Teichoic Acid Cross Linking Staphylococcus Aureus

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Teichoic acids are temporal and spatial regulators ofpeptidoglycan cross-linking in Staphylococcus aureusMagda L. Atilanoa,1, Pedro M. Pereirab,1, James Yatesa, Patricia Reedb, Helena Veigab, Mariana G. Pinhob,2,3,and Srgio R. Filipea,2,3

aLaboratory of Bacterial Cell Surfaces and Pathogenesis and bLaboratory of Bacterial Cell Biology, Instituto de Tecnologia Qumica e Biolgica, Universidade

Nova de Lisboa, 2781-901 Oeiras, Portugal

Edited by Richard P. Novick, New York University School of Medicine, New York, New York, and approved September 20, 2010 (received for review April1, 2010)

The cell wall of Staphylococcus aureusis characterized by an ex-

tremely high degree of cross-linking within its peptidoglycan (PGN).

Penicillin-binding protein 4 (PBP4) is required for the synthesis of

this highly cross-linked peptidoglycan. We found that wall teichoic

acids, glycopolymers attached to the peptidoglycan and importantfor virulence in Gram-positive bacteria, act as temporal and spatial

regulators of PGN metabolism, controlling the level of cross-linking

by regulating PBP4 localization. PBP4 normally localizes at the di-

vision septum, but in the absence of wall teichoic acids synthesis, it

becomes dispersed throughout the entire cell membrane and is un-

able to function normally. As a consequence, the peptidoglycan of

TagO null mutants, impaired in wall teichoic acid biosynthesis, hasa decreased degree of cross-linking, which renders it more suscep-

tible to the action of lysozyme, an enzyme produced by different

host organisms as an initial defense against bacterial infection.

cell division | cell wall | penicillin-binding proteins | transpeptidases |lysozyme

The cell wall of Gram-positive bacteria is a highly complexnetwork composedmainly of peptidoglycan(PGN) and teichoicacids (TAs), both essential to the maintenance of the structuralintegrity and shape of the bacterial cell. Peptidoglycan is a hetero-geneous polymer of glycan chains cross-linked by short peptides ofvariable length and amino acid composition (1). Teichoic acids are

phosphate-rich glycopolymers that can be either covalently linkedto PGN (wall teichoic acids, or WTAs) or anchored to the cyto-plasmic membrane (lipoteichoic acids, or LTAs) (2, 3).

Despite decades of study, it is still not well understood whypathogenic and nonpathogenic bacteria have TAs at their cellsurface. TAs contribute to a variety of processes, including re-sistance to environmental stresses, such as heat (4) or low os-molarity (5), to antimicrobial peptides (6), antimicrobial fattyacids (7), cationic antibiotics (8), and lytic enzymes produced bythe host, including lysozymes (9, 10). TAs also act as receptors forphage particles (11), and they can bind cationic groups (particu-larly magnesium ions), providing a reservoir of ions close to thebacterial surface that may be important for the activity of differentenzymes (12). More recently, TAs have been proposed to be in-

volved in cell division and morphogenesis (13). Lack of LTAsynthesis in rod-shaped Bacillus subtilis cells causes defects in theformation of the division septum and in cell separation (14),whereas lack of WTAs results in round cells (15). Moreover,enzymes involved in LTA synthesis localize predominantly at thedivision sites of bacteria (14), and enzymes involved in the syn-thesis of WTAs localize in helical patterns (16) similar to thepreviously described patterns of PGN synthesis observed duringelongation ofB. subtilis cells (17). The observation that mutantslacking LTAs have altered autolysis rates (5, 18) or that in-terference with the synthesis of WTA in B. subtilis triggers thetranscription of several genes involved in PGN synthesis (19),constitutes additional, indirect evidence that suggests a link be-tween TAs and cell morphogenesis through an effect on the

biosynthesis of PGNs, the correct assembly of which is requiredfor proper cell division and morphogenesis.

