Dynamic Localization of a Transcription Factor in Bacillus subtilis: theLicT Antiterminator Relocalizes in Response to Inducer Availability

Fabian M. Rothe, Christoph Wrede, Martin Lehnik-Habrink, Boris Görke, Jörg Stülke

Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University Göttingen, Göttingen, Germany

Bacillus subtilis transports �-glucosides such as salicin by a dedicated phosphotransferase system (PTS). The expression of the�-glucoside permease BglP is induced in the presence of the substrate salicin, and this induction requires the binding of the anti-terminator protein LicT to a specific RNA target in the 5= region of the bglP mRNA to prevent the formation of a transcriptionterminator. LicT is composed of an N-terminal RNA-binding domain and two consecutive PTS regulation domains, PRD1 andPRD2. In the absence of salicin, LicT is phosphorylated on PRD1 by BglP and thereby inactivated. In the presence of the inducer,the phosphate group from PRD1 is transferred back to BglP and consequently to the incoming substrate, resulting in the activa-tion of LicT. In this study, we have investigated the intracellular localization of LicT. While the protein was evenly distributed inthe cell in the absence of the inducer, we observed a subpolar localization of LicT if salicin was present in the medium. Upon ad-dition or removal of the inducer, LicT rapidly relocalized in the cells. This dynamic relocalization did not depend on the bindingof LicT to its RNA target sites, since the localization pattern was not affected by deletion of all LicT binding sites. In contrast,experiments with mutants affected in the PTS components as well as mutations of the LicT phosphorylation sites revealed thatphosphorylation of LicT by the PTS components plays a major role in the control of the subcellular localization of this RNA-binding transcription factor.

The heterotrophic soil bacterium Bacillus subtilis lives in an en-vironment that strongly fluctuates with respect to nutrient

availability. To cope with this challenge, B. subtilis is able to selectfrom a large variety of potential substrates the carbon source thatis most advantageous for growth and propagation. In B. subtilisand many other bacteria, the phosphoenolpyruvate (PEP)-depen-dent sugar phosphotransferase system (PTS) is the key player inthe regulated transport and catabolism of carbohydrates (1, 2).The PTS comprises a family of carbohydrate-specific transportproteins collectively called enzymes II (EIIs). The driving force fortransport is provided by phosphorylation of the substrate duringits uptake. The phosphoryl groups are derived from PEP and aredelivered to the EIIs via the sequential (de)phosphorylation of thetwo general PTS proteins enzyme I (EI) and histidine protein(HPr) at histidine residues. The EIIs are composed of the twocytoplasmic EIIA and EIIB domains and of the EIIC domain,which forms the membrane channel. HPr donates the phosphorylgroups to a histidine residue in the IIA domain, from which it istransferred to a cysteine or histidine residue in the EIIB domainand subsequently to the translocated sugar.

The expression of the functions required for transport and uti-lization of a particular PTS sugar is usually strictly controlled bydedicated regulatory proteins. The regulation of PTS operons maytake place at the level of transcription initiation by repressors oractivators or by controlling transcript elongation by RNA-bindingantitermination proteins that modulate RNA structures (1, 3).BglG-type transcriptional antitermination proteins represent awidespread family of regulators that control and are controlled byPTS proteins (1, 4, 5). The antitermination proteins bind a con-served RNA antiterminator sequence (RAT) in their target RNAsand thereby prevent the formation of a transcriptional terminatorleading to expression of the target genes (6, 7). The antiterminatorproteins are composed of an N-terminal RNA-binding domainfollowed by two iterative PTS regulatory domains (PRDs), whichreceive information about carbohydrate availability from the PTS

proteins (4, 8). B. subtilis contains four antiterminator proteins ofthe BglG family, including the well-characterized LicT protein.LicT regulates expression of the bglPH operon, encoding the�-glucoside-specific EII transporter BglP and the phospho-�-glu-cosidase BglH, and of the bglS gene, which codes for a �-D-gluca-nase (9, 10, 11). In the absence of �-glucosides such as salicin, BglPinactivates LicT by its phosphorylation on histidine residues 100and 159, located in the first PRD (PRD1). The phosphoryl groupsare transferred back to BglP, if a substrate becomes available.However, to gain antitermination activity, LicT additionally re-quires phosphorylation by HPr, which takes place at histidine res-idues 207 and 269, located in the second PRD (PRD2) (12, 13).The latter phosphorylation/dephosphorylation is involved in car-bon catabolite regulation downregulating LicT when preferredPTS sugars become available. Structural analysis suggests thatphosphorylation of PRD2 stabilizes the active LicT dimer throughcontacts at the PRD interfaces (14, 15). In contrast, phosphoryla-tion of PRD1 leads to inactivation of LicT, presumably due to itsmonomerization (1, 8). Comparable regulatory mechanisms, in-volving antagonistically acting phosphorylations catalyzed by acognate EII and HPr, were also proposed for the homologousproteins SacT and GlcT from B. subtilis and BglG from Escherichiacoli (16, 17, 18).

