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J Bacteriol. May 2007; 189(9): 3525–3531.
Published online Feb 16, 2007. doi:  10.1128/JB.00044-07
PMCID: PMC1855886

Role of PBP1 in Cell Division of Staphylococcus aureus[down-pointing small open triangle]


We constructed a conditional mutant of pbpA in which transcription of the gene was placed under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter in order to explore the role of PBP1 in growth, cell wall structure, and cell division. A methicillin-resistant strain and an isogenic methicillin-susceptible strain, each carrying the pbpA mutation, were unable to grow in the absence of the inducer. Conditional mutants of pbpA transferred into IPTG-free medium underwent a four- to fivefold increase in cell mass, which was not accompanied by a proportional increase in viable titer. Examination of thin sections of such cells by transmission electron microscopy or fluorescence microscopy of intact cells with Nile red-stained membranes showed a morphologically heterogeneous population of bacteria with abnormally increased sizes, distorted axial ratios, and a deficit in the number of cells with completed septa. Immunofluorescence with an antibody specific for PBP1 localized the protein to sites of cell division. No alteration in the composition of peptidoglycan was detectable in pbpA conditional mutants grown in the presence of a suboptimal concentration of IPTG, which severely restricted the rate of growth, and the essential function of PBP1 could not be replaced by PBP2A present in methicillin-resistant cells. These observations suggest that PBP1 is not a major contributor to the cross-linking of peptidoglycan and that its essential function must be intimately integrated into the mechanism of cell division.

Penicillin-binding proteins (PBPs) are enzymes involved in the last stages of peptidoglycan biosynthesis. There are four native PBPs, PBP1 to PBP4, in Staphylococcus aureus and an additional PBP, PBP2A, in methicillin-resistant S. aureus (MRSA) strains (1, 5, 11). This extra PBP is encoded by the exogenous gene mecA and, in contrast to the native PBPs, shows low affinity for β-lactams, ensuring continued cell wall synthesis in the presence of otherwise lethal concentrations of these antibiotics (11, 25).

The primary amino acid structure of PBP1 shows a high degree of similarity to the sequences of PBP2B and SpoVD from Bacillus subtilis, PBP2X from Streptococcus pneumoniae, and PBP3 from Escherichia coli (33). All of these proteins are high-molecular-weight (HMW) class B PBPs, composed of a C-terminal domain with conserved transpeptidase motifs and an N-terminal domain of as-yet-unknown function (6, 7). Several lines of evidence indicate that PBP2B of B. subtilis, the pneumococcal PBP2X, and PBP3 of E. coli are involved in cell division (2, 20, 27, 34), consistent with their location in division and cell wall (dcw) synthesis clusters on the chromosome (28).

An earlier study by Pucci and colleagues has determined the chromosomal location of S. aureus pbpA in a dcw cluster together with other determinants, such as mraY, divIB, ftsA, and ftsZ (28), and a role for PBP1 in cell division of S. aureus has been proposed (22).

PBP1 of S. aureus was reported to be essential for growth because disruption of the chromosomal pbpA copy was lethal unless additional copies of the gene were present on a plasmid (33). However, the specific role(s) of PBP1 and its cellular address has not been investigated in detail.

The purpose of the studies described here was to construct a pbpA conditional mutant and use it to better define the essentiality of PBP1 for growth, to determine the contribution of PBP1 to the chemical structure of peptidoglycan, and to explore its role in cell division of S. aureus.


Bacterial strains, media, and general methods.

The strains and plasmids used in this study are listed in Table Table1.1. The mecA-negative strain COL-S was constructed by introducing plasmid pSR (14), encoding the chromosomal cassette recombinase ccrA and ccrB genes, into strain COL and selecting for susceptibility to oxacillin, essentially as described previously (14), in order to excise the staphylococcal cassette chromosome mec (SCCmec) from the chromosome. S. aureus strains were grown at 37°C in tryptic soy broth (TSB) (Difco Laboratories, Detroit, MI) with vigorous aeration: 25-ml cultures were incubated in 250-ml Erlenmeyer flasks on a horizontal water bath shaker at a speed of 180 rpm. Alternatively, bacteria were plated on tryptic soy agar (Difco Laboratories). E. coli strains were grown at 37°C in Luria-Bertani (LB) broth (Difco Laboratories) with aeration or on LB agar (Difco Laboratories). Bacterial growth was monitored by measuring the optical density of S. aureus cultures at 620 nm (OD620). Erythromycin and chloramphenicol were used at a concentration of 10 μg/ml for the selection of pMGP1- and pSK5632-derived S. aureus mutants, respectively. Conditional mutants were always grown in the presence of erythromycin and IPTG (isopropyl-β-d-thiogalactopyranoside) (Sigma, St. Louis, MO) at a concentration of 500 μM unless otherwise stated. DNA manipulations were performed according to standard procedures (17). The high-fidelity PfuTurbo DNA polymerase (Stratagene, Heidelberg, Germany) was used to generate PCR fragments for cloning; these were sequenced to ensure that no mutations had been introduced. E. coli strain DH5α was used for plasmid construction and propagation.

