Localization and Interactions of Teichoic Acid Synthetic Enzymes in Bacillus subtilis

The thick wall of gram-positive bacteria is a polymer meshworkcomposed predominantly of peptidoglycan (PG) and teichoic acids,both of which have a critical function in maintenance of thestructural integrity and the shape of the cell. In Bacillussubtilis 168 the major teichoic acid is covalently coupled toPG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradationover the lateral wall has been shown to occur in a helical pattern.However, the spatial organization of WTA assembly and its relationshipwith cell shape and PG assembly are largely unknown. We havecharacterized the localization of green fluorescent proteinfusions to proteins involved in several steps of WTA synthesisin B. subtilis: TagB, -F, -G, -H, and -O. All of these localizedsimilarly to the inner side of the cytoplasmic membrane, ina pattern strikingly similar to that displayed by probes ofnascent PG. Helix-like localization patterns are often attributableto the morphogenic cytoskeletal proteins of the MreB family.However, localization of the Tag proteins did not appear tobe substantially affected by single disruption of any of thethree MreB homologues of B. subtilis. Bacterial and yeast two-hybridexperiments revealed a complex network of interactions involvingTagA, -B, -E, -F, -G, -H, and -O and the cell shape determinantsMreC and MreD (encoded by the mreBCD operon and presumably involvedin the spatial organization of PG synthesis). Taken together,our results suggest that, in B. subtilis at least, the synthesisand export of WTA precursors are mediated by a large multienzymecomplex that may be associated with the PG-synthesizing machinery.

INTRODUCTION

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INTRODUCTION

In most bacteria, the rigid cell wall (CW) is responsible forproviding shape and structural integrity to the cell. The thickCW of gram-positive bacteria is a multilayered structure composedpredominantly of peptidoglycan (PG) (also called murein) andanionic polymers, particularly teichoic acids (TA) (for recentreviews, see references 5, 47, and 54). The highly cross-linkedPG polymer (poly-N-acetylglucosamine and N-acetylmuramic acid)network is an essential determinant of cell shape and is responsiblefor protection from the cellular turgor pressure. Many rolesfor TA have been proposed, including cell shape maintenance(61, 68), resistance to antimicrobial peptides (1, 35, 36),biofilm formation (27), acid tolerance (9) and, efficient releaseof secreted proteins into the culture medium (49).

TA are either covalently bound to the PG (wall TA [WTA]) oranchored to the cytoplasmic membrane (lipo-TA). In the gram-positivemodel organism Bacillus subtilis, WTA is present in quantitiesroughly equal to those of PG and constitutes the major classof anionic polymers (26). The type of WTA polymer varies betweenstrains. In B. subtilis 168, the major WTA consists of a poly-glycerol-phosphate[poly-(Gro-P)] chain of 45 to 60 subunits and a "PG linkageunit" of N-acetylglucosamine-β-(1-4)-N-acetylmannosamine(GlcNAc-ManNAc). WTA is covalently linked to the PG througha phosphodiester bond between the anomeric carbon of GlcNAcin the PG linkage unit and the 6-hydroxyl of MurNAc in the PGchain.

In B. subtilis 168 the genes responsible for WTA synthesis aretagABDEFGHO and mnaA (Fig. 1). According to proposed models,WTA biosynthesis occurs predominantly at the cytoplasmic sideof the membrane (5). Initially, TagO is thought to couple GlcNActo the membrane-embedded lipid undecaprenyl-pyrophosphate. Thenext step in the pathway, thought to be catalyzed by TagA, isthe addition of ManNAc, which is made from the epimerizationof GlcNAc by MnaA (59, 60), to produce the lipid-linked GlcNAc-ManNAcdisaccharide. Recent in vitro biochemical studies from the Brownlaboratory have established that TagB then functions as the"Tag primase," which couples the first glycerol-phosphate toManNAc (7), followed by TagF acting as the "Tag polymerase,"which adds the additional glycerol-phosphates extending thepoly-(Gro-P) chain (56). TagD activates glycerol-phosphate throughlinkage to CTP, making CDP-glycerol, the substrate for bothTagB and TagF (50), and TagE glucosylates glycerol-phosphatesubunits in the poly-(Gro-P) chain (51). Finally, TagG and TagHare thought to constitute a two-component ABC-type transporterthat mediates the translocation of the TA polymer across themembrane. TagH constitutes the nucleotide binding domain (NBD),and TagG is the membrane-spanning domain and presumed translocasecomponent of the ABC-type transporter TagGH (40). After translocation,the WTA polymer is covalently attached to the PG through anunknown mechanism (40). As all of these synthetic reactionsare believed to take place at the membrane in an ordered fashion(5), it could be envisaged that these proteins form a complexin which the lipid-linked WTA precursor molecule is passed throughthe chain of biosynthetic steps, but evidence for such a complexhas not yet been provided.