To investigate whether the synthesis of WTAs is directly re-quired for building a PGN macromolecule with the correctstructure, we used Staphylococcus aureus as a model organism. S.aureus is a Gram-positive bacteria and a prominent pathogen inthe community and healthcare settings, well known for its viru-lence and antibiotic resistance (20, 21). The main advantage ofusing S. aureus for studying PGNs is that it has only four peni-cillin-binding proteins (PBPs 14) instead of 12 or 16 that are

present in the traditional model organisms Escherichia coli andB. subtilis, respectively (22). PBPs are enzymes involved in thefinal stages of PGN biosynthesis, which synthesize glycan chains(via transglycosylation reactions) and cross-link different glycanchains through short peptides (via transpeptidation reactions).The level of PGN cross-linking varies between bacterial species,and S. aureus has an unusually high degree of cross-linking (23),which is due mainly to the action of PBP4 (24, 25), and seems torequire the long and flexible pentaglycine crossbridge that S.aureus cells use to connect two stem peptides from differentglycan strands (23, 26). PBP4 is not essential for S. aureus via-bility. However, it has an important role in antibiotic resistance,as it is essential for the expression of-lactam resistance incommunity-acquired methicillin-resistant strains (25). Interest-ingly, inactivation of PBP4 has been reported in laboratory stepmutants and clinical isolates with intermediate vancomycin re-sistance (2729), which have decreased levels of PGN cross-linking. This suggests that modulation of the degree of cross-linking is important for resistance to different antibiotics.

In this study, we describe a previously uncharacterized linkbetween WTA biosynthesis and PGN biosynthesis. We foundthat WTAs act as temporal and spatial regulators of PGN me-tabolism, controlling the level of cross-linking by directing PBP4to the division septum. We also showed that the highly cross-linked PGN that results from the regulated action of PBP4 ismore resistant to enzymatic degradation by lysozyme. This in-creased resistance may be advantageous to S. aureus bacteriaduring interactions with host organisms that produce lysozyme asan initial defense against bacterial infection.

Author contributions: M.L.A., P.M.P., M.G.P., and S.R.F. designed research; M.L.A., P.M.P.,

J.Y., and P.R. performed research; M.L.A., P.M.P., M.G.P., and S.R.F. analyzed data; H.V.

contributed new reagents/analytic tools; and M.L.A., P.M.P., M.G.P., and S.R.F. wrote the

paper.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

The authors declare no conflict of interest.

1M.L.A. and P.M.P. contributed equally to this work.

2M.G.P. and S.R.F. contributed equally to this work.

3To whom correspondence may be addressed. E-mail: [email protected] or sfilipe@

itqb.unl.pt.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1004304107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1004304107 PNAS | November 2, 2010 | vol. 107 | no. 44 | 1899118996

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Results

TagO Null Mutant Has Decreased Levels of Peptidoglycan Cross-

Linking. To test whether the presence of WTAs is required tobuild a PGN macromolecule with the correct structure, we de-leted the tagO gene from the chromosome of the S. aureusNCTC8325-4 strain, leaving no antibiotic resistance markers, toproduce the strain NCTCtagO. TagO is the first enzyme in theteichoic acid biosynthetic pathway, catalyzing the transfer ofGlcNAc-1phosphate from cytoplasmic UDP-linked precursorsto the C55-P lipid anchor bactoprenol (30). NCTCtagO did notproduce detectable levels of TAs (Fig. S1) and showed severalphenotypes previously described for staphylococcal mutants im-paired in WTA biosynthesis (4), such as a larger cell diameterthan wild-type cells, aggregation in clusters (Fig. S1), tempera-ture sensitivity, and resistance to infection by phage 80.

As a first approach to detecting changes in PGN structure due tothe deletion oftagO, we purified PGN from NCTCtagO and itsparental strain NCTC8325-4 and tested its susceptibility to the actionof lysozyme, an enzyme that cuts PGN between the N-acetylmuramicacid and N-acetylglucosamine residues of the glycan chains. S. aureusis known for itsintrinsic abilityto resist lysozyme due to modificationsof its PGN, such as O-acetylation ofN-acetylmuramic acid residuesand attachment of teichoic acids, which prevent access of the enzyme

to its substrate (10, 31). We treated cell walls from both the wild-typeand the TagO mutant with hydrofluoric acid, which removes teichoicacids and O-acetyl groups, and incubated the resulting naked PGNwith lysozyme. The PGN of NCTCtagO was more susceptible tolysozyme than that of NCTC8325-4 (Fig. 1D), suggesting differencesin PGN structure between the two strains.