Strains and plasmids

Construction of pbpA conditional mutants.

An 850-bp fragment of pbpA was amplified from COL DNA by PCR using primers pbp1spacF (5′-GTACCCGGGACGATAATGTAAAGGTAG-3′ [SmaI site underlined]) and pbp1spacR (5′-TAGAGATCTGCATCCATGACAACCGC-3′ [BglII site underlined]). The amplified pbpA fragment and plasmid pMGPI (25) were digested with SmaI and BglII and ligated to create plasmid pMGPA. Plasmid pMGPA was introduced into RN4220 by electroporation as previously described (15). The correct insertion of pMGPA into the chromosome of RN4220 via a single reciprocal crossover (a Campbell-type recombination event) was confirmed by PCR and Southern blotting. The inserted plasmid was then transferred to COL-S or COL by transduction using phage 80α as described (23), generating COL-SspacP1 and COLspacP1, respectively.

Growth of pbpA conditional mutants and depletion of PBP1.

Overnight cultures were diluted to an OD620 of 0.05 in fresh TSB with IPTG (500 μM) and incubated at 37°C in an orbital shaker at 180 rpm for four generations. This “refreshment” step allowed the cells to leave stationary phase. Cultures were then washed with fresh TSB to remove IPTG and diluted to an OD620 of 0.05 in medium without IPTG and with different inducer concentrations (35, 50, 80, and 500 μM).

To deplete the conditional mutants of PBP1, an extra incubation step was introduced into the procedure. After the initial “refreshment” growth step, the cultures were diluted to an OD620 of 0.05 in medium without IPTG, incubated at 37°C in an orbital shaker at 180 rpm, and monitored carefully by OD620 measurements. Just before a complete halt in the increase of the OD, the bacteria were rediluted to an OD620 of 0.05 in fresh growth media containing different concentrations of IPTG, and their rates of growth were monitored.

Complementation assay.

A 2.9-kb fragment that included the complete pbpA coding sequence and 300 bp upstream of it was amplified from COL DNA by PCR using primers pbp1FPstI (5′-GTATACTGCAGCAACAACCAC-3′ [PstI site underlined]) and pbp1RBamHI (5′-CAGGGATCCTCTTCTTAATCCAGAC-3′ [BamHI site underlined]). The amplified pbpA fragment and plasmid pSK5632 (8) were digested with PstI and BamHI and ligated, generating pSKP1. The replicative plasmid pSKP1 was introduced into RN4220 by electroporation and subsequently transferred to COLspacP1 by transduction, generating COLspacP1pSKP1.

Analysis of pbpA transcription by real-time RT-PCR.