In recent years it has been learned that bacterial cells exhibit ahighly complex spatial organization which is often dynamic (19).

Received 25 January 2013 Accepted 27 February 2013

Published ahead of print 8 March 2013

Address correspondence to Jörg Stülke, jstuelk@gwdg.de.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00117-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.00117-13

2146 jb.asm.org Journal of Bacteriology p. 2146–2154 May 2013 Volume 195 Number 10

For instance, a rapidly growing number of bacterial proteins arereported to localize in rings, foci, helices, or clusters at specificintracellular sites. These architectures, which often undergo a spa-tiotemporal dynamics, underlie various cellular activities, includ-ing protein complex formation and cross talk between cellularmachineries. Recently, it has been suggested that the PTS is alsospatially organized. Depending on the experimental setup and thegrowth conditions, the general PTS enzymes, EI and HPr, werefound to localize to the cell poles, clustered in the cytoplasm ordispersed throughout the cell in E. coli (20, 21, 22). An even morecomplex spatiotemporal localization pattern was reported for theE. coli BglG protein, which controls the �-glucoside utilizationgenes in E. coli and is the functional counterpart of LicT. When notoccupied with transport, the cognate PTS transporter BglF phos-phorylates and concomitantly sequesters BglG at the inner face ofthe cytoplasmic membrane (23). Following the addition of a sub-strate for BglF, the BglG protein is released and migrates to the cellpoles, where it undergoes HPr-dependent activation (20, 24),which involves its phosphorylation (18). Subsequently, BglG mi-grates to the cytoplasm, where it catalyzes antitermination at thebgl operon. Sequestration by the cognate transport protein at themembrane has also been observed for a few other transcriptionalregulators controlling carbohydrate utilization genes. Similar toBglG, the transcriptional activator protein MalT, controllingmaltose utilization in E. coli, is inactivated through sequestrationby the resting maltose transporter (25). In contrast, the Mlc re-pressor protein, which controls multiple PTS functions in E. coli,is recruited to the membrane through interaction with the trans-locating glucose transporter (26). An unusual mode of membranesequestration has recently been revealed for the transcriptionalactivator protein MtlR in B. subtilis (27). MtlR, which controls themannitol utilization genes, contains two PRDs followed by EIIB-and EIIA-like domains. On the one hand, the activity of MtlR iscontrolled by multiple PTS-catalyzed phosphorylations at itsPRD2 and the EIIB-like domain. However, to gain activity, MtlRmust also interact via its C-terminal EIIA-like domain with thetranslocating mannitol-specific EII.

In this study, we investigated the localization of the antitermi-nator protein LicT in B. subtilis. We observed that inactive LicT isnot sequestered at the membrane by its cognate transporter BglPbut is evenly distributed in the cytoplasm. Upon activation, LicTrapidly relocalizes to subpolar regions independent of the pres-ence of its RNA targets. In conclusion, LicT undergoes a dynamicrelocalization in the cell that correlates with its activity and de-pends on its phosphorylation status. The localization of LicT dras-tically differs from that reported for its homolog BglG in E. coli,suggesting species-specific localization traits. This conclusion issupported by our observation that EI of the PTS is evenly distrib-uted in the B. subtilis cell independent of PTS transport activity,while it was reported to localize to the cell poles in E. coli.

MATERIALS AND METHODSBacterial strains and growth conditions. The B. subtilis strains used inthis study are listed in Table 1. E. coli DH5� (28) was used for plasmidconstructions and transformation using standard techniques (28).

Luria-Bertani (LB) broth was used to grow E. coli and B. subtilis. Whenrequired, media were supplemented with antibiotics at the following con-centrations: ampicillin, 100 �g ml�1 (for E. coli); spectinomycin, 150 �gml�1; kanamycin, 7.5 �g ml�1; phleomycin, 6 �g ml�1; chlorampheni-col, 5 �g ml�1; and erythromycin plus lincomycin, 2 and 25 �g ml�1,respectively (for B. subtilis).

B. subtilis was grown in C minimal medium supplemented with auxo-trophic requirements (at 50 mg l�1) as indicated below (29), and carbonsources were used at a concentration of 0.5% (wt/vol) unless stated oth-erwise. The basal medium used in this study, CSE, is C minimal mediumsupplemented with succinate and glutamate (29). Sporulation (SP) plateswere prepared by the addition of 15 g l�1 Bacto agar (Difco) to sporulationmedium (8 g of nutrient broth per liter, 1 mM MgSO4, and 13 mM KCl,supplemented after sterilization with 2.5 �M FeSO4, 500 �M CaCl2, and10 �M MnCl2).

