Interplay of transcriptional and small RNA-dependent controlmechanisms regulates chitosugar uptake in Escherichia coliand Salmonella
Jacqueline Plumbridge,1* Lionello Bossi,2
Jacques Oberto,3 Joseph T. Wade4,5 andNara Figueroa-Bossi2**1UPR9073-CNRS† (associated with Université Diderot,Sorbonne Paris Cité), Institut de BiologiePhysico-Chimique, 13, Pierre et Marie Curie, 75005Paris, France.2UPR3404-CNRS, Centre de Génétique Moléculaire,Gif-sur-Yvette, 91198 (associated with Université ParisXI, 91405 Orsay), France.3UMR8621-CNRS Institut de Génétique etMicrobiologie, Université Paris XI, 91405 Orsay, France.4Wadsworth Center, New York State Department ofHealth, Albany, NY 12208, USA.5Department of Biomedical Sciences, School of PublicHealth, University of Albany, Albany, NY 12201, USA.
Summary
Escherichia coli and Salmonella can use chitin-derived oligosaccharides as carbon and nitrogensources. Chitosugars traverse the outer membranethrough a dedicated chitoporin, ChiP, and are trans-ported across the cytoplasmic membrane by the chi-tobiose transporter (ChbBCA). Previous workrevealed that synthesis of the chitoporin, ChiP,requires transcription of the chbBCARFG operon. Asequence from the chbBC portion of the transcript wasshown to act as a decoy target for a regulatory smallRNA, ChiX, that normally blocks chiP expression. ChiXis destabilized and degraded upon pairing with chbBCRNA. Here, we show that the chiP gene, like thechbBCARFG operon, is also downregulated at thetranscriptional level by the NagC repressor. NagCrepression is critical in maintaining chiP mRNA levelslow enough, relative to ChiX, to allow full silencing bythis sRNA. We also show that pairing of ChiX to chbBCRNA downregulates chbC under uninduced condi-tions, that is, when ChiX is in excess to the decoy
sequence. Hence, under these conditions, chbBC RNAis not just a decoy, but a true target of ChiX regulation.Altogether these findings underscore the importanceof stoichiometry in dictating the strength of the sRNAresponse and in differentiating the regulator from theregulatory target.
Introduction
Chitin, the polymer of N-acetylglucosamine is the mostabundant nitrogen-containing polysaccharide on earth.Many microorganisms (bacteria, archaea and fungi)secrete chitinases, a class of enzymes that break downchitin into shorter oligosaccharides like chitobiose andchitotriose. E. coli and Salmonella can use chitin break-down products as sole source for both carbon and nitro-gen. However, they do not secrete chitinases and mustthus rely on other organisms to generate chitosugars innature (Francetic et al., 2000).
The initial steps in the chitobiose/triose utilizationpathway involve the passage of the sugars across theouter membrane, followed by the active transport acrossthe cytoplasmic membrane. The first step requires a dedi-cated porin named ChiP (YbfM), while the second step isachieved through the function of a phosphotransferase(PTS) system encoded by the first three genes of thechbBCARFG operon (Keyhani and Roseman, 1997;Plumbridge and Pellegrini, 2004; Kachroo et al., 2007)(Fig. 1A and B). The two systems are co-ordinately regu-lated and specifically induced when bacteria grow in thepresence of chitosugars (Plumbridge and Pellegrini,2004; Kachroo et al., 2007; Figueroa-Bossi et al., 2009;Rasmussen et al., 2009). The regulatory coupling isachieved through an interplay of transcriptional and post-transcriptional mechanisms.
Under uninduced conditions, transcription of the chboperon is repressed by the NagC repressor, which binds totwo sites in the chb promoter region. Expression of chiPgene is prevented by a constitutively made small RNA(sRNA), ChiX [also called MicM (Overgaard et al., 2009;Rasmussen et al., 2009) or RybC (Mandin and Gottesman,2009)], which pairs with a sequence overlapping the Shine-Dalgarno motif of chiP mRNA, blocking translation and
stimulating degradation of this mRNA (Fig. 1A)(Figueroa-Bossi et al., 2009; Overgaard et al., 2009). Inaddition, the repression of chiP translation by ChiX inducespremature Rho-dependent transcription termination withinthe chiP gene, preventing expression of the chiQ gene(encoding a putative lipoprotein of unknown function)located immediately downstream of chiP and normallycotranscribed with the latter (Bossi et al., 2012).