The PGN muropeptide composition of NCTCtagO andNCTC8325-4 was then analyzed by HPLC, which revealed a re-duced level of PGN cross-linking in NCTCtagO when comparedwith the parental strain NCTC8325-4, with the concomitant ac-cumulation of monomeric and dimeric muropeptides (Fig. 1A andFig. S2). This result suggests that TAs may be involved in the laterstages of PGN maturation, which include the introduction of extracross-links between the glycan strands.

Decreased Levels of Peptidoglycan Cross-Linking in a TagO NullMutant Result from Delocalization of PBP4. The secondary (high-level) cross-linking ofS. aureus results mainly from the activity of

PBP4, as inactivation ofpbpD gene, encoding PBP4, leads to thedisappearance of the highly cross-linked muropeptide species (24,25) that typically elute as a broad peak at the end of the HPLCchromatogram (Fig. 1A, arrow). Interestingly, the composition ofPGN purified from NCTCtagO was very similar to that of thePGN from NCTCpbpD (Fig. 1A). This observation led us to testif PBP4 was altered in the tagO null mutant. Sequencing of thepbpD gene (including its promoter region) in NCTCtagO did notidentify any mutations, and Western blot analysis with specific

anti-PBP4 antibodies showed that levels of PBP4 were identical inNCTCtagO and in its parental strain NCTC8325-4 (Fig. 1B).Furthermore, binding of Bocillin-FL (a fluorescent derivative ofpenicillin V and a substrate analog for PBPs) to the staphylo-coccal PBPs showed that the binding of PBP4 to this substrateanalog was not altered in NCTCtagO cells when compared withthe parental strain NCTC8325-4 (Fig. 1C).

We previously showed that inhibition of cell-wall synthesis inS. aureus by-lactam antibiotics results in delocalization of PBP2(32). Therefore, we decided to test whether the lack of high-levelpeptidoglycan cross-linking in the tagO background was theresult of incorrect localization of PBP4. For that purpose, weconstructed S. aureus RN4220 strains expressing a C-terminalYFP fusion to PBP4 from its native chromosomal locus and under

the control of its native promoter. RN4220 background was usedbecause it is the onlyS. aureus strain that can be efficientlytransformed with foreign DNA. The transformation efficiency ofNCTC8325-4 is extremely low, and the tagO mutation renders itresistant to phages such as 80, preventing transduction andimpairing the introduction of additional constructs into thisbackground. When the PBP4YFP fusion was expressed in theRN4220 parental background (RNPBP4YFP), it localized to thedivision septum (Fig. 2), where cell-wall synthesis has beenreported to take place in S. aureus (33). However, when the samefusion was expressed in the tagO background (RNtagO-PBP4YFP), PBP4 was observed all around the cellular mem-brane, with no specific accumulation at the division septum (Fig.2). This effect was not general to all staphylococcal PBPs, as PBP1did not lose its septal localization in a tagO background

(Fig. S3).To quantify the extent of delocalization of PBP4 in the absence

of the TagO protein, we calculated the ratio offluorescence

Fig. 1. Highly cross-linked muropeptides, resulting from PBP4 activity, are less abundant in the peptidoglycan of a S. aureus tagO mutant. (A) HPLC analysis

of mutanolysin-digested PGN of the parental strain NCTC8325-4 and mutants NCTCtagO and NCTCpbpD. Arrow points to highly cross-linked muropeptide

species, which are less abundant in the mutants lacking TagO and PBP4. (IV) Muropeptide species from monomers to pentamers. ( B) Western blot analysis,

using a specific anti-PBP4 antibody, of membrane proteins from the same three strains showing that PBP4 is expressed at wild-type levels in the NCTCtagO

background. (C) Analysis of membrane proteins labeled with Bocillin-FL and separated by SDS/PAGE, showing that PBP4 is able to bind Bocillin-FL in the

NCTCtagO background. (D) Lysozyme hydrolysis of purified PGN from NCTC8325-4, NCTCtagO, and NCTCpbpD, followed by monitoring the decrease in

absorbance at OD600nm, and showing that PGN with a lower degree of cross-linking had an increased susceptibility to lysozyme.

