The small RNA RyhB activates the translation of shiAmRNA encoding a permease of shikimate, a compoundinvolved in siderophore synthesis
Karine Prévost, Hubert Salvail,Guillaume Desnoyers, Jean-François Jacques,Émilie Phaneuf and Eric Massé*Département de Biochimie, Groupe ARN, Université deSherbrooke, Sherbrooke, Québec, Canada.
Summary
RyhB is a small RNA (sRNA) that downregulatesabout 20 genes involved in iron metabolism. It isexpressed under low iron conditions and pairs withspecific mRNAs to trigger their rapid degradation bythe RNA degradosome. In contrast to this, anotherstudy has suggested that RyhB also activates severalgenes by increasing their mRNA level. Among theseactivated genes is shiA, which encodes a permeaseof shikimate, an aromatic compound participating inthe biosynthesis of siderophores. Here, we demon-strate in vivo and in vitro that RyhB directly pairsat the 5�-untranslated region (5�-UTR) of the shiAmRNA to disrupt an intrinsic inhibitory structurethat sequesters the ribosome-binding site (Shine-Dalgarno) and the first translation codon. This is thefirst demonstration of direct gene activation by RyhB,which has been exclusively described in degradationof mRNAs. Our physiological results indicate that thetransported compound of the ShiA permease, shiki-mate, is important under conditions of RyhB expres-sion, that is, iron starvation. This is demonstrated bygrowth assays in which shikimate or the siderophoreenterochelin correct the growth defect observed for aryhB mutant in iron-limited media.
Introduction
Most organisms depend on the metal iron (Fe) for many oftheir enzymatic reactions. Indeed, because it is a transi-tion metal, iron can easily exchange electrons with differ-ent substrates. One of the best examples is respiration, inwhich iron is critical for electron transport between allcomponents of the respiratory chain. However, respirationalso generates hydrogen peroxide, which, in the presence
of excess iron, will generate reactive oxygen species suchas hydroxyl radical (OH·) through the Fenton reaction(reviewed in Imlay, 2006). These reactive oxygen speciesattack DNA, RNA, proteins, as well as cellular membranesand destroy them. Thus, to minimize its harmful effects,bacteria and eukaryotes maintain the intracellular ironlevel within a strict optimal range. To achieve this balance,bacteria tightly regulate the acquisition of iron accordingto cellular needs.
In Escherichia coli, the master regulator Fur (Ferricuptake regulator) directly senses the intracellular ironlevel and regulates expression of iron acquisition genesaccordingly (Ernst et al., 1978; Hantke, 1981; reviewed inHantke, 2001). When iron is replete, the activated Furbinds to the promoter region (Fur box) of regulated genesand represses the transcription. Conversely, when iron issparse, Fur releases the promoter, allowing expression ofthe Fur-regulated genes. An important number of Fur-regulated genes in E. coli is involved in the biosynthesisand transport of siderophores (enterochelin or enterobac-tin in E. coli ), which are low-molecular weight moleculeswith a strong affinity for iron. Therefore, when iron is low,Fur becomes inactive and allows the expression of sid-erophore biosynthesis genes. The siderophores are thensecreted in the environment to bind iron. The iron-siderophore complexes are recovered by the cell via outermembrane receptors and inner membrane transporters.Once inside the cell, the iron is released from the sidero-phore by esterases (Greenwood and Luke, 1978; Brick-man and McIntosh, 1992). When the intracellular iron hasreached a sufficient level, Fur becomes activated andrepresses the siderophore biosynthesis genes. Interest-ingly, Fur regulates other cellular processes such as acidtolerance (Hall and Foster, 1996), protection against reac-tive oxygen species (Niederhoffer et al., 1990; Tardat andTouati, 1993), metabolic pathways (Stojiljkovic et al.,1994), as well as toxin and virulence factor production(Litwin and Calderwood, 1993). In addition, Fur regulatesthe small regulatory RNA (sRNA) RyhB (Massé and Got-tesman, 2002).
RyhB is a 90-nucleotide sRNAthat is highly expressed inthe absence of iron. It base-pairs with specific mRNAs andtriggers degradation of both the sRNA and mRNA with thehelp of the multiprotein complex RNA degradosome(Massé et al., 2003). A recent microarray experiment has
determined that RyhB downregulates close to 20 mRNAs,which all encode iron-using proteins (Massé et al., 2005).This mRNA regulation pathway has been suggested toprotect the cell against iron-limited conditions (Masséet al., 2005; Massé and Arguin, 2005). Indeed, we recentlyobserved that ryhB mutants were defective in adapting toiron-limited media as compared with wild-type strains(Jacques et al., 2006). Surprisingly, RyhB expression hasalso been linked to activation of many mRNAs such asshiA, ftnA, ompX and ygdQ (Massé et al., 2005). However,the mechanism of activation of these genes by RyhB is notyet understood.
RyhB is part of a larger group of sRNAs that pair withtheir cognate mRNA targets. This group, which includesDsrA, OxyS, SgrS, MicA and RprA, depends on the RNAchaperone Hfq to function properly (Gottesman, 2005).Characterization of Hfq demonstrated that the chaperoneis crucial for sRNA-mRNA pairing as well as for stability ofcertain sRNAs (Moller et al., 2002; Massé et al., 2003). Forexample, RyhB, which is stable in wild-type cells, becomesextremely unstable in an hfq mutant (Massé et al., 2003). Ithas been suggested that Hfq binds to RyhB at the samesequence that is cleaved by the RNA degradosome (Mollet al., 2003). Hfq could participate in activities such asmodifying the RNA structure, bringing RNAs closer toincrease interaction, and finally keeping sRNAs protectedagainst degradation (Moller et al., 2002).
