Dual Roles of FmtA in Staphylococcus aureus Cell Wall Biosynthesisand Autolysis

Aneela Qamara and Dasantila Golemi-Kotraa,b

Departments of Biologya and Chemistry,b York University, Toronto, Ontario, Canada

The fmtA gene is a member of the Staphylococcus aureus core cell wall stimulon. The FmtA protein interacts with �-lactamsthrough formation of covalent species. Here, we show that FmtA has weak D-Ala-D-Ala-carboxypeptidase activity and is capableof covalently incorporating C14-Gly into cell walls. The fluorescence microscopy study showed that the protein is localized to thecell division septum. Furthermore, we show that wall teichoic acids interact specifically with FmtA and mediate recruitment ofFmtA to the S. aureus cell wall. Subjection of S. aureus to FmtA concentrations of 0.1 �M or less induces autolysis and biofilmproduction. This effect requires the presence of wall teichoic acids. At FmtA concentrations greater than 0.2 �M, autolysis andbiofilm formation in S. aureus are repressed and growth is enhanced. Our findings indicate dual roles of FmtA in S. aureusgrowth, whereby at low concentrations, FmtA may modulate the activity of the major autolysin (AtlA) of S. aureus and, at highconcentrations, may participate in synthesis of cell wall peptidoglycan. These two roles of FmtA may reflect dual functions ofFmtA in the absence and presence of cell wall stress, respectively.

The fmtA gene was identified as a methicillin resistance factor inStaphylococcus aureus by Komatsuzawa et al. (23). Inactivation

of fmtA leads to increased sensitivity of methicillin-resistant S.aureus strains (MRSA) to Triton X-100 and �-lactams and de-creases the level of highly cross-linked peptidoglycan (PG) (22,23). Cell wall inhibitors such as oxacillin, methicillin, cefoxitin,phosfomycin, and bacitracin cause upregulation of the transcrip-tion levels of fmtA (22).

Several studies on the genome-wide S. aureus response to cellwall inhibitors have demonstrated that their biological activitycauses upregulation of fmtA (3, 26, 27, 41, 46). In addition, inac-tivation of genes involved with cell wall biosynthesis, such asmurF, leads to higher levels of fmtA (41). Hence, fmtA is consid-ered part of the core cell wall stimulon (26).

The function of fmtA is not known. The fmtA gene has beenlinked to biofilm formation and autolysis, two interconnectedprocesses in bacteria whereby the ability of S. aureus to producebiofilms is linked to its ability to undergo a controlled autolysisprocess (2, 25). Inactivation of fmtA diminishes the ability of S.aureus to form biofilms (4, 44) and enhances autolysis (4, 23).Boles et al. showed the cell wall of the S. aureus fmtA mutant to lackwall teichoic acids (WTAs) (4). Teichoic acids are phosphate-richglycopolymers referred to as WTAs (Fig. 1) when connected to thepeptidoglycan (PG) or as lipoteichoic acids (LTAs) when con-nected to the cytoplasmic membrane (51, 54). They make up asmuch as 60% of the total cell mass in Gram-positive bacteria andhave been implicated in a variety of processes, including biofilmproduction and autolysis (13, 21, 33, 39, 49). Recent reports haveimplicated WTAs in spatial and temporal regulation of the S. au-reus major autolysin (AtlA) and penicillin-binding protein 4(PBP4) (1, 40).

The primary structure of FmtA harbors two of the three con-served motifs of PBPs, SXXK and S(Y)XN (where X indicates anyamino acid) while lacking the KTG motif. The protein forms co-valent species with �-lactam compounds; however, this interac-tion is weak, suggesting that FmtA is intrinsically resistant to�-lactam inactivation (10). The other four native PBPs of S. au-reus are sensitive to �-lactam. The methicillin-resistant S. aureus

strains have acquired an additional PBP, PBP2a, which also inter-acts poorly with �-lactams (36).

S. aureus remains a major concern for public health due toresistance to a wide range of antibiotics. Understanding how itcopes with cell wall stress would provide insights toward newtreatment strategies. Further, genome-wide screening studieshave shown that inhibition of nonessential genes could be a suc-cessful strategy in sensitizing S. aureus to antibiotics that otherwisehave failed in clinical settings due to resistance (5, 20, 28, 43).

Here we investigate the relationship of fmtA to cell wall biosyn-thesis. Our new findings shed light on the role of fmtA in S. aureusmethicillin resistance, autolysis, and biofilm formation.

MATERIALS AND METHODSMaterials and chemical reagents. Growth media were purchased fromEMD Bioscience. Enzymes and chemicals were purchased from Sigmaand New England BioLabs. Lipoteichoic acids were purchased from Invi-voGen. Amplex Red (AR) was purchased from Molecular Probes, Inc.14C-Gly was purchased from Perkin Elmer. Monoclonal anti-FmtA serumwas purchased from GeneScript Corporation. Sterile 96-well tissue cul-ture plates were purchased from Corning Incorporated. Safranin was pur-chased from bioMérieux Canada.

