Small RNA-Mediated Activation of Sugar Phosphatase Mrna Regulates Glucose Homeostasis

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Small RNA-Mediated Activation of Sugar Phosphatase mRNA Regulates Glucose Homeostasis

Kai Papenfort,1,2 Yan Sun,3 Masatoshi Miyakoshi,1 Carin K. Vanderpool,3,* and Jo¨ rg Vogel1,* 1Institute for Molecular Infection Biology, University of Wu¨ rzburg, Wu¨ rzburg 97070, Germany 2Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA 3Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA *Correspondence: [email protected] (C.K.V.), [email protected] (J.V.) http://dx.doi.org/10.1016/j.cell.2013.03.003

SUMMARY glycolytic flux, their intracellular levels must be tightly regulated since high levels are toxic and strongly impair cell growth (Irani Glucose homeostasis is strictly controlled in all and Maitra, 1977; Kadner et al., 1992) and cause DNA damage domains of life. Bacteria that are unable to balance (Bucala et al., 1985; Lee and Cerami, 1987). Similarly, many non- intracellular sugar levels and deal with potentially metabolizable sugars can cause phosphosugar stress. For toxic phosphosugars cease growth and risk being example, the glucose analog a-methyl-glucoside (a-MG) is outcompeted. Here, we identify the conserved haloa- efficiently imported by PtsG, the major glucose transporter of a cid dehalogenase (HAD)-like enzyme YigL as the pre- Escherichia coli and Salmonella (Jahreis et al., 2008). -MG-6- phosphate accumulates in the cytoplasm and can terminate viously hypothesized phosphatase for detoxification bacterial growth (Pikis et al., 2006; Rogers and Yu, 1962). of phosphosugars and reveal that its synthesis Given their importance for carbohydrate uptake and is activated by an Hfq-dependent small RNA in metabolism, glucose uptake genes are subject to extensive tran- Salmonella typhimurium. We show that the scriptional control (Jahreis et al., 2008). Moreover, high phos- glucose-6-P-responsive small RNA SgrS activates phosugar levels trigger destabilization of the ptsG messenger YigL synthesis in a translation-independent fashion RNA (mRNA), reducing its half-life by 10-fold to less than by the selective stabilization of a decay intermediate 30 s (Kimata et al., 2001; Morita et al., 2003). This posttranscrip- of the dicistronic pldB-yigL messenger RNA (mRNA). tional repression of ptsG is mediated by an RNA-based control Intriguingly, the major endoribonuclease RNase E, circuit involving the regulatory RNA SgrS (Vanderpool and Got- previously known to function together with small tesman, 2004). Activated by its cognate transcription factor RNAs to degrade mRNA targets, is also essential SgrR in response to phosphosugar stress (Vanderpool and Got- tesman, 2007), SgrS represses the ptsG mRNA at the level of for this process of mRNA activation. The exploitation translation, sequestering its ribosome binding site (RBS) via a of and targeted interference with regular RNA turn- short base-pairing interaction (Kawamoto et al., 2006; Vander- over described here may constitute a general route pool and Gottesman, 2004). Like many small regulatory RNAs for small RNAs to rapidly activate both coding and (sRNAs) of E. coli and Salmonella, SgrS depends upon the noncoding genes. RNA chaperone Hfq (Vogel and Luisi, 2011) for both its activity and stability. The Hfq-dependent base pairing by SgrS prevents INTRODUCTION the translation of PtsG protein, while the simultaneous recruit- ment of the major endoribonuclease RNase E via Hfq actively Control of glucose homeostasis is a key regulatory function that promotes ptsG mRNA decay (Morita et al., 2005). has been described in all kingdoms of life (Jahreis et al., 2008; Although SgrS can restrict the synthesis of new glucose Polakof et al., 2011; Smeekens et al., 2010). Uptake of glucose is transporters to alleviate phosphosugar stress, this mechanism accompanied by a phosphorylation event that generates the phos- offers no immediate remedy for the continued accumulation of phosugar glucose-6-phosphate (G6P). The charged phosphate sugar phosphates by existing PtsG transporters since protein group is vital for retention of intracellular glucose since it prevents half-lives normally exceed those of mRNAs (Maier et al., 2011). diffusion back across the cell membrane. In higher organisms, Indeed, we have discovered that the SgrS-induced reduction glucose is phosphorylated by hexokinases after uptake (Polakof of PtsG protein levels is slow; treatment of Salmonella with et al., 2011; Smeekens et al., 2010), whereas bacteria commonly a-MG decreased PtsG protein levels only 2-fold after 80 min generate G6P during membrane translocation of glucose by the (i.e., almost three generations) (Figure 1A). How can this slow phosphotransferase system (PTS) (Jahreis et al., 2008). reduction in protein levels restore glucose homeostasis and pro- Although G6P and other phosphosugars are key metabolic in- tect cells from the growth-inhibitory effects of phosphosugar termediates required to produce ATP and NADH and maintain accumulation?

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Figure 1. YigL Is Induced by SgrS and Required to Counteract Phosphosugar Stress (A) YigL and PtsG proteins or SgrS RNA were probed on western or northern blots, respectively, prior to (0 min) and following a-MG treatment (10–80 min) of Salmonella enterica wild-type (WT) and DsgrS in exponential growth phase. (B) Overnight growth of selected E. coli gene disruption strains (as indicated) on sugar-supplemented minimal media plates show that loss of YigL, unlike other HAD phosphatases (OtsB, YniC, YidA, YfbT, and YbiV), phenocopies the growth defect of DsgrS in the presence of a-MG or 2-deoxyglucose. (C) Salmonella wild-type or DsgrS strains carrying inducible pBAD expression plasmids were stressed with a-MG in logarithmic phase. Subsequent ectopic expression (induced with L-arabinose) of YigL fully rescues DsgrS bacteria; the active-site mutant YigLK191A fails to rescue. Error bars indicate the SD of three independent biological replicates. See also Figures S1, S2, and S7.

In eukaryotes, carbohydrate dephosphorylation is a prerequi- networks employ noncoding RNAs to rapidly adjust to stress. site for efficient efflux (Berg et al., 2002). Likewise, there is RNA activation mechanisms like the one we describe here will evidence to suggest that unidentified bacterial phosphatases immediately impact target protein levels, in contrast with dephosphorylate carbohydrates prior to efflux (Haguenauer sRNA-mediated mRNA repression, where the greater stability and Kepes, 1972; Thompson and Chassy, 1983; Winkler, of proteins relative to mRNAs dictates a delay. This study may 1971). However, despite decades of work on the regulation of therefore illustrate a general strategy for posttranscriptional sugar metabolism, the phosphatases involved and their physio- regulation that provides multiple temporal layers of target logical roles have remained unclear. Here, we identify the control. conserved YigL protein as one of these hypothesized phosphatases, demonstrate its central role in phosphosugar stress atten- RESULTS uation and glucose homeostasis, and reveal a mechanism of posttranscriptional control whereby SgrS activates the yigL SgrS Activates the Phosphatase-Encoding yigL Gene mRNA to achieve stress relief. upon Phosphosugar Stress Physiologically, the sRNA-based control of yigL provides a A recent screen for SgrS targets in Salmonella (Papenfort et al., crucial mechanism for rapid growth rescue and bacterial survival 2012) identified several repressed mRNAs; e.g., the PTS-encod- during phosphosugar stress and demonstrates how regulatory ing ptsG and manXYZ mRNAs, whose regulation is conserved