Transformation and enzyme assays. Chromosomal DNA of B. subti-lis was isolated using the DNeasy tissue kit (Qiagen) according to thesupplier’s protocol. B. subtilis was transformed with plasmids and chro-mosomal DNA according to the two-step protocol (30). Transformantswere selected on SP plates containing antibiotics as described above. Forenzyme assays, cells were harvested in the exponential growth phase at anoptical density at 600 nm (OD600) of 0.6 to 0.8.

Quantitative studies of lacZ expression in B. subtilis were performed asfollows. Cells were grown in LB medium and harvested at an OD600 of 0.6to 0.8. �-Galactosidase specific activities were determined with cell ex-tracts obtained by lysozyme treatment as described previously (30). Oneunit of �-galactosidase is defined as the amount of enzyme which pro-duces 1 nmol of o-nitrophenol per min at 28°C.

Plasmid constructions. To facilitate the fusion of B. subtilis proteinsto the green and yellow fluorescent proteins (GFP and YFP, respectively),we constructed the plasmids pGP1870 and pGP1871, respectively. For thispurpose, we amplified the gfp and yfp gene fragments using the primerpairs ML220/ML221 and ML222/ML223 and plasmids pSG1151 (31) andpIYFP (32) as the templates, respectively. The amplicons were digestedwith HindIII and cloned into the vector pUS19 (33). The vectors pGP1870and pGP1871 allow the construction of GFP/YFP fusions and integrationof the plasmid into the chromosome via Campbell-type recombinationbetween the cloned gene fragment and the chromosomal copy of the gene(see http://www.subtiwiki.uni-goettingen.de/wiki/index.php/PGP1870and http://www.subtiwiki.uni-goettingen.de/wiki/index.php/PGP1871).

To fuse the LicT protein to the green and yellow fluorescent proteins,we amplified the 3= 600 bp of the licT gene lacking a stop codon using theprimer pair FR114/FR115 and chromosomal DNA of B. subtilis 168 as thetemplate and cloned the amplicon between the BamHI and SalI sites ofpGP1870 and pGP1871, respectively. The resulting plasmids werepGP1292 (licT-gfp) and pGP1296 (licT-yfp). Integration of these plasmids

TABLE 1 B. subtilis strains used in this study

Strain Genotype Source or referencea

168 trpC2 Laboratory collectionGM1112 sacXY�3 sacB�23 sacT�4 bglP::Tn10 (cat::ery)

amyE(sacB::lacZ Phlr)9

GP427 trpC2 �licTS::ermC 7GP475 trpC2 �bglP::ermC GM1112 ¡ 168GP1225 trpC2 licT-gfp spc pGP1292 ¡ 168GP1229 trpC2 licT-yfp spc pGP1296 ¡ 168GP1236 trpC2 �bglP::erm licT-gfp spc GP475 ¡ GP1225GP1242 trpC2 amyE::(bglP-lacZ phl) licT-gfp spc GP1225 ¡ QB5335GP1243 trpC2 amyE::(bglP-lacZ phl) licT-yfp spc GP1229 ¡ QB5335GP1245 trpC2 amyE::(bglP-lacZ phl) �licTS::ermC GP427 ¡ QB5335GP1257 �ptsH::cat licT-gfp spc pGP1292 ¡ MZ303GP1258 trpC2 licT (H100A)-gfp spc pGP1306 ¡ 168GP1259 trpC2 licT (H207A)-gfp spc pGP1307 ¡ 168GP1260 trpC2 licT (H100A/H207A)-gfp spc pGP1308 ¡ 168GP1261 trpC2 �RATbglS::cat This workGP1262 trpC2 �RATbglP::kan This workGP1263 trpC2 �RATbglS::cat �RATbglP::kan GP1262 ¡ GP1261GP1264 trpC2 �RATbglS::cat �RATbglP::kan licT-gfp spc pGP1292 ¡ GP1263GP1266 trpC2 bglP-cfp ermC This workGP1267 trpC2 ptsH-cfp ermC This workGP1268 trpC2 ptsI-cfp ermC This workMZ303 �ptsH::cat 16QB5335 trpC2 amyE::(bglP-lacZ phl) 36a Arrows indicate construction by transformation.

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into the chromosome of B. subtilis leads to the in-frame fusion of the gfp oryfp alleles to the entire licT gene lacking its stop codon. To fuse mutantvariants of LicT to GFP, PCR-based mutagenesis (34) was performed toobtain the alleles coding for LicT(H100A), LicT(H207A), and LicT(100A207A). For this purpose, the licT gene was amplified using the outerprimer pair FR162/FR115 and the mutagenesis primers FR164 ([forLicT(H100A)] and FR163 [for LicT(H207A)]. The mutated alleles werecloned between the BamHI and SalI sites of pGP1870, giving pGP1306and pGP1307. To obtain the double mutant allele, pGP1306 was used asthe template in the mutagenesis PCR and FR163 as the mutagenesisprimer. After cloning of the fragment into pGP1870, the resulting plasmidwas pGP1308.