Construction of FmtA�27S127A mutant. FmtA�27 refers to the ma-ture protein sequence. The signal peptide sequence (27 amino acids at theN terminus) was not included in the cloning process of fmtA (10). Thisfacilitated the isolation of the protein from the cytoplasm. The matureprotein lacks any cysteine residues in the primary structure. Hence, isola-tion of this otherwise periplamismic protein from the cytoplasm shouldnot have any effect on the folding of the protein (10).

Mutation of Ser127 to Ala was carried out using a QuikChange site-

Received 24 January 2012 Accepted 29 April 2012

Published ahead of print 7 May 2012

Address correspondence to Dasantila Golemi-Kotra, dgkotra@yorku.ca.

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

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.00187-12

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directed mutagenesis kit (Stratagene). The pET24a (�)::fmtA27 vector(10) was amplified using Pfu Turbo DNA polymerase and a pair of muta-genic primers, DirS127A (5=-CGATGTTTTTAATAGGTGCAGCTCAAAAATTTTC-3= ) and RevS127A (5=-GAAAATTTTTGAGCTGCACCTATTAAAAACATCG-3= ) (the mutated nucleotides are italicized). The PCRproduct was digested with the DpnI restriction endonuclease and used totransform Escherichia coli BL21(DE3). The nucleotide sequence of themutant variants was verified by DNA sequencing. The purifications ofFmtA�27 and the S12A mutant were carried as described by Fan et al. (10).

DD-Carboxypeptidase assay. Carboxypeptidase activity of FmtA�27

against the substrate surrogate N�,Nε-diacetyl-L-Lys-D-Ala-D-Ala (thesurrogate for the peptidoglycan donor strand) was monitored with a flu-orescence-based assay (17). A typical 100-�l assay mixture consisted of 6mM tripeptide–10 �M FmtA�27– 0.1 M Tris (pH 8.5) buffer. The surro-gate for the peptidoglycan acceptor strand, Gly-Gly peptide, was added toone set of reaction mixtures at a 1:10 ratio (donor to acceptor).

Transpeptidase activity assays. Transpeptidase activity of FmtA�27

was investigated using radioactive 14C-labeled glycine (14C-Gly) as theacceptor. Cross-linking experiments were carried out using cell wall iso-lated from S. aureus RN4220. A typical 100-�l reaction mixture consistedof 50 �g of cell wall, FmtA�27 at different concentrations (0 to 30 �M),and 1 mM 14C-Gly, all in 0.1 M Tris (pH 8.5) buffer. In a control reaction,albumin (15 �M) or ovalbumin (15 �M) was incubated with cell wall (50�g) and 14C-Gly (1 mM).

Reaction mixtures were incubated for 2 h at 37°C. Following incuba-tion, samples were spun down at 25,000 � g for 30 min at 4°C. Pelletscontaining cell walls were washed twice with 0.1 M Tris (pH 8.5) buffer toremove any nonspecifically bound 14C-Gly. The incorporated radioactiv-ity was recorded by a Tri-carb liquid scintillation analyzer (Packard). Thesame experiments were carried out with FmtA�27S127A at 30 �M.

Preparation of S. aureus cell walls with and without wall teichoicacid. Cell walls from S. aureus RN4220 were isolated as previously re-ported (7). Removal of WTAs from cell walls was carried out using hy-drofluoric acid (HF) based on a protocol by Vialle et al. (47). The cell wallpreparation resulting in peptidoglycan with no WTAs attached is referredto as peptidoglycan (PG). Binding of FmtA�27 to cell walls was performedas previously described (10).

Investigation of the FmtA�27 hydrolase activity. FmtA�27 (2.5 �M)was incubated with PG (1 mg/ml) in 50 mM sodium phosphate (pH 7.0)buffer (1 ml) at 37°C for different times (0.5, 1, 2, 3, 4, and 5 h). Thechange in absorbance was monitored at 450 nm. Mutanolysin from Strep-tomyces globisporus was used as a positive control.

In another experiment, PG (1 mg/ml)–50 mM phosphate (pH 7.0)buffer was incubated with 10 �M FmtA�27 in a 20-�l reaction mixture. Incontrol experiments, PG was digested with mutanolysin and undigested

PG. The reaction mixture was loaded into a C18 reverse-phase high-per-formance liquid chromatography (HPLC) column (Persuit; Varian) (5 �;250 by 4.6 mm). A linear gradient of 0% to 30% methanol in a 100 mMphosphate (pH 2.8) buffer was used to elute any peptidoglycan fragments.

Investigation of FmtA�27 interaction with WTA by the use of acidicnPAGE/H�. Interaction of FmtA�27 with WTAs was investigated withnative polyacrylamide gel electrophoresis (nPAGE)/H�. A typical reac-tion mixture (15 �l) consisted of 10 �g of FmtA�27 (15 �M final concen-tration) and 5 �g of WTA in the presence of NaCl at different concentra-tions (100, 300, or 500 mM), all in 50 mM sodium phosphate (pH 7.0)buffers. Another reaction mixture containing 8 �g of PBP2a (15 �M finalconcentration) was also prepared using a method similar to that describedabove. Bovine serum albumin (BSA), ovalbumin, RNaseA, and carbonicanhydrase (10 �g each) were used as negative controls. The PAGE gelswere stained with Coomassie blue.