In the presence of chitobiose/triose, a metabolite ofchitobiose, probably monodeacetylated chitobiose-6P(Verma and Mahadevan, 2012), causes the ChbR regu-latory protein to activate transcription of the chb operon.The polycistronic chb transcript includes a sequence(between chbB and chbC) that functions as a decoy targetfor ChiX. As the chb transcript accumulates, ChiX pairingwith the intercistronic chbBC mRNA elicits the degrada-tion of the sRNA resulting in the progressive relief of chiPrepression (Figueroa-Bossi et al., 2009; Overgaard et al.,2009). Although this was shown to be the primary mecha-
nism responsible for a roughly 100-fold increase in chito-porin expression in the presence of chitobiose orchitotriose in Salmonella, chiP expression could be furtherinduced by the chitosugars in strains deleted for chiXgene. This increase was of the order of threefold butsuperimposed on the translational derepression, thiseffect amounted to a massive accumulation of both ChiPprotein and mRNA (Figueroa-Bossi et al., 2009). Inactiva-tion of the ChbR activator protein completely abolishedthe effect, suggesting that: (i) the increase resulted fromactivation of chiPQ transcription and (ii) the ChbR proteinwas involved in the activation mechanism in Salmonella(Figueroa-Bossi et al., 2009). Detection of a sequencereminiscent of a ChbR binding site in the region upstreamfrom the Salmonella’s chiP promoter (−188 to −169) ten-tatively supported this interpretation. Intriguingly,however, examination of the chiP region in the Escheri-chia coli genome found no trace of a ChbR box at thecorresponding position (Fig. 1C).
Fig. 1. Metabolism and organization ofgenes for use of chitobiose.A. The chiPQ and chbBCARFG operons withtarget sites for sRNA ChiX indicated.B. Metabolism of chitobiose. Chitobiose/triosecrosses the outer membrane via thechitoporin encoded by chiP, it isphosphorylated by the chitobiose PTS(chbBCA) as it crosses the inner membraneand enters the cytoplasm, where it ismetabolized to GlcNAc6P by the products ofthe chbFG genes and then to fructose-6P bythe products of the nagAB genes (Keyhaniand Roseman, 1997; Plumbridge andPellegrini, 2004; Kachroo et al., 2007; Vermaand Mahadevan, 2012).C. Sequence alignment of the regionsupstream of chiP in E. coli K12 andS. enterica serovar Typhimurium between theRho-independent terminator for glnS and thestart codon for chiP. NagC (yellow), CAP(bright green), ChbR (cyan) and GalR (olivegreen) consensus binding sites are indicatedby shading. The mapped transcription start(+1) sites for chiP in Salmonella(Figueroa-Bossi et al., 2009) and E. coli(Rasmussen et al., 2009) are shown in bolditalic and consensus −10 and −35 sequencesconserved in both organisms in italic.Sequences, which can base pair with theChiX sRNA are shown in blue italic.
A bioinformatics survey identified the presence of aNagC binding site, between the −35 and −10 promoterhexamers, which was conserved in both Salmonella andE. coli (Oberto, 2010) (Fig. 1C). NagC is the repressor ofthe divergent nagE–nagBACD operon, which encodesproteins involved in N-acetylglucosamine (GlcNAc) trans-port and metabolism. N-acetylglucosamine 6-phosphate(GlcNAc6P), the product of the nagE-encoded PTS trans-porter, and also a downstream product of chitobiosemetabolism (Fig. 1B) functions as the allosteric effector ofNagC inactivating the protein and leading to derepressionof the nag and chb operon (Plumbridge, 1991; Plumbridgeand Pellegrini, 2004). This suggested that, like thechbBCARFG operon, the chiPQ operon might be underthe negative control of the NagC repressor. Thus, at thebeginning of this study our objective was twofold: (i) inves-tigate ChbR involvement in chiP regulation in E. coli andSalmonella and (ii) assess the role of NagC in this regu-lation in both organisms.
Results
NagC binds upstream of chiP in E. coli and Salmonellabut ChbR only binds in Salmonella
DNase I footprinting confirmed that NagC bound to a siteupstream of chiP overlapping the −10 and −35 regions ofthe chiP promoter (site NagC1) in S. enterica serovarTyphimurium LT2 (henceforth referred to as Salmonella)as well as in E. coli (Fig. 2). DNase I footprinting alsolocated a second NagC operator site further upstream(NagC2) in both E. coli and Salmonella (Figs 2 and 1C)but only at higher concentrations of NagC.
Co-operative binding of NagC to two sites is requiredfor regulation of most NagC controlled operons in E. coli(nagEBACD, glmUS, chbBCARFG, fimB), although thegalP gene is controlled via a single operator with highaffinity (El Qaidi et al., 2009). The distance between thetwo NagC sites upstream of chiP is 223 bp in E. coli and235 bp in Salmonella. In both E. coli and Salmonella the
Fig. 2. Binding of NagC, ChbR, GalR and GalS to the regulatory regions of E. coli and Salmonella chiP.A and B. Salmonella DNA fragment corresponding to −290 to +29 relative to the chiP +1 (PCR fragment ChiP1-ChiP2) was labelled at position−290 (ChiP1) (A) and at +29 (ChiP2) (B).C and D. E. coli DNA fragment corresponding to −304 to +29 relative to the chiP +1 (PCR fragment YbfM1-YbfM3) labelled at −304 (C) and at+29 (D). The marker (M) is pBR322 digested with MspI and is labelled relative to the chiP transcription start sites (+1) for each gel. Thelabelled DNA (about 1 nM) was mixed with decreasing amounts of the proteins indicated above each lane. When 4 concentrations of NagC,ChbR, GalR or GalS were tested on each DNA (indicated by a wedge covering 4 lanes), the concentrations were 100, 50, 25 and 12.5 nM(calculated for monomeric protein). The concentrations of ChbR tested on the E. coli DNA (Gel C, lanes 2 and 3; Gel D, lanes 1 and 2) were200 and 100 nM. Regions protected by NagC, ChbR, GalR and GalS are indicated.