Cell 153, 426–437, April 11, 2013 ª2013 Elsevier Inc. 427 between E. coli and Salmonella (Rice and Vanderpool, 2011; response to sugar stress caused by naturally occurring sugars Vanderpool and Gottesman, 2004), and the sopD mRNA encod- entering glycolysis. ing a Salmonella-specific virulence factor (Papenfort et al., 2012). However, SgrS also rapidly upregulated the yigL mRNA, encod- SgrS Mediates Discoordinate Expression of the ing a protein that belongs to the large and ubiquitous family of pldB-yigL Operon mRNA haloacid dehalogenase (HAD)-like phosphatases (Figure S1 In Salmonella and other bacteria (Figures 2A and S2C), yigL is available online; Koonin and Tatusov, 1994). Preferred sub- located between pldB (phospholipase 2) and yigM (putative strates of these enzymes are phosphorylated sugars, but which transport protein). The pldB and yigL genes are cotranscribed one acts on G6P, a key sugar intermediate in glycolysis, is (Kro¨ ger et al., 2012; Figure S3A). However, SgrS specifically unknown. Intriguingly, in vitro experiments testing substrate regulated yigL and not pldB in our previous target screen (Pa- specificity of purified YigL reported high affinity toward several penfort et al., 2012). To validate putative discoordinate operon phosphosugars including G6P (Kuznetsova et al., 2006), sug- expression, we monitored SgrS-dependent changes in tran- gesting a role for YigL activation in the SgrS-controlled phospho- script abundance from the individual cistrons. SgrS strongly sugar stress response. elevated the expression of yigL (14.5-fold) and had no effect Treatment of Salmonella wild-type and DsgrS strains with on the two flanking genes (Figure 2B). Since transcription of a-MG showed that YigL synthesis was activated under stress pldB and yigL is coupled, the SgrS-mediated activation of yigL conditions and that SgrS was essential for this activation must be posttranscriptional. In support of this hypothesis, acti- (Figure 1A, lanes 1–5 versus 6–10). Further experiments using vation of yigL did not occur in the absence of Hfq (Figure S3B), an E. coli K12 DyigL strain revealed that the absence of yigL re- an essential RNA chaperone for SgrS activity (Kawamoto et al., sults in an a-MG-induced growth defect that transiently 2006; Papenfort et al., 2012). mimicked the previously observed (Vanderpool and Gottesman, Primer extension analysis using a yigL-specific primer re- 2004) phenotype of the DsgrS mutant (Figure S2A). Thus, vealed that the yigL transcript stabilized by SgrS had a 50 end SgrS-dependent activation of the phosphatase YigL may be within the upstream pldB gene (Figure S4A), suggesting that it particularly relevant during the early phase of the phosphosugar represents a processed form of the pldB-yigL mRNA. A primer stress response. specific for the 30 end of pldB gave the same result (Figure 2C, lanes 5 and 6), corresponding to a 50 end at position U+841 (rela- In Vivo Relevance of the YigL Phosphatase tive to the pldB +1 position). This stable 50 end is within the pldB Several conserved bacterial HAD phosphatases in addition to reading frame, 191 nt upstream of the yigL start codon (Fig- YigL (namely, OtsB, YniC, YidA, YfbT, and YbiV) have affinity ure S4B), and is unlikely to result from de novo transcription; for phosphorylated sugar substrates in vitro (Kuznetsova et al., the region lacks canonical promoter motifs and differential 50 2006). To examine their relevance for combating glucose-related rapid amplification of complementary DNA ends (RACE) indi- phosphosugar stress in vivo, we tested corresponding E. coli cated a 50 monophosphate end, which typically results from mutants for growth defects in the presence of a-MG or another RNA processing (Figure S5A). Note that the RNA processing glucose analog, 2-deoxyglucose. These stressors exclusively must be SgrS independent since the same 50 end is observed restricted the growth of the DsgrS and DyigL strains in wild-type and DsgrS bacterial strains (Figures 2D and S4A). (Figure 1B). Thus, the SgrS-activated YigL protein is likely the In sum, the data suggest that SgrS activates YigL synthesis by hypothesized enzyme that dephosphorylates glucose-derived increasing the abundance of a decay intermediate of the dicis- phosphosugars (Winkler, 1971). tronic pldB-yigL mRNA. To further understand the role of YigL during phosphosugar stress, yigL expression was uncoupled from SgrS and phospho- RNase E Cleavage of mRNA Is a Prerequisite for sugar stress (plasmid pBAD-YigL, Figure 1C). Induction of yigL in SgrS-Mediated YigL Activation DsgrS cells challenged with a-MG promoted rapid recovery from RNase E, an endoribonuclease with a preference for A/U-rich the stress (Figure 1C). A lysine 191 to alanine (K191A) mutation in single-stranded regions (Belasco, 2010; Carpousis et al., 2009; the enzyme’s catalytic center (Koonin and Tatusov, 1994) fully Mackie, 2013), is responsible for the majority of mRNA turnover abrogated recovery from a-MG stress (Figure 1C), further sup- in Salmonella and E. coli. To test whether RNase E generated the porting our model that SgrS activates production of YigL to yigL mRNA species for SgrS-mediated activation, we utilized a dephosphorylate toxic sugar compounds. Salmonella rne-ts strain that produces an RNase E protein that To address whether YigL activity was more generally required is rapidly inactivated when cells are shifted to 44C(Figueroa- to deal with phosphosugars, we tested phenotypes of sgrS and Bossi et al., 2009). Northern blots showed that inactivation of yigL mutants in a pgi (phosphoglucose isomerase) mutant back- RNase E indeed suppressed SgrS-induced accumulation of pro- ground in which impaired glycolysis causes an accumulation of cessed yigL mRNA (Figure S5B, lanes 7 and 8) and also led to G6P and chronic phosphosugar stress (Kimata et al., 2001). accumulation of putative full-length pldB-yigL mRNA (Fig- Growth of sgrS/pgi and yigL/pgi mutant strains on glucose or ure S5B, lanes 6 and 8), which implicates RNase E as a key factor trehalose (PTS sugars that enter glycolysis as G6P) was strongly in general turnover and SgrS-specific activation of yigL. In sup- inhibited (Figure S2B), whereas strains with corresponding single port of this hypothesis, basal processing of pldB-yigL mRNA mutations had no growth phenotype. Further, all strains grew (Figure 2D, lanes 6 and 8) and SgrS-mediated accumulation of well on maltose and xylose, non-PTS sugars that would not yigL mRNA (Figure 2D, lanes 3, 4, 7, and 8) and YigL protein syn- cause G6P accumulation. Thus, YigL is also required for the thesis (Figure 2E, lanes 3, 4, and 8) were dependent on functional

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Figure 2. SgrS Promotes Discoordinate Expression of the pldB-yigL Operon (A) The pldB-yigL-yigM locus of Salmonella. Gene synteny is observed in other species including E. coli (Figure S2C). Wavy lines represent the predicted mRNAs from this region (Kro¨ ger et al., 2012).