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FIG. 1. Overview of WTA biosynthesis. The biosynthetic steps in the formation of WTA precursors are shown. Arrows indicate catalytic steps, and the enzymes responsible are shown. The precursor is assembled at the cytoplasmic membrane prior to transport by TagGH and linkage to the PG. The question mark indicates an unknown enzymatic reaction.

Until recently it was thought that WTA was an indispensablecomponent of the CW, as mutations in any of the tag genes, excepttagE, were lethal in B. subtilis (see, e.g., references 6, 40,and 59). However, careful analysis has revealed that in factWTA is dispensable for growth in B. subtilis and Staphylococcusaureus, provided that the first committed step catalyzed byTagO is deleted (20, 21). The cause for the lethality of downstreammutations probably lies in the sequestration of metabolitessuch as undecaprenyl-pyrophosphate, which is also required asa lipid linker for the translocation of PG precursors (20).Nevertheless, loss of WTA in B. subtilis is associated withloss of rod shape and swelling, nonuniform thickening of thePG layer, aberrant placement of the division septum, and reducedgrowth rate, indicating that WTA plays an important role inmaintaining the shape and structural integrity of the bacterialcell (20).

A number of shape mutants (namely, Rod mutants) that show arounded phenotype in B. subtilis have been identified. Two ofthe early rod genes to be identified were mapped to tag genes.rodC1 was a temperature sensitive mutation in tagF, and rodDwas a mutation in tagE (29). The other two classes of Rod mutationswere mapped to rodA and mreD (rodB), which are both believedto play a role in PG synthesis (28, 67). mreB morphogenes havecome to the fore since the discovery that they encode actinhomologues. B. subtilis contains three MreB isoforms (MreB,Mbl, and MreBH) that assemble into helical filaments aroundthe cell periphery (19, 32, 65) and colocalize in what appearsto be a single helical structure (11, 18). Use of fluorescentderivatives of the antibiotics vancomycin and ramoplanin, whichlabel newly incorporated PG precursors, has revealed an underlyinghelical pattern of PG synthesis in B. subtilis (15, 63). Whetherthis pattern is influenced by the MreB cytoskeleton is at presentcontroversial. Nevertheless, it has been suggested that theMreB isoforms are involved in the positioning of PG synthasesknown as penicillin-binding proteins (PBPs) (24), a PG hydrolase(LytE) (11), and/or other membrane-associated cell morphogenesisproteins, such as MreC and MreD (22, 23, 38, 41, 66). Conversely,it has been suggested that MreB requires MreC/D for proper localizationin B subtilis (17). MreC and MreD (encoded by the mreBCD operon)are essential membrane proteins that also localize in a helix-likepattern and that are believed to function in the same morphogeneticpathway as MreBs. MreC has been recently shown to interact withhigh-molecular-weight PBPs in B. subtilis (66) and in Escherichiacoli (38) by bacterial two-hybrid (B2H) and yeast two-hybrid(Y2H) experiments, respectively, and in Caulobacter crescentusby affinity chromatography (22). Altogether, it is currentlybelieved that MreBCD form an essential membrane-bound complexinvolved in the spatial organization of the PG synthesis machineryalong the cylindrical CW (5, 54).

Several studies have attempted to determine the localizationof WTA on the surface of B. subtilis cells (12, 52, 57, 58).It has been suggested that they localize at division septa andeither in patches or uniformly along the cell cylinder, butnone of these studies were conclusive. One recent study addressedthe location of the "Tag primase" TagB and showed that it localizesto the cytoplasmic membrane in a disperse pattern (7). Sincea considerable body of recent work has suggested that CW biogenesismay occur in a helical manner governed by the MreB (actin-like)proteins, we decided to make a more systematic study of Tagprotein localization and of its relationship with the MreB cytoskeleton.Imaging of functional green fluorescent protein (GFP) fusionsto TagO, TagB, TagF, TagG, and TagH revealed a similar set ofmembrane-associated localization patterns. Fluorescence waspresent at the division sites and in a pattern consistent witha helix along the sidewall. For TagG and TagH at least, it wasreduced at mature cell poles. We then addressed the protein-proteininteractions of Tag proteins by using B2H and Y2H assays (30,34). Extensive interactions between the WTA biosynthetic proteinsas well as with the putative translocase TagGH were revealed.Interactions between several of the Tag proteins and the shapedeterminants MreC and MreD were also detected. Our findingssuggest that the synthesis and translocation of WTA precursorsare mediated by a large multienzyme complex (consisting of atleast TagO, TagA, TagB, TagF, TagG, and TagH) that localizesat sites of the cytoplasmic membrane similar to those whereinsertion of nascent PG takes place, possibly through interactionswith the bacterial cytoskeleton proteins MreC and MreD.