Coregulation of a sugar porin and PTS transporter 3
upstream site (NagC2) was of lower affinity than thedownstream, promoter proximal site in the footprint, whichis not the case for other NagC regulated genes (e.g.Plumbridge, 1995; Plumbridge and Pellegrini, 2004). For-mation of a DNA loop has been demonstrated or deducedto be required for NagC regulation at these sites, since itallowed NagC to bind to the lower affinity but promoter-proximal site. It can also be noted that the distancesbetween the NagC sites at chiP are longer than at otherNagC controlled genes (Cournac and Plumbridge, 2013),which will not favour loop formation.
ChbR bound to the region upstream of the chiP gene inSalmonella at the sequence resembling the previouslyidentified ChbR consensus (Plumbridge and Pellegrini,2004; Kachroo et al., 2007) (Fig. 2A and B) at a concen-tration (50 nM) comparable to its affinity at chbB in E. colimeasured under similar conditions (Plumbridge andPellegrini, 2004). However, no binding of ChbR to the chiPupstream region of E. coli was detected (Fig. 2C and D)and the sequence protected in Salmonella, unlike theNagC site, is not conserved in E. coli (Fig. 1C). It can alsobe noted that there is just one copy of the ChbR consen-sus sequence at chiP whereas there are tandem ChbRsites upstream of chbB in both E. coli and Salmonella (13positions are identical in a stretch of 19 bases in the fourChbR sites upstream of chbB in E. coli and Salmonella.Eleven of these positions are identical in the chiP ChbRsite; Fig. S1A). Moreover, the location of the ChbR site atSalmonella chiP at about −178 is far upstream for a typicalprokaryotic activator. At chbB the proximal ChbR siteoverlaps the −35 sequence, so that ChbR is likely to makedirect contact with RNA polymerase. Altogether, theseobservations suggested that the residual chiP inductionby chitobiose in the ΔchiX background is unlikely to bedue to direct activation by ChbR at the chiP promoter.
The chiP gene is under the transcriptional control of theNagC repressor
As the next step in our comparative study, we examined theeffects of nagC and chbR mutations on regulation of chiP–lacZ translational fusions. To have the Salmonella andE. coli measurements closely comparable, we reproducedin E. coli the exact same translational fusion previouslyused in the characterization of chiP in Salmonella(Figueroa-Bossi et al., 2009). This fusion joins the lacZopen reading frame to the 8th codon of chiP. In spite of this,the absolute values of β-galactosidase activity were repro-ducibly threefold to fivefold higher throughout this study inSalmonella as compared with E. coli. The reason for thisdifference remains elusive. Nevertheless, the relative vari-ations of β-galactosidase activity, resulting from mutationsor conditions affecting chiP expression, were comparablein E. coli and Salmonella and allowed a number of conclu-sions to be made. In both bacteria the expression from thechiP fusion in the wild-type background is extremely low,but increases at least 100-fold in the presence of chitobi-ose (Fig. 3). As observed previously for Salmonella, loss ofthe ChiX sRNA produces a high level of chiP–lacZ expres-sion, which is further enhanced by growth on chitobiose inboth bacteria. From the data in Fig. 3, it is apparent thatinactivation of the ChbR repressor completely preventsresidual chiP induction by chitobiose in ΔchiX backgroundin E. coli as it does in Salmonella. Given that the E. colisequence lacks the ChbR binding site and shows nobinding to the purified protein in vitro (see above), thesefindings suggest that the ChbR requirement for the residualinduction of chiP by chitobiose is independent of ChbRbinding to the chiP promoter region.
Inactivation of the NagC repressor causes chiP expres-sion to increase no more than twofold to threefold in ΔchiX
Fig. 3. Effect of mutations in chiX, nagC, chbR on expression of a chiP–lacZ fusion in E. coli and Salmonella. The bars showβ-galactosidase activities (Miller units) with standard deviations measured in MOPS minimal glycerol medium with or without 0.1% chitobiosein bacteria carrying the mutations indicated (the genotypes of strains used are listed in Table S1).
background. Interestingly, however, the effect of nagCinactivation is amplified in the wild-type backgroundwhere the increase is 8- and 14-fold in Salmonella andE. coli respectively. Presumably, the twofold to threefolddecrease in chiP mRNA levels is sufficient to ensure thatthe ChiX : chiP mRNA ratio is high enough for full silenc-ing by the sRNA (Levine et al., 2007; Levine and Hwa,2008). It thus appears that NagC plays a key role insetting chiP expression levels under uninduced condi-tions. In the ΔchiX background, the nagC mutation pro-duces the same level of chiP expression in the presenceor absence of chitobiose. Since this level corresponds tothe fully chitobiose-induced level of the nagC+ ΔchiXstrain, these results strongly suggest that residual chiPinduction in ΔchiX bacteria grown on chitobiose resultsfrom a relief of NagC repression.