(B) Quantitative real-time PCR analysis of pldB, yigL, and yigM transcript levels in response to SgrS overproduction (plasmid pPL-SgrS). RNA levels in cells with control vector were set at a value of 1.0 to calculate fold-expression changes. Error bars indicate the SD of three independent measurements. (C) Primer extension analysis detects SgrS-induced processing of the pldB-yigL mRNA after sRNA induction for 15 and 30 min from a pBAD plasmid in Salmonella DsgrS cells (lanes 1–6). The pldB-yigL-speciﬁc extension product at U+841 (compare with A) was visualized using a radiolabeled primer. Lanes 7–10 prove the expected lack of signal in a DpldB-yigL strain. (D) RNase E is required for SgrS-mediated yigL activation. Primer extension reveals no processing at U+841 in an RNase E-temperature sensitive strain (rne-TS, lanes 5–8) at 44C when RNase E is inactive. (E) Western or northern blots show YigL or SgrS expression in the same context of RNase E activity as above. See also Figures S3 and S5.

RNase E. Collectively, these data demonstrate that RNase-E- (Figure 3A, lane 4), which carries a G176C point mutation in the dependent processing 200 nt upstream of the yigL start codon seed (Papenfort et al., 2012). Combined with the observed precedes SgrS-mediated activation of yigL mRNA. Intriguingly, requirement for Hfq, these experiments strongly suggested although RNase E was previously known to facilitate sRNA- that SgrS activated the yigL transcript via direct base pairing. mediated transcript decay, it now turns out to also have an A series of translational gfp fusions (Urban and Vogel, 2007) essential role in target mRNA activation. was constructed to localize the SgrS binding site within pldB- yigL mRNA (Figure 3B). The full-length fusion, pldB(+1)-yigL::gfp, SgrS Activates the yigL Transcript by Seed Pairing initiates at the +1 transcriptional start site of pldB and was acti- SgrS is an 240 nt sRNA with two main functional domains: a vated 4-fold by SgrS (Figures 3B and 3C). Truncation to the 50-located open reading frame that encodes the 40-amino-acid processing site in pldB(+841)-yigL::gfp (Figures 3B and S4B) still SgrT peptide that can block glucose import (Wadler and Vander- allowed SgrS-dependent activation (Figure 3C), suggesting that pool, 2007), and a 30-located conserved seed region that base SgrS binds downstream of the RNase E site. Further truncations pairs with all known SgrS targets (Kawamoto et al., 2006; Papen- revealed that the putative SgrS binding site resides between fort et al., 2012; Rice and Vanderpool, 2011; Vanderpool and nucleotides 914 and 963 (compare SgrS-dependent induction Gottesman, 2004). Complementation of a Salmonella DsgrS of +963 and +914 reporters, Figures 3B and 3C). The RNAhybrid strain with plasmids carrying various sgrS alleles demonstrated algorithm (Rehmsmeier et al., 2004) predicted an RNA duplex of that SgrT played no role in yigL regulation (Figure 3A, compare the SgrS seed with nucleotides 935–955 within pldB (Figure 3D). lanes 5 and 2), whereas the conserved seed region was crucial This interaction involves a stable 6+12 bp RNA helix (MFE for regulation as evidenced by loss of YigL activation by SgrS* of 34.6 kcal/mol) that engages the critical seed position that,

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A C 5

ECO UGA

SgrS SgrS

SgrS*

SgrS vector YigL 3

1 2 GroEL 1 2 3 4 5 fold-induction 1

0 +1 +841 +914 +963 B pldB yigL gfp D yigL* +932 +960 G , +1 5 , -AGG U C UUAAC -3 ACGCCA GCUCAGUCGCGC –69 +1 1017 |||||| | ||| ||||||| | UGUGGU UGAGUCAGUGUG , 3 ,- GGA U A UACUA -5 +841 190 163 +841 SgrS* +914 +914 E yigL::gfp yigL*::gfp

+963 .

ctr. ctr

SgrS SgrS SgrS* +963 SgrS* GFP

GroEL 123 4 5 6

Figure 3. SgrS Binds in the pldB Coding Sequence (A) Western blot of YigL protein in Salmonella DsgrS carrying the indicated expression plasmids. YigL is equally upregulated by Salmonella and E. coli SgrS (SgrSEco), independent of a functional sgrT ORF (premature stop codon in SgrSUGA). However, a G/C mutation in the SgrS seed region (SgrS*, see also below) abrogates upregulation, suggesting activation by base pairing. (B) Overview of the pldB-yigL::gfp reporters employed to identify the SgrS-pldB interaction site. Scissors indicate the RNase E-dependent processing at U+841. (C) Regulation of the different pldB-yigL::gfp reporters by SgrS. The SD of three independent biological replicates is indicated by error bars. (D) The SgrS-pldB RNA interaction. Numbering for pldB is relative to A of start codon AUG, and for SgrS counting from +1 site. Vertical arrows denote compensatory nucleotide changes. Note that the yigL* mutation lies in pldB ORF. (E) Western blot showing activation of gfp reporters for yigL or yigL* mutant by SgrS or compensatory SgrS* RNA, respectively, validates base pairing in vivo. See also Figure S5.

when mutated in SgrS*, impairs YigL activation (see Figure 3A). ability of SgrS to increase YigL protein levels. However, activa- The base-pairing interaction was validated in vivo with a tion was restored when ribosomes were prevented from reach- compensatory C947G point mutation in the pldB(+1)-yigL*::gfp ing the SgrS site by the introduction of a premature stop codon reporter, which restores activation by SgrS* (Figure 3E, lanes in pldB. Together, these data suggest that activation of yigL by 3, 5, and 6). Thus, SgrS activates YigL synthesis by seed pairing SgrS requires initial RNase E cleavage within the upstream in pldB, far upstream of the yigL start codon. open reading frame (ORF) to remove ribosomes and allow ac- The demonstration that SgrS directly base pairs with the pldB cess to the target site. coding sequence provides a potential rationale for the requirement of RNase E for activation of YigL expression. During SgrS Pairing Stabilizes the yigL mRNA translation of the dicistronic pldB-yigL mRNA, ribosomes could Seeking to understand how SgrS activates YigL expression, we prevent SgrS binding or displace bound SgrS, owing to the could eliminate that SgrS inﬂuenced yigL at the level of mRNA strong RNA helicase activity of 70S ribosomes (Takyar et al., synthesis because both its transcription (with pldB; Figure 2B) 2005). Moreover, the physical coupling of the transcription and and the RNA processing required for activation (Figure 2D) translation machineries (Svetlov and Nudler, 2012) may further were SgrS-independent events. Consequently, we tested restrict access of SgrS to the translated full-length pldB-yigL whether SgrS activated yigL by interfering with mRNA decay, mRNA. To test whether processing within pldB serves to free measuring mRNA stability as a function of base pairing. In the the SgrS binding site, we manipulated ribosome occupancy on absence of sRNA pairing, the yigL mRNA exhibited a half-life the chromosomally encoded pldB-yigL mRNA and tested the of 1.5 min (Figure 4A, pBAD or pBAD-SgrS*), whereas pairing effect on SgrS-mediated activation of YigL (Figure S4C). As pre- with SgrS increased the half-life to 9 min (Figure 4A, pBAD- dicted by our model, an increase in ribosome occupancy in pldB SgrS), revealing that mRNA stabilization underlies SgrS activa- by an improved Shine-Dalgarno sequence strongly impaired the tion of YigL synthesis.