COL and COLspacP1 were grown in TSB and/or TSB supplemented with 35, 50, 80, and 500 μM IPTG. Samples were collected at an OD620 of 0.8 (corresponding to the OD at which COLspacP1 grown without IPTG stopped growing), and total RNA was isolated as described previously (31). An on-column DNase digestion using RNase-free DNase (QIAGEN, Valencia, CA) was performed to remove residual DNA. RNA was isolated from three independent cultures. The transcription levels of pbpA were determined by two-step real-time reverse transcriptase PCR (RT-PCR) using the relative standard curve method (user bulletin no. 2; Applied Biosystems Inc.). cDNA was generated by RT with 1 μg of DNase I-treated total RNA and TaqMan reverse transcription reagents with random hexamers (Applied Biosystems) in a total volume of 100 μl. The reaction mixture was incubated at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min. Real-time PCR was carried out with an ABI Prism 7900 sequence detection system (Perkin-Elmer Applied Biosystems). PCRs were performed, and mixtures included 1× iTaq SYBR green Supermix with ROX (Bio-Rad), 200 nM (each) forward and reverse primers, 5 μl of 1:5 dilutions of cDNA, and water to a final volume of 25 μl. Primers were as follows: PBP1forwardRT (5′-TTTTAGCATACAGTCAGCGACCA-3′) and PBP1reverseRT (5′-TCCAGGCTCGTATGTGTTTTGA-3′) were used for pbpA amplification, and primers ptaForRT (5′-AGAAGCAATCATTGATGGCGA-3′) and ptaRevRT (5′-ACCTGGCGCTTTTTTCTCAG) were used for pta amplification. The following conditions were used: 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. Each PCR plate contained two replicates of each sample cDNA and standard curves for each gene. These standard curves were made with six serial twofold dilutions of COL chromosomal DNA (from 2.5 ng to 78.1 pg). The specificity of the amplified products was verified by analysis of the dissociation curves generated by the ABI 7900 software based on the specific melting temperature for each amplicon. The melting curves of the PCR products were obtained by stepwise increases in the temperature from 60°C to 90°C. For all experiments, the amount of target (pbpA) and endogenous control (housekeeping gene pta) was determined from the respective standard curve by conversion of the mean threshold cycle values. Normalization was then obtained by dividing the quantity of pbpA by the quantity of pta. The normalized values of pbpA of COLspacP1 grown with the different IPTG concentrations were then divided by the normalized value of pbpA in COL and expressed as an n-fold difference relative to COL.

Electron microscopy.

Strain COL was grown in TSB, and COLspacP1 was grown in TSB or TSB with 500 μM IPTG. When COLspacP1 grown in the absence of IPTG stopped growing (as indicated by no increase in the OD620 determined at three consecutive 10-min intervals), samples for electron microcopy were collected, centrifuged, fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.0), and processed for electron microscopy according to the procedure of Ryter et al. (30), as modified by Tomasz et al. (32).

Fluorescence microscopy.

COLspacP1 was grown in TSB or TSB with 500 μM IPTG. Samples were collected at an OD620 of 0.8, corresponding to the OD at which COLspacP1 grown without IPTG stopped growing. Cell membranes of live cells were stained with Nile red (Molecular Probes Inc., Eugene, OR) at a final concentration of 8 μg/ml for 5 min at room temperature without agitation. Fluorescence microscopy was performed with a Leica DMRA2 microscope coupled to a CoolSNAP HQ Photometrics camera (Roper Scientific, Tucson, AZ).

Immunofluorescence microscopy.

Strain LH607 was grown to an OD620 of 0.7, and a sample was harvested and prepared for immunofluorescence essentially as described previously (26). Briefly, cells were fixed with the fixative Histochoice (Amresco) and lysed for 1 min on a polylysine-treated slide with lysostaphin (Sigma) at a final concentration of 10 μg/ml. Immunolabeling was performed overnight at 4°C with anti-PBP1 antiserum diluted 1:800 in 2% bovine serum albumin in 1× phosphate-buffered saline. Cells were washed and incubated for 1 to 2 h at room temperature with anti-rabbit immunoglobulin G-fluorescein isothiocyanate conjugate (Sigma) diluted 1:500 in 2% bovine serum albumin in 1× phosphate-buffered saline. Cells were again washed, and Vectashield mounting medium (Vector Laboratories) was added. Cells were visualized by phase-contrast and fluorescence microscopy in a Leica DMRA2 microscope coupled to a CoolSNAP HQ Photometrics camera.

Peptidoglycan composition.

A COL culture and a COLspacP1 culture previously depleted of PBP1 were used to inoculate 500 ml of TSB (COL) and TSB supplemented with 50 μM and 500 μM IPTG (COLspacP1). Cultures were rediluted to an OD620 of 0.025 and grown to an OD620 of 0.3 and were harvested for the preparation of cell walls. Cell walls were isolated, peptidoglycan purified, and digested with muramidase, and the muropeptide composition was determined by reversed-phase high-performance liquid chromatography (HPLC), as previously described (3).


PBP1 is essential for growth of both MSSA and MRSA.