In this work, we describe a mechanism where the sRNARyhB activates the expression of shiA, which encodes fora shikimate permease. Our data suggest that shiA is poorlytranslated because the ribosome binding site (RBS) isunavailable within the shiA 5′-untranslated region (5′-UTR). An inhibitory structure in shiA mRNA is predicted toblock translation initiation, which correlates with instabilityof the mRNA. However, when RyhB is expressed, it pairswith the 5′-UTR of shiA mRNA to prevent the formation ofthis inhibitory structure. Increased production of the ShiApermease could facilitate the acquisition of shikimate, acompound involved in the biosynthesis of siderophores (fora review see Herrmann and Weaver, 1999). In fact, ourphysiological experiments demonstrate that shikimatecorrects the adaptation defect of a ryhB mutant in iron-limited media. Our data suggest a previously unexpectedmechanism of adaptation to iron starvation. To our knowl-edge, this is the first time that a sRNA involved in activedegradation of mRNA targets is shown to also activatemRNA expression.
Results
Determination of the transcriptional +1 of the shiAmRNA
With the use of microarrays, we have previously shownthat RyhB expression leads to increased levels of several
transcripts (Massé et al., 2005). Among the candidatetranscripts, shiA was further investigated to determinewhether its regulation was directly controlled by RyhB.Because the promoter of the shiA gene has not beencharacterized yet, we performed a primer extensionanalysis under conditions where RyhB was induced, thatis, in the presence of the iron chelator 2,2′-dipyridyl (dip)in the media (Massé and Gottesman, 2002). The resultsshown in Fig. 1 demonstrate two weak transcriptionalstart sites in the absence of the inducer (–dip) while astrong single transcription start signal is visible in thepresence of the inducer (+dip). Densitometry of the tran-scription start signals demonstrates a twofold effect ofRyhB on the shiA signal (data not shown).
RyhB does not activate the promoter of shiA
There are at least two potential mechanisms that maycontribute to RyhB-mediated increase of shiA mRNA.First, the upregulation of shiA mRNA levels could resultfrom a more active promoter. Second, accumulation ofshiA could result from increased stability of the mRNA. Totest the first of these two possibilities, we investigated thepotential effect of RyhB on the transcription initiation ofshiA. We fused the shiA promoter to the reporter genelacZ (see Fig. S1 for details on lacZ fusions). The resultingfusion, shiADint′–lacZ, has the full promoter and regula-tory region of shiA from -200 to +6 with respect to thetranscriptional +1 site, yet it lacks the RyhB base-pairingregion. The fusion was inserted in single copy into thechromosome (see Experimental procedures for details).As shown in Fig. 2, in the presence of the iron chelator2,2′-dipyridyl (+dip) there is no significant difference onthe level of shiADint′–lacZ activity with or without RyhBexpression. In addition, we transformed the arabinose-inducible pBAD-ryhB into a strain carrying the shiADint′–lacZ fusion. The addition of arabinose to induce RyhBfrom pBAD-ryhB did not affect shiADint′–lacZ expressionsignificantly (data not shown). These results suggest thatRyhB does not act on the promoter of shiA.
The sRNA RyhB increases shiA mRNA stability
The second possibility for activation of shiA mRNA couldbe increased stability of the mRNA. To test this, we mea-sured the half-life of the shiA mRNA under conditionswhere RyhB was expressed or not. As shown in Fig. 3A,shiA mRNA has a half-life of about 3 min in the wild-typecells (+dip) after addition of rifampicin. This is in contrastto the ryhB::cat cells (+dip), which show that shiA mRNAis already degraded at time point zero as no single bandis observed. The same results are obtained when weanalysed shiA stability in conditions where RyhB is notexpressed (–dip). These data strongly indicate that
increased shiA mRNA levels depends directly on RyhBexpression.
To compare the shiA mRNA levels in the presence andabsence of RyhB, we performed a primer extension onsamples extracted the same way as above. This allowed usto determine the shiA mRNA level at the time when rifampi-cin is added. As shown in Fig. 3B, the shiA transcript is stillsignificantly present after 2 and 4 min in wild-type cells ascompared with ryhB::cat cells, which has already lost itssignal at time 2 min. The results from the Northern blot andthe primer extension suggest that the level of shiA mRNAis a consequence of increased mRNA stability. Accordingto our densitometry results (Fig. 3C), shiA mRNA has ahalf-life of 2 min in the presence of RyhB, but of less thana minute in the absence of RyhB.
The 5�-UTR of shiA mRNA potentially forms aninhibitory structure that blocks translation initiation
The result from Fig. 1 allowed us to predict the possiblesecondary structure of the shiA mRNA from the transcrip-tional +1 site. We used the bioinformatic application mfold(http://www.bioinfo.rpi.edu/applications/mfold/) to gener-ate the potential secondary structures of shiA mRNA fromposition +1 to +86, which covers the RBS and up to thefirst three codons of the open reading frame (ORF). Asshown in Fig. 4A, a potential secondary structure in theshiA mRNA could spontaneously block translationinitiation. In this structure, the first 51 nucleotides of the5′-UTR of shiA mRNA can clearly interact with the RBSand the first codon of the ORF. Thus, it is likely thattranslation initiation of the shiA mRNA is blocked whenthis structure is formed.
Fig. 1. Determination of the transcriptional +1 site of the shiAmRNA by primer extension. Total RNA extracted from wild-typecells (EM1055) treated with 2,2′-dipyridyl (+dip) or without (–dip)was used with radiolabelled oligo EM242. The same oligo wasused for determination of the sequence of the shiA gene (lane1–4). The +1 signal (represented by arrows) observed in thepresence of 2,2′-dipyridyl was twofold stronger than the signalwithout 2,2′-dipyridyl as measured by densitometry. The putative-35 and -10 regions of the shiA promoter are indicated on thesequence on the left. This experiment has been performed twicewith similar results.
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Fig. 2. The lacZ fusion to shiA promoter, shiADint′–lacZ, isinsensitive to RyhB expression. Experiments have been performedin the presence or absence of 2,2′-dipyridyl (+dip or –dip) inwild-type (ryhB+) or ryhB::cat (ryhB-) cells. These experimentshave been performed in triplicate.