18992 | www.pnas.org/cgi/doi/10.1073/pnas.1004304107 Atilano et al.
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measured at the septum versus the fluorescence measured at thelateral wall (Fig. 2). If a fluorescent protein or dye (such as NileRed membrane stain) is homogeneously distributed over the en-tire cell membrane, the intensity of the fluorescent signal at theseptum (which contains two membranes) should be approxi-mately twice the fluorescence at the lateral membrane. It followsthat, if a fluorescent protein is specifically accumulated at the

division septum, then the ratio offl

uorescence at the septumversus thelateral wall should be higher than 2. When this ratio wascalculated for PBP4YFP in the parental strain RNPBP4YFP, weobtained an average value of 4.0 1.52 whereas a value of 1.6 0.27 was obtained for the tagO mutant RNtagOPBP4YFP,indicating that the specific accumulation of PBP4 at the septum inwild-type cells was completely lost if the TagO protein was absent.Furthermore, complementation of RNtagOPBP4YFP, withplasmid encoded TagO (but not with the empty plasmid vector),restored the correct localization of PBP4 at the septum (Fig. 2).

PBP4 Is Recruited to the Division Septum Later than TagO. Thesimplest explanation for the dependence of PBP4 on TagO forseptal localization would be that TagO localizes to the divisionseptum and recruits PBP4 by proteinprotein interaction. In ac-

cordance, we found that a TagO

GFP fusion localized at the di-vision septum (Fig. 3A and Fig. 4D). To study the dynamics ofPBP4 and TagO recruitment to the septum, namely to determineif they arrived at the septum at the same time, we constructedstrain RNTagOPBP4, which expresses both TagOGFP andPBP4mCherry fusion proteins. We analyzed over 3,000 cells anddetermined the localization of TagO and PBP4 in 900 cells thatwere in the initial stages of septum synthesis. In this sub-population, TagO arrived at the septum before PBP4 (TagO wasseen as two septal spots corresponding to a septal ring whereasPBP4 was not yet present at the septum) or was found ahead ofPBP4 (TagO was found across the entire septum whereas PBP4was still seen as two septal spots) in 44.3% of the cells (Fig. 3 B).PBP4 arrived at the septum before TagO or was found ahead ofTagO in only 4.4% of the cells. In 51.2% of thecells, both proteins

had the same localization. The fact that TagO and PBP4 re-cruitment to the septum occurs at different times suggests thatPBP4 is not recruited to the septum by direct proteinproteininteraction with TagO. Further indication came from studies witha bacterial two-hybrid system (34), which failed to detect any in-teraction between PBP4 and TagO (Fig. S4).

Synthesis of Teichoic Acids, and Not TagO Protein Itself, Is Required

for PBP4 Recruitment to the Division Septum. To elucidate whetherit was the presence of the TagO protein at the septum or theactivity of the TagO protein (also implying the presence of tei-choic acids) at the septum that was required for PBP4 re-cruitment, we constructed a series of strains expressing TagOproteins with single amino acid changes, with the aim of selectingmutantswithloss of TagO activity (i.e., lack of TA production)butwith the ability to correctly localize at the septum when fused toGFP protein (suggesting that the protein may be correctly folded).Four conserved residues of TagO (D87, D88, G152, N198) wereindividually substituted with alanine residues, and a double mu-tant in which D87 and D88 were simultaneously substituted withalanines was also constructed. The different TagO proteins (wildtype and mutants) were expressed from the replicative pMADplasmid (35) under the control of the native tagO promoter in theRNtagO background and tested for their ability to catalyze TAsynthesis (Fig. 4B). Expression of TagOD87Aand TagOD87A/D88A