MATERIALS AND METHODS

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MATERIALS AND METHODS

General methods. The strains and plasmids used in this study are listed in Table1 (for a full list of plasmids used in the two-hybrid studies,see Table S2 in the supplemental material). B. subtilis cellswere made competent for transformation with DNA either by themethod of Kunst and Rapoport (39) or by the method of Anagnostopoulosand Spizizen (2) as modified by Jenkinson (31). DNA manipulationsand E. coli DH5 ${alpha}$ or XL1-Blue transformations were carried outusing standard methods (53). Solid medium used for growing B.subtilis was nutrient agar (Oxoid), and liquid medium was eithercasein hydrolysate (CH) medium (62) or S medium (33) supplementedwith 1% (vol/vol) CH (S⁺ medium) and with xylose (0.5%). WhereSMM was added, the above media were made at 2x concentrationand diluted 50:50 with a 2x SMM solution (1 M sucrose, 33.7mM maleic acid, 40 mM MgCl₂, pH 7.0). Antibiotics were usedat the following concentrations: kanamycin, 5 µg/ml; chloramphenicol,5 µg/ml; erythromycin, 1 µg/ml; lincomycin, 25 µg/ml;and/or spectinomycin, 50 µg/ml. Media used for growingE. coli strains were 2x TY (53) and nutrient agar supplementedwith ampicillin (100 µg/ml) or kanamycin (5 µg/ml),as required.

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TABLE 1. Bacterial strains and plasmids used in this study

Construction of GFP fusions. Primers and restriction endonucleases used are listed in TableS1 in the supplemental material. For the gfp-tagG and gfp-tagHfusions, approximately one-third of each of the promoter-proximalparts of the tagG and tagH genes was amplified by PCR and clonedinto pSG4902 (69). Transformation of the resulting plasmids(Table 1) into B. subtilis, with selection for chloramphenicolresistance, resulted in strains that carried a gfp fusion totagG or tagH at the chromosomal locus as the only copy of thegene of interest and under the control of the P_xyl promoter.Correct integration at the chromosomal locus was confirmed byPCR.

For tagB-gfp, tagF-gfp, and tagO-gfp fusions, approximatelyone-third of the 3'-proximal parts of each of the tagB, tagF,and tagO genes was amplified by PCR and cloned into pSG1151(43), generating plasmids pSG5879, pSG5880, and pSG5881, respectively.The plasmids were checked by sequencing and transformed intoB. subtilis 168, with selection for chloramphenicol resistanceto produce strains carrying a gfp fusion to tagB (strain 3798),tagF (strain 3799), and tagO (strain 3800) at the chromosomallocus under control of the native promoter.

For the gfp-tagB and gfp-tagF fusions, the full-length codingregions of tagB and tagF were PCR amplified and cloned intoplasmid pSG1729 (43) to generate plasmids pSG4535J and pSG4537J.The resulting inserts were sequenced, and the plasmids pSG4535Jand pSG4537J were transformed into wild-type B. subtilis 168cells, with selection for spectinomycin resistance, to givestrains 2575J and 2580J, which contain both the natural copyof tagF and tagB and a second, xylose-dependent copy of tagFor tagB, respectively, fused to gfp at the ectopic amyE locus.

To test the functionality of the GFP-TagF fusion, B. subtilisstrain EB247 (56), harboring the tagF1 (rodC1) temperature-sensitivemutation, was transformed by plasmid pSG4535J to produce strain2577J, which contains the tagF1 mutation at the chromosomallocus and the xylose-inducible gfp-tagF fusion at amyE. Disruptionof amyE was confirmed for all pSG1729 derivates using a starchplate assay (14), and the correct integration of the insertsat the amyE locus was confirmed by PCR.

To disrupt the native mbl gene in the strains containing theGFP fusions, chromosomal DNA of strain 2505 [ ${Omega}$ (mbl::spc)] (32)was used to transform strains 3110 and 3512, to produce strains3530 and 3506, respectively. For mreB disruptions, chromosomalDNA of strain 3725 ( ${Omega}$ neo3427 ${Delta}$ mreB) (25) was used to transformstrains 3110 and 3512, to produce strains 3742 and 3741, respectively.For mreBH disruptions, chromosomal DNA of strain 2535 [ ${Omega}$ (mreBH::spc)](11) was used to transform strains 3110 and 3512, to producestrains 3747 and 3746, respectively.

Microscopy. Microscopy was performed essentially as described previously(55). Image acquisition was done as described previously (42)using Metamorph v 6.0 software (Universal Imaging Corp., Downingtown,PA). Images from a single focal plane were deconvolved usingthe "No Neighbors" algorithm from the Metamorph software package.Overlays of micrographs were assembled using Metamorph beforeexporting the images to Adobe Photoshop v 6.0.