Investigation of FmtA�27 interaction with WTA by trypsin diges-tion. FmtA�27 (40 �M) was incubated either with or without WTAs (1.25�g in 12.5 mM sodium phosphate [pH 5.5] buffer and 0.2% sodium azide;80-�l reaction volume) for 1 h at 37°C. The mixture was subjected totrypsin digestion based on a protocol from a ProteoExtract kit (CalBiochem) (trypsin assay concentration � 0.2 �g/�l) and further incu-bated at 37°C. Aliquots of 10 �l were removed at defined time intervals,and the digestion was terminated with 10 �l of sodium dodecyl sulfate(SDS) loading dye, followed by heating at 60°C for 5 min. Samples wereanalyzed by 15% Tris-Tricine SDS-PAGE. Each experiment was repeatedthree times.

In the case of limited trypsin digestion, the samples were subjected totrypsin only for 15 min and the protein samples were analyzed by matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) massspectroscopy (MS) at the Advanced Protein Technology Center, Hospitalfor Sick Children, Toronto, Canada.

CD spectroscopy studies of FmtA�27 in the presence of WTAs.Far-UV (200 to 260 nm) circular dichroism (CD) spectra of FmtA�27 (14�M in 50 mM sodium phosphate [pH 7.0] buffer), in the presence ofWTAs, were recorded at 22°C using a Jasco J-810 instrument and a cuvettewith a 1.0-mm path length. The average of three scans was recorded andcorrected for the signals from the buffer, PG fragment, and WTAs.

Autolysis assays. S. aureus RN4220 cells were grown in tryptic soybroth (TSB) overnight at 37°C. Cells were diluted 200-fold in fresh TSBmedium and grown with shaking at 37°C to an optical density at 620 nm(OD620) of 0.8. Cells were harvested by centrifugation (6,300 � g, 10 min)and washed twice with ice-cold water. The pellet was resuspended in 1�phosphate-buffered saline (PBS) (pH 7.0) to an OD620 of 1.00 and dis-pensed into a 96-well plate (200 �l per well). Cells were incubated at 37°Cin the presence of various concentrations of FmtA�27, with agitation at200 rpm. Autolysis was measured as a decrease in OD620 over time. Inthe case of the S. aureus �tarO mutant and its parental strain (EBII16),Mueller-Hinton broth (MHB) medium was used and FmtA�27 waskept at 0.2 �M.

Biofilm assay. Cultures of S. aureus RN4220 in TSB, or of the �tarOmutant and EBII16 strain in MHB, were grown overnight and normalizedby optical density. The cell cultures were next diluted 50-fold into freshTSB supplemented with 0.2% glucose. Wells of a sterile, 96-well flat-bottomed tissue culture plate (Corning Incorporated) were filled with200-�l aliquots of the diluted culture. FmtA�27 was added to the assay atvarious final concentrations (0 to 6 �M). Cultures were incubated for 24h at 37°C in the closed plate and assayed for biofilm formation accordingto the method of Christensen et al. (6). Adherent cells were fixed withBouin fixative (Sigma) and stained with 0.1% Safranin (bioMérieux, Can-ada) for 10 min. Wells were then washed with running tap water. Imagesof the inverted plates were taken using a digital camera. Biofilms weredissolved using 30% glacial acetic acid for 15 min, and relative biofilmformation was also assayed by reading absorbance at 562 nm using a platereader.

FIG 1 Schematic illustration of S. aureus cell envelope. PG, peptidoglycan,WTA, wall teichoic acid; LTA, lipoteichoic acid; D-Ala, D-alanine; GlcNAc,N-acetylglucosamine; ManNAc, N-acetylmannosamine; P, phosphate.

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Construction of gfp-fmtA�27 fusion. The fmtA�27 amplicon was li-gated into a pcDNA3.1/NT-GFP-TOPO vector (Invitrogen). To facilitatesubcloning of gfp-fmtA chimera into the pET24a vector, a silent mutationwas introduced by QuikChange site-directed mutagenesis (Stratagene) toremove the NdeI restriction site in the gfp gene. The following mutagenicprimers were used: Dir- (5=-CGTTATCCGGATCACATGAAACGGCATGAC-3=) and Rev (5=-GTCATGCCGTTTCATGTGATCCGGATAAG-3=). Introduction of the mutation was confirmed by DNA sequencinganalysis. Another set of primers was designed to amplify the gfp-fmtA�27

fusion gene for the final cloning to the pET24a vector: Dir (5=-AGCCATATGGCCAGCAAAGGAGAAGAAC-3=) and Rev- (5=-AGCGAATTCTTATTATTGAACAATAACACCCTTCG-3=) (NdeI and EcoRI restrictionsites are italicized). The successful cloning of the fusion gene into pET24awas also confirmed by DNA sequencing analysis.

The final construct, gfp-fmtA�27::pET24a, was used to transform E. coliBL21(DE3) cells. Expression and purification of green fluorescent pro-tein-FmtA�27 (GFP-FmtA�27) was carried out as described before (10). Inaddition, the activity of the chimeric FmtA�27 was investigated using flu-orescent penicillin (Bocillin) and binding to cell walls isolated from S.aureus EBII16 and �tarO mutant, as previously described (10).