Final proof that the ChbR binding site at −180 in Sal-monella is not implicated in chitobiose induction of chiPcame from replacing the ChbR binding site in Salmonellawith 11 bp derived from the equivalent region in E. coli incis in the chiP–lacZ fusion construct on the chromosome.The induction by chitobiose was normal in both chiX+ andchiX backgrounds (Fig. S1B).
Taken together, the above data allow us to draw aplausible scenario for chiP transcriptional regulation withNagC playing an important role in the regulatory switch.The NagC repressor inhibits chiP transcription underuninduced conditions through binding to the chiP pro-moter. In the presence of chitobiose, the chbBCARFGoperon is induced leading to inactivation of ChiX by thechbB-C intergenic region and a first level of chiP induc-tion. The subsequent metabolism of chitobiose by theenzymes of the chb operon, ChbFG (Verma andMahadevan, 2012) generates GlcNAc6P, which inacti-vates NagC leading to full-scale induction of both the chiPand chb operons. Thus the ChbR requirement for chiPinduction can be simply explained based on its essentialrole in activation of the chb operon.
Binding of GalR and GalS proteins in the regionupstream from the E. coli chiP promoter
Interestingly, the region occupied by ChbR in Salmonellacorresponds to a GalR consensus site in E. coli. Bindingof GalR to this site was initially identified by genomicchromatin immunoprecipitation (ChIP-chip) assays (J.Wade, unpubl. data). We confirmed that GalR does bindto this site by DNase I footprinting (site called GalR,Fig. 2C and D, lanes 9–12). In addition, the GalS isore-pressor was also found to bind to that site and GalS alsobound weakly to two other sites, nearer to the promoter(labelled GalS1 and 2, Fig. 2C and D, lanes 13–16).However, mutations in neither galR nor galS had anyeffect on chiP–lacZ expression (Fig. 4) and did not affect
repression by NagC. Growth on galactose actuallydecreased expression threefold compared with the samestrain in glycerol, presumably because of cataboliterepression exerted via the consensus CAP (cataboliteactivating protein = CRP cAMP receptor protein) sitelocated at position −70.5 from the transcription start site(Fig. 1C). The chiP promoter is subject to strong catabo-lite repression in both E. coli and Salmonella. Growth onglucose reduces expression of chiP–lacZ fusion in chiXstrains about 12-fold in E. coli and sixfold in Salmonellarespectively (Fig. S2). GlcNAc also produces strong cat-abolite repression. Consequently, the expression of chiPin wild-type and chiX strains is similar in glycerol andGlcNAc. The relief of NagC repression by growth onGlcNAc is masked by the catabolite repression generatedby GlcNAc (Fig. S2).
Stoichiometry dictates whether chbBC RNA is theregulator or the regulatory target of the ChiX sRNA
Under inducing conditions, base-pairing between ChiXand a sequence from the chbB-chbC portion of the accu-mulating chb transcript directs ChiX into a decay pathway.Interestingly, chbBC RNA integrity is also affected by thepairing interaction, as revealed by the occurrence ofRNase E cleavages in the proximity of the pairing site(Figueroa-Bossi et al., 2009). This suggested that besidescontrolling ChiX sRNA levels, chbBC RNA could be itselfa target of ChiX regulation. To investigate this possibilitywe analysed the effects of the chiX deletion on theexpression of the chbC gene downstream of the ChiXtarget. We compared the expression of lacZ gene fusionswith chbB (i.e. upstream of the ChiX pairing site) and with
Fig. 4. Effect of mutations in nagC, galR and galS on expressionof a chiP–lacZ fusion in E. coli. The bars show β-galactosidaseactivities (Miller units) with standard deviations measured in MOPSminimal glycerol medium with or without 0.2% galactose in bacteriacarrying the mutations indicated (strains used are listed inTable S1).
Coregulation of a sugar porin and PTS transporter 5
chbC (i.e. downstream of the ChiX pairing site). Sinceinsertion of the lac material would interfere with expres-sion of the distal chbR regulator gene, required for tran-scription of the chbBCARFG operon, this analysis wasperformed with the chbBC region (including the promoterand regulatory sequences) placed in trans; namely at theλ attachment (attλ) site in E. coli and at the att site of theFels-1 prophage in Salmonella. Results showed chbB–lacZ expression to be totally unaffected by ChiX under allconditions tested (Fig. 5A and B). In contrast, the chiXdeletion caused the basal level of expression of a trans-lational chbBC–lacZ protein fusion to increase 15-fold and8-fold in E. coli and Salmonella respectively (Fig. 5A andC). Significantly, this effect was observed under unin-duced conditions, but not in the presence of chitobiose(Fig. 5B and D), indicating that when the chb operon ismaximally transcribed, ChiX is no longer able to down-regulate chbC expression. Combined with the previousfindings (Figueroa-Bossi et al., 2009; Overgaard et al.,2009), these results suggest that whether chbBC RNAworks as a regulator or as a regulatory target depends onits stoichiometry relative to ChiX sRNA (see Discussion).