If the putative secondary structure of the shiA mRNAblocks translation of shiA, it could explain the observedinstability of the mRNA. When RyhB base-pairs with themRNA and prevents formation of the secondary struc-ture, translation of shiA may stabilize the message. Toinvestigate the role of RyhB in increasing the shiAmRNA stability, we sought a potential pairing betweenthe two RNAs that might explain the positive regulation
of shiA by RyhB. We determined a possible pairingbetween the shiA 5′-UTR and the sRNA RyhB (seeFig. 4B). RyhB pairs with 38 bases of shiA over a total of51 nucleotides. Notably, we identified the presence of a‘core’ region consisting of 12 nucleotides that pair con-secutively (Fig. 4B). It is possible that RyhB pairingwould displace the inhibitory structure of the shiA mRNAand thereby relieve the block in the translation initiationregion. Thus, the resulting increased translation maystabilize the RNA strand by protecting it against ribonu-clease activity.
RyhB activates shiA at a post-transcriptional level
The model described in the previous section suggeststhat RyhB turns on the translation of shiA. To investigatethis further, we inserted the regulatory region ofshiA gene (from -200 to +25 nucleotides with respect tothe initiator codon) upstream of a transcriptional andtranslational lacZ reporter gene carried by pFRD andpRS1551 plasmids respectively. Both fusions wereinserted in single copy into the chromosome with thehelp of l phage (see Experimental procedures fordetails). We then compared the effect of RyhB on theexpression of shiA′–lacZ (transcriptional fusion) andshiA′–′lacZ (translational fusion). Figure 5 (right) showsthat the transcriptional shiA′–lacZ fusion is activatedabout twofold in conditions of RyhB expression (wild-type +dip) as compared with the same conditions butwithout RyhB (ryhB::cat +dip). In contrast with this, thetranslational fusion shiA′–′lacZ (Fig. 5, left) is activatedeightfold in the presence of RyhB (wild-type +dip com-pared with ryhB::cat +dip). Because the effect of RyhBon the translational fusion is the strongest, this suggeststhat RyhB activates shiA translation.
RyhB pairs in vivo with shiA mRNA to initiate translation
The above data indicate that RyhB potentially pairswith shiA mRNA. To demonstrate the direct interactionbetween both RNAs, we first designed a mutated RyhBthat harbours a significant modification (seven mutatednucleotides) in the sequence necessary for pairingwith shiA mRNA (see Fig. 4C for sequence details).The resulting RyhB7 construct, expressed from anarabinose-inducible pBAD promoter (pBAD-ryhB7), wastested on both transcriptional (shiA′–lacZ) and transla-tional (shiA′–′lacZ) fusions as shown in Fig. 6A and Brespectively. The effect of RyhB7 on both fusions wasvery similar to the control vector pNM12 (ryhB– ryhB7–),which suggests that the modified RyhB7 does not inter-act with shiA mRNA. The wild-type RyhB, expressedfrom pBAD-ryhB, was able to activate both shiA′–lacZ(twofold) and shiA′–′lacZ (eightfold). Next, we modified
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Fig. 3. The sRNA RyhB increases shiA mRNA stability.A. Northern blots of shiA mRNA from wild-type and ryhB::catstrains. Cultures were grown until an OD600 of 0.5, at which point2,2′-dipyridyl was added (+dip) or not (–dip) for 50 min. Rifampicin(rif) was added (250 mg ml-1 final) at time 0 to block transcriptionand total RNA was extracted. The upper part of the shiA mRNAsignal is partially covered by the 16S rRNA, which has a slightlylarger size. 16S rRNA was used as a loading control.B. Primer extension with oligo EM242 specific to shiA mRNA ontotal RNA extracted from cells grown as in (A) +dip. The left panelshows shiA expression in a wild-type background. The right panelshows shiA in a ryhB::cat background. Primer extension on 16SrRNA was performed as control.C. Analysis by densitometry of the primer extension signals in B.
both shiA transcriptional and translational fusions tointroduce compensatory mutations allowing pairing withRyhB7 (Fig. 4C). The resulting shiA7′–lacZ and shiA7′–′lacZ were first tested in the presence of wild-type RyhBas shown in Fig. 6. No effect of wild-type RyhB wasobserved on these two fusions (similar to pNM12). This
is in contrast with the modified RyhB7, which clearlyactivated both shiA7′–lacZ (two- to threefold) andshiA7′–′lacZ (five- to sixfold). Taken together, theseresults demonstrated that direct RNA–RNA pairing withRyhB is crucial for the mechanism of translation activa-tion of shiA mRNA.
Fig. 4. The 5′-UTR of shiA mRNA potentially forms an inhibitory structure that blocks translation initiation.A. Determination of the potential secondary structure of shiA mRNA from nucleotide +1 to +86 by the software mfold. The sequence shownincludes the putative ribosome binding site (RBS) and the translation initiator codon (AUG) of shiA.B. RyhB sRNA potentially pairs at the 5′-UTR of shiA. The interaction includes 38 pairing over 51 nt. The core represents a 12 nucleotidesconsecutive pairing between RyhB and shiA mRNA.C. Pairing between mutated RyhB7 and shiA7. The modified nucleotides of RyhB or shiA mRNA are in light font.D. The mutations in shiA7 mRNA apparently do not affect the predicted secondary structure of the transcript from nucleotides +1 to +86. Themutated nucleotides are indicated by arrows.