Investigation of GFP-FmtA�27 localization to S. aureus by fluores-cence microscopy. S. aureus EBII16 and �tarO mutant strains weregrown overnight in MHB growth media. Cells were diluted 200-fold infresh MHB and grown to an OD600 of �0.6. Cells were harvested bycentrifugation at 9,300 � g for 10 min. The cells were washed with 1�phosphate-buffered saline (PBS) and resuspended in 1� PBS buffer togive a final OD600 of 1.2.

In a typical binding assay, 100 �l of 5 �M GFP-FmtA�27 protein so-lution was mixed with 400 �l of washed S. aureus cells to yield a finalconcentration of 1 �M GFP-FmtA�27. Reaction mixtures were incubatedfor 10 min at room temperature. Staphylococcal cells with bound proteinwere sedimented by centrifugation at 16,000 � g for 3 min. The cells werewashed with PBS and resuspended in PBS. A drop of bacterial suspensionwas placed on a polylysine-coated glass slide and immediately analyzedusing epifluorescence microscopy performed with a laser-scanning con-focal microscope (Fluoview FV300). Similar experiments were carried outat GFP-FmtA�27 concentrations of 2, 5, and 10 �M. Prior to analysis bymicroscopy, mixtures were incubated with 1 �M protein for 0.5, 1, and 2h to measure binding time of GFP-FmtA�27 to S. aureus cells.

To assess the effect of oxacillin and vancomycin on the localization ofFmtA, S. aureus EBII16 and �tarO mutant strains were exponentiallyexposed to either oxacillin (final concentration, 4 �g/ml) or vancomycin(final concentration, 20 �g/ml) for 1 h. Aliquots were sampled at the finaldose. Sequential dilutions were developed on MHB agar plates to deter-mine the number of viable cells. The rest of the cell cultures were pro-cessed as described above.

RESULTSFmtA�27 displays DD-carboxypeptidase activity. In this study, weworked with the mature FmtA protein, FmtA�27, which lacks thesignal peptide sequence (10). FmtA�27 catalyzed the removal ofthe D-Ala from the diacetyl tripeptide substrate (a surrogate forthe PG donor strand). The rate of D-Ala removal increased in thepresence of Gly-Gly, which is a surrogate for the PG acceptorstrand. Different ratios of donor to acceptor were investigated,and the optimum ratio was 1:10. Under these conditions, the cat-alytic efficiency of DD-carboxypeptidase activity of FmtA was mea-sured to be 0.745 � 106 M1 s1. The FmtA�27S127A mutantwas completely devoid of this activity.

Potential transpeptidase activity of FmtA�27 was investigatedby monitoring the catalytic incorporation of 14C-Gly into S. au-reus cell walls. Albumin and ovalbumin, proteins unrelated to PGbiosynthesis, served as negative controls in these experiments (Fig.2A). FmtA�27 covalently incorporated 14C-Gly into the cell walls,

but albumin and ovalbumin did not. The 14C labeling of cell wallsby FmtA�27 depended on the concentration of the protein (Fig.2A). The level of 14C-Gly incorporation into the cell walls by theFmtA�27S127A mutant was the same as the background levels (inthe absence of FmtA�27) (Fig. 2B).

FmtA�27 interacts with S. aureus WTAs. FmtA�27 binds tocell walls (10). To identify the cell wall component essential forFmtA�27 recruitment, we investigated the binding of FmtA�27 tocell walls digested with either lysostaphin or mutanolysin. Lyso-staphin cleaves the pentaglycine bridge in PG and decreases thedegree of PG cross-linking (glycan strands are less cross-linked).Mutanolysin cleaves the glycan strands after N-acetylmuramicacid and produces different oligomers of muropeptides.

Digestion of cell walls with mutanolysin resulted in as much asan 80% decrease of FmtA�27 binding to cell walls (Fig. 3), whereasdigestion of cell walls with lysostaphin resulted in a 40% decreaseof FmtA�27 binding (Fig. 3). The persistence of FmtA�27 bindingto cell walls digested by lysostaphin was investigated further. Welooked at the components of cell wall present in our cell wallpreparations and took note that WTAs were not removed fromthe cell wall preparations. WTAs were removed from the cell wallpreparations by HF. The newly prepared cell walls are referred toas peptidoglycan (PG) here.

FmtA�27 failed to bind to PG (Fig. 4). This experiment wasrepeated with cell walls isolated from the �tarO mutant. The tarOgene is involved in the first step of WTA biosynthesis, and, as such,S. aureus tarO mutant cell walls lack WTAs (8). The in vitro bind-

FIG 2 Investigation of the 14C-Gly incorporation into cell wall isolated fromS. aureus RN4220 by FmtA�27. (A) FmtA�27 at different protein concentra-tions and albumin and ovalbumin (15 �M) were incubated with cell wall (50�g [100-�l final volume]) isolated from S. aureus RN220 in the presence of14C-Gly (1 mM). (B) Comparison of the activity of FmtA�27 to the activity ofthe FmtA�27S127A mutant. The concentration of each of the two proteins was15 �M. Error bars represent standard deviations calculated from at least threeindependent experiments.