Previous, unsuccessful attempts to disrupt pbpA in the methicillin-susceptible S. aureus (MSSA) strain RN4220 indicated the essentiality of this gene in MSSA (33). In order to confirm this observation and to test the essentiality of PBP1 in MRSA, a conditional mutant of pbpA was constructed by placing the gene under the control of the IPTG-inducible Pspac promoter (12). The integrative plasmid pMGPA, containing the ribosome-binding site and the first 277 codons of pbpA, was introduced into the MSSA strain RN4220 by electroporation, creating a strain bearing a single functional copy of pbpA under the control of the Pspac promoter (Fig. (Fig.1).1). The Pspac-pbpA chromosomal fusion was next transferred by transduction to the background of strain COL, an MRSA strain, and its isogenic MSSA derivative, COL-S, from which SCCmec had been removed by precise excision (14), generating the isogenic strains COLspacP1 and COL-SspacP1, respectively.

FIG. 1.
Construction of the pbpA conditional mutant. An 850-bp fragment of pbpA containing the ribosome-binding site was cloned downstream of the Pspac promoter in the integrative vector pMGPI. The resulting plasmid, pMGPA, was introduced into S. aureus RN4220 ...

Neither of these conditional mutants were able to grow in the absence of IPTG (Fig. (Fig.2A),2A), and once the strains had been depleted of PBP1, their growth rates were proportional to the concentration of IPTG in the medium, as shown for COLspacP1 in Fig. Fig.2B2B.

FIG. 2.
Essentiality of pbpA expression for S. aureus growth and transcription of pbpA in the mutant strain COLspacP1 grown with different inducer concentrations. (A) Representative growth curves of pbpA conditional mutants COL-SspacP1 ([open diamond]) and COLspac ...

The pbpA gene is located in a division and cell wall (dcw) synthesis cluster together with a number of other essential determinants, including, downstream of it, the mraY, divIB, ftsA, and ftsZ genes (28). For this reason, it was important to test whether the growth defect of the conditional mutants was due to a polar effect. The replicative plasmid pSKP1, containing the complete coding sequence of pbpA, was introduced into COLspacP1, and the strain was grown in the absence of IPTG. Growth of COLspacP1 in IPTG-free medium was successfully reestablished when pbpA was present in a plasmid (Fig. (Fig.2C2C).

The abundance of the pbpA transcripts in COLspacP1 grown with different IPTG concentrations was also analyzed by real-time RT-PCR. For each sample, pbpA values were normalized to the values of the housekeeping gene pta, used as the endogenous control, and expressed as an n-fold difference relative to COL. The successful induction of pbpA transcription was confirmed, and the amount of pbpA transcript was found to vary according to the inducer concentration present in the medium (Fig. (Fig.2D).2D). The mutant grown with the optimal IPTG concentration (500 μM) showed an increase of approximately 1.4 times the level of pbpA transcript in the parental strain, COL.

Deficit in growth rate in the pbpA conditional mutant.

The complementation experiments clearly show that the dependence of growth of the conditional pbpA mutants on the IPTG inducer is related to the production of PBP1. By increasing the concentration of IPTG in the medium, it was possible to increase the growth rate of the pbpA conditional mutants. Nevertheless, we were never able to fully restore the growth rate of the parental strain in the conditional mutants even by supplying the highest concentrations of the inducer. The reasons for this are not clear. The possibility of insufficient expression of pbpA from the Pspac promoter was disproved by the analysis of pbpA transcription, which actually showed an increase in the level of pbpA transcript in COLspacP1 grown with optimal IPTG concentration. In E. coli and B. subtilis, many of the genes in the dcw cluster are cotranscribed in the form of long polycistronic messages, and this coordinate expression is important for normal growth (9, 10, 13, 18, 29). In the case of the S. aureus conditional mutants described here, pbpA is expressed from its native locus but not under the control of its native promoter. Furthermore, even though we have shown that the growth defect in COLspacP1 was unlikely to be caused by a polar effect, the dcw cluster is interrupted by integration of the Pspac-pbpA-bearing construct (Fig. (Fig.1).1). The inability to fully restore normal growth to the conditional mutant may be interpreted in the context of the genomic channeling hypothesis, according to which the clustering of dcw genes would favor the cotranslational assembly and function of cell division and peptidoglycan precursor synthesis complexes (19).

Changes in cell morphology during residual growth of COLspacP1 in IPTG-free medium.