Hfq is required for shiA inhibition of translation
An important factor for many RNA-dependent processesis the RNA chaperone Hfq. To investigate the potentialrole of Hfq on the expression of shiA, we introduced thetranscriptional and translational fusions into an hfq mutant(hfq1::kan, ryhB::cat) and hfq wild-type (hfq2::kan,ryhB::cat) strains. The hfq2::kan allele contains akanamycin-resistance cassette that is inserted down-stream of the hfq gene and leaves the product active (Tsuiet al., 1994). To limit the effect of RyhB, both strains areryhB mutant. As shown in Fig. 7A, the expression ofshiA′–lacZ transcriptional fusion is only slightly affected(twofold) by the hfq mutant. However, the translationalshiA′–′lacZ fusion is activated 10-fold in an hfq mutantstrain as compared with wild type. This suggests that theRNA chaperone Hfq is required for inhibition of shiAtranslation. Because both mutant and wild-type hfq strainscarry the inactivated ryhB::cat allele, we can conclude thatthis effect is not related to RyhB.
While the previous experiment investigates the role ofHfq on shiA in the absence of RyhB, we also tested theeffect of RyhB on our fusions in both hfq1::kan andhfq2::kan backgrounds. As shown in Fig. 7B, the expres-sion of RyhB in an hfq1::kan mutant slightly activates theshiA′–lacZ transcriptional fusion (hfq– dip– compared withhfq– dip+). Interestingly, in the absence of Hfq, RyhB stillactivates significantly the shiA′–′lacZ translational fusion(compare hfq– dip– to hfq– dip+). This indicates that evenin the absence of Hfq, RyhB can pair to shiA mRNA andactivate its expression.
RyhB and shiA RNAs interact in vitro with the aid of thechaperone Hfq
Our genetic data suggest that shiA mRNA is regulated byHfq even in the absence of RyhB sRNA (see Fig. 7). Tostudy the potential Hfq–shiA interaction, we performed invitro gel electrophoresis mobility shift assays (EMSAs)with radiolabelled shiA mRNA and Hfq hexamer. Theresults (Fig. 8A) demonstrate that Hfq interacts at lowconcentration with shiA mRNA as 50% of the mRNA isshifted with only 2 nM of Hfq hexamer.
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Fig. 5. RyhB activates shiA at a post-transcriptional level asdemonstrated by b-galactosidase assays. The transcriptional fusion(shiA′–lacZ) expression increases twofold under conditions of RyhBexpression (ryhB+) with 2,2′-dipyridyl (dip+). The translationalfusion (shiA′–′lacZ) increases sevenfold in conditions of RyhBexpression (ryhB+, dip+). No effect is observed in ryhB::cat cells(ryhB-) with (dip+) or without (dip–) 2,2′-dipyridyl.
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Fig. 6. RyhB pairs in vivo with the 5′-UTR of shiA mRNA to initiatetranslation as demonstrated by b-galactosidase assays.A. The wild-type transcriptional fusion (shiA′–lacZ) is specificallyactivated by wild-type RyhB (pBAD-ryhB) while the mutatedtranscriptional fusion shiA7′–lacZ is specifically activated by themutated RyhB7 (pBAD-ryhB7). See Fig. 4C for potential pairingbetween shiA7 and RyhB7. The plasmid pNM12 is used as acontrol (ryhB– ryhB7–).B. The wild-type translational fusion (shiA′–′lacZ) is specificallyactivated by wild-type RyhB (pBAD-ryhB) while the mutatedtranslational fusion (shiA7′–′lacZ) is specifically activated by themutated RyhB7 (pBAD-ryhB7). The plasmid pNM12 is used as acontrol (ryhB– ryhB7–).
Furthermore, the results also indicate that Hfq binds withhigh affinity to shiA mRNA. This association suggests thatHfq binding modulates the interaction between RyhB andshiA. To test this, we performed a gel EMSA with radiola-belled shiA and RyhB in the presence or absence of Hfq.Asshown in Fig. 8B, RyhB and shiA interact with each other toform a binary complex (shiA–RyhB) after 15 min of incu-bation in the absence of Hfq (–Hfq). In contrast with this,the presence of Hfq (Fig. 8B, +Hfq) correlates with therapid formation of a ternary complex (shiA–RyhB–Hfq)within the first minute of incubation. The kinetics of complex
formation are represented in the graph in Fig. 8C. Thisgraph demonstrates clearly that Hfq stimulates the rate ofcomplex formation between RyhB and shiA.
Both the shikimate and the siderophore enterochelincorrect the growth defect of ryhB::cat cells iniron-depleted media
Our data suggest that expression of the permease ofshikimate is activated by RyhB when iron is limited. Inaddition, we previously demonstrated that ryhB::cat cellsare defective in adapting to iron-depleted media (Jacqueset al., 2006). Because it is an intermediate of the sidero-phore biosynthesis pathway (see Fig. S3 for shikimatepathway), we investigated a possible physiological role ofshikimate in iron-depleted media. To test this, we moni-tored the growth of wild-type and ryhB::cat cells in Chelex-treated M63 media in the presence or absence of 0.1%shikimate (see Experimental procedures for details).Results are shown in Fig. 9A. As demonstrated previouslywhen iron is depleted, ryhB::cat cells have a significantlag before starting logarithmic growth, compared withwild-type cells (Jacques et al., 2006). In the presence of0.1% shikimate, however, the ryhB::cat cells start logarith-mic growth as rapidly as wild-type cells. Thus, shikimatetotally corrects the adaptation defect of ryhB::cat cells inabsence of iron. In addition, we have performed the sameexperiment with 10-fold less shikimate with similar results(data not shown).
The addition of shikimate in the media may help thesynthesis of end products of the shikimate pathway. One ofthese end products is the siderophore enterochelin(Fig. S3). Thus, we looked for the effect of enterochelin onthe defective growth of ryhB::cat cells under the sameconditions (iron-depleted media) as above. Figure 9Bdemonstrates the corrected growth defect of ryhB::catmutant in the presence of iron-free enterochelin. In additionto this, as shown in Fig. S3, the formation of the sidero-phore enterochelin through the shikimate pathway gener-ates the intermediary 2,3-dihydroxybenzoate (DHB),which can also function as a weak siderophore (Hancocket al., 1977). As shown in Fig. 9C, DHB partially correctsgrowth of ryhB::cat cells. Thus, both enterochelin and DHBcorrect the growth defect of ryhB::cat cells in iron-depletedmedia. This suggests that ryhB::cat defective growth is dueto reduced enterochelin production and iron acquisition.This supports the idea that ryhB::cat growth defect is linkedto the synthesis of siderophores through the shikimatepathway and not to aromatic amino acids and cofactors.