did not result in the production of detectable amounts of TAs;expression of TagOD88Aand TagOG152Aled to the production ofsignificantly reduced amounts of TAs (less than 25% of wild-typelevels); and expression of TagON198Aresulted in a small decreasein the amount of TAs produced when compared with expressionof wild-type TagO protein (74% of wild-type levels). The locali-zation of PBP4YFP was then determined in cells expressing ei-ther TagOwt or the different TagO mutants, and we found thatPBP4 was unable to localize correctly at the division septum in thefour mutants with significantly reduced levels of WTAs (Fig. 4A).Moreover, there is a direct correlation between the amount of

Fig. 2. Septal localization of PBP4 is lost in a tagO null mutant. Microscopy

images and quantification of septum versus lateral membrane fluorescence

(fluorescence ratio, FR) of PBP4YFP in a wild-type background (RNPBP4YFP),

a tagO background (RNtagOPBP4YFP), and a tagO mutant com-

plemented with plasmid-encoded tagO (RNtagOPBP4YFPptagO). Also

shown are RNPBP4YFP cells labeled with membrane dye Nile Red, which ishomogeneously distributed in the cell membrane. Quantification was per-

formed in 100 cells displaying closed septa for each strain. Horizontal lines

correspond to average FR values. FR values over 2 indicate preferential

septal localization whereas FR values equal to or under 2 indicate that

a protein is dispersed over the cell surface. p values < 107. Scale bar: 1 m.

Fig. 3. TagO protein is recruited to the division septum before PBP4. (A) The

strain RNTagOPBP4, expressing simultaneously TagOGFP and PBP4

mCherry protein fusions, was analyzed by fluorescence microscopy. (Top)

Results showing that TagO reaches the division septum before PBP4 in sev-

eral cells. (Bottom) Cells in which PBP4 and TagO colocalize at the septum.

Scale bar: 1 m. (B) Localization of TagOGFP and PBP4mCherry was ana-

lyzed in 902 cells in the early stages of septum formation. Localization of

each protein was assigned to three sequential stages: scattering around the

entire membrane; localization in a ring around the division plane, usually

seen as two spots; and localization over the entire closed septum, usuallyseen as a line across the cell. In 44% of the cells, TagO was found at the

septum ahead of PBP4, meaning that either TagO is already at the sep-

tum, seen as two spots, whereas PBP4 is still scattered around the cell

membrane or that TagO is already across the entire septum, whereas PBP4 is

still in a ring around the division septum.

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WTA production and the fraction of PBP4 recruited to the di-vision septum (Fig. 4C).

It was possible that PBP4 delocalization in strains expressingTagO proteins with lower or no activity was not due to the lack ofTAs at the septum, but to the fact that mutated TagO proteinswere degraded or had lost their septal localization. We thereforeselected TagOG152Afor further localization studies because it wasstill able to produce small amounts of WTAs, implying that theprotein was probably correctly folded. However, TagOG152A

showed some of the phenotypes characteristic oftagO mutants,such as phage resistance, decreased cross-linking, or cell cluster-ing (Fig. S5), implying that the amount of WTA produced was not

sufficient to fully complement the phenotype of RNtagO. WhenTagOG152Awas fused to GFP and expressed in RN4220, theprotein correctly localized to the septum, similarly to TagOwt (Fig.4D). Furthermore, introduction of the G152A mutation in TagOGFP did not result in reduced levels of expression of the fluo-rescent protein (Fig. S5). The fact that TagOG152Ahad loweractivity but maintained correct folding and localization, and wasunable to recruit PBP4 to the septum, strongly suggests that thepresence of WTA, andnot thepresence of the TagO protein itself,was required for PBP4 localization.

Discussion

Teichoic acids have been reported to be involved in cell growth,cell division, and morphogenesis (4, 5, 36), but their exact role inthese processes remains unknown. In this study, we show that

WTAs have a fundamental role in PGN metabolism as theymodulate the degree of cross-linking by temporally and spatiallyregulating the recruitment of PBP4 to the site of cell-wall syn-thesis, the division septum.