B2H plasmid construction and assay. The method used was that of Karimova et al. (34), adapted asdescribed by Daniel et al. (16). The coding sequence of eachgene was amplified by PCR from the wild-type strain 168 genomicDNA using the primers listed in Table S1 in the supplementalmaterial and inserted into pKT25, pUT18C, and pUT18. For a fulllist of plasmids used in this study, see Table S2 in the supplementalmaterial.

Y2H plasmid construction and assay. Coding sequences of each gene were amplified by PCR from thewild-type strain 168 genomic DNA using the primers listed inTable S1 in the supplemental material and cloned as translationalfusions with the Gal4 DNA-binding (BD) and Gal4 activation (AD)domains in the recipient vectors pGBDU-C1 (bait, Ura⁺) and pGAD-C1(prey, Leu⁺), respectively (30). Two-hybrid bait and prey constructionswere introduced into the Saccharomyces cerevisiae haploid strainsPJ69-4a and PJ69-4 ${alpha}$ , respectively, by gap repair (44). The two-hybridassays were performed according to a previously described matingstrategy (48). Interaction phenotypes were scored by replicaplating the diploids onto plates selecting for the expressionof the HIS3 and ADE2 interaction reporters. Control matingswith empty pGBDU and pGAD vectors were used to detect self-activationand as negative controls for interaction.

RESULTS

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RESULTS

Localization of the putative WTA transporter TagGH in a helix-like pattern. Lipid-linked WTA precursors are thought to be transported acrossthe membrane by a dedicated ABC-type transporter, TagGH. TagGand TagH are predicted to have 6 transmembrane spans and 1 transmembranespan, respectively, with both N termini in the cytosol (TMHMMv 2.0; http://www.cbs.dtu.dk/services/TMHMM/) (37). To examinethe sites of WTA export, i.e., the sites of nascent WTA insertion,N-terminal gfp fusions to tagG and tagH were constructed. Thegfp-tagG and gfp-tagH fusions were integrated into the chromosomeat the tagGH locus, replacing the respective wild-type copyof the gene. This generated two strains containing either gfp-tagG(strain 3512) or gfp-tagH (strain 3110) under the control ofthe xylose-inducible promoter P_xyl. Both strains displayed xylose-dependentgrowth (not shown), indicating that expression of gfp-tagG andgfp-tagH could replace that of the essential tagG and tagH genes,respectively. In the presence of inducer, these strains showedno morphological defects, further indicating that the GFP fusionswere functional. GFP-TagG and GFP-TagH were both expressed asfull-length fusion proteins, as shown by Western blotting usinga specific anti-GFP antibody (data not shown), and showed similarlocalization patterns (Fig. 2A and C). They localized to ongoingdivision sites, and a weak, uneven fluorescence signal was detectedover the cell cylinder. Strikingly, little fluorescence wasdetected in old cell poles, and, consistent with this, a gapbetween the fluorescence signal at the division site and thatin the cylinder emerged as cells completed division (Fig. 2A and C).Out-of-focus light in the GFP fluorescence images was reducedby two-dimensional (2D) deconvolution (see Materials and Methods),which was applied to single images of cells expressing eitherGFP-TagG or GFP-TagH. Interestingly, in both the raw and thedeconvolved images of GFP-TagG (Fig. 2A and B, respectively)or GFP-TagH (Fig. 2C and D), the cylindrical staining was reminiscentof the helix-like patterns seen in B. subtilis cells stainedwith fluorescent probes for nascent PG (15, 63) or expressinga GFP fusion to the CW hydrolase LytE (11), which were shownto be dependent on the MreB isoforms Mbl and MreBH, respectively,and of the helical configuration displayed at the membrane byGFP fusions to MreC and MreD (41). To test if the helix-likedistribution of TagG and/or TagH (and thus WTA export) was alsoinfluenced by the actin-like cytoskeleton, the GFP-TagG andGFP-TagH fusions were examined in mreB, mreBH, and mbl singlemutant strains. These strains were grown under conditions (CHmedium supplemented with SMM) previously shown to allow thesemutants to grow with near-wild-type morphology (11, 25). Underthese conditions, the localization pattern of either GFP-TagGor GFP-TagH in any of the three mreB-like single mutants (Fig.2E to J) was not significantly different from that displayedin wild-type cells (Fig. 2B and D). mreC and mreD are essentialunder normal growth conditions, but viability can also be rescuedby high concentrations of magnesium (41). However, mreC andmreD mutants are unstable and display severe shape defects underthese conditions (41), unlike the mreB-like mutants, in whichthe rod shape is restored by Mg²⁺ (11, 25). Thus, a possibleeffect of MreC and/or MreD on the positioning of the WTA transporterTagGH could not be tested. Indeed, in a ${Delta}$ mreC or a ${Delta}$ mreD background,any change in the localization patterns of TagGH could be attributedeither to the lack of MreCD proteins or to a secondary effectresulting from the shape defects.