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C D

Figure 4. Posttranscriptional Activation of yigL Is Independent of Translation Initiation (A) RNA half-life measurements show that base pairing by SgrS stabilizes the yigL mRNA. Decay of yigL mRNA was followed after stop of transcription in Salmonella DsgrS expressing SgrS or pairing-deficient SgrS* induced from pBAD plasmids 10 min before the half-life assay. SgrS RNA was probed in the pBAD- SgrS sample. Error bars indicate the SD of three independent biological replicates. (B) The pldB-ompX::gfp reporter and minigene constructs, and location of RNase E cleavage (scissors) and SgrS binding sites. The minigene (30 end of pldB) produces a noncoding RNA containing the SgrS site. Ribosome occupancy of the SgrS site was restored in ‘‘pldB mini+21aa ORF’’ with a short artificial reading frame. (C) Successful regulation of pldB-ompX::gfp reporters by SgrS proves that activation is determined by sequences in pldB (compare to Figure 3C). The SD of three independent biological replicates is indicated by error bars. (D) Quantitative real-time PCR analysis of pldB minigene activation, with or without an artificial 21aa ORF, in a Salmonella DsgrS background. Fold activation was determined 5, 10, and 15 min after induction of pBAD-SgrS relative to empty pBAD vector. Error bars indicate the SD of three independent measurements. See also Figures S4, S5, S6, and S7.

The prototypical mechanism of sRNA-mediated mRNA synthesis, still permitted mRNA stabilization by SgrS (Fig- activation is ‘‘anti-antisense’’ pairing in which the sRNA disrupts ure S6). Thus, activation seemed to rely upon a translation- an intrinsic inhibitory structure of the mRNA that normally independent mRNA stabilization that was determined solely by sequesters the target’s RBS and thus impairs initiation of protein the RNA duplex formed between SgrS and the 30 coding synthesis. Since translation protects against ribonucleases, the sequence of pldB. steady-state level of the activated target mRNA may also in- In support of a translation-independent mechanism, SgrS also crease (Fro¨ hlich and Vogel, 2009; Gottesman and Storz, 2011). stabilized a 120 nt untranslated RNA generated by a synthetic To identify a potential translational activation mechanism that minigene that comprises 64 bp of pldB sequence around the would explain the stabilization of yigL mRNA, we initially focused SgrS target site (Figures 4B and 4D). Thus, SgrS posttranscrip- on potential cis-regulatory elements downstream of the critical tionally activates its target through base pairing far upstream of processing site in pldB-yigL. Unexpectedly, however, we the start codon, without directly regulating translation. Impor- observed that SgrS could activate reporter constructs contain- tantly, SgrS activation of the minigene RNA was abolished by ing fusions of pldB to an ompX::gfp reporter (i.e., lacking the introduction of a 21 aa translated ORF covering the SgrS target RBS of yigL) to about the same degree as the corresponding site (Figures 4B and 4D). This further supports the model that yigL::gfp reporters (compare Figures 4B and 4C to Figures 3B RNase E cleavage in the pldB ORF is required to free the and 3C). Further, inactivation of the yigL start codon (AUG/ mRNA of elongating ribosomes and allow SgrS access for AGG mutation), although fully abrogating reporter protein pairing and stabilization of the processed yigL mRNA.

Cell 153, 426–437, April 11, 2013 ª2013 Elsevier Inc. 431 A sis, levels of the synthetic pldB minigene transcript increase by 5-fold in the temperature-sensitive rne-ts strain shifted to the nonpermissive temperature (Figure 5A). This argues that RNase E determines mRNA stability in this region. Moreover, when the relevant fragment of the pldB mRNA (nucleotides 742–983; Fig- ure S5C) was exposed to puriﬁed RNase E in vitro, two stable cleavage products were generated (Figure S5D): one precisely matched the pldB cleavage in vivo at positions U+840/841 (Fig- ures 2C and S5A), whereas the second cleavage site (+948 BCto +955 region) occurred further downstream within the SgrS binding site in pldB (Figures S5C and 3D). This result strongly suggested that SgrS pairing at this position could protect the mRNA from cleavage. Next, we employed the RNase E in vitro cleavage assay and radiolabeled RNAs to compare the accumulation of pldB or SgrS processing products over time in the presence of increasing concentrations of unlabeled SgrS or pldB, respectively (Figures 5B and 5C; also see Figure S5F for processing products). Again, RNase E produced two stable cleavage products from the pldB transcript (Figure S5F). Of note, RNase E also targeted the sRNA, cleaving at nucleotides 177–179 and 203 of the Salmonella SgrS sequence (Figures S5E and S5F). Simulta- neous addition of SgrS and RNase E to the pldB cleavage reaction reduced the accumulation of the downstream pldB cleavage product corresponding to nucleotides 948–955 of the full-length pldB transcript by 2-fold after 60 min of incubation (Figure S5F). Cleavage at this site was further decreased in the D presence of increasing SgrS concentrations (Figures 5B and 5C). The presence of pldB RNA likewise impacted SgrS decay as it inhibited cleavage at positions +177–179 (Figures 5B ad 5C), which are engaged in base pairing with pldB (Figure 3D) and all other known SgrS targets (Kawamoto et al., 2006; Papen- fort et al., 2012; Rice and Vanderpool, 2011; Vanderpool and Gottesman, 2004). In summary, our data suggest that SgrS com- petes with RNase E for access to pldB at position 948–955 and that successful SgrS-pldB base pairing counteracts endonucleolytic decay by a mutual protection mechanism. Figure 5. SgrS Antagonizes pldB Cleavage by RNase E (A) Northern blot of noncoding pldB minigene RNA in Salmonella DsgrS and Efﬁcient Export of Toxic Sugar Compounds Requires DsgrS/rneTS (RNase E inactive at 44C) strains. Cells were grown at 28Cto SgrS-Mediated Activation of YigL

OD600 of 1.0, split (time point 0 min), and cultivated further at the indicated To evaluate the physiological importance of the posttranscrip- temperatures. tional activation of yigL, we examined the ability of bacteria to (B) In vitro synthesized, radiolabeled pldB RNA (see Figure S5C) was incubated overcome sugar stress and export the accumulated phosphory- with RNase E for 60 min in the absence or presence of nonlabeled SgrS RNA lated sugars as a function of SgrS-pldB base pairing. We utilized (ratios as indicated). The autoradiograph shows the pldB degradation prod- a ucts after denaturing electrophoresis. Size markers are to the left. An arrow previous observations that -MG induces rapid accumulation of indicates the 948–955 cleavage in the pldB RNA (see Figure S5C). SgrS in wild-type bacteria and its subsequent removal from the (C) Analogous to (B), but radiolabeled SgrS RNA incubated with RNase E and medium leads to a rapid decline in SgrS levels (Vanderpool unlabeled pldB RNA. The 177–179 cleavage product (see Figure S5E) is and Gottesman, 2004); this latter relief of stress involves efﬂux indicated. of neutral a-MG (Rogers and Yu, 1962; Sun and Vanderpool, (D) Quantiﬁcation of signals from (B) and (C), based on triplicate experiments. 2011; Winkler, 1971). Thus, we compared the kinetics of SgrS in- Error bars indicate the SD of three independent replicates. See also Figures S4, S5, and S6. duction and depletion between four E. coli strains: wild-type, the DyigL mutant, a pldB* strain with a chromosomal point mutation that disrupts base pairing with SgrS, and a repaired pldB strain Target Activation through Direct Competition with that served as wild-type control for pldB* (Figure S7A). All strains RNase E displayed comparable levels of SgrS after sugar-stress induction The above results raised the possibility that SgrS pairing with the (Figure 6A). In stark contrast to wild-type cells, however, removal pldB region protects the processed yigL mRNA from further of a-MG failed to lower SgrS levels in DyigL cells (Figure 6A, RNase E-mediated mRNA decay. Consistent with this hypothe- compare DyigL to WT and pldB). Similarly, mutation of the

432 Cell 153, 426–437, April 11, 2013 ª2013 Elsevier Inc. A B

Figure 6. Posttranscriptional Activation of YigL Is Required for a-MG Efﬂux (A) Northern blot of SgrS RNA to follow phosphosugar stress recovery of E. coli cells mutated for SgrS-pldB base pairing. Samples were taken from untreated () cells, cells treated with a-MG for 15 min, and treated cells at 10, 20, and 40 min after wash and reinoculation into fresh media. In strain pldB*, a chromosomal point mutation in the SgrS site in pldB prevents base pairing. The pldB strain serves as isogenic control to pldB*. (B) Export of a-MG is retarded when yigL is missing or SgrS cannot activate it (pldB* mutant). The above strains were treated with radiolabeled [14C] a-MG and then diluted (time point 0 min) to follow a-MG efﬂux for 50 min. Error bars indicate the SD of three independent biological replicates. See also Figure S7.