FmtA Interacts with WTAs

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ing assays with cell walls isolated from the �tarO mutant showedthat these cell walls could not recruit FmtA�27; the protein re-mained predominately in the unbound fraction (Fig. 4).

The WTA-binding properties of FmtA�27 were further studiedusing WTAs isolated from S. aureus RN4220. Incubation ofFmtA�27 with WTAs resulted in a lack of electrophoretic resolu-tion of the protein by the use of an nPAGE/H� gel (Fig. 5). Weconcluded that FmtA�27 could not enter the gel in the presence ofWTAs; however, addition of NaCl to the reaction mixture facili-tated resolution of FmtA�27 by the nPAGE/H� procedure. At 500mM NaCl, the protein was resolved completely (Fig. 5A). Thepresence of salt alone had no effect on the electrophoretic resolu-tion of FmtA�27 by nPAGE/H� (Fig. 5B). These results stronglyindicate that FmtA�27 interacts with WTAs. Similar experimentswere carried out with PBP2a, a penicillin-binding protein that hastranspeptidase activity. PBP2a displayed an affinity for the WTAsand behaved in the same way as FmtA (Fig. 5C). Proteins unre-lated to cell wall metabolism, including RNaseA, and carbonicanhydrase, did not show any binding to WTAs, while BSA showeda weak affinity for teichoic acids (Fig. 6).

WTAs induce a conformational change in FmtA�27 struc-ture. The CD experiments showed that FmtA�27 undergoes adose-dependent conformational change in the presence ofteichoic acids (Fig. 7). The conformational change is associatedwith a reduction in FmtA�27 �-helix content, as interpreted froma decrease in the CD signal at 222 nm. The reduction in �-helixcontent was calculated to be 6% in the presence of WTAs at 6.25�g/ml, 14% in the presence of WTAs at 12.5 �g/ml, and 21% inthe presence of WTAs at 25 �g/ml (Fig. 7).

The interpretation of the effect of WTAs on FmtA�27 second-ary structure was supported by trypsin digestion experiments (Fig.8). The digestion profiles of FmtA�27 differed in the presence andabsence of WTAs. Three major fragments were released upon in-cubation of FmtA�27 with trypsin; however, when WTAs (6.25�g/ml) were present, six fragments were produced (Fig. 8). These

observations provide further evidence that WTAs interact withFmtA�27.

To identify the specific region in the protein involved withbinding to WTAs, FmtA�27 was subjected to a 15-min trypsindigestion in the absence and presence of WTAs. Analysis of thereleased peptides by MALDI-TOF MS (see Fig. S1S in the supple-mental material) showed a notable difference: a peptide with amass of 1,369 Da was not detected when the digestion was carriedout in the presence of WTA. Instead, a peptide with a mass of 853Da exhibited more abundance. The peptide with a mass of 1,369Da mapped to amino acids 312 to 323 in the C-terminal portion ofFmtA�27, and the peptide with a mass of 853 Da mapped to aminoacids 155 to 163 of FmtA�27.

FmtA�27 induces autolysis of S. aureus. Binding of FmtA�27

to WTA suggests that it could affect processes regulated by WTA.One of the important processes controlled by WTA and LTA isautolysis. The activity of AtlA, the major autolysin of S. aureus, hasbeen shown to be controlled by teichoic acids (14, 40, 50, 51). Weinvestigated the potential effect of FmtA�27 on autolysis of S. au-reus RN4220 by incubating cells with different concentrations ofFmtA�27. We discovered that FmtA�27 exogenously added to S.aureus cells affected autolysis of cells in a dual mode. At concen-trations of up to 0.1 �M, FmtA�27 induced autolysis over 1 h,whereas at concentrations higher than 0.2 �M, an enhancedgrowth of bacteria was observed instead (Fig. 9A). A similar ob-servation was also made for the �tarO mutant, which lacks WTAs(8) (Fig. 9B).

FmtA�27 induces biofilm formation in S. aureus. Controlledautolysis is essential for formation of biofilms (15, 19); therefore,FmtA�27-induced autolysis suggests that FmtA�27 might be in-volved in biofilm production in S. aureus. Indeed, recent reportsshow that inactivation of FmtA leads to cell biofilm formationdeficiency (4, 44). Our study showed that biofilm production inthe S. aureus RN4220 strain increases with the FmtA�27 concen-tration up to 0.1 �M and that it decreases at FmtA concentrationsabove 0.2 �M (Fig. 10), much like the dual effect observed forFmtA-induced autolysis.

We investigated the WTA effect on the ability of FmtA�27 toinduce biofilm formation by comparing biofilm formation of the�tarO mutant with that of the wild-type strain in the presence orabsence of 0.2 �M FmtA�27. These studies showed that, although

FIG 3 FmtA�27 binding to cell wall. Cell wall (40 �g) was digested with lyso-staphin (A) or mutanolysin (B) at different time intervals. Binding of FmtA�27

(2.5 �M) to cell wall products was investigated with 12.5% SDS-PAGE. Sam-ples represent the control (C), unbound (U), wash (W), and pellet (P) frac-tions. The SDS-PAGE gels were stained with Coomassie blue.