In conclusion, it appears that in addition to silencingchiP, ChiX is directly responsible for setting the basallevels of the main component of the chitobiose PTS trans-porter. ChiX might act by inhibiting chbC translation (itstarget sequence lies 45 nucleotide upstream of chbC ini-tiating AUG) and/or by stimulating cleavage of chbCmRNA by RNase E (see above). Preliminary findings
suggest that RNase E is not required for setting the basallevel of chbC expression, implying that ChiX is directlyaffecting chbC translation. Finding that chbB–lacZexpression is unaffected by the chiX deletion suggeststhat the action of the sRNA does not affect the level of thechbR regulator. Consistent with this interpretation, dele-tion of chiX and/or nagC had only small effects (maximum2× derepression) on β-galactosidase activity of a chbR–lacZ translational fusion in cis in Salmonella (Fig. S1C).
Discussion
We have shown that the chiP gene, encoding a special-ized porin for chitobiose and chitotriose, is under thecontrol of the N-acetylglucosamine (GlcNAc) operonrepressor, NagC, in both E. coli and Salmonella. NagCbinds to two sites upstream of the chiP gene, one span-ning the interval between the −35 and −10 hexamers ofthe promoter, the other more than 200 bp upstream fromthe promoter. However, in both organisms repression ofchiP by NagC is inefficient resulting in a relatively highbasal level of chiP transcription in the absence of chitobi-ose (Figueroa-Bossi et al., 2009). This could be a conse-quence of the low affinity of the repressor for the upstreamNagC site so that cooperative binding of NagC to the twooperators is unlikely. Repression by NagC probably relieson just the proximal NagC site. Despite this prevalent chiPtranscription, synthesis of ChiP protein is prevented by thesmall RNA ChiX, which blocks translation by base-pairing
Fig. 5. Effect of the chiX mutation onchbB–lacZ and chbBC–lacZ transcriptional(op) and translational (pr) fusions in E. coliand Salmonella. The bars give theβ-galactosidase activities (Miller units) withstandard deviations measured in MOPSminimal glycerol medium with out (A, C) orwith (B, D) 0.1% chitobiose (strains used arelisted in Table S1).
to the Shine-Dalgarno region of chiP mRNA (Figueroa-Bossi et al., 2009; Overgaard et al., 2009; Bossi et al.,2012). Possibly stronger NagC repression is not usefulwithin the present context of post-transcriptional regula-tion by the sRNA. Binding of NagC to the upstream site,which could produce greater repression via DNA loopformation, has thus been lost. However, full chiP inductionin the presence of chitobiose or chitotriose requires therelief of both NagC-dependent transcriptional repressionand ChiX-dependent translational repression. As sug-gested by previous work and confirmed and extendedhere, this is achieved through a mechanism that coupleschiP expression to the expression of the chbBCARFGoperon encoding the chitobiose PTS transporter and thefirst two enzymes of the sugar’s catabolic pathway. Onone hand, accumulation of chbBCARFG mRNA and itspairing to ChiX causes the latter to be diverted from chiPrepression and degraded. On the other hand, the activityof ChbG and ChbF enzymes leads to the production ofGlcNAc-6P, the inducer for NagC (Fig. 6) (Plumbridge,1991; Verma and Mahadevan, 2012). The latter step iscritical for the initiation of the entire induction cascade, asthe chbBCARFG operon itself is repressed by NagC(Plumbridge and Pellegrini, 2004). In addition, activationof chbBCARFG transcription requires the ChbR activatorprotein bound to another catabolic intermediate, probablymonoacetylated Chb6P synthesized by ChbG encoding achito-oligosugar deacetylase (Verma and Mahadevan,2012).
The presence of a sequence resembling a ChbRbinding site in the region upstream from the chiP promoterin Salmonella but not in E. coli led us to test whetherChbR bound this site and played a direct role in chiPinduction in Salmonella. Intriguingly, while footprint analy-sis confirmed such binding, we obtained no evidence for aChbR involvement in chiP induction other than its indirectrole as activator of chbBCARFG transcription. Deletingthe chbR gene in a strain already deleted for nagC andchiX had no significant effect on chiP derepressed levels(Fig. 3). Furthermore, replacement of the ChbR bindingsite in the Salmonella chiP promoter region with thesequence found at the corresponding position in E. colidid not affect chiP induction by chitobiose to any signifi-cant extent. Presently, the role of ChbR binding site in theSalmonella’s chiP promoter region, if any, remainsunknown.