SgrS target site strongly reduced recovery from phosphosugar and Storz, 2011). For the large class of Hfq-dependent sRNAs stress (Figure 6A, pldB*; Figure S7A). Thus, the absence of (Chao et al., 2012; Vogel and Luisi, 2011), negative regulation YigL or disruption of YigL activation through SgrS both equally is typically at the level of translation, but recent studies have cause chronic SgrS induction and impaired recovery from phos- uncovered repression mechanisms in which mRNA stability phosugar stress. control is equally important or the primary mechanism (Des- To understand why the activation of YigL is required for stress noyers et al., 2009; Pfeiffer et al., 2009). Despite a burgeoning recovery, we monitored efflux rates of 14C-radiolabeled a-MG list of examples of translational activation, positive regulation after removal of external phosphosugar (Figure 6B). Whereas by sRNAs is less understood (Fro¨ hlich and Vogel, 2009). Here, intracellular a-MG levels declined rapidly in wild-type E. coli we describe a pathway of suboperonic gene regulation in which (Figure 6B, WT and pldB), the DyigL strain showed a dramatic the sRNA SgrS intercepts and stabilizes an intermediate of delay in a-MG efflux (Figure 6B). Importantly, the single point mRNA turnover to selectively upregulate a downstream cistron mutation in pldB* that disrupts binding of SgrS and stabilization of a polycistronic mRNA (Figure 7). This mechanism constitutes of the yigL mRNA displays a similar defect in efflux rate as the an activator arm of the RNA-based response to sugar stress, DyigL strain. (Note that although the rapid a-MG export in which enables the cell to deal with problematic sugars more wild-type cells supports an active efflux mechanism, the slow directly than the comparatively slow repression of sugar uptake decrease of intracellular a-MG levels in DyigL and pldB* cells functions. seems passive and might largely be attributed to growth and The fact that the SgrS RNA utilizes two oppositely acting arms the nonspecific activities of the other HAD phosphatases.) specializing in sugar import (repression arm) and export (activa- Taken together, we have identified YigL as a phosphatase tion arm) is a striking example of regulatory logic that is enabled involved in regulating glucose homeostasis by promoting sugar by the generic ability of Hfq-dependent sRNAs to act as both re- efflux and revealed posttranscriptional activation through SgrS pressors and activators of mRNAs. A further example is McaS as an essential feature of this stress response. Moreover, iden- sRNA, which contributes to the switch from a sessile to a motile tification of this arm of the glucose efflux system in bacteria hints lifestyle of E. coli by simultaneously repressing a major biofilm that the necessity for carbohydrate-export systems has been transcription factor and activating a positive regulator of motility broadly conserved. (Jørgensen et al., 2012; Thomason et al., 2012). The SgrS RNA, now established as a molecule with three separate companion DISCUSSION functions—gene repression (ptsG, manX, sopD), gene activation (yigL), and template for the SgrT peptide—that all collaborate to Regulatory small RNAs that act through short base pairing, control one of the most fundamental parts of metabolism, offers including eukaryotic microRNAs and bacterial sRNAs, are best unique opportunities to understand the kinetics and threshold known as repressors of target mRNAs (Bartel, 2009; Gottesman behavior in RNA-based regulatory circuits.

Cell 153, 426–437, April 11, 2013 ª2013 Elsevier Inc. 433 recognize sRNA-mRNA duplexes for nearby cleavage (Bandyra et al., 2012; Corcoran et al., 2012), it was unexpected that base pairing of SgrS stabilizes the yigL transcript. However, mRNA turnover by RNase E proceeds in discrete steps that are determined by the availability of a preferred downstream cleavage site (Belasco, 2010; Carpousis et al., 2009; Mackie, 2013). We posit that by masking a sensitive cleavage site around nucleotides 948–955 of the pldB mRNA, SgrS interferes with the stepwise 50-to-30 progression of RNase E through the coding sequence of pldB; this slows the decay of the 50 truncated pldB-yigL mRNA intermediate and increases YigL production (Figure 7). Intriguingly, although general mRNA turnover normally marks the end of a transcript’s lifetime, in this case it is essential for positive regulation by a trans-encoded riboregulator. Here, it constitutes the first step in an ordered activation process— partial mRNA decay first, followed by selective stabilization of an mRNA degradation fragment, resulting in increased protein synthesis—which may constitute a general principle of small RNA-mediated target activation. The operonic structure of bacterial genomes is thought to ensure that functionally related genes are coordinately ex- pressed when they are required. However, transcriptional and posttranscriptional mechanisms can mediate condition-dependent uncoupling of individual cistron expression (Adhya, 2003). These mechanisms include favorable RNase E cleavage events (Nilsson et al., 1996), trans-regulation by Hfq-dependent sRNAs in intergenic regions of polycistronic mRNAs (Desnoyers et al., 2009; Møller et al., 2002; Opdyke et al., 2011; Reichenbach et al., 2008; Urban and Vogel, 2008), and sRNA-guided RNase III processing that stabilizes the adjacent cistrons (Opdyke Figure 7. Model of YigL Activation within the Phosphosugar Stress et al., 2011). Our results with SgrS suggest that the global RNase Response E activity, which continuously generates thousands of mRNA in- During membrane translocation glucose is phosphorylated by importing PTS (green barrel), a process that impairs efflux and activates the sugar for termediates, may provide a rich source to uncouple cistrons by downstream metabolic tasks. Accumulation of phosphosugars is toxic and subsequent sRNA-mediated stabilization. However, many such activates SgrS RNA (through the SgrR transcription factor). Aided by Hfq types of regulation may be missed with the current bio- protein, SgrS targets the pldB coding sequence and blocks sustained 50-to-30 computational tools since these typically ignore target sites endonucleolytic turnover of the pldB-yigL discistronic transcript by RNase E. that (as for yigL) are located in mRNA coding sequences. Initial mRNA decay is essential for SgrS-mediated activation. Stabilization of the 0pldB-yigL mRNA fragment increases the levels of HAD phosphatase YigL, A Phosphatase for Bacterial Glucose Detoxification which dephosphorylates the accumulated sugars to facilitate their export via undefined efflux systems (gray barrel). and Homeostasis Organisms must regulate their metabolic flux so that substrate transport and catabolism provide sufficient precursors for A Translation-Independent Pathway of mRNA Activation biomass and energy production while minimizing buildup of by Small RNA potentially toxic metabolic intermediates. When normal control Our genetic and biochemical evidence indicates a mechanism of mechanisms fail, metabolite accumulation can cause impaired trans target activation by direct mRNA stabilization. First, unlike growth or even lysis (Bucala et al., 1985; Irani and Maitra, canonical anti-antisense mechanisms, the yigL RBS is dispens- 1977; Kadner et al., 1992; Lee and Cerami, 1987; Lee et al., able for activation by SgrS (Figure 4C). Second, the activation re- 2009). The mechanisms counteracting such perturbations of quires functional RNA decay (Figure 2D) but not translation, as sugar homeostasis have often remained a mystery, but regulato- both an mRNA with a mutated yigL start codon (Figure S6) and ry RNAs have increasingly been implicated in these processes the noncoding synthetic RNA (Figure 4D) are stabilized. Finally, (Beisel and Storz, 2010). The recent discovery of phosphosugar SgrS protects the critical pldB-yigL region from RNase E cleav- stress control by SgrS RNA has begun to shed light on a robust age in vitro (Figures 5B, S5D and S5F). Interestingly, the activa- regulatory circuit that adjusts sugar influx to needs by repression tion is context dependent as the critical RNase E site cannot of major sugar importers. This report now provides evidence that easily be moved elsewhere in the native pldB mRNA without in addition to controlling sugar influx, SgrS also indirectly pro- loss of regulation (Figure S7B). motes efflux of overly abundant sugars by inducing a sugar Considering that sRNAs, including SgrS, recruit RNase E via phosphatase whose activity is required to produce the neutral Hfq (Aiba, 2007; Pre´ vost et al., 2011) and that RNase E can sugar substrates for an as-yet-undefined efflux pump.