COLspacP1 was grown without IPTG, and the OD, viable titer, and morphology of the bacteria were tested. A parallel culture supplemented with the optimal concentration of IPTG was used as a control. After resuspension in IPTG-free medium, COLspacP1 continued to increase in OD, from an initial OD620 of 0.05 to 0.8 (Table (Table2).2). However, the initial viable titer showed only a minor increase, completely disproportionate with the 16-fold increase in OD. In the control culture, the increase in viable titer showed a precise parallel with the increase in OD.

Inhibition of cell division and abnormal morphology in COLspacP1 cells grown without IPTG

We used transmission electron microscopy to examine the morphology of cells in populations of strain COL and the pbpA conditional mutant COLspacP1 grown in the absence or presence of IPTG in the medium. The morphological differences between these bacteria were quite striking. The parental COL cells formed a homogeneous population of bacteria, with an average ratio of 1.18 ± 0.01 between the equatorial (septal) and longitudinal axes of the cells (Fig. 3A, B, and E). In contrast, three subpopulations with distinct morphologies could be identified among cells of COLspacP1 grown in the absence of inducer. The first of these, subpopulation 1, representing approximately 22% of the cells evaluated, maintained both the average axis ratio and overall dimensions found in the parental COL population (Fig. 3C and F). The second and most abundant, subpopulation 2 (63% of the cells), also maintained the parental axis ratio but showed an increase of about 45% in cell size (Fig. 3C, D, and F and Table Table2).2). The third, subpopulation 3, representing about 15% of the cells, showed an altered average axis ratio (1.7 ± 0.08), with an increased longitudinal axis (Fig. 3C and F).

FIG. 3.
Morphology of the parental strain COL and its pbpA conditional mutant, COL-spacP1, grown in the absence of IPTG. Strain COL was grown in TSB, and COLspacP1was grown either in TSB or in TSB supplemented with 500 μM IPTG. Thin sections were processed ...

Evaluation of the morphology of the COLspacP1 mutant grown in the presence of the optimal inducer concentration presented a picture similar to that of strain COL: cells showed homogeneous morphology, and subpopulations 2 and 3 were absent (Fig. (Fig.3E3E).

The existence of the distinct subpopulations when COLspacP1 was grown in the absence of inducer was confirmed by fluorescence microscopy using the dye Nile red to stain membranes of live cells (Fig. (Fig.3G),3G), and quantitative evaluations of the morphologies by this method (data not shown) gave results identical to those illustrated in Fig. Fig.3F3F.

We do not presently know whether the aberrant morphologies observed in bacteria “growing” with inhibited pbpA transcription correspond to cells with different degrees of the deleterious effects caused by depletion of PBP1 or whether they reflect the heterogeneity of the population and represent cells that were at different stages of the cell cycle when cell division was blocked by the lack of PBP1.

PBP1 and the formation of septa.

Both COL and COLspacP1 grown without IPTG showed the same proportion of cells with signs of septation (around 80%). However, cells with complete septa represented around 90% of the COL population (Fig. 3A and H), while cells with complete septa were present in only about 15% of the COLspacP1 mutant population grown without IPTG (Fig. 3C and H). Interestingly, we note that depletion of pbpA does not seem to perturb division site selection, as most cells in the mutant population show correct placement (at midcell) of the septum even though cells with completely formed septa were rarely observed in cultures of COLspacP1 in the absence of inducer (Fig. (Fig.3).3). Moreover, the rare COLspacP1 cells with complete septa showed thickened equatorial cell walls. The incomplete septa may have been initiated when PBP1 was still available but were not completed due to the depletion of PBP1. Another possibility that we cannot presently exclude is that PBP1 may function only at a late stage in septum formation.

In any event, the inhibition of cell division eventually leads to cells with increased size, which appear to be enveloped by a cell wall of normal thickness. The source of this cell wall material is not clear; it may result from cell wall synthesis that continues in the absence of PBP1 or may involve the redistribution of cell wall from the incomplete septa produced when PBP1 was still available.

Localization of PBP1.

The striking changes in cell division detected in the pbpA conditional mutant raised the question of whether PBP1 localized to division sites in S. aureus. We therefore examined the subcellular localization of PBP1 by immunofluorescence using a rabbit anti-PBP1-raised polyclonal antibody. To prevent unspecific binding of the antibody, the protein A mutant strain LH607 was used in these experiments. The fluorescence signal was localized in the septum (Fig. (Fig.4),4), as was the case for COLspacP1 grown with 500 μM IPTG, whereas the same mutant strain depleted of PBP1 showed only background fluorescence (data not shown).

