Recent advances have been made in the understanding of the small RNAs that modulate the intracellular iron usage in different organisms such as, Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, Shigella flexneri and cyanobacteria.