Strain construction by long flanking homology PCR mutagenesis.To replace the RNA antiterminator (RAT) sequences of the bglP and licSgenes with terminatorless kanamycin and chloramphenicol resistancecassettes, we generated PCR products using oligonucleotides (see Table S1in the supplemental material) to amplify DNA fragments flanking theRAT sequences and intervening antibiotic resistance cassettes as describedpreviously (35). These PCR products were directly used to transform B.subtilis strains.

To fuse the PTS components to the cyan fluorescent protein (CFP), weperformed a fusion PCR with amplicons of the B. subtilis chromosomalbglP gene, of the cfp ermC cassette amplified from plasmid pBP20 (K.Gunka and F. M. Commichau, unpublished data), and of the downstreambglH gene. In a similar way, we obtained a fusion PCR product that al-lowed the construction of a ptsH-cfp strain. To avoid interference with theexpression of the downstream bglH and ptsI genes, respectively, the ter-minator downstream of the ermC gene was omitted in the PCR. For thegeneration of a ptsI-cfp strain, we used the same procedure, but in this casethe ermC terminator was included.

Western blotting. For Western blot analysis, proteins were separatedby 12% SDS-PAGE and transferred onto polyvinylidene difluoride(PVDF) membranes (Bio-Rad) by electroblotting. Rabbit anti-GFP poly-clonal antibodies (Biozol, Eching, Germany; 1:10,000) served as primaryantibodies. The antibodies were visualized by using anti-rabbit immuno-globulin alkaline phosphatase secondary antibodies (Promega) and theCDP-Star detection system (Roche Diagnostics), as described previously(29).

Microscopy. For fluorescence microscopy, cells were grown in CSE,CSE-salicin (0.1% [wt/vol]), or CSE-sorbitol (0.5% [wt/vol]) medium toan OD600 of 0.3 to 0.4, harvested, and resuspended in phosphate-bufferedsaline (pH 7.5; 50 mM). Fluorescence images were obtained with an Ax-ioskop 40 FL fluorescence microscope, equipped with digital cameraAxioCam MRm and AxioVision Rel 4.8 software for image processing(Carl Zeiss, Göttingen, Germany) and Neofluar series objective at a pri-mary magnification of �100. The applied filter sets were the YFP HC-Filterset (BP, 500/24; FT, 520; LP, 542/27; AHF Analysentechnik, Tübin-gen, Germany) for YFP detection, the eGFP HC-Filterset (BP, 472/30; FT,495; LP, 520/35; AHF Analysentechnik) for eGFP detection, filter set 47(BP, 436/20; FT, 455; LP, 480/40; Carl Zeiss) for CFP visualization, andfilter set 49 (G, 365; FT, 395; LP, 445/50; Carl Zeiss) for 4=,6-diamidino-2-phenylindole (DAPI) visualization. The overlays of fluorescent andphase-contrast images were prepared for presentation with Adobe Pho-toshop Elements 8.0 (Adobe Systems, San Jose, CA).

RESULTSLocalization of PTS proteins involved in salicin uptake. Salicin istaken up by a phosphotransferase system composed of the generalcomponents enzyme I and HPr and the �-glucoside-specific per-mease BglP. To investigate the localization of these proteins, weconstructed a set of strains expressing these PTS componentsfused to the cyan fluorescent protein at their C termini (Table 1).These fusion proteins were encoded at the authentic loci of thePTS components. Prior to the investigation of protein localiza-tion, we tested whether the fusion proteins were biologically ac-

tive. For this purpose, we compared the growth of these strains onminimal medium containing glucitol, glucose, or salicin to that ofwild-type B. subtilis 168. All strains grew well with the non-PTScarbon source glucitol. In contrast, the strain expressing the HPr-CFP protein (GP1267) did not grow on glucose and salicin,whereas the bacteria with enzyme I-CFP (GP1268) or BglP-CFP(GP1266) grew well in the presence of these carbon sources (datanot shown). These results indicate that the tagged enzyme I andBglP proteins were active in the transport of glucose (enzyme I)and salicin (enzyme I and BglP), whereas the tagged HPr proteinwas unable to participate in PTS-dependent sugar uptake. Thissuggests that the HPr-CFP protein was inactive, and therefore, thefollowing experiments were performed only with the strains ex-pressing active fusion proteins.

To localize enzyme I and BglP, the strains GP1268 and GP1266were grown in CSE minimal medium with salicin or glucitol as thecarbon source. For enzyme I-CFP, a bright fluorescence that wasevenly distributed in the cell was observed under both conditions(Fig. 1A). This result is in good agreement with the establishedconstitutive expression of the ptsHI operon (36) and with the ab-sence of a membrane anchor in enzyme I. For BglP, the result wasdifferent. This protein gave bright signals in the membrane regionof the cell when the bacteria were grown in the presence of theBglP substrate, salicin. In contrast, only background signals weredetected during growth with glucitol (Fig. 1B). These observationsprecisely matched our expectations since BglP is known to be themembrane-bound permease for salicin uptake, and its expressionis strongly dependent on presence of the inducer salicin (9).