The synthesis of PGN in S. aureus occurs mainly through theaction of PBPs 14. PBP1 is a monofunctional transpeptidase,essential for cell viability and required for septation and cellseparation at the end of cell division (37). It localizes to the di-vision septum through a mechanism that is independent of itsability to bind its substrate (38). PBP2 is an essential bifunctionaltransglycosylase and transpeptidase that plays a central role in theability of bacteria to express their resistance to antibiotics (39) and

localizes to the division septum in a way that is dependent on itsability to recognize the translocated substrate (32). PBP3 andPBP4 are nonessential, monofunctional transpeptidases whoselocalization has not yet been studied in detail (22).

We have shown here that in wild-type cells PBP4 can be foundat the septum ofS. aureus, similarly to PBP1 and PBP2. How-ever, in a different way from these two proteins, recruitment ofPBP4 to the septum is dependent upon the synthesis of WTAs.In S. aureus strains lacking TagO, the first enzyme in the teichoicacid biosynthesis pathway, PBP4 no longer accumulates specifi-cally at the division septum, but instead is dispersed over theentire cell membrane. Concomitantly with PBP4 delocalization,the level of PGN cross-linking in tagO mutants is severely de-creased, a phenotype also observed by Schlag et al. (40) whilethis manuscript was in preparation.

Fig. 4. Synthesis of teichoic acids, and not TagO protein itself, is required for PBP4 recruitment to the division septum. (A) Quantification of septum versus

lateral membrane fluorescence (fluorescence ratios, FR) and fluorescence microscopy images for PBP4YFP protein fusion in tagO mutants complemented

with wild-type TagO protein (RNtagOPBP4YFPptagOwt) or with different TagO mutants (RNtagOPBP4YFPptagOD87A, D88A, D87A/D88A, G152A, and N198A).

Quantification was performed in 100 cells that displayed closed septa for each strain. Horizontal lines correspond to average FR values. FR values over 2

indicate septal localization, and FR values equal to or under 2 indicate that a protein is dispersed over the cell surface. p values < 107. Scale bar: 1 m. ( B)

WTAs were isolated from RNtagOptagO and RNtagOptagOD87A, D88A, D87A/D88A, G152A, and N198A and analyzed by native PAGE stained with alcian blue/silver

stain. Mutations of the aspartic acids and glycine residues led to a decrease or absence of the WTAs. ( C) Comparison between the levels of WTA and the

degree of PBP4 localization to the division septa (calculated as described in Materials and Methods) indicates a strong correlation between the amount of

WTA present in the cell and the ability of PBP4 to localize at the septum. (D) Fluorescence microscopy images of RNTagOwtGFP and RNTagOG152AGFP showing

that the TagO

G152A

GFP fusion localizes to the division septum, similarly to the GFP fusion to the wild-type TagO protein. Scale bar: 1 m.

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Recruitment of PBP4 to the septum does not seem to occur viaa direct proteinprotein interaction with TagO because (i) PBP4and TagO do not interact in a bacterial two-hybrid screening, ( ii)the two proteins do not colocalize in 49% of the cells in earlystages of septum synthesis, and (iii) the presence of (inactive)TagO protein properly localized at the septum is not sufficient tokeep PBP4 at that location. Instead, recruitment of PBP4 to theseptum seems dependent on the septal synthesis of WTAs. If thissynthesis is abolished, either by complete removal of TagO or by

generating TagO point mutants, which lose their activity whilemaintaining correct localization at the septum, then PBP4 loses itsseptal localization and becomes unable to perform its function inthe synthesis of highly cross-linked PGN. The fact that an intactPBP4 is unable to perform its function when incorrectly localizedmay be due to the substrate being found only at the septum or tothe lateral PGN exhibiting a different structure when comparedwith the septal PGN, which may not allow the addition of furthercross-links between the glycan strands.