Unexpectedly, the upstream region of DNA correspond-ing to the ChbR site in Salmonella is occupied by a bonafide GalR binding site in E. coli, as shown by chromatinimmunoprecipitation experiments in vivo and DNaseI foot-printing in vitro. This seemed to imply another linkbetween galactose and amino sugar metabolism asexemplified by NagC participating with GalR and GalS inthe repression of galP in E. coli (El Qaidi et al., 2009).
However, mutations in neither galR nor galS had anyeffect on chiP expression. The observation that the chiPgenomic context is conserved in many Enterobacteriacea(Fig. S3A) prompted us to search for GalR consensusbinding sites (using the E. coli galE, galP, galR, galS andmglB GalR binding sites as input for the search matrix). AGalR site was found in 6 species (E. coli, Shigella, Citro-bacter, Chronobacter, Klebsiella and Raoultella), whichotherwise show a low sequence conservation in theregion (Fig. S3B). A recent chromosome conformationcapture (3C) study with GalR (Qian et al., 2012) showedthat this protein mediates long-range interactionsbetween known GalR regulatory targets and other sitesmatching the GalR consensus. These contacts, observedin stationary cells, might include the chiP GalR site.However, we have no explanation of why thesesequences have evolved to bind GalR in some speciesbut ChbR in Salmonella and probably in Serratia where aChbR consensus site was also found at a similar locationbut in the opposite orientation relative to Salmonella(Fig. S3B).
In the region upstream of the chiP ORF, the NagC1binding site and a consensus CAP binding site, stand outas being completely conserved in 13 species (Fig. 1C,Fig. S3B). Similarly putative −10 and −35 promotersequences, corresponding to the mapped 5′ end of thechiP mRNA in E. coli (Rasmussen et al., 2009) and Sal-monella (Figueroa-Bossi et al., 2009) are also clearlydetected in all 13 species. The ChiX pairing sequence isalso conserved in 7 of the species and compensatoryspecies-specific variations might account for ChiX bindingin other species (Horler and Vanderpool, 2009). Thesequences upstream of the CAP site are less well con-served but a Rho-independent terminator downstream ofglnS and sequences resembling the NagC2 site are eachdetectable in 10 of the strains (Fig. S3B). The strongconservation of the NagC1 site clearly implies that NagCregulation of chiP is important for these bacteria.
In the course of this study we have examined whetherthe mechanism responsible for ChiX sRNA degradationhad any effect on expression of the chb operon. Wediscovered that the deletion of chiX caused sharpincreases in the basal levels of expression of chbBC–lacZtranslational fusions in E. coli and Salmonella (15-fold andeightfold respectively; Fig. 5A and C). This suggested thatChiX pairing with the RNA sequence from the chbB-chbCintercistronic spacer – the very mechanism responsiblefor ChiX inactivation during chitobiose induction – down-regulates chbC expression under uninduced conditions.As the ChiX base-pairing site in the chbB-chbC intergenicregion is 45 bp upstream of the chbC translational initia-tion codon this raises the question of how translationalregulation, without any concomitant effect on a transcrip-tional fusion, is achieved. Although the ChiX pairing site is
Coregulation of a sugar porin and PTS transporter 7
outside the region covered by the 30S ribosomal subunitduring initiation (Huttenhofer and Noller, 1994), ChiXannealing could still perturb the efficiency of the initiationprocess, for example, by targeting a translation enhancerelement (Sharma et al., 2007). Alternatively, formation ofa secondary structure between the Shine-Dalgarno motifand the ChiX pairing sequence (as predicted by the Mfoldprogram; Zuker, 2003) might bring the ChiX pairingsequence inside the 30S subunit interaction window,allowing ChiX to directly interfere with translation initiation.
In contrast to its effect on the basal level of chbCexpression, the chiX deletion had no effect onβ-galactosidase accumulation following ectopic inductionof the chbBC–lacZ fusion with chitobiose (Fig. 5B and D).It thus appears that the choice as to whether ChiX acts aregulator or is targeted for decay depends on the relativeconcentration of its cognate pairing sequence. This isconsistent with the proposed threshold-linear responsemodel of sRNA-mediated regulation (Levine et al., 2007;Levine and Hwa, 2008). Under uninduced conditions, thetranscriptional rate of ChiX exceeds that of both chiP andchbBC. Both these target mRNAs are thus degradedupon pairing with the sRNA. Conversely, in the presenceof chitobiose the vast accumulation of the chb transcript,presumably combined with the increase in chiP transcrip-tion, causes the entirety of the ChiX pool to turnover. Theexcess chiP and chb mRNAs are free to be translated intoproteins, which accumulate.