Localization of the antitermination protein LicT. As statedabove, the detection of BglP depended on the presence of salicin in

FIG 1 Localization patterns of the PTS proteins involved in salicin uptake.Shown are fluorescence microscopy images of cells harboring a ptsI-cfp(GP1268) (A) or a bglP-cfp fusion (GP1266) (B) in the chromosome. Cells ofGP1268 (EI-CFP) (A) and GP1266 (BglP-CFP) (B) were grown in CSE mini-mal medium with salicin (sal) or glucitol (gluc) and prepared for microscopyin the logarithmic phase of growth. Scale bars, 2 �m.

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the medium. This induction is controlled by the transcriptionalantiterminator LicT, which in the presence of salicin binds theRAT sequence in the bglP leader mRNA in order to allow tran-script elongation beyond the intrinsic terminator located in theleader region (9, 11). This prompted us to test the localization ofthe antiterminator protein LicT as well. For this purpose, we con-structed strains with LicT fused to either the green or the yellowfluorescent protein (GP1225 and GP1229, respectively). To assessthe activity of the LicT fusion proteins, we constructed a set ofstrains carrying a fusion of the LicT-regulated bglP promoter to apromoterless lacZ gene encoding �-galactosidase. The resultingstrains were cultivated in minimal medium without an addedsugar or in the presence of salicin or salicin and glucose. As shownin Table 2, no �-galactosidase activity was detectable with thewild-type strain in minimal medium without salicin. In the pres-ence of salicin, the expression was induced to about 1,840 U permg of protein. If both glucose and salicin were available, only 113U of activity was detected. In the absence of a functional licT gene(GP1245), no bglP expression was observed under all conditionstested. These results are in excellent agreement with previous ob-servations (37). They show the salicin-dependent induction aswell as glucose-mediated carbon catabolite repression of LicT ac-tivity. Moreover, it is obvious that a functional licT gene is essen-tial for the expression of bglP. The strains expressing the LicTfusion proteins exhibited an expression pattern of bglP that wasvery similar to that of the strain carrying the nontagged wild-typeLicT protein (Table 2). Therefore, the fluorescently labeled LicTproteins have retained full activity in antitermination as well as thephysiological control of their activity by PTS components.

To study the localization of LicT, we grew strain GP1225 ex-pressing LicT-GFP in CSE minimal medium without an addedsugar or in the presence of glucitol, salicin, or glucose. The resultsare shown in Fig. 2. Generally, the fluorescence was much weakerthan observed for the other fusions used in this study. This is ingood agreement with the rather weak level of licT transcription

(38). In the absence of any sugar as well as in the presence of thenoninducing carbon sources glucitol and glucose, LicT was evenlydistributed throughout the cell. In contrast, we observed an accu-mulation of the protein in the subpolar regions when the bacteriawere grown in the presence of the inducer salicin, i.e., when LicTwas active (Fig. 2C). Similar results were obtained with LicT fusedto YFP (data not shown). These results suggest a correlation be-tween LicT activity and its intracellular localization.

Dynamic relocalization of LicT upon a nutrient shift. Asshown above, the localization of LicT is controlled by the avail-ability of the inducer salicin and is thus correlated to its activity inantitermination. If, in turn, the localization would reflect the ac-tivity of the protein, one would expect a rapid relocalization whenthe nutrient supply changes. To address this issue, we cultivated B.subtilis GP1225 expressing LicT-GFP in CSE minimal medium tothe mid-logarithmic phase and transferred the bacteria to freshmedium containing the inducer salicin. The localization of LicT-GFP was analyzed prior to the shift and 5, 15, and 30 min after theaddition of salicin. As shown in Fig. 3A, LicT was evenly distrib-uted throughout the cell in the CSE medium. This result is in goodagreement with the results described above (Fig. 2A). Five minutesafter the nutrient shift, LicT started to concentrate in the subpolarregions of the cells. This process continued, and after 15 min thegreat majority of cells had LicT in the subpolar regions. The samelocalization was also observed 30 min after the nutrient shift.

Next, we tested whether the LicT localization would also alterwhen cells are shifted from a medium with the inducer salicin to amedium without salicin. For this purpose, GP1225 was grown tothe mid-logarithmic phase in CSE medium containing salicin.Then, the cells were washed and resuspended in fresh CSE me-dium containing glucitol. Again, the LicT localization was ob-served before and after the shift. As shown in Fig. 3B, LicT exhib-ited the subpolar localization typical for the active protein duringgrowth with salicin. After the shift to glucitol-containing medium,the localization changed and eventually the protein was again ho-mogeneously distributed in the cells as observed before for inac-tive LicT irrespective of the noninducing carbon source available.However, this delocalization of LicT was slower; only after 15 mindid most cells exhibit the homogeneous distribution of LicT. Theprocess was finished in the last sample analyzed 30 min after themedium shift.