On the basis of the results described in this work, we proposethat teichoic acid synthesis functions not only as a spatial cue, butalso as a temporal cue, for PBP4 recruitment to the divisionseptum. Fig. 5 illustrates a model in which the initial cell-wallsynthetic machinery is recruited to the division septum in the

early stages of its formation. TagO, and most likely theremaining enzymes involved in WTA biosynthesis, are recruitedto the septum and initiate WTA biosynthesis, which functions asa temporal indication that early PGN biosynthesis is completeand that PGN can be further processed to become highly cross-linked. PBP4 (which, as we have shown, arrives at the septumlater than TagO) is then recruited to the septum, where it takesover the last steps of PGN synthesis, performing the finalweaving of the PGN mesh. Importantly, it is likely that re-cruitment of PBP4 is not mediated by fully synthesized/matureWTAs, which are present throughout entire surface ofS. aureus(but may not yet be present at the septum), but rather by animmature form of WTA corresponding to an intermediate ofWTA biosynthesis, which is encountered only at the septum.

One hypothesis would be that the addition ofD-alanyl groups tothe WTA backbone, catalyzed by the enzymes encoded in thedltABCD operon, would be essential for binding of PBP4 to theWTA because the natural substrate of PBP4 is the D-alanyl-D-alanine terminus of the peptidoglycan muropeptide precursor.However, we have deleted the dltABCD operon from RN4220and shown that it has no significant effect on localization ofPBP4 (Fig. S6).

The fact that synthesis of WTA and PGN share the same lipidcarrierbactoprenolfor their precursors led us to think of analternative model for PBP4 delocalization. Bactoprenol is usuallyfound in limiting amounts in the cell. Therefore, inhibition ofWTA synthesis could increase the availability of bactoprenol forPGN synthesis, leading to an increase in the metabolicflux towardPGN synthesis. This could then result in the increased synthesis,and possibly delocalization, of lipid II (bactoprenol linked toa dissacharide-pentapeptide with a pentaglycine crossbridge), thesubstrate of PBPs, which could be the driving force for PBP4delocalization. We have tested this hypothesis by purifying thelipid-linked PGN precursors (Fig. S7). The fact that there was nodetectable accumulation of lipid II in the tagO mutant led us torule out changes in lipid II concentrations as the cause for PBP4delocalization. Therefore, although we cannot formally rule outthe possibility that delocalization of PBP4 results not from theabsence of WTA intermediates, but from other cellular changesthat are themselves caused by the depletion of WTA inter-mediates, we currently favor the model depicted in Fig. 5.

Interestingly, while this manuscript was in preparation, Schlagand colleagues (40) reported that WTAs are involved in target-ing the bifunctional autolysin Atl to the septum. The authorspropose an exclusion strategy in which mature WTAs, which are

present throughout the mature cell wall but absent (or in lowerconcentration) at the septum, would prevent binding of Atl tothe old cell wall but not to the septal region. Therefore, WTAsmay play a key role not only in the regulation of the secondarycross-linking of PGN, but also in regulating the cleavage of thePGN macromolecule, coordinating (or temporally and spatiallyrestricting) both its synthesis and degradation/autolysis.

Why does S. aureus require such fine-tuning of the level of thecross-linking of its PGN? One possibility may be that careful reg-ulation of the timing of PGN cross-linking may be required toensure the covalent attachment of different molecules to the PGN.Delaying the production of highly cross-linked PGN would permitthe introduction of bulky glycopolymers, such as WTAs or largeproteins, through the assembled PGN. Afterward, staphylococcal

cells would promote cross-linking of PGN to high levels, which, aswe have shown, renders it more resistant to lysozyme, an enzymeproduced by hosts as a defense against bacterial pathogens.

Materials and MethodsBacterial Strains and Growth Conditions. The bacterial strains used in this

study are listed in Table S1, and the details of their construction are de-

scribed in SI Materials and Methods. Primers used in this study are listed in

Table S2. Strains and plasmids used in the bacterial two hybrid assays are

listed in Table S3. S. aureus strains were grown at 30 C in tryptic soy broth

medium (Difco) supplemented with appropriate antibiotics when required

(erythromycin 10 g/mL or kanamycin 50 g/mL and neomycin 50 g/mL;

Sigma-Aldrich) and transformed by electroporation as previously de-

scribed (41). E. colistrains were grown at 37 C in LuriaBertani medium

(Difco) supplemented with 100 g/mL of ampicillin (Sigma-Aldrich).