Discussion
This study revealed three major observations relatedto sRNA-mediated regulatory mechanisms and iron
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Fig. 7. Hfq is required for intrinsic shiA inhibition of translation asdemonstrated by b-galactosidase assays.A. The transcriptional fusion (shiA′–lacZ) is slightly activated in theabsence of Hfq (hfq–) as compared with wild-type Hfq (hfq+). Thetranslational fusion (shiA′–′lacZ) is activated eightfold in theabsence of Hfq (hfq–) as compared with wild-type Hfq (hfq+). TheshiA′–lacZ and shiA′–′lacZ fusions are carried in either the hfq–(hfq1::kan, ryhB::cat) or the hfq+ (hfq2::kan, ryhB::cat) background.These strains all carry the ryhB::cat allele.B. RyhB still activates shiA′–lacZ and shiA′–′lacZ fusions even inthe absence of Hfq. These experiments have been performed threetimes.
metabolism. First, although the sRNA RyhB has beenexclusively studied in active degradation of many mRNAs,here we demonstrate for the first time that it also activatestranslation of at least one mRNA. Second, we showedthat iron starvation activates, through RyhB, the expres-sion of the ShiA permease of shikimate, a compoundrequired for siderophore biosynthesis. While previousattempts have failed to find an effector of shiA expression(Whipp et al., 1998), our data indicate that shiA is regu-lated at the translational level by RyhB. Third, we demon-strate that shikimate and enterochelin, and, to a lesserextent, DHB eliminate the growth defect of ryhB::cat cellsin iron-limited conditions. This suggests that one essential
role of RyhB is to activate the synthesis of siderophoresthrough shikimate acquisition.
Remarkably, shiA translation activation by RyhB isreminiscent of DsrA and rpoS. Previous work demon-strated a mechanism of gene activation by the sRNA DsrAthat regulates the translation of rpoS mRNA, whichencodes the stationary-phase transcription factor sS
(Sledjeski et al., 1996; Majdalani et al., 1998; Lease andBelfort, 2000). In the absence of DsrA, the translationinitiation of rpoS mRNA is inhibited by an intrinsic second-ary structure. The 5′-UTR covers the RBS and the firstcodon of rpoS. In contrast, when DsrA is expressed, itpairs to the inhibitory 5′-UTR of rpoS to allow translation
Fig. 8. RyhB and shiA RNAs interact in vitrowith the aid of the chaperone Hfq.A (inset). A representative electrophoresismobility shift assay with 5 nM of radiolabelledshiA RNA in the presence of increasingconcentration of Hfq hexamer [1 (0 nM), 2(1 nM), 3 (1.5 nM), 4 (2 nM), 5 (3 nM), 6(3.5 nM), 7 (4 nM), 8 (5 nM), 9 (7.5 nM), 10(10 nM)].A. Densitometry of the shiA–Hfq complexformation analysed from inset.B. Time-course of formation of shiA–RyhBcomplex in the absence (–Hfq, left) orpresence (+Hfq, right) of Hfq protein.Radiolabelled shiA mRNA at 5 nM, in theabsence or presence of 2 nM of Hfq hexamer,is mixed with 40 nM of RyhB at time 0. Thepresence of Hfq (+Hfq) allows rapid formationof a ternary complex (shiA–RyhB–Hfq). TheshiA+RyhB lane represents shiA mRNA in thepresence of 32-fold excess RyhB. TheshiA+Hfq lane shows the shiA mRNA in thepresence of Hfq only, before the addition ofRyhB.C. Densitometry of shiA–RyhB complexformation in the absence (–Hfq) or presence(+Hfq) of Hfq as measured in B.
initiation. It has also been observed that the stability ofrpoS mRNA increases in the presence of DsrA, most likelybecause of active translation, which prevents RNA deg-radation (Lease and Belfort, 2000).
Hfq can bind rpoS 5′ region with high affinity (Mikuleckyet al., 2004). However, contrary to shiA, the expression ofrpoS mRNA is reduced in the absence of Hfq (Brown andElliott, 1996). This divergence may depend on the orga-nization of shiA and rpoS mRNAs. Whereas shiA is asingle transcript, rpoS is part of the nlpD–rpoS operon(Takayanagi et al., 1994). Thus, rpoS is downstream of anactively translating gene, which may prevent the bindingof Hfq, because of ribosome activity.
Interestingly, whereas DsrAactivates translation of rpoS,it also represses translation of hns mRNA. Nevertheless,even if DsrA blocks translation of hns, there is no evidencethat this mRNA is actively degraded by the RNA degrado-
some. This is in contrast with the negative regulation ofRyhB, which blocks translation of its targets and facilitatestheir degradation (Massé et al., 2003; Morita et al., 2006).
Surprisingly, even though the translation activationmechanisms by DsrA and RyhB sRNAs are very similar,they differ in their negative regulation. The mechanisticdifferences between both sRNAs, however, are stillobscure. This difference between both sRNAs mecha-nism of repression may indicate that RyhB must eliminateits target mRNAs more rapidly than DsrA.