Taken together, our results demonstrate that LicT changes itsintracellular localization in response to the presence of the inducersalicin and the accompanying change in LicT activity.

Role of the LicT RNA targets for the localization of LicT. Asreported above, active LicT concentrates in the subpolar regions

TABLE 2 Activities of LicT fusion proteins

StrainRelevantgenotype

�-Galactosidase activity (U/mg of protein)a

CSE CSE-Sal CSE-Sal/Glc

QB5335 Wild type 0.5 � 0.5 1,840 � 61 113 � 10GP1245 �licT 2 � 1 2 � 0 1.5 � 0.5GP1242 licT-gfp 3 � 1 1,620 � 100 75 � 14GP1243 licT-yfp 2.5 � 0.5 1,490 � 85 90 � 13a All measurements were performed at least in triplicate. Values are means � standarddeviations. Sal, salicin; Glc, glucose.

FIG 2 The cellular localization of LicT correlates with its activity. Shown are fluorescence micrographs of GP1225 cells expressing a LicT-GFP fusion in themid-logarithmic phase grown in CSE minimal medium without carbon sources (A) or with glucitol (gluc) (B), salicin (sal) (C), or glucose (glc) (D). Scale bar,2 �m.

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of the cells, whereas inactive protein is homogeneously distrib-uted. The active protein binds its RNA targets to prevent tran-scription termination of the controlled bglS and bglPH mRNAs(11). It is therefore conceivable that the active protein follows thelocalization of its target molecules. If this were the case, one wouldexpect loss of inducer-dependent intracellular distribution of LicTin a strain that is devoid of LicT’s target sites. To test this hypoth-esis, we deleted the regions corresponding to the RAT/terminatorcontrol regions of bglS and the bglPH operon in a way that did stillallow expression of these genes (Fig. 4A). The resulting strain,GP1264, was grown in CSE medium with salicin or glucitol, andthe localization of LicT-GFP was investigated. As shown in Fig. 4B,the intracellular distribution of LicT in this strain was indistin-guishable from that in the wild type; i.e., the protein was concen-trated in the subpolar regions during growth with salicin, whereasit was evenly distributed throughout the cell during growth withglucitol. This observation excludes the possibility that binding ofLicT to its target RAT sequences modulates the localization of theprotein.

Role of the PTS components in inducer-dependent LicT lo-calization. As demonstrated above, the localization of LicT corre-lates perfectly with its activity in antitermination. However, thelocalization of LicT does not follow its target RNA molecules andmust therefore depend on other factors. It is well established thatthe activity of LicT is controlled by multiple PTS-dependent phos-phorylation events. In the absence of the inducer, BglP phosphor-ylates LicT to inhibit its activity, whereas HPr can phosphorylateLicT in the absence of preferred carbon sources to stimulate theactivity (12, 13). To analyze the implication of the PTS compo-nents in LicT localization, we constructed strains with deletions ofeither the bglP or the ptsH gene.

The mutant strains were grown in CSE minimal medium in thepresence of salicin or glucitol. For the bglP mutant GP1236, weobserved a subpolar localization of LicT in both media (Fig. 5A).Thus, the bglP mutant does not seem to be able to sense the pres-ence or absence of the inducer salicin; indeed, the transduction of

this information to LicT is the task of BglP. Thus, in the bglPmutant, the LicT protein is constitutively active (10), and it isalways localized as the active protein. In the ptsH mutant lackingthe general PTS protein HPr, no phosphorylation of LicT is pos-sible since HPr can directly phosphorylate LicT, but it serves alsoas a phosphoryl group donor in the phosphorylation chain fromBglP to LicT (9). In this mutant, the intracellular distribution ofLicT was similar to that observed in the bglP mutant; i.e., LicTaccumulated in the subpolar regions of the cells (Fig. 5B).

The results obtained with the PTS mutants suggest that it is notthe activity of LicT that is important for the intracellular localiza-tion, since LicT is constitutively active in bglP mutants but com-pletely inactive in ptsH mutants. Thus, specific phosphorylationevents seem to control the localization of LicT.

Impact of the different phosphorylation sites on LicT local-ization. The conserved histidine residues in PRD1 and PRD2 arephosphorylated by BglP and HPr, respectively. To study the im-pact of these phosphorylation events in more detail, we con-structed a set of strains that express LicT variants with mutationsin His-100 (PRD1, H100A), His-207 (PRD2, H207A), or bothresidues. To exclude the possibility that the mutations affected thestability and thus the intracellular accumulation of the LicT-GFPfusion proteins, we cultivated the different strains in CSE minimalmedium without an added carbohydrate or with salicin or gluci-tol. The amounts of LicT-GFP were analyzed by a Western blotusing antibodies that recognize GFP. As shown in Fig. 6, the dif-ferent LicT variants were expressed in each medium. Therefore,the mutations did not affect the stability of the mutant LicT-GFPfusion proteins.