434 Cell 153, 426–437, April 11, 2013 ª2013 Elsevier Inc. Carbohydrate efflux has been reported in many bacterial spe- and is given with the SD of three independent biological replicates as indi- cies (Haguenauer and Kepes, 1972; Reizer and Saier, 1983; cated by error bars. Thompson and Chassy, 1982), including E. coli (Huber et al., 1980; Liu et al., 1999; Winkler, 1971), but its physiological impor- RNA Half-Life Measurements tance remains somewhat enigmatic. The a-MG-6-phosphate After growth to OD600 of 1.0, cells were induced for pBAD-SgrS expression for 30 min and treated with rifampicin (500 mg/ml final concentration) to stop tran- phosphatase required for a-MG efflux was postulated decades scription. RNA was prepared at the time points indicated in the figure legends. ago but remained unidentified (Hagihira et al., 1963; Winkler, Transcript decay was quantified with quantitative real-time PCR (yigL)or 1971). Similarly, unknown phosphatases were implicated in northern blot (SgrS). efflux of 2-deoxyglucose (Thompson and Chassy, 1982) and methyl-b-D-thiogalactoside (Reizer and Saier, 1983). Our data RNase E Cleavage Assay suggest that YigL is one of these ‘‘missing’’ phosphatases In vitro synthesized pldB and/or SgrS RNAs were denatured and allowed and is required at least for the removal of phosphorylated to renature prior to incubation (750 nM RNA concentration) in reaction 2-deoxyglucose and a-MG as an immediate response to reduce buffer for 30 min at 30 C. RNase E (100 nM, purified catalytic domain; gift from Ben F. Luisi) was added for the indicated time before the RNA intracellular stress. Indeed, when yigL is preinduced from a het- was separated in a denaturing gel. Reaction conditions, RNA preparation, erologous promoter prior to stress, cells produce no significant and visualization of cleavage events are detailed in Extended Experimental stress response (Figure S7C). Procedures. We have thus shown that bacteria rely on a biochemical activity that is fundamentally similar to mammals where G6P formed a-Methyl-Glucoside Efflux Assay in the liver from gluconeogenic precursors and/or glycogen re- Overnight cultures in minimal MOPS medium with 0.4% glycerol were diluted quires dephosphorylation by G6Pase for controlled release of 1:200 into fresh medium and cultivated at 37 CtoanOD600 of 0.2. The cells 14 glucose into the bloodstream (Chou et al., 2010). As such, the were incubated with [ C]aMG (ARC; 3.3 mM; 1 mCi/ml) at room temperature SgrS-mediated activation of yigL is an exciting example of how for 20 min and then diluted 1:200 with fresh medium. At the indicated times, 20 ml samples were filtered (0.45 mm pore size) and cellular radioactivity the investigation of a small RNA-based regulatory circuit can was read with a scintillation counter. lead to the discovery of a new metabolic function. It is worth noting that in E. coli alone, numerous other HAD phosphatases SUPPLEMENTAL INFORMATION of unknown function exist. For many, it is known (Kaasen et al., 1992) or inferred (Kuznetsova et al., 2006; Roberts et al., 2005) Supplemental Information includes Extended Experimental Procedures, seven that the physiological substrates for these enzymes are sugar figures, and three tables and can be found with this article online at http://dx. phosphates. Taken together with our results, these observations doi.org/10.1016/j.cell.2013.03.003. imply that HAD family phosphatases play important roles in carbohydrate utilization and metabolic homeostasis that we are only ACKNOWLEDGMENTS just beginning to uncover. We thank B.L. Bassler and B.F. Luisi for comments on the manuscript, B. Plaschke for excellent technical assistance, D. Podkaminski for help with EXPERIMENTAL PROCEDURES cloning and growth curves, B.F. Luisi for the gift of RNase E preparations, S.A. Gorski for help with the manuscript, and K. Fo¨ rstner for help with phylo- Detailed protocols for all sections are described in Extended Experimental genetic analysis. K.P. is supported by a postdoctoral fellowship from the Procedures. Human Frontiers in Science Program. The Vanderpool lab received support from the American Cancer Society (research scholar grant ACS2008-01868), Bacterial Strains and sRNA Plasmids and the Vogel lab received support from DFG Priority Programs SPP1258 For strains, oligonucleotides, and plasmids, see Tables S1, S2, and S3. (Vo875/2-2) and SPP1316 (Vo875/6-1), the BMBF NGFN program (grant Standard growth was at 37C in Luria-Bertani (LB) broth or where 01GS0806), and the Bavarian BioSysNet program. indicated in M9 minimal media supplemented with glycerol (0.2%), ampicillin (100 mg/ml), kanamycin (50 mg/ml), chloramphenicol (20 mg/ml), or L-arabinose Received: August 1, 2012 (0.2%), where appropriate. 2-Deoxyglucose (Sigma, #D8375) and a-MG Revised: January 26, 2013 (Sigma, #M9376) were added at a final concentration of 0.5%. Liquid cultures Accepted: March 1, 2013 were monitored by optical density at 600 nm (OD600). Published: April 11, 2013

Gene Expression Analysis and Reporter Regulation REFERENCES Protocols for transcript detection by northern blot analysis or quantitative real- time PCR, for the study of RNA processing in vivo by primer extension or 50 Adhya, S. (2003). Suboperonic regulatory signals. Sci. STKE 2003, pe22. RACE, and for GFP and FLAG fusion protein detection on western blots Aiba, H. (2007). Mechanism of RNA silencing by Hfq-binding small RNAs. Curr. have been described elsewhere (Papenfort et al., 2009; Urban and Vogel, Opin. Microbiol. 10, 134–139. 2007). Equal loading was validated by detection of GroEL or OmpA proteins Bandyra, K.J., Said, N., Pfeiffer, V., Go´ rna, M.W., Vogel, J., and Luisi, B.F. (western blot) or 5S ribosomal RNA (rRNA; northern blot). Hybridization probes (2012). The seed region of a small RNA drives the controlled destruction and oligonucleotides for quantitative real-time PCR or primer extension are of the target mRNA by the endoribonuclease RNase E. Mol. Cell 47, given in the Extended Experimental Procedures. 943–953. Target regulation using gfp reporter fusions was studied using a well- established two-plasmid system (Urban and Vogel, 2007)inSalmonella Bartel, D.P. (2009). MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233. DsgrS cells. GFP ﬂuorescence of cells was determined at OD600 of 2.0, using a Victor3 plate reader (Urban and Vogel, 2007). Fold induction is Beisel, C.L., and Storz, G. (2010). Base pairing small RNAs and their roles in the ratio of the cellular GFP signal from SgrS-expressing to control cells global regulatory networks. FEMS Microbiol. Rev. 34, 866–882.