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FIG. 2. Localization of the ABC transporter TagGH. Fluorescence images of cells expressing GFP-TagG (A and B), GFP-TagH (C and D) in a wild type background or in the absence of Mbl (E and H), MreB (F and I), or MreBH (G and J) are shown. Cells were grown to mid-exponential stage at 30°C in CH medium (or CH supplemented with SMM for the mutants [see text]) with 5 mM K₂HPO₄/KH₂PO₄ (pH 7.5) and 0.5% xylose and imaged on wet agarose slides. Panels B and D show 2D deconvolutions of panels A and C, respectively. Panels E to J show 2D deconvolutions of raw images that are not included in the figure. In panels A and C, white arrowheads represent ongoing division sites and white arrows indicate regions of reduced fluorescence between the division septum and the lateral CW. White arrowheads in B and D indicate the absence of TagGH from polar regions. Bar, 5 µm.

Localization of the WTA biosynthetic enzymes TagO, TagB, and TagF to the membrane. The first step of WTA biosynthesis is thought to be catalyzedby TagO (Fig. 1), which is predicted to have 11 transmembranespans with its C terminus located in the cytosol (TMHMM v 2.0)(37). To examine the subcellular localization of TagO, a gfpfusion was made to the C terminus of the gene. The fusion wasintegrated into the chromosome at the tagO locus, under controlof the native tagO promoter (strain 3800). The TagO-GFP fusion,which was the only copy of TagO present in the cells, was judgedto be functional, as cells displayed a wild-type morphologywhereas cells with tagO disrupted become spherical (20). TagO-GFPlocalized to the membrane, in a nonuniform pattern along thecylindrical region, and also to the division sites (Fig. 3A).Deconvolution (Fig. 3D) revealed that this pattern was alsoconsistent with a helix-like organization, similar to that displayedby the TagG- and TagH-GFP fusions (Fig. 2B and D, respectively).Again, the fluorescence intensity appeared to be reduced atolder cell poles (Fig. 3A), although this effect was less clearthan for the TagGH proteins.

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FIG. 3. Localization of TagO, TagB, and TagF. Fluorescence images of cells expressing TagO-GFP (A), TagB-GFP (B), and TagF-GFP (C) are shown. Panels D to F show 2D deconvolutions of the images in panels A to C, respectively. Cells were grown to mid-exponential stage at 30°C in CH medium with 5 mM K₂HPO₄/KH₂PO₄ (pH 7.5) and imaged on wet agarose slides. Expression of the fusion proteins was under control of the native promoters. Bar, 5 µm.

TagB, the "Tag primase," was previously reported to localizeto the cell periphery and septa using a TagB-GFP fusion clonedat an ectopic locus, under control of an inducible promoterand in the presence of the wild-type tagB copy (7). To testwhether a similar localization of TagB was observed at its endogenouslevels of expression, we integrated a tagB-gfp fusion in placeof wild-type tagB at the chromosomal locus (strain 3798). Cellsexpressing the tagB-gfp fusion as the only copy of the essentialtagB gene in the cell were viable and displayed normal growthand morphology, indicating that the fusion protein was functional(data not shown). Expression of TagB-GFP from the native tagBpromoter produced a very weak fluorescence signal, the majorityof which was associated with the division sites in either rawor deconvolved images (Fig. 3B and, E, respectively). Here deconvolutionunderscored the weakness of the signal and did not improve theresolution as much as for the other GFP fusions described. Afluorescence signal at the membrane along the cell cylinderwas occasionally seen, but it was not as strong and/or uniformas that previously reported for the (overexpressed) inducibleTagB-GFP fusion (7). Thus, at endogenous expression levels andunder the growth conditions tested, TagB mainly localizes todivision sites, with perhaps only a few molecules present inthe sidewall.

The polymerization of the poly-(Gro-P) chain, catalyzed by the"Tag polymerase" TagF, is a key step in WTA biosynthesis (56).To establish the sites of WTA polymerization in B. subtilisand their relationship with the sites of WTA nucleation (i.e.,TagB localization) and of WTA export (i.e., TagGH localization),N- and C-terminal GFP fusions were constructed for tagF. ThetagF-gfp fusion was integrated into the chromosome at the tagFlocus, placing it under control of the native promoter (strain3799). This fusion was judged to be functional, as it couldreplace the essential tagF gene in the cell without any morphologicaldefects (not shown). Despite the fact that TagF has no predictedmembrane-spanning sequences, the TagF-GFP fusion (Fig. 3C) displayeda pattern of localization similar to those of TagO-GFP (Fig.3A), GFP-TagG (Fig. 2A), and GFP-TagH (Fig. 2C). Again, deconvolutionrevealed a pattern that could be consistent with a helix-likeorganization along the sidewall (Fig. 3F), although the possiblehelicity of TagF was not as striking as that for the membraneproteins TagG and TagH (see above). As for the TagO-GFP fusion,a possible reduction in fluorescence at old cell poles was notespecially evident. The N-terminal gfp-tagF fusion was integratedat an ectopic locus (amyE) under the control of P_xyl, in additionto the wild-type copy of tagF (strain 2575J). This fusion wasjudged to be functional, as it could complement the temperature-sensitiverodC1 (tagF1) mutation (strain 2577J; data not shown). The inducibleGFP-TagF fusion gave a very bright signal that localized uniformlyaround the cell periphery (not shown), in a pattern similarto that described by Bhavsar et al. for the inducible TagB-GFPfusion (7). Thus, both TagB and TagF localize nonuniformly alongthe cell cylinder at their endogenous levels but are more uniformlydistributed around the membrane when overexpressed.