FIG 4 The effect of removal of WTAs on binding of FmtA�27 to cell wall. (A)Cell walls (100 �g) of S. aureus RN4220 (WT) and the tarO deletion strain(�tarO) were incubated with FmtA�27 at 2.5 �M for 2 h at 4°C and analyzed by12.5% SDS-PAGE. (B) Cell walls with removed WTA [CW (-TA)] (100 �g)isolated from S. aureus RN4220 were incubated with FmtA�27 (15 �M). Ex-perimental conditions were the same as described for panel A. Samples repre-sent control (C), unbound (U), wash (W), and pellet (P) fractions. The SDS-PAGE gels was stained with Coomassie blue.

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FmtA�27 induced maximum biofilm formation in the S. aureuswild-type strain, it was entirely unable to induce this process in the�tarO mutant strain despite similar conditions (Fig. 10C).

FmtA�27 localizes in the division septum. To investigate lo-calization of FmtA to S. aureus, we fused FmtA�27 to the C termi-nus of GFP. GFP-FmtA�27 expressed well in E. coli (25 mg/liter),and the protein was purified using the protocol for FmtA�27 pu-rification (10). The GFP tag showed no effect on the interaction ofFmtA with �-lactams or cell walls (see Fig. S2S in the supplemen-tal material). We therefore concluded that GFP-fused FmtA�27 isindistinguishable from wild-type FmtA�27.

Gründling and Schneewind reported that the GFP proteinalone does not bind to S. aureus cells (16). Our fluorescence mi-croscopy studies showed that GFP-FmtA�27 bound at the divisionseptum in the dividing S. aureus RN4220 cells, where cell wallsynthesis is reported to occur in S. aureus (33). In nondividingcells, GFP-FmtA�27 was localized as two dots corresponding to thering of the future division plane (Fig. 11). A localization patternsimilar to that of GFP-FmtA�27 has been reported for PBP4 of S.aureus (1). In the �tarO mutant, GFP-FmtA�27 was observed allover the cell wall but with no specific accumulation at the divisionseptum (Fig. 11).

Exposure of S. aureus to either oxacillin or vancomycin (eachof which inhibits the cross-linking step in peptidoglycan biosyn-thesis by binding, respectively, to the transpeptidase domain ofPBPs or the D-Ala-D-Ala portion of the peptidoglycan) did nothave any effect on the localization of GFP-FmtA�27. A similarobservation was made for PBP4 (1). In the case of PBP2, however,

the presence of these antibiotics caused delocalization of the PBP2from the division site, suggesting that PBP2 requires substratebinding for its correct localization (35).

DISCUSSION

The fmtA gene was identified as a methicillin resistance factor(23), and it is present in all S. aureus strains. It is part of the corecell wall stimulon (26). Its expression level is elevated by the pres-ence of cell wall inhibitors and knockout of genes that are directlyinvolved with peptidoglycan biosynthesis (3, 23, 26, 27, 42, 46).Furthermore, inactivation of fmtA results in a lack of WTAs on S.aureus cell walls and deficiency in biofilm production (4, 44).

The function of FmtA is not known. In an earlier study, weshowed that FmtA is capable of forming covalent species with�-lactams involving the serine-active site residue of the SXXKconserved motif (10). The �-lactam binding activity of FmtA sug-gests that it is a PBP. Of the four well-known PBPs of S. aureus,PBP1 is a monofunctional transpeptidase that is essential for via-bility of S. aureus (48). PBP2 is a bifunctional enzyme with bothtransglycosylase and transpeptidase activity, and it is essential for

FIG 5 FmtA�27 interaction with WTAs analyzed by nPAGE/H�. (A) FmtA�27 (15 �M) was incubated with 5 �g of WTAs in the presence of differentconcentrations of NaCl in a 15-�l reaction volume. Samples were analyzed with nPAGE/H�. (B) FmtA (15 �M) was incubated with different concentrations ofNaCl, and samples were analyzed with nPAGE/H�. (C) PBP2a (15 �M) was incubated with 5 �g of WTA in a 15-�l reaction volume in the absence or in thepresence of 500 mM NaCl. The nPAGE gels were stained with Coomassie blue.

FIG 6 Investigation of the interaction between TAs and protein unrelated topeptidoglycan biosynthesis. Lipoteichoic acids (LTAs; 5 �g) were incubatedwith bovine serum albumin (BSA; pI 4.6), ovalbumin (pI 4.5 to 4.6), RNaseA(pI 9.63), or carbonic anhydrase (pI 5.88 to 6.55) at 10 �g each in a 15-�lreaction volume. Samples were analyzed by nPAGE/H�. Protein bands werestained with Coomassie blue.

FIG 7 Investigation of FmtA�27 interaction with WTAs by CD. The CD spec-tra of FmtA�27 (14 �M) were collected in the absence and in the presence ofdifferent concentrations of WTAs. The CD spectra were recorded using 50 mMsodium phosphate buffer (pH 7.0) at 22°C. The data points represent averagesof the results of three scans and were corrected for buffer contribution.