While RyhB clearly enhances the translation of shiA, italso increases the mRNA level of shiA significantly (Figs 1and 3A and B). This is unlikely related to promoter activitybecause the data in Fig. 2 show that RyhB does not affectthe shiA promoter expression. Thus, we explain this bythe presence of ribosomes, which procure the shiA tran-scripts protection from ribonucleases attack (Deana and
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Fig. 9. Shikimate (Shik), or dihydroxybenzoate (DHB), or the siderophore enterochelin (Ent) corrects the growth defect of ryhB::cat cells iniron-limited media. Wild-type and ryhB::cat cells were grown overnight at 37°C in Chelex-treated M63 media without iron at 0.2% glucose. Thenext day, cells were diluted 1/100 in fresh Chelex-treated M63 media without iron at 0.05% glucose in the presence or absence of (A) 600 mMshikimate, (B) 1 mM enterochelin and (C) 33 mM DHB. The cultures were incubated at 37°C and OD600 was determined during growth. Thephenotype of ryhB::cat cells is suppressed by a plasmid carrying the wild-type ryhB gene (data not shown). These graphs are representativeof experiments that have been performed at least three times.
Belasco, 2005; Kaberdin and Blasi, 2006). We tested thispossibility by monitoring the effect of RyhB on a transcrip-tional shiA′–lacZ fusion harbouring a mutated first AUGcodon in the shiA ORF, thereby eliminating the translationinitiation. Surprisingly, RyhB still enhances the expressionof this fusion by two- to threefold (data not shown), whichsuggests another mechanism. A different possibility is thatthe presence of RyhB paired on shiA 5′-UTR could blockaccess to a ribonuclease or disrupt a structure that isprone to ribonuclease attack. Further work is needed toclarify this observation.
While performing this study, we considered a mecha-nism where activation of shiA translation would be acti-vated by a RyhB-dependent mRNA cleavage. In thismodel, RyhB would generate a single cleavage in theinhibitory 5′-UTR of shiA, allowing ribosomes access to themRNA. This is unlikely, as the results of the primer exten-sion show in Figs 1 and 3B; the shiA transcript keeps thesame +1 in the presence or absence of RyhB. In addition,we observed the same transcriptional start site in aryhB::cat background (data not shown), which suggeststhat RyhB is not involved in processing of the shiA tran-script. Therefore, we have no indication that RyhB inducesa specific cleavage to activate shiA mRNA translation.
Our results suggest that RyhB may not be the onlyfactor that regulates shiA expression. The RNA chaper-one Hfq, which fluctuates according to both cellulargrowth rate and phase, potentially modulates ShiA level(Vytvytska et al., 1998; Ali Azam et al., 1999). As shown inFig. 7, Hfq repression of shiA is mostly at the translationlevel (10-fold). It is not clear, however, if this repression isdue to direct binding of Hfq on the translation initiation siteof shiA or if Hfq promotes an inhibitory RNA structure(Fig. 4A and see working model in Fig. 10). Because shiAstructure is already organized for intrinsic repression, wefavour the hypothesis that Hfq helps the 5′-UTR folding on
the translation initiation sequence. Notably, the Hfqprotein binds on single-stranded AU-rich sequences(Zhang et al., 2002). We sought for such a site in shiA5′-UTR and found that the largest loop in the foldedmRNA corresponded to the criteria for an Hfq binding site.Deletion of nine nucleotides in this AU-rich stretch(leaving a 5-nucleotide loop) suggests that it is importantfor normal regulation of shiA translation (see supplemen-tary Fig. S2). Another possibility is that an unknown Hfq-dependent sRNA acts negatively on the shiA mRNAstability and/or translational activity. In such a case, theabsence of Hfq reduces the activity of this sRNA, whichwould increase the shiA mRNA level and translation.
Shikimate is an intermediary metabolite of aromaticamino acids, folic acid, ubiquinone and siderophore bio-synthesis (Herrmann and Weaver, 1999). Interestingly,the shikimate pathway is only present in bacteria, proto-zoans and plants. Because higher eukaryotes do notshare this pathway, it is an interesting target for antibioticsand herbicides. Our work demonstrates that this pathwayis more complex than previously thought. Although theshikimate pathway was already linked to biosynthesis ofmany compounds, it is now clear that iron plays an impor-tant role in its regulation. We expect that a similar mecha-nism also exists in other species that use the shikimatepathway for cell growth.
As our growth assays demonstrate, it is now possible totest compounds that may be involved in iron metabolism(Fig. 9). While the growth of ryhB::cat cells is alwaysslower than wild-type cells, it is not clear why the ryhB::catcells vary in growth from one culture to another (compareFig. 9A–C). In iron-depleted media, the addition of shiki-mate could help the growth of ryhB::cat cells by increasingthe synthesis of siderophores. This is surprising becausethe translation of the shikimate permease ShiA is notactivated in ryhB::cat cells. However, it has been previ-ously suggested that E. coli may have another shikimatetransport system (Knop et al., 2001). It is possible that, inthe context of iron starvation, the constitutive shikimatetransport is not sufficient to meet the cellular needs. Thus,translational activation of shiA by RyhB would result inboosting the shikimate acquisition specifically during con-ditions of iron-depletion. Because E. coli has developedmore than one system of acquisition, it suggests thatshikimate is crucial for the cell. Future studies shouldclarify the relationship between both shikimate acquisitionsystems.
Experimental procedures
Strains and plasmids
Derivatives of E. coli MG1655 were used in all experiments.DH5a strain was used for routine cloning procedures. E. coliexpression strain BL21 (DE3) was used for overproduction
RBS AUG
Translation ONTranslation OFF
RyhB
5'
shiA mRNA shiA mRNA
RBS AUG
Fig. 10. Working model of RyhB translation activation of shiAmRNA. In the presence of Hfq, shiA mRNA forms an inhibitorystructure that blocks the translation initiation (OFF). In conditions ofRyhB expression, the sRNA opens the inhibitory structure in shiAmRNA, which allows translation to proceed (ON). Consequently,the shikimate permease is more expressed.