Wall Teichoic Acid Analysis. WTAs were extracted by alkaline hydrolysis from

overnight cultures, analyzed by native polyacrylamide gel electrophoresis,

and visualized by combinedalcian blue/silver staining, as previously described

(42). ImageJ software was used to quantify the percentage of WTA pro-

duced by each strain (43). The signal intensity of each lane was quanti fied

and normalized against the corresponding value for the wild type (consid-

ered as 100%).

Peptidoglycan Purification and Analysis. PGN from NCTC8325-4, NCTCtagO,

and NCTCpbpD was prepared from exponentially growing cells as pre-

viously described (44) and as detailed in SI Materials and Methods.

Detection of PBPs. Membrane protein extracts were prepared from expo-

nentially growing cells as previously described (28) and as detailed in SI

Materials and Methods.

Fig. 5. Model for the role of teichoic acids synthesis in PBP4 recruitment

to the septum. The early cell-wall synthetic machinery assembles at the

division site, leading to the synthesis of new PGN, with low levels of cross-

linking (Left). TagO (together with other WTA synthetic enzymes) is

recruited to the septum by an unknown mechanism, leading to the syn-

thesis of intermediate molecules in TA biosynthesis (Center). These inter-

mediates (or another cellular component dependent on TA biosynthesis)

function as a temporal and spatial cue for PBP4 recruitment to the division

septum, allowing the synthesis of highly cross-linked PGN to occur in

a regulated manner (Right).

Atilano et al. PNAS | November 2, 2010 | vol. 107 | no. 44 | 18995

MICROBIOLOGY
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6/6

Fluorescence Microscopy. S. aureus strains were grown to midexponential

phase and observed by fluorescence microscopy on a thin layer of 1%

agarose in PBS. When necessary, cells were stained with membrane dye Nile

Red (3 g/mL; Molecular Probes). Images were obtained using a Zeiss Axio

Observer.Z1 microscope equipped with a Photometrics CoolSNAP HQ2

camera (Roper Scientific using Metamorph software (Meta Imaging series

7.5) and analyzed using Image J software (43).

Fluorescence ratio (FR) was determined by quantifying thefluorescence at

the center of the division septa (only cells with closed septa were considered

for this analysis) divided by the fluorescence at the lateral wall. Average

background fluorescence was subtracted from both values. Quantificationwas performed for at least 100 cells with complete septa for each strain. The

percentage of PBP4 localized at the division septa (Fig. 4C) was calculated

from the ratio of the FR value obtained for each RNtagOPBP4YFPptagOmut

strain divided by the FR value obtained for RNtagOPBP4YFPptagOwt.

ACKNOWLEDGMENTS. We thank Dr. D.-J. Scheffers for critical reading ofthe manuscript, Dr. H. Komatsuzawa and Dr. M. Sugai (Hiroshima University,Japan) for the generous gift of PBP1 and PBP4 antibodies, and Dr. G. Kar-imova (Institut Pasteur, France) for the generous gift of the BTH plasmids.This work was funded by Fundao para a Cincia e Tecnologia throughresearch Grants POCI/SAU-IMI/56501/2004 and PTDC/SAU-MII/75696/2006(to S.R.F.), POCI/BIA-MIC/67845/2006 and PTDC/BIA-MIC/099151/2008 (toM.G.P.) and fellowships SFRH/BD/28440/2006 (to M.L.A.), SFRH/BD/41119/

2007 (to P.M.P.), SFRH/BPD/23838/2005 (to J.Y.), SFRH/BPD/23812/2005(P.R.), and SFRH/BD/38732/2007 (H.V.), and a European Molecular BiologyOrganization long-term fellowship (ALTF 1042-2007) (to J.Y.).

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Teichoic Acid Cross Linking Staphylococcus Aureus

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