Our current view of the regulation of chitobiose metabo-lism is depicted in Fig. 6. Transcriptional activationappears to occur in two stages. First, expression of theinner membrane transporter chbCBA is activated directlyby the ChbR protein, which allows entry of Chb-6P andgeneration of the specific ChbR-inducing signal (by actionof ChbG enzyme; Verma and Mahadevan, 2012). Activa-tion of chbBCA expression by ChbR is essential to relieveChiX repression of chiP. Second, as the flux of chitobiosethrough the ChbBCA transporter and ChbFG enzymesincreases, the level of GlcNAc6P, the inducer for NagC,increases and relieves repression by NagC of both chiPand chb operons. This double control, presumably requir-ing higher levels of GlcNAc6P to derepress NagC than the
monodeaceylated Chb6P to activate ChbR, ensures thatonly when sufficient chitobiose is present are bothoperons maximally expressed. On the other hand, theinitial priming reaction to inactivate ChbR must dependupon the low basal level of the chbBCA-encoded trans-porter and the chbG-encoded deacetylase, to allow entryof Chb-6P and generation of the ChbR-inducing signal.Interestingly, this basal level of chbBCA expression is setby the reciprocal action of ChiX sRNA on chbBCA mRNA.This downregulation of the transporter might be expectedto slow down the response time for the detection of chi-tobiose in the environment but can ensure that the chiPand chb genes are only expressed in amounts compatiblewith the environmental supply of chitosugars.
Experimental procedures
Bacterial strains and growth conditions
Bacterial strains used are listed in Table S1. Escherichia colistrains are all derivatives of MC4100 and Salmonella entericaserovar Typhimurium strains are derived from MA3409, aderivative of strain LT2 cured for the Gifsy-1 prophage(Figueroa-Bossi et al., 1997). Bacteria were grown in LB ascomplex medium or in synthetic minimal MOPS medium(Neidhardt et al., 1974) supplemented with 0.4% glycerol, towhich was added, where indicated, 0.1% chitobiose forβ-galactosidase assays. Cultures were sampled throughoutexponential growth and β-galactosidase activities (Millerunits; Miller, 1972) reported for culture ODs betweenA650 = 0.5 and 0.8. Results are the mean with standard devia-tion for 2 to 6 independent cultures.
Genetic techniques
Generalized transduction was performed using phages P1virin E. coli and P22, HT 105/1 int-201 (Schmieger, 1972) inSalmonella. E. coli deletion mutations alleles (made byrecombineering), chbR::cat and nagC::tet were describedpreviously (Plumbridge and Pellegrini, 2004; Pennetier et al.,2008). ΔgalS::FRTkan and ΔgalR::FRTkan were from theKeio collection (Baba et al., 2006). Salmonella strains carry-ing an in-frame lacZ fusion to the 8th codon of the chiP geneat the chiP locus (chiP91-pCE40), a chiX deletion Δ[chiX-ybaP]::cat, or transposon insertions in chbR (chbR::Tn5-
Fig. 6. Model for chiP and chbBCARFG regulation. (A) Under uninduced conditions and (B) Induced by the presence of chitobiose. Threeregions of the E. coli or Salmonella chromosome are shown: (i) the chiX gene encoding the ChiX sRNA, (ii) the nagDCAB–nagE–glnS–chiPQregion encoding the genes of the nag operon for the uptake (nagE) and metabolism (nagBA) of GlcNAc and the nagC transcriptionalrepressor, glnS encoding glutaminyl-tRNA synthetase and chiPQ, encoding the OM chitoporin (chiP) and a putative lipoprotein (chiQ), (iii) thechbBCARFG operon coding for a PTS transporter (chbBCA); enzymes for the degradation of chitobiose (chbFG) and the chbR transcriptionalactivator. Red lines with bars correspond to repression or inactivation while green lines with arrows correspond to induction or activation.Wavy lines indicate mRNA levels and when dotted indicate that the transcript is unstable. In the absence of chitosugars (uninduced) the chb,chiP and nag operons are repressed by NagC. Translation of the chiP mRNA is repressed by base-pairing with the ChiX sRNA. In thepresence of chitobiose (induced), ChbR bound to its inducing signal (the product of the action of the ChbG enzyme, probably monoacetylatedChb6P) activates expression of the chbBCARFG operon and sequesters the ChiX sRNA so relieving the translational repression of chiP.Further metabolism of chitobiose generates GlcNAc6P, the inducing signal for NagC leading to loss of NagC repression and full expression ofchbB and chiP.
Coregulation of a sugar porin and PTS transporter 9
TPOP) and nagC (nagC::Tn5-TPOP) were described in aprevious study (Figueroa-Bossi et al., 2009). The chbB–lacZtranscriptional and translational fusions, to the 15th codon ofthe chbB ORF, carried by a λ lysogen at attλ in E. coli weredescribed previously (Plumbridge and Pellegrini, 2004).
Other strains were made by similar chromosomal engi-neering (recombineering) techniques using the λ red recom-bination method (Datsenko and Wanner, 2000; Murphy et al.,2000; Yu et al., 2000) implemented as in Datsenko andWanner (2000). Donor DNA fragments were generated byPCR using plasmid DNA or chromosomal DNA or DNA oligo-nucleotides as templates. The primers used to make theseconstructs are listed in Table S2. Amplified fragments wereelectroporated into the appropriate strains expressing λ redoperon from conditional plasmid pKD46 (Datsenko andWanner, 2000). Electroporation was carried out using a Bio-Rad MicroPulser under the conditions specified by the manu-facturer. Recombinant colonies were selected on LB platescontaining the appropriate antibiotic. Constructs were verifiedby PCR and DNA sequence analysis (performed by GATCcompany or MWG).