of recombinant Hfq protein. EM1055 (wild type, MG1655derivative) and EM1238 (ryhB::cat) strains have beendescribed earlier (Massé and Gottesman, 2002). For cellscarrying pNM12 or pBAD-ryhB, and pBAD-ryhB7, ampicillinwas used at a final concentration of 50 mg ml-1. Transcrip-tional and translational fusions were constructed by insertinga polymerase chain reaction (PCR) product (chromosomalDNA as template) into pFRD (Repoila et al., 2003) andpRS1551 (Simons et al., 1987) respectively. Figure S1shows details of the lacZ fusions and Table 1 describes theoligos used in this study. PCR fragments containing -200 to+25 relative to the shiA start codon (oligos EM182–EM183)and -200 to -77 relative to the shiA start codon (oligosEM182–EM203) were digested by EcoRI and BamHI andligated into EcoRI/BamHI-digested pFRD to generate shiA′–lacZ and shiADint′–lacZ respectively. A PCR fragment from-200 to +26 relative to the shiA start codon (oligos EM182–EM208) was digested by EcoRI and BamHI and ligated intoEcoRI/BamHI-digested pRS1551 to generate shiA′–′lacZ.Other constructs were generated by a three-step PCRmutagenesis. Briefly, the shiA′–lacZ construct was used astemplate for two independent PCR reactions (oligosEM194–EM290 and EM195–EM289). The two PCR prod-ucts were mixed to serve as the template for a third PCRreaction (oligos EM194–EM195). The resulting PCR productwas digested by EcoRI and BamHI and ligated into EcoRI/BamHI-digested pFRD to generate shiA7′–lacZ. The sameprocedure was used to generate shiA7′–′lacZ, but withshiA′–′lacZ as the original template and pRS1551 as vector.Transcriptional and translational fusions were delivered insingle copy into the bacterial chromosome at the l att siteas described previously (Simons et al., 1987). Stablelysogens were screened for single insertion of recombinantl by PCR (Powell et al., 1994).
To generate pBAD-ryhB7, the original vector pBAD-ryhB(Massé et al., 2003) was used as a template for two PCRreactions (oligos EM168–EM308 and EM169–EM307). Thetwo PCR products were mixed to serve as the template for athird PCR reaction (oligos EM168–EM169). The resulting
PCR product was digested by MscI and EcoRI and insertedinto MscI/EcoRI-digested pNM12 (Majdalani et al., 1998).
The construct pET21b–hfq was generated by PCR usingthe oligonucleotides EM123 and EM124 and genomic DNAfrom E. coli strain EM1055 as template. The resulting PCRproduct was digested with NdeI and HindIII and cloned intocorresponding sites in pET21b. The resulting construct wastransformed into the BL21 strain for Hfq overexpression with1 mM IPTG. For confirmation, all strains and plasmids con-structs were verified by sequencing.
Preparation of the Chelex-treated M63 media
To remove traces of iron, we treated the M63 media (preparedwithout iron) with 6% Chelex 100 resin (Sigma, St-Louis, MO)and stirred overnight at 4°C. The media was then filtered toremove the resin. The culture flasks were treated with concen-trated HCl and rinsed thoroughly with fresh deionized waterbefore use. Depending on the experiment, 0.1% shikimate(6 mM, final), 33 mM 2,3-dihydroxybenzoate, 1 mM iron-freeenterochelin (EMC microcollections, Tubingen, Germany)600 mM of each aromatic amino acids (tryptophan, phenylala-nine and tyrosine) and 37 mM of each cofactor precursors(4-aminobenzoate and 4-hydroxybenzoate) were added to themedia before treatment with Chelex. Shikimate was added insubstantial amounts because the expression of the shikimatepermease shiA is reduced in the ryhB::cat background.
Beta-galactosidase assays
Kinetic assays for b-galactosidase activity were performed asdescribed previously (Majdalani et al., 1998) using a Spec-traMax 250 microtitre plate reader (Molecular Devices,Sunnyvale, CA). Briefly, overnight bacterial cultures wereincubated in Luria–Bertani (LB) media with ampicillin at a finalconcentration of 50 mg ml-1 at 37°C and diluted 1000-fold into50 ml of fresh LB media with ampicillin at 37°C. Cultures weregrown with agitation to an OD600 of 0.5 before inducing RyhB
expression by adding 250 mM of 2,2′-dipyridyl (for strainsEM1055 or EM1238), or 0.1% arabinose (for strains carryingthe pBAD-ryhB or pBAD-ryhB7 or the control vector pNM12).Specific activities were calculated using the formula Vmax/OD600; these are approximately 25-fold lower than standardMiller units. The results reported represent data of at leastthree experimental trials.
RNA extraction and Northern blot analysis
Total RNA was extracted from cells at the indicated time usingthe hot phenol procedure (Aiba et al., 1981). RNA sampleswere stored in DEPC water at -80°C until further use. ForNorthern blot analysis, the total RNA was loaded on a poly-acrylamide (3 mg RNA). After migration, the RNA was trans-ferred to a Hybond-XL membrane (Amersham, Piscataway,NJ) and cross-linked with UV (1200 Joules). The membranewas prehybridized with 50% formamide, 5 ¥ SSC, 5 ¥ Den-hardt, 1% SDS and 100 mg ml-1 sheared salmon sperm DNAfor 4 h at 60°C. Then, the radiolabelled RNA probe wasadded directly in the prehybridization buffer with the mem-brane and incubated for 16 h at 60°C. Before exposure on aphosphor screen, the membrane was washed three timeswith 1 ¥ SSC/0.1% SDS and twice with 0.1 ¥ SSC/0.1% SDS.The phosphor screen exposure was revealed on a Storm 860(Molecular Dynamics).
Primer extension
Transcriptional +1 of shiA mRNA or control 16S rRNA weredetermined by primer extension according to the SuperscriptII protocol (Invitrogen, Burlington, ON). Briefly, 40 mg of totalRNA (for shiA mRNA) or 2 mg (for 16S rRNA) were incubatedwith 2 pmol of 32P-radiolabelled EM242 or EM345 oligo,respectively, in 1 ¥ First-strand buffer. After a 5 min incuba-tion at 65°C, followed by 1 min on ice, 200 units of Super-script II were added to the reaction. Reverse transcriptionwas allowed to proceed for 50 min at 43°C before the enzymewas inactivated at 70°C for 15 min. After treatment with 20 mgof RNase A, the reaction was precipitated and then migratedon a denaturing 8% polyacrylamide gel.