For the determination of LicT localization, the bacteria weregrown in CSE minimal medium supplemented with salicin or glu-citol. For the constitutively active LicT-H100A protein (GP1258),we observed a subpolar localization pattern of LicT under bothconditions (Fig. 7A). This result is in excellent agreement with theLicT localization in the bglP mutant, in which H-100 cannot bephosphorylated. The inactive Lic-H207A protein was found to be

FIG 3 Dynamic relocalization of LicT after a nutrient shift. (A) Cells of GP1225 (licT-gfp) were grown in CSE minimal medium without any carbohydrate,washed at time zero, and resuspended in CSE medium containing salicin. (B) GP1225 was grown in CSE minimal medium with salicin. At time zero, the cells werewashed and resuspended in CSE with glucitol. Fluorescence microscopy images were taken at time zero and at 5, 15, and 30 min after the addition of the newmedium. The scale bars correspond to 2 �m.

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evenly distributed under both conditions (Fig. 7B). Similarly, theinactive LicT-H100A/H207A double mutant protein was evenlydistributed both in the presence and absence of salicin (Fig. 7C).

Taken together, our data support the idea that the phosphor-ylation of LicT is crucial for its localization: all mutant proteinsthat are no longer responsive to the presence of salicin in theiractivity do also exhibit an unregulated localization. Similarly, inthe PTS mutants that result either in constitutive LicT activity orin its complete inactivity, no response of LicT localization to thenutrient supply was observed. However, while the inactive LicTmutant proteins were always found throughout the cytoplasm, asubpolar localization was observed for the LicT protein that wasinactive due to the absence of HPr. This difference suggests notonly that the phosphorylation events are required for the correctintracellular recruitment of LicT but also that the integrity of theprotein is important (see Discussion).

DISCUSSION

In this work, we provide the first analysis of the intracellular lo-calization of PTS components and of a PTS-controlled transcrip-tion factor in B. subtilis.

Our results demonstrate that the general PTS protein enzyme Iis evenly distributed in the cytoplasm. This is in good agreementwith the annotation of this protein as a soluble PTS protein. How-ever, contradictory results have been reported for the localization

of enzyme I in E. coli. Similar to our results, E. coli enzyme I wasfound to be distributed in the cytoplasm by Neumann et al. (22),whereas a spotty distribution was reported by Patel et al. (21).Finally, polar localization of enzyme I was observed by Lopian etal. (20). It is difficult to judge the reason for these obvious differ-ences. One factor may be the overexpression of enzyme I and theuse of different fluorescence tags. In this work, we expressed thefluorescence-tagged PTS proteins from their own promoter, thusavoiding any effect of artificial expression.

A study on the subcellular localization of the �-glucoside-spe-cific antitermination protein BglG indicated that this protein isrecruited to the membrane by its cognate permease and sensor,BglF, and that BglG is released into the cytoplasm if the inducerand substrate salicin becomes available (23). Our results suggestthat the localization of LicT in B. subtilis does also depend on thefunctional �-glucoside permease BglP: when no substrate is avail-able, and BglP phosphorylates LicT on the PRD1 and thereby in-activates the protein, LicT is homogeneously distributed through-out the cytoplasm. In contrast, lack of phosphorylation of PRD1when salicin is available (phosphate flux from BglP to the sub-strate) or the bglP gene is deleted results in subpolar localization ofLicT. It should be noted that no membrane association of LicT wasdetected under all conditions tested in this work. Thus, the local-ization pattern of LicT differs from that of its E. coli homologBglG. However, both proteins relocalize upon addition of the in-

FIG 4 Effect of the LicT RNA targets on the subcellular localization of LicT. (A) Construction of strain GP1264 lacking the RNA binding sites of LicT. The RNAtargets of LicT (RAT) and the overlapping terminator strucures (t) present in the leader regions of the bglS gene or the bglPH operon were replaced with achloramphenicol (cat) or a kanamycin resistance (aphA3) gene, respectively. (B) Fluorescence microscopy images of GP1264 (licT-gfp �RAT). The cells weregrown in CSE minimal medium with salicin or glucitol and prepared for microscopy in the logarithmic phase of growth. Scale bar, 2 �m.

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ducer, and in both cases this relocalization is achieved after about15 min (Fig. 3) (20). It is interesting that a very recent study on theB. subtilis PRD-type transcription factor MtlR revealed that thisprotein is recruited to the membrane by its cognate mannitol-specific EIIB domain of the mannitol permease MtlA. Moreover,this membrane association was found to be important for theactivity of MtlR (27). Thus, PRD-type transcription factors seemgenerally to require specific localization patterns for their activity;however, the precise localization differs from one protein to theother.