Cell 153, 426–437, April 11, 2013 ª2013 Elsevier Inc. 435 Belasco, J.G. (2010). All things must pass: contrasts and commonalities in Koonin, E.V., and Tatusov, R.L. (1994). Computer analysis of bacterial haloacid eukaryotic and bacterial mRNA decay. Nat. Rev. Mol. Cell Biol. 11, 467–478. dehalogenases defines a large superfamily of hydrolases with diverse speci- Berg, J.M., Tymoczko, J.L., and Stryer, L. (2002). Glycolysis is an energy- ficity. Application of an iterative approach to database search. J. Mol. Biol. conversion pathway in many organisms. In Biochemistry (New York: W H 244, 125–132. Freeman). Kro¨ ger, C., Dillon, S.C., Cameron, A.D.S., Papenfort, K., Sivasankaran, S.K., Bucala, R., Model, P., Russel, M., and Cerami, A. (1985). Modification of DNA Hokamp, K., Chao, Y., Sittka, A., He´ brard, M., Ha¨ ndler, K., et al. (2012). The by glucose 6-phosphate induces DNA rearrangements in an Escherichia coli transcriptional landscape and small RNAs of Salmonella enterica serovar plasmid. Proc. Natl. Acad. Sci. USA 82, 8439–8442. Typhimurium. Proc. Natl. Acad. Sci. USA 109, E1277–E1286. Carpousis, A.J., Luisi, B.F., and McDowall, K.J. (2009). Endonucleolytic initia- Kuznetsova, E., Proudfoot, M., Gonzalez, C.F., Brown, G., Omelchenko, M.V., tion of mRNA decay in Escherichia coli. Prog. Mol. Biol. Transl. Sci. 85, 91–135. Borozan, I., Carmel, L., Wolf, Y.I., Mori, H., Savchenko, A.V., et al. (2006). Genome-wide analysis of substrate specificities of the Escherichia coli haloa- Chao, Y., Papenfort, K., Reinhardt, R., Sharma, C.M., and Vogel, J. (2012). An cid dehalogenase-like phosphatase family. J. Biol. Chem. 281, 36149–36161. atlas of Hfq-bound transcripts reveals 30 UTRs as a genomic reservoir of Lee, A.T., and Cerami, A. (1987). Elevated glucose 6-phosphate levels are regulatory small RNAs. EMBO J. 31, 4005–4019. associated with plasmid mutations in vivo. Proc. Natl. Acad. Sci. USA 84, Chou, J.Y., Jun, H.S., and Mansfield, B.C. (2010). Glycogen storage disease 8311–8314. type I and G6Pase-b deficiency: etiology and therapy. Nat. Rev. Endocrinol. Lee, S.J., Trostel, A., Le, P., Harinarayanan, R., Fitzgerald, P.C., and Adhya, S. 6, 676–688. (2009). Cellular stress created by intermediary metabolite imbalances. Proc. Corcoran, C.P., Podkaminski, D., Papenfort, K., Urban, J.H., Hinton, J.C., and Natl. Acad. Sci. USA 106, 19515–19520. Vogel, J. (2012). Superfolder GFP reporters validate diverse new mRNA tar- Liu, J.Y., Miller, P.F., Willard, J., and Olson, E.R. (1999). Functional and gets of the classic porin regulator, MicF RNA. Mol. Microbiol. 84, 428–445. biochemical characterization of Escherichia coli sugar efflux transporters. Desnoyers, G., Morissette, A., Pre´ vost, K., and Masse´ , E. (2009). Small RNA- J. Biol. Chem. 274, 22977–22984. induced differential degradation of the polycistronic mRNA iscRSUA. EMBO J. Mackie, G.A. (2013). RNase E: at the interface of bacterial RNA processing and 28, 1551–1561. decay. Nat. Rev. Microbiol. 11, 45–57. Figueroa-Bossi, N., Valentini, M., Malleret, L., Fiorini, F., and Bossi, L. (2009). Maier, T., Schmidt, A., Gu¨ ell, M., Ku¨ hner, S., Gavin, A.C., Aebersold, R., and Caught at its own game: regulatory small RNA inactivated by an inducible tran- Serrano, L. (2011). Quantification of mRNA and protein and integration with script mimicking its target. Genes Dev. 23, 2004–2015. protein turnover in a bacterium. Mol. Syst. Biol. 7, 511. Fro¨ hlich, K.S., and Vogel, J. (2009). Activation of gene expression by small Møller, T., Franch, T., Udesen, C., Gerdes, K., and Valentin-Hansen, P. (2002). RNA. Curr. Opin. Microbiol. 12, 674–682. Spot 42 RNA mediates discoordinate expression of the E. coli galactose Gottesman, S., and Storz, G. (2011). Bacterial small RNA regulators: versatile operon. Genes Dev. 16, 1696–1706. roles and rapidly evolving variations. Cold Spring Harb. Perspect. Biol. 3, Morita, T., El-Kazzaz, W., Tanaka, Y., Inada, T., and Aiba, H. (2003). Accumu- a003798. lation of glucose 6-phosphate or fructose 6-phosphate is responsible for Hagihira, H., Wilson, T.H., and Lin, E.C. (1963). Studies on the glucose- destabilization of glucose transporter mRNA in Escherichia coli. J. Biol. transport system in Escherichia coli with alpha-methylglucoside as substrate. Chem. 278, 15608–15614. Biochim. Biophys. Acta 78, 505–515. Morita, T., Maki, K., and Aiba, H. (2005). RNase E-based ribonucleoprotein Haguenauer, R., and Kepes, A. (1972). NaF inhibition of phosphorylation and complexes: mechanical basis of mRNA destabilization mediated by bacterial dephosphorylation involved in -methyl-D glucoside transport in E. coli K 12. noncoding RNAs. Genes Dev. 19, 2176–2186. A pH dependant phenomenon sensitive to uncoupling agents. Biochimie 54, Nilsson, P., Naureckiene, S., and Uhlin, B.E. (1996). Mutations affecting mRNA 505–512. processing and fimbrial biogenesis in the Escherichia coli pap operon. Huber, R.E., Pisko-Dubienski, R., and Hurlburt, K.L. (1980). Immediate J. Bacteriol. 178, 683–690. stoichiometric appearance of beta-galactosidase products in the medium Opdyke, J.A., Fozo, E.M., Hemm, M.R., and Storz, G. (2011). RNase III partic- of Escherichia coli cells incubated with lactose. Biochem. Biophys. Res. ipates in GadY-dependent cleavage of the gadX-gadW mRNA. J. Mol. Biol. Commun. 96, 656–661. 406, 29–43. Irani, M.H., and Maitra, P.K. (1977). Properties of Escherichia coli mutants Papenfort, K., Podkaminski, D., Hinton, J.C., and Vogel, J. (2012). The ances- deficient in enzymes of glycolysis. J. Bacteriol. 132, 398–410. tral SgrS RNA discriminates horizontally acquired Salmonella mRNAs through Jahreis, K., Pimentel-Schmitt, E.F., Bru¨ ckner, R., and Titgemeyer, F. (2008). a single G-U wobble pair. Proc. Natl. Acad. Sci. USA 109, E757–E764. Ins and outs of glucose transport systems in eubacteria. FEMS Microbiol. Papenfort, K., Said, N., Welsink, T., Lucchini, S., Hinton, J.C., and Vogel, J. Rev. 32, 891–907. (2009). Specific and pleiotropic patterns of mRNA regulation by ArcZ, a Jørgensen, M.G., Nielsen, J.S., Boysen, A., Franch, T., Møller-Jensen, J., and conserved, Hfq-dependent small RNA. Mol. Microbiol. 74, 139–158. Valentin-Hansen, P. (2012). Small regulatory RNAs control the multi-cellular Pfeiffer, V., Papenfort, K., Lucchini, S., Hinton, J.C., and Vogel, J. (2009). adhesive lifestyle of Escherichia coli. Mol. Microbiol. 84, 36–50. Coding sequence targeting by MicC RNA reveals bacterial mRNA silencing Kaasen, I., Falkenberg, P., Styrvold, O.B., and Strøm, A.R. (1992). Molecular downstream of translational initiation. Nat. Struct. Mol. Biol. 16, 840–846. cloning and physical mapping of the otsBA genes, which encode the osmoreg- Pikis, A., Hess, S., Arnold, I., Erni, B., and Thompson, J. (2006). Genetic ulatory trehalose pathway of Escherichia coli: evidence that transcription is requirements for growth of Escherichia coli K12 on methyl-alpha-D-glucopyr- activated by katF (AppR). J. Bacteriol. 174, 889–898. anoside and the five alpha-D-glucosyl-D-fructose isomers of sucrose. J. Biol. Kadner, R.J., Murphy, G.P., and Stephens, C.M. (1992). Two mechanisms for Chem. 281, 17900–17908. growth inhibition by elevated transport of sugar phosphates in Escherichia Polakof, S., Mommsen, T.P., and Soengas, J.L. (2011). Glucosensing coli. J. Gen. Microbiol. 138, 2007–2014. and glucose homeostasis: from fish to mammals. Comp. Biochem. Physiol. Kawamoto, H., Koide, Y., Morita, T., and Aiba, H. (2006). Base-pairing require- B Biochem. Mol. Biol. 160, 123–149. ment for RNA silencing by a bacterial small RNA and acceleration of duplex Pre´ vost, K., Desnoyers, G., Jacques, J.F., Lavoie, F., and Masse´ , E. (2011). formation by Hfq. Mol. Microbiol. 61, 1013–1022. Small RNA-induced mRNA degradation achieved through both translation Kimata, K., Tanaka, Y., Inada, T., and Aiba, H. (2001). Expression of the block and activated cleavage. Genes Dev. 25, 385–396. glucose transporter gene, ptsG, is regulated at the mRNA degradation step Rehmsmeier, M., Steffen, P., Hochsmann, M., and Giegerich, R. (2004). Fast in response to glycolytic flux in Escherichia coli. EMBO J. 20, 3587–3595. and effective prediction of microRNA/target duplexes. RNA 10, 1507–1517.