Interaction network of the WTA biosynthetic machinery. The similarity between the localization patterns of TagO, TagF,TagG, and TagH, which act at different steps of the WTA biosyntheticpathway (see Fig. 1), suggested common sites of action for allthese WTA-synthesizing enzymes and prompted us to address thepossibility that WTA might be synthesized by a membrane-associatedmultienzyme complex. Potential binary interactions between allthe Tag proteins involved in WTA synthesis (TagABDEFGHO) (Fig.1) were tested in vivo using both the Y2H (30) and the B2H (34)systems.

For the Y2H assay, fusions of the Gal4 BD ("bait") and the Gal4AD ("prey") to full-length copies of the Tag proteins were constructed.Bait and prey fusions were combined in yeast diploid cells,and their ability to interact was tested (Fig. 4A). TagB expressedas bait (BD-TagB) exhibited a self-activation phenotype (a inFig. 4A) and so was uninformative in these assays. A stronginteraction was detected between the two components of the putativeABC-type transporter, TagG and TagH, in the orientation BD-TagH/AD-TagGalthough not in the reverse orientation of the AD and BD fusions.This lack of reciprocal interaction, which might be due to conformationalchanges in the proteins fused to the AD and BD domains, is notunusual in Y2H assays (for instance, see Table 2 in reference64). The "Tag primase" TagB interacted with the "Tag polymerase"TagF. Self-interactions were detected with TagD and TagF, suggestingthat these proteins dimerize or oligomerize in vivo. Tests suchas that of AD-TagD/BD-TagO in Fig. 4A, in which only one ofthe two independent matings reporting an interaction showeda positive result and where the interaction was not seen inthe reciprocal experiment, were disregarded.

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FIG. 4. Two-hybrid interaction assays. (A and B) Y2H interactions. Matrices of Y2H interactions occurring between TagABDFGHO (A) and between TagABDFGHO and MreCD (B) are shown. The indicated proteins were expressed as baits (BD, Gal4 BD fusion) and/or as preys (AD, Gal4 AD fusion). Pairs of independent diploid yeast cells (1 and 2) expressing the various BD/AD combinations were arrayed as indicated and subjected to selection for expression of the HIS3 interaction reporter as described in Materials and Methods. The empty bait and prey vectors (BD and AD) were used to detect self-activation and as negative controls for interaction (–). The BD-TagB fusion protein exhibited a strong self-activation phenotype (a). Positive interactions are boxed or circled in red. (C and D) B2H interactions. Matrices of bacterial two-hybrid interactions occurring between TagABDEFGHO (C) and crosses of TagABDEFGHO with MreC and MreD (D) are shown. C refers to a fusion to the C terminus of T18 in the high-copy pUT18C vector. Other fusions are made to the C terminus of T25 (low-copy pKT25 vector) or to the N terminus of T18 (high-copy pUT18 vector). Transformations were carried out and plates incubated at 30°C for a maximum of 36 h.

To further investigate the interactions between the WTA proteins,the B2H system was used. In the B2H system, the T25 and theT18 fragments of the catalytic domain of Bordetella pertussisadenylate cyclase are fused to full-length copies of the Tagproteins. One potential advantage of this system is that itdoes not require the interaction partners to be associated withthe DNA for the interaction to be reported and is thereforemore suited for membrane proteins (34) (see Discussion). Asshown in Fig. 4C, TagA, TagB, TagH, and TagF all showed strongself-interactions. TagD also showed self-interaction, albeitwith a slightly weaker readout. TagO, TagA, TagB, TagF, andTagH all gave positive interaction signals with each other,and these interactions were reciprocal, with the exception ofTagO, for which only the T25-TagO fusion gave a positive result.In previous observations with the FtsW protein of E. coli (34)it was suggested that T18 fusions to proteins that contain 10or more transmembrane spans (FtsW and TagO contain 10 and 11,respectively) cannot insert correctly into the cytoplasmic membrane.TagH also showed a strong and reciprocal interaction with TagG.TagE, the last enzyme acting on the WTA precursor prior to export,showed interactions only when fused to T18. TagE interactedwith TagA and TagH, as well as TagB and TagO, but for the lattertwo only when TagE was fused to the C-terminal end of T18. Thelack of reciprocity means that the TagE results should be interpretedwith caution.