Construction of chromosomal lacZ gene fusions (in cis)
Unless specified otherwise, in-frame fusions of lacZ to genesof interest were constructed by the procedure of Ellermeieret al. (2002). This involved: (i) inserting an FRT-kan-FRT(KanR) module amplified from plasmid pKD13 in the targetgene – most often combined with the generation of an inter-nal deletion – (ii) excising the kan cassette with Flp recom-binase (leaving a ‘scar’ sequence with a single FRT site) and(iii) inserting lac-containing plasmid pCE40 at the FRT site viaFlp-mediated recombination (Ellermeier et al., 2002). Theprocedure allowed us to reproduce in E. coli the exact samechiP–lacZ fusion previously used in the study of chiP regula-tion in Salmonella (Figueroa-Bossi et al., 2009), and to con-struct lacZ fusions to chbR and chbBC genes in Salmonella.Details of primers and templates used in the first step areprovided in Table S2.
Construction of chbBC–lacZ transcriptional andtranslational fusions (in trans)
To study chbB-chbC regulation using lacZ fusions, thesegene fusions were placed at a separate chromosomal loca-tion from the chbBCARFG operon (in trans). In E. coli thiswas achieved by placing the construct in a λ prophage(inserted at the primary attλ site), as previously described forchbB–lacZ protein and transcriptional fusions (Plumbridgeand Pellegrini, 2004) whereas in Salmonella the fusion wasinserted in the Fels1 prophage. The DNA fragment corre-sponding to the regulatory region of the chb operon of E. coli,the chbB gene and the first 12 amino acids of chbC wassynthesized using the oligonucleotides Chb1E to R-chbC-E(739 bp) with EcoRI extensions and was inserted intopRS414 and pRS415 (Simons et al., 1987) digested withEcoRI to give plasmids carrying the translational and tran-scriptional fusions respectively. A fragment carrying the catcassette from pKD3 amplified with oligonucleotides ps1-ps2(Datsenko and Wanner, 2000) was inserted into the unique
HindIII site of the chbBC–lacZ plasmids within the Chb1Eoligonucleotide. A DNA fragment encompassing the cat-chbBC–lacZ’ fragment was amplified (using Advantage HD)with the oligonucleotides RBP22 and Lac22. This fragmentwas electroporated into MC4100 lysogenized with the λRS45vector phage (Simons et al., 1987) and carrying pKD46expressing the λ red genes (Datsenko and Wanner, 2000).Bacteria, which had incorporated the chbBC promoter frag-ment into the phage, were selected as blue on LBcm Xgalplates. The CmRes cassette was then removed by Flp medi-ated excision by treatment with pCP20 (Datsenko andWanner, 2000). To construct a similar chbBC–lacZ transla-tional fusion in Salmonella, a DNA fragment including the chboperon promoter (from −136) and extending to the distal FRTsite of a chbC::kan insertion was amplified from strainMA9998 (Table S1) with primers ppI24 and ppI25 (Table S2)and inserted in place of the Fels-1 prophage by recombineer-ing. The insert was converted to a lacZ fusion as described inthe previous section.
DNase I footprinting
DNA fragments for footprinting covering the 5′ chiP regulatoryregions of E. coli and Salmonella were synthesized by PCRwith one of the oligos previously labelled with [γ-32P]-ATP andpolynucleotide kinase. Footprinting was carried out asdescribed previously (El Qaidi et al., 2009). The bindingbuffer was 25 mM HEPES, 100 mM K glutamate pH 8.0 con-taining 0.5 mg ml−1 BSA. Dilutions of the purified proteins[NagC, a generous gift of Mitchel Lewis, ChbR (Plumbridgeand Pellegrini, 2004) and GalR or GalS (El Qaidi et al., 2009)]were mixed with labelled DNA (final concentration about1 nM) in a total volume of 40 μl for 15 min at RT and thentreated with DNase I (0.05 μg ml−1 for 1 min). The reactionwas stopped with phenol (100 μl) and 200 μl buffer (0.5 M Naacetate pH 5.0 2.5 mM EDTA containing 10 μg ml−1 herringsperm DNA) added. After phenol extraction the labelled DNAwas precipitated and analysed on a 6% (or 8%, Fig. 2A)denaturing (7 M) urea polyacrylamide gel followed byphosphoimagery.
Acknowledgements
We thank Anne Stringer for performing qPCR validation ofGalR ChIP-chip data. This work was supported by funds fromthe CNRS and Université Paris 7, Denis Diderot, by grantANR-09-Blan-0399 (GRONAG) to J.P., by grant ANR-BLAN07-1 187785 (SalsARN) to L.B. and by the ‘Initiatived’Excellence’ program from the French Government (Grant‘DYNAMO’, ANR-11-LABX-0011-01).
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Supporting information
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Coregulation of a sugar porin and PTS transporter 11