436 Cell 153, 426–437, April 11, 2013 ª2013 Elsevier Inc. Reichenbach, B., Maes, A., Kalamorz, F., Hajnsdorf, E., and Go¨ rke, B. (2008). Thomason, M.K., Fontaine, F., De Lay, N., and Storz, G. (2012). A small RNA The small RNA GlmY acts upstream of the sRNA GlmZ in the activation of glmS that regulates motility and biofilm formation in response to changes in nutrient expression and is subject to regulation by polyadenylation in Escherichia coli. availability in Escherichia coli. Mol. Microbiol. 84, 17–35. Nucleic Acids Res. 36, 2570–2580. Thompson, J., and Chassy, B.M. (1982). Novel phosphoenolpyruvate- Reizer, J., and Saier, M.H., Jr. (1983). Involvement of lactose enzyme II of dependent futile cycle in Streptococcus lactis: 2-deoxy-D-glucose uncouples the phosphotransferase system in rapid expulsion of free galactosides from energy production from growth. J. Bacteriol. 151, 1454–1465. Streptococcus pyogenes. J. Bacteriol. 156, 236–242. Thompson, J., and Chassy, B.M. (1983). Regulation of glycolysis and sugar phosphotransferase activities in Streptococcus lactis: growth in the presence Rice, J.B., and Vanderpool, C.K. (2011). The small RNA SgrS controls sugar- of 2-deoxy-D-glucose. J. Bacteriol. 154, 819–830. phosphate accumulation by regulating multiple PTS genes. Nucleic Acids Res. 39, 3806–3819. Urban, J.H., and Vogel, J. (2007). Translational control and target recognition by Escherichia coli small RNAs in vivo. Nucleic Acids Res. 35, 1018–1037. Roberts, A., Lee, S.Y., McCullagh, E., Silversmith, R.E., and Wemmer, D.E. Urban, J.H., and Vogel, J. (2008). Two seemingly homologous noncoding (2005). YbiV from Escherichia coli K12 is a HAD phosphatase. Proteins 58, RNAs act hierarchically to activate glmS mRNA translation. PLoS Biol. 6, e64. 790–801. Vanderpool, C.K., and Gottesman, S. (2004). Involvement of a novel transcrip- Rogers, D., and Yu, S.H. (1962). Substrate specificity of a glucose permease of tional activator and small RNA in post-transcriptional regulation of the Escherichia coli. J. Bacteriol. 84, 877–881. glucose phosphoenolpyruvate phosphotransferase system. Mol. Microbiol. 54, 1076–1089. Smeekens, S., Ma, J., Hanson, J., and Rolland, F. (2010). Sugar signals and molecular networks controlling plant growth. Curr. Opin. Plant Biol. 13, Vanderpool, C.K., and Gottesman, S. (2007). The novel transcription factor 274–279. SgrR coordinates the response to glucose-phosphate stress. J. Bacteriol. 189, 2238–2248. Sun, Y., and Vanderpool, C.K. (2011). Regulation and function of Escherichia Vogel, J., and Luisi, B.F. (2011). Hfq and its constellation of RNA. Nat. Rev. coli sugar efflux transporter A (SetA) during glucose-phosphate stress. Microbiol. 9, 578–589. J. Bacteriol. 193, 143–153. Wadler, C.S., and Vanderpool, C.K. (2007). A dual function for a bacterial Svetlov, V., and Nudler, E. (2012). Unfolding the bridge between transcription small RNA: SgrS performs base pairing-dependent regulation and encodes and translation. Cell 150, 243–245. a functional polypeptide. Proc. Natl. Acad. Sci. USA 104, 20454–20459. Takyar, S., Hickerson, R.P., and Noller, H.F. (2005). mRNA helicase activity of Winkler, H.H. (1971). Efflux and the steady state in alpha-methylglucoside the ribosome. Cell 120, 49–58. transport in Escherichia coli. J. Bacteriol. 106, 362–368.

Cell 153, 426–437, April 11, 2013 ª2013 Elsevier Inc. 437