In B. subtilis, MreC and MreD proteins localize in a helix-likepattern (41), and MreC was recently shown to interact with severalhigh-molecular-weight PBPs, including PbpA and PbpH (66), suggestingthat MreCD may help to position the PG machinery (22-24, 41,66). To address whether they might also be involved in positioningthe WTA biosynthetic machinery, the Y2H and B2H systems wereused to test for interactions between MreCD and the Tag proteins.In the Y2H assay, interactions between MreD and TagH and TagOwere detected (Fig. 4B). In the B2H, interactions between bothMreC and MreD and TagH, TagO, TagA, TagB, and TagF were detected(Fig. 4D). The MreB proteins of B. subtilis do not seem to befunctional in standard two-hybrid experiments (our unpublishedresults) and were therefore excluded from the test matrices.

DISCUSSION

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DISCUSSION

Subcellular sites of synthesis and export of WTA. We have characterized the localization of functional GFP fusionsto TagB, TagF, TagG, TagH, and TagO. All of the fusions showedlocalization at division sites and, to a greater or lesser extent,along the lateral sides of the cells. In general, the fluorescenceintensity for fusions under native control was relatively low,making imaging difficult, with TagB being weakest and TagGHstrongest. Since TagGH comprises the putative ABC-type transporterfor WTA, the GFP fusions to TagGH probably identify the sitesof export of WTA precursors. Both TagG and TagH localized tonew septa in dividing cells and unevenly, in a helix-like pattern(see below), along the length of the cell (Fig. 2A to D). Thefluorescence signal at the poles was reduced in separating cells(Fig. 2A and C) and was essentially absent from poles aftercell separation (Fig. 2B and D). We note that the helix-likepattern is best observed for GFP fusions to proteins containingat least one transmembrane span. The virtual absence of TagGHin the newly completed cell pole suggests that the export andhence the incorporation of WTA into the crosswalls occurs duringdivision (septum formation) and is absent after cell separationand formation of the cell poles. This may provide the molecularbasis for the observation that turnover of WTA at the cell poleis significantly slower than that in the cell cylinder, as detectedusing both phage binding assays (3, 4, 12) and concanavalinA binding (46). If WTA and PG synthesis are coupled, this isalso consistent with the observation that the turnover of PGis very slow at cell poles (12, 57). Interestingly, the fusionsto the other proteins, of which that of TagO was strongest,did not show such a striking reduction in signal at mature cellpoles (Fig. 3A and D). It is therefore possible that TA synthesiscan occur at the old cell poles, uncoupled from export.

All of the localization patterns, especially those of TagGHand TagO, were highly reminiscent of the staining seen withfluorescent probes that label the topology of nascent PG insertion,which has been shown to occur at the septum and in a helix-likepattern over the lateral wall (15, 63). This suggested thatnascent WTA might also be incorporated into the lateral wallfollowing an underlying helical tract. Early studies using radiolabelingsuggested that in B. subtilis new WTA polymers are incorporatedonly into newly synthesized PG strands (45). Although we cannotprovide conclusive evidence that the sites of export of PG precursorscorrespond exactly to the sites of export of WTA precursors,this seems plausible in the light of our observations and wouldallow for rapid incorporation of the WTA into the CW, withoutthe long WTA polymers having to diffuse laterally along theouter surface of the membrane to reach the sites where nascentPG strands are about to be inserted.

MreB proteins control cell shape determination, and they arebelieved to achieve this by directing the spatial organizationof the sites of PG insertion. This prompted us to analyze whetherthe helix-like pattern of localization displayed by the WTAtransporter TagGH was also directed by the MreB cytoskeleton.No significant difference in the localization of either TagGor TagH was found in the mreB, mbl, or mreBH single mutant backgrounds(Fig. 2E to J) relative to the wild-type background (Fig. 2A and B).Although this raises the possibility that the actin-like cytoskeletondoes not position the sites of WTA export, it seems more likelythat the system is redundant and that more than one MreB homologuecan act as a helical guide for WTA export.