Radiolabelled RNAs generated by in vitro transcription
The radiolabelled antisense RNA probes, used in Northernblots, were transcribed with T7 RNA polymerase (Roche,Germany) from a PCR product. The same approach was alsoused for generating radiolabelled sense transcripts, used inbinding assays. Transcription was performed in T7 transcrip-tion buffer (40 mM Tris-HCl at pH 8.0, 6 mM MgCl2, 10 mMdithiothreitol, 2 mM spermidine), 400 mM NTPs (A, C, and G),10 mM UTP, 5 ml of a-32P-UTP (3000 Ci mmol-1), 20 unitsRNAGuard (Amersham), 20 units T7 RNA polymerase and0.5 mg of DNA template. After 4 h of incubation at 37°C, themixture was treated with 2 units of Turbo DNAse from Ambion(Austin, TX) and extracted once with phenol-chloroform. Non-incorporated nucleotides were removed with a G-50 Sepha-dex column, and the probe was added directly to theprehybridization buffer.
Hfq purification
Hfq was purified according to an adaptation of the methoddescribed in Zhang et al. (2002). Briefly, overnight culture ofBL21 with pET21b–hfq was grown in LB medium with50 mg ml-1 ampicillin and 30 mg ml-1 chloramphenicol. Theculture was grown at 37°C with agitation until the OD600
reached 0.6. Hfq expression was induced by adding 5 mMIPTG (Bioshop, Burlington, ON) in the media for 3 h. Cellswere pelleted by centrifugation and resuspended in 25 ml ofbuffer C (Zhang et al., 2002), and then lysed by sonication at25% amplitude during 4 min (5 s sonication followed by a 5 spause on ice) with a Branson digital sonifier. The lysate wastreated with 100 units of Turbo DNase (Ambion) for 20 min at37°C. After centrifugation, the supernatant was incubated at80°C for 10 min with light agitation, and centrifuged again toremove insoluble material. The supernatant was then placedon a poly A sepharose column (Pharmacia, Uppsala,Sweden), with the flowthrough reloaded twice. The columnwas washed with 35 ml of buffer C containing 1 M NH4Cl. Theproteins were eluted in 2 ml fractions using 35 ml of buffer Ccontaining 1 M NH4Cl and 8 M urea. Fractions containing Hfqwere pooled and dialysed three times at room temperatureagainst 1 l of buffer C containing 0.25 M NH4Cl. Glycerol wasadded to a final concentration of 10%. The concentration ofthe protein was assayed using the BCA method (from Pierce,Rockfort, IL).
Electrophoretic mobility shift assay of target mRNAsand RyhB with purified Hfq
Five nM of 32P-labelled shiA transcript (generated from invitro transcription of PCR product with oligos EM295 andEM328) was heated at 90°C with 0.1 mg/reaction yeasttRNA for 2 min. Then, the RNA was slowly cooled until thetemperature has reached 37°C. The samples were incu-bated for 10 min at 37°C with or without purified Hfq in 10 mlof binding buffer (10 mM Tris–HCl, pH 7.5, 5 mM Mgacetate, 100 mM NH4Cl and 0.5 mM DTT). After cooling onice for 1 min, the loading buffer was added and thensamples were loaded on a 5% native polyacrylamide gel(run in TBE 1 ¥).
Electrophoresis mobility shift assay kinetics of binaryand ternary complexes assay
A mastermix of 5 nM 32P-labelled transcript and0.05 mg/reaction of yeast tRNA and another one with 40 nMunlabelled transcript and 0.05 mg/reaction yeast tRNA wereheated as described above. Both mastermixes were dividedin two secondary mixes, and 10 ml of binding buffer/reactionwas added into each, with or without 20 nM of purified Hfq.After 10 min at 37°C, secondary mixes were pooled in thefollowing manner: labelled transcript + unlabelled transcriptwithout Hfq, and labelled transcript + unlabelled transcript witha fixed concentration of Hfq. Aliquots of both final reactionswere taken at time points 0, 1, 2.5, 5, 15 and 30 min. Aftercooling on ice for 1 min, loading buffer was added andsamples were loaded on a 5% native polyacrylamide gel (runin TBE 1 ¥).
We thank Mélina Arguin for purified Hfq protein and technicalassistance. We are grateful to Susan Gottesman, SimonLabbé, Cari Vanderpool and Frederieke Brouwers for usefulcomments on the manuscript. This work was funded by anoperating grant MOP69005 to E.M. from the Canadian Insti-tute for Health Research (CIHR). E.M. is a Canadian Institutesfor Health Research (CIHR) new investigator scholar.
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Supplementary material
The following supplementary material is available for thisarticle:Fig. S1. Details of transcriptional and translational lacZfusions used in this study. The transcriptional fusions containa translational stop between shiA (blue) and lacZ (yellow)ORFs that is represented by a gap. The mut7 mutations(detailed in Fig. 4C and D) in the transcriptional and transla-tional fusions are represented by XXX.Fig. S2. Deletion of AU-rich sequence in shiA 5-UTR allowsfull expression of shiA. In this figure, both transcriptionalshiA′–lacZ and translational shiA′–′lacZ fusions are eitherwild-type (wt) or harbouring the AU-rich sequence deletion(DlH).Fig. S3. The shikimate pathway. Black arrows representintermediates of the pathway. Grey arrows demonstrate thefeedback inhibition from the aromatic amino acids.
This material is available as part of the online article from:http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2958.2007.05733.x(This link will take you to the article abstract).
Please note: Blackwell Publishing is not responsible for thecontent or functionality of any supplementary materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding author forthe article.
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