FIG. 4.
PBP1 localization by immunofluorescence. Panels show selected specimens from the same field. White arrows indicate the localization of the florescence signal in septa. Phase contrast (PHC) images are shown in the first column, and florescence (FL) images ...

PBP1 and the chemical composition of peptidoglycan.

The results in the preceding sections have shown that PBP1 is essential for growth and division. As cell wall synthesis in S. aureus is believed to occur mainly in the septum (26), where PBP1 is localized, we wanted to examine the composition of the peptidoglycan in the conditional mutant. For this purpose, COLspacP1 was grown with a suboptimal (50 μM) or optimal (500 μM) IPTG concentration, and the peptidoglycan composition was analyzed by reversed-phase HPLC. The growth rate of the culture grown at the suboptimal IPTG concentration was drastically reduced (see Fig. Fig.2B),2B), and yet the same conditions caused only minor alterations in peptidoglycan composition compared to the composition of the parental strain COL, namely, a decrease in the proportion of peak 1, representing the unsubstituted disaccharide pentapeptide monomer (Fig. (Fig.5).5). However, this appears to be a nonspecific alteration often observed in other mutants in the background of strain COL, some in genes not directly involved with cell wall synthesis (4; also our unpublished results). Thus, reduction in the level of pbpA expression sufficient to cause a significant decrease in growth rate did not cause detectable alteration in the composition of the peptidoglycan of the conditional mutant. This is in sharp contrast to the profound changes in peptidoglycan composition and cross-linking that have been described for mutants of the S. aureus pbpD or pbpB gene (16, 25).

FIG. 5.
Peptidoglycan HPLC profiles for strains COL and COLspacP1 grown with optimal (500 μM) and suboptimal (50 μM) IPTG concentrations. The deficit of PBP1 did not have a significant impact on peptidoglycan composition. The only alteration observed ...

Although we cannot exclude the possibility that alterations in the composition of the peptidoglycan in the pbpA conditional mutant do occur but were not detected by the particular analytical technique used, our observations suggest that PBP1 does not play a major role in the cross-linking of S. aureus peptidoglycan. This is also consistent with the surprising finding reported in this communication that, in contrast to the case of PBP2, PBP1 remains essential even in the background of the MRSA strain COL, which carries a constitutively expressed PBP2A. PBP2 is the only bifunctional class A HMW PBP in S. aureus (7, 21), and its transpeptidase activity is essential for growth in MSSA but can be replaced by PBP2A in MRSA, where its transglycosylase activity is required for optimal expression of resistance but not for growth (24, 25). The fact that PBP1, a monofunctional class B HMW PBP, carrying only the transpeptidase motifs (33), is essential for growth in both MSSA and MRSA implies that its essential function cannot be replaced by PBP2A, suggesting that its primary function may not be that of a transpeptidase.

The observations described in this communication show that the main phenotypic consequences of PBP1 depletion in S. aureus occur at the level of septum formation. This finding, together with the localization of PBP1 in the septum, strongly suggests that the essential role of PBP1 in S. aureus is linked to some specific function of this protein in cell division. The nature of this function is currently under investigation.


We thank Rita Sobral and Ana Madalena Ludovice for helpful discussions and H. Komatsuzawa and M. Sugai for the generous gift of PBP1 antibody. We also thank R. A. Skurray for kindly providing plasmid pSK5632 and T. Ito and K. Hiramatsu for plasmid pSR. Real-time PCR and electron microscopy were performed at the Genomic Resource Center and at the Bio-Imaging Resource Center of The Rockefeller University.

Partial support for this study was provided by a grant (2 RO1 A1045738-06) from the National Institute of Health, U.S. Public Health Service, to A.T. and by contracts from Fundação para a Ciência e a Tecnologia, Portugal: POCTI/BIA-MIC/58416/2004 to H.L. and POCI/BIA-BCM/56493/2004 to M.G.P. S.F.F.P. was supported by grant SFRH/BD/9185/2002 from Fundação para a Ciência e Tecnologia.


[down-pointing small open triangle]Published ahead of print on 16 February 2007.


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