Evidence for a WTA-synthesizing multienzyme complex at the membrane. Using the B2H and Y2H systems, a complex network of interactionshas been found between the WTA synthetic enzymes (TagO, TagA,TagB, and TagF), all of which also interacted with TagH, theNBD of the ABC-type transporter. Furthermore, TagA, TagB, TagD,TagH, and TagF all showed self-interactions, suggesting thatthey dimerize or oligomerize in vivo. All of these protein-proteininteractions are believed to be physiologically relevant inview of the WTA biosynthetic pathway (Fig. 1) and of the similarlocalization patterns displayed by the Tag proteins in vivo(Fig. 2 and 3). Such a multienzyme complex would allow WTA tobe rapidly synthesized at the cell membrane prior to (or simultaneouslywith) export through the TagGH transporter. The B2H system detectedmore interactions than the Y2H system. Both systems rely onthe reconstitution of function by the interaction of two independentprotein domains fused to candidate interacting proteins. Inthe Y2H system an additional requirement is that the interactingfusion proteins are correctly targeted to the yeast nucleus,where they have to bind DNA in order for an interaction to berecorded. In the B2H system the readout is provided by the responseto a cyclic AMP-activated signaling cascade, which precludesthe need for the interacting partners to be near the transcriptionmachinery. Hence, the B2H system appears more suited for membrane(and membrane-associated) proteins such as the Tag proteins,which could explain the larger number of interactions detectedin our B2H assays compared to our Y2H assays (34).

TagG and TagH, the respective membrane-spanning domain and NBDof the predicted ABC transporter that translocates TA precursors,displayed a strong interaction in both the Y2H and the B2H assays.Interestingly, TagG did not show a positive interaction withany of the other proteins tested, supporting the idea that theinteractions detected by these systems reflect a physiologicallyrelevant specificity. TagH also showed a self-interaction, whereasTagG did not, which was unexpected since dimerization is a commonfeature in the structure of ABC transporters (8). However, itis important to note that a lack of interaction in two-hybridassays does not exclude a possible interaction in the nativecellular system. Thus, it is possible that TagG forms a dimerin B. subtilis, which either cannot be detected in our assaysor requires other factors (such as TagH) to be formed. Our analysissuggests that TagH plays a key role in WTA synthesis, presumablyby linking the enzymes involved in the synthesis of the WTAprecursors to the translocase TagG and providing the energyfor active export of WTA.

TagA, TagB, TagF, TagH, and TagO were found to interact withMreC and MreD in the B2H system (Fig. 4D). The interactionsbetween MreD and both TagH and TagO were further confirmed bythe Y2H system (Fig. 4B). Interestingly, interactions of MreCwith high-molecular-weight PBPs were recently detected usingthe same B2H system (66). This suggests that, in B. subtilisat least, MreC and MreD may have a role in positioning (or coupling)both the WTA-synthesizing machinery and the PG-synthesizingmachinery along the cylindrical wall. In E. coli, MreC has beenshown to interact with both MreB and MreD, and MreC and MreBboth showed self-interactions in a B2H assay (38). In B. subtilis,MreB, Mbl and MreBH are currently not functional in standardtwo-hybrid experiments (i.e., the expected self- and cross-interactionsbetween them are not detected in either Y2H or B2H assays [ourunpublished results]), but the three isoforms colocalize ina single helical structure (11), and direct interactions betweenMreC and Mbl and between MreB, Mbl, and MreBH were recentlydetected at the cell membrane by bimolecular fluorescence complementation(18). Thus, if MreC and MreD are positioned by an interactionwith MreB and/or MreBH and/or Mbl and if they are in turn positioningthe PG and the WTA synthetic machineries as suggested by thedirect interactions detected between MreC/D and both PBPs (66)and Tag proteins (Fig. 4B and D), then lack of a single MreBisoform would not lead to a loss of localization of either PBPs(55) or Tag proteins (Fig. 2E to J). It is tempting to speculatethat through (co-)localizing all these proteins along an organizedhelical path, the bacterial actin cytoskeleton spatially coordinatesPG and WTA synthesis to ensure that new tracts of PG containingWTA are inserted in the lateral wall. Obviously, the interactionssuggested here should be tested biochemically in due course,e.g., via coimmunoprecipitation or affinity chromatography experiments.

In summary, the results presented here suggest that WTA, likePG (i.e., all major CW polymers) is laid down in helical tractsand that WTA is incorporated into the PG at the same place andat the same time that the PG precursors are inserted into theCW. This may define discrete and highly organized sites on themembrane where PG and WTA coupled synthesis takes place, allowingthe balanced composition and structure of the CW that is necessaryto ensure controlled CW growth in rod-shaped bacteria.

ACKNOWLEDGMENTS

We thank Eric Brown for providing B. subtilis strain EB247.

R.C.-L. was funded by a long-term fellowship from the HumanFrontier Science Programme Organization (HFSPO) during partof this work. R.C-L. and P.N. are supported by the InstitutNational de la Recherche Agronomique (INRA). Work in the Erringtonlab was supported by a grant from the HFSPO. D.-J.S. was fundedby a VENI fellowship from the Research Council for Earth andLife Sciences from The Netherlands Organization for ScientificResearch (NWO).

FOOTNOTES

* Corresponding author. Mailing address: Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle NE2 4HH, United Kingdom. Phone: (44) 191 222 8126. Fax: (44) 191 222 7424. E-mail: jeff.errington{at}ncl.ac.uk

Published ahead of print on 21 December 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

REFERENCES

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