The determinants of intracellular protein localization are onlypoorly understood. While transmembrane domains can easily beidentified in a protein, it is not known which factors drive thedifferential localization of LicT in the presence or absence of the

inducer salicin. For example, the peripheral membrane proteinDivIVA binds primarily to sites that exhibit a negative membranecurvature (39, 40). In this case, it was suggested that DivIVA mul-timers bridge the curvature (40). Another factor that often deter-mines the localization of proteins is recruitment by other proteinsor macromolecules. In B. subtilis, this is best studied for the assem-bly of the spore coat, which depends on the initial ATP-drivenpolymerization of SpoIVA on the spore surface (41, 42). Similarly,protein-dependent recruitment was proposed for the inactiveBglG and the active MtlR transcription factors. In the case of LicT,the localization determinants are unknown. Interestingly, LicTactive in transcriptional antitermination localizes to the same re-gions of the cell as the ribosomes (43, 44). Given the couplingbetween transcription and translation in bacteria, it is tempting tospeculate that the RNA molecules to which LicT binds recruit theprotein to the subpolar regions of the cell. Surprisingly, our resultsindicate that differential LicT localization is observed even in astrain devoid of the bglPH and bglS RAT RNA regions (Fig. 4B).One might argue that LicT may bind other RAT sequences in theabsence of its cognate targets. However, as shown previously, LicTis unable to bind any of the other RAT sequences present in B.subtilis (45). Thus, phosphorylation is likely to be the driving forcefor LicT (re)localization.

Our analyses with PTS mutants and LicT phosphorylation sitemutants support the idea that the PTS-dependent phosphoryla-tion of LicT controls its subcellular localization. Indeed, muta-tions affecting enzyme I or HPr resulted in the absence of phos-phorylation of the PRD1 of LicT, and both mutations did alsoresult in the prevention of the homogeneous distribution of LicTthat is normally observed when LicT is phosphorylated on PRD1in the absence of the inducer salicin. The loss of phosphorylationof both PRD1 and PRD2 gave contradicting results: while subpo-lar LicT localization was observed in the ptsH mutant lacking HPr,the nonphosphorylatable LicT-H100A/H207A protein was ho-mogeneously distributed in the cytoplasm. Though counterintui-tive, these observations are not unprecedented: phosphorylation-dependent protein localization or protein-protein interactionssometimes require not only the presence or absence of the phos-phoryl group; the requirements for correct localization/interac-tion may extend to the correct amino acid sequence of the pro-teins. This was reported for the localization of E. coli HPr, whichdepends on the presence of the phosphorylation site (His-15) forproper polar localization (20). Similarly, the interaction betweenthe B. subtilis HPr protein and the CcpA transcription factor de-pends on the integrity of the His-15 phosphorylation site (46, 47).Finally, the presence or absence of the phosphorylation site His-15

FIG 5 Effects of PTS components on the LicT localization pattern. The B.subtilis �bglP mutant strain expressing a LicT-GFP fusion (GP1236) (A) andthe �ptsH mutant with a LicT-GFP fusion (GP1257) (B) were grown in CSEminimal medium containing 0.1% salicin or 0.5% glucitol. The fluorescencepictures were taken in the mid-logarithmic phase of growth. The scale barscorrespond to 2 �m.

FIG 6 Accumulation of LicT mutant proteins. The B. subtilis strains GP1225 (licT-gfp; wild type), GP1258 (licT-H100A-gfp), GP1259 (licT-H207A-gfp), andGP1258 (licT-H100A/H2007A-gfp) were grown in CSE minimal medium, in CSE with salicin, or in CSE with glucitol at 37°C to the mid-log phase. The crudeextract obtained from 0.1 OD unit at 600 nm of each culture was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis andblotting onto a polyvinylidene difluoride membrane, the GFP tag was detected using rabbit polyclonal antibodies.

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in HPr and its regulatory paralog Crh is a major determinant forthe specificity of the regulatory interaction between Crh and themethylglyoxal synthase MgsA (48).

While the field of intracellular protein localization in bacteria isstill in its infancy, it is becoming more and more obvious thatproteins are very dynamic and that they relocalize depending onthe growth conditions. The analysis of the driving forces and themolecular mechanisms behind this movement of proteins will bea major challenge for future research.

ACKNOWLEDGMENTS

We thank Vanessa Otten, Oliver Schilling, and Dominik Tödter for theirhelp with some experiments and Sabine Lentes for technical assistance.

This work was supported by grants from the Federal Ministry of Edu-cation and Research SYSMO network (PtJ-BIO/0313978D) and the DFG(SFB860) to J.S. F.M.R. was supported by a fellowship from the GermanNational Academic Foundation.

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