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viability of S. aureus (37). PBP3 and PBP4 are believed to bemonofunctional and dispensable transpeptidases. Furthermore,PBP2 and PBP4 were shown to work together to synthesize ahighly cross-linked peptidoglycan (24).

In this report, we show that FmtA has DD-carboxypeptidaseactivity; it catalyzes the removal of the last D-Ala residue from theN�,Nε-diacetyl-L-Lys-D-Ala-D-Ala peptide (a surrogate for the PGdonor strand). However, it shows low enzymatic activity. In thepresence of Gly-Gly (a surrogate for the PG acceptor strand), the

catalytic efficiency of FmtA�27DD-carboxypeptidase activity was

determined to be 0.745 � 106 M1 s1. In comparison, the cat-alytic efficiency of the DD-carboxypeptidase activity of PBP4 of S.aureus was determined to be 15.7 M1 s1 (30). The activity ofother well-studied PBPs has also been reported to be higher, rang-ing from 10 M1 s1 for PBP5 of E. coli to 2.9 � 104 M1 s1 forPBP3 of N. gonorrhoeae, according to a study using the same sub-strate as used in our study (42).

The low enzymatic activity of FmtA could be due to three mainreasons: (i) it lacks the third conserved motif, the KTG motif (thismotif has been shown to be involved in substrate binding andcatalysis) (9, 29, 52); (ii) the L-Lys-D-Ala-D-Ala is not the FmtAnative substrate; (iii) the DD-carboxypeptidase activity is reminis-cent of that of a PBP from which FmtA had evolved; and (iv) theenzyme may require interaction with a ligand in the periplasmicspace to become active. Several proteins involved with either pep-tidoglycan biosynthesis or its remodeling have been shown to be-come active in the presence of either the native substrate or aparticular ligand. For example, PBP2a of S. aureus becomes activeupon binding to PG (12), E. coli PBP1a and PBP1b (two lipopro-teins) are activated through interactions with membrane-boundproteins LpoA and LpoB (32, 45) and PBP5 of E. coli requiresbinding to the membrane to become active (38).

In spite of the poor activity of the enzyme observed using PGsurrogates, we discovered that FmtA�27 is capable of incorporat-ing 14C-Gly into isolated S. aureus cell walls. This observationsuggests that FmtA may have transpeptidase activity, which agreeswith previous reports that inactivation of fmtA is associated with adecrease in the level of the highly cross-linked peptidoglycan andan increase in the sensitivity of S. aureus to oxacillin (22, 23).

The reported link between FmtA and the level of cross-linked

FIG 8 Investigation of FmtA�27 interaction with WTAs by trypsin digestion.FmtA�27 (40 �M) was digested with trypsin in the absence (TA) and pres-ence (�TA) of WTAs (1.25 �g, 80 �l). Aliquots (10 �l) were removed atdifferent time intervals and quenched by addition of loading buffer and heat-ing at 60°C. Samples were analyzed with 15% Tris-Tricine SDS-PAGE.

FIG 9 FmtA�27 effect on autolysis of S. aureus RN4220. (A) Autolysis of S.aureus RN4220 cells in the presence of different concentrations of FmtA�27

and 1� PBS buffer, monitored for 1 h (empty circles) and 2 h (filled circles).Error bars indicate standard deviations. (B) The effect of 0.2 �M FmtA�27 onautolysis of S. aureus tarO parental strain (WT) and tarO-deleted mutantstrain (�tarO).

FIG 10 Stimulation of biofilm formation by FmtA�27 in S. aureus RN4220.(A) Detection of biofilm formation of S. aureus RN4220 plates in the presenceof various concentrations of FmtA�27 following 24 h of growth in polystyreneflat-bottomed tissue culture plates. (B) Quantification (A562) of biofilmgrowth with various concentrations of FmtA�27. Data represent the averages ofthe results of at least three independent experiments. Error bars indicate stan-dard deviations. (C) Detection of biofilm formation of S. aureus wild-typestrain (WT) and �tarO mutant strain (�tarO) in the presence of 0.2 �MFmtA�27 after 24 h of growth in polystyrene flat-bottomed tissue cultureplates.

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PG should be noted, as the cross-linked sites in PG are reported tobe targets of teichoic acid attachments in Streptococcus pneu-moniae and Bacillus licheniformis (11, 53). Interestingly, inactiva-tion of fmtA leads to a lack of teichoic acid attachment in S. aureus(4). In light of these reports, it is possible that the native functionof FmtA might be involved with teichoic acid attachment throughmediation of cross-linked PG synthesis or marking the teichoicacid attachment sites in PG. In this study, we demonstrated thatFmtA interacts specifically with WTAs. Proteins unrelated to cellwall biosynthesis (BSA, ovalbumin, RNaseA, and carbonic anhy-drase) showed no interactions with WTAs, suggesting that thenegative charge on WTAs does not enable indiscriminate proteinbinding.

WTAs are proposed to be temporal and spatial regulators ofpeptidoglycan cross-linking that operate by mediating localiza-tion of proteins associated with PG biosynthesis and remodeling,such as PBP4 and AtlA (1, 40). Although a direct interaction be-tween these two proteins and WTAs has not been demonstrated,our study shows that interaction of WTAs with PG-associatedproteins is not limited to FmtA. PBP2a, the major source of resis-tance to �-lactams in S. aureus, interacts with WTAs in the same

way as FmtA. This finding suggests that interaction of PG-associ-ated proteins, such as PBPs, with WTAs could be more commonthan previously thought.

The interaction of FmtA with teichoic acids led us to investi-gate two processes in S. aureus that are closely linked to teichoicacids, autolysis and biofilm production. We observed that exoge-nous FmtA�27 induced autolysis and biofilm production in S. au-reus at concentrations lower than 0.1 �M. However, atFmtA�27concentrations higher than 0.2 �M, S. aureus exhibitedenhanced growth and deficiency in biofilm production. These ob-servations corroborate recent reports that S. aureus �fmtA mu-tants have lost their ability to precisely control the autolysis activ-ity of autolysins (4) and their ability to effectively form biofilms (4,44). The ability of S. aureus to form biofilms is intimately linked toits ability to undergo controlled autolysis (2, 25). We thereforepropose that the effect FmtA has on biofilm formation is linked toits ability to induce controlled autolysis.

The FmtA role in autolysis and, hence, biofilm formation doesnot result from a direct role of FmtA in autolysis, as FmtA does nothave autolysis activity (see Fig. S3S in the supplemental material).It is possible that binding of FmtA to WTAs may activate S. aureusautolysins. The major autolysin of S. aureus is AtlA (30), which isinvolved in the separation of daughter cells after cell division.Studies have shown that WTAs control the activity of AtlA (14, 40,50, 51) and that modulation of the WTA negative charge affectsAtlA activity (33, 39). Binding of FmtA to WTAs might affect theactivity of AtlA by modulating the negative charge of WTAs; how-ever, we cannot exclude the possibility of interactions betweenFmtA and AtlA.

The dual mode of FmtA activity for S. aureus, induction andinhibition of autolysis, suggests that FmtA might serve two differ-ent roles in S. aureus, both dependent of the FmtA concentrationin the cell; at low concentrations (�0.1 �M), FmtA may be in-volved in positively controlling the autolysis process, and at higherprotein concentrations (�0.2 �M), FmtA may repress the activityof autolysins and may be involved in PG biosynthesis.

In this study, we discovered that FmtA localizes at the cell di-vision septum, most likely by binding to the cross wall (Fig. 11).PBP4 and AtlA also localize in the cell division septum (1, 40). Thelocalization data clearly show that FmtA is initially localized at thedivision poles and then moves toward the center of the bacteriumas the cross-wall is synthesized (Fig. 11B). The presence of cell wallinhibitors did not cause delocalization of the protein, demonstrat-ing that PG does not mediate the FmtA recruitment to the divisionseptum. Our finding that FmtA interacts with WTAs suggests thatWTAs might recruit FmtA to the cell wall. However, no matureWTAs have been shown to be present in the cross-wall region (40).It is plausible that FmtA can be recruited either by a precursor ofWTAs in the cross-wall region or by another target. Based on theeffect that FmtA has on autolysis, we cannot exclude the possibil-ity that FmtA interacts with AtlA; AtlA also localizes in the cross-wall region (40). Incidentally, the structural genes of atlA andfmtA are close to each other in S. aureus genomes and are sepa-rated by three structural gene loci, SA0906, SA0907, and SA0908(gene numbering per the S. aureus N315 strain), with no knownfunctions.

On a final note, it is intriguing that one autolysin and two PBPs(FmtA and PBP4) colocalize in the same region of the cell wallduring the process of cell division. It is proposed that PG-synthe-sizing enzymes and autolysins form a complex, which would be

FIG 11 Fluorescence microscopy imaging of FmtA�27 localization in S. au-reus. Pictures show localization of GFP-FmtA�27 at the cell septum in S. aureusRN4220 (WT strain) and loss of septal localization in the �tarO strain. S.aureus RN4220 cultures and �tarO mutant strains were incubated with 1 �MGFP-FmtA�27 at room temperature (RT) for 10 min. Pictures were taken withan epifluorescence microscope after removing unbound GFP-FmtA�27. Fluo-rescence (GFP), differential interference contrast (DIC), and overlap of GFPand DIC images are shown. Red arrows identify the GFP-FmtA�27 protein in S.aureus cells. Bar, 1 �m. (B) A conceptual model of FmtA location in S. aureusbased on GFP-FmtA localization studies.

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significantly more efficient in PG biosynthesis (18). Our localiza-tion studies, and those performed with AtlA and PBP4, suggestthat AtlA, FmtA, and/or PBP4 may work cooperatively to mediatePG remodeling during normal cell growth conditions. In the pres-ence of cell wall inhibitors, FmtA could repress autolysis and me-diate growth, probably by enhancing PG biosynthesis.

ACKNOWLEDGMENTS

This work was supported by a grant to D.G.-K. from the Canadian Insti-tutes of Health Research.

The �tarO deletion strain (EBII44) and its parental strain (EBII16)were kindly provided by Eric D. Brown’s laboratory at McMaster Univer-sity, Hamilton, Ontario, Canada.

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