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-phosphoglucomutase deficiency on cell wall properties and fitness in Streptococcus gordonii
Département de Microbiologie Fondamentale, Bâtiment Biophore, Université de Lausanne, Quartier UNIL-Sorge, CH-1015 Lausanne, Switzerland
Correspondence
Vladimir Lazarevic
vladimir.lazarevic{at}unil.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-phosphoglucomutase, which converts glucose 6-phosphate to glucose 1-phosphate, is encoded by pgm. The pgm transcript is monocistronic and is initiated from a 
A-like promoter. Mutants with a gene disruption in pgm exhibited an altered cell wall muropeptide pattern and a lower teichoic acid content, and had reduced fitness both in vitro and in vivo. In vitro, the reduced fitness included reduced growth, reduced viability in the stationary phase and increased autolytic activity. In vivo, the pgm-deficient strain had a lower virulence in a rat model of experimental endocarditis.
-phosphoglucomutase; PSD, post-source decay; WTA, wall teichoic acidThe GenBank/EMBL/DDBJ accession number for the nucleotide sequence of Streptococcus gordonii pgm reported in this paper is DQ234767.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-Phosphoglucomutase (PGM) converts glucose 6-phosphate to glucose 1-phosphate, which is the precursor of UDP-glucose. UDP-glucose serves as a glucosyl donor in several metabolic pathways. In Gram-positive bacteria, UDP-glucose may be used as the precursor of wall teichoic acids (WTAs), lipoteichoic acids, membrane glycolipids, capsules and exopolysaccharides (Boels et al., 2001
; Lazarevic et al., 2005).
PGM deficiency has pleiotropic effects, the most common being morphological changes and slower growth (Hardy et al., 2000; Buchanan et al., 2005; Lazarevic et al., 2005). In the soil bacterium Bacillus subtilis, altered cell morphology of PGM-deficient mutants correlates with a deficiency in membrane glycolipids and is reversible by addition of Mg2+ (Lazarevic et al., 2005). Moreover, these mutants have a reduced ability to form biofilms and this may be attributed to their inability to synthesize a hypothetical glucose-containing exopolysaccharide. Finally, changes in WTA content and structure, resulting from an inability to synthesize UDP-glucose, confer resistance to some phages.
In Streptococcus pneumoniae, an important cause of respiratory infections, PGM loss is associated with a reduced virulence, possibly due to a reduction in capsule production (Hardy et al., 2000, 2001). Similarly, in Streptococcus iniae, a fish pathogen that can cause invasive infection in humans, PGM deficiency correlates with diminished capsule production and reduced virulence, which might be due to an increased susceptibility to phagocytic clearance (Buchanan et al., 2005).
Streptococcus gordonii is a primary colonizer of the tooth surface, where it contributes to the formation of dental plaque. The bacterium may enter the bloodstream and, in susceptible individuals, cause infective endocarditis (Douglas et al., 1993). However, the use of S. gordonii strains harbouring foreign immunogenic peptides on their surface has been considered as an immunization technique (Pozzi et al., 1992). In the present work, we assessed the physiological implications of PGM in S. gordonii by studying the effects of PGM deficiency on in vitro and in vivo fitness, as well as on cell wall properties.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, used as a host for plasmid clones, was grown at 37 °C, either in Luria–Bertani (LB) broth (Difco) with aeration or on LB agar plates. Growth of the cultures was followed by monitoring OD600 with a Novaspec II spectrophotometer (Pharmacia LKB). When appropriate, erythromycin was added to the media at a final concentration of 5 µg ml–1 for S. gordonii. For E. coli transformants, ampicillin was used at a concentration of 50 µg ml–1. Bacterial stocks were stored at –80 °C in broth supplemented with 10 % (v/v) glycerol.
Construction of a pgm knockout mutant.
The protein sequence deduced from the B. subtilis pgcA gene encoding PGM was used to search for homology against the unfinished S. gordonii Challis genomic sequence available at The Institute for Genomic Research website (http://www.tigr.org). The 1977 bp region comprising the S. gordonii orthologue of B. subtilis pgcA was designated pgm, and it was PCR-amplified and sequenced; no discrepancies were found.
A 1038 bp pgm segment was PCR-amplified using oligonucleotides PGM_5' and PGM_3'_B, and cloned into pBAD-TOPO vector (Invitrogen) to obtain plasmid pBAD-TOPO_PGM. The 29 bp internal part of the cloned pgm segment (residues 584–612 with respect to the putative pgm translational start) was excised by HindIII/SalI and replaced by the 1083 bp erythromycin-resistance cassette, which includes its own promoter. The latter was obtained by the HindIII/SalI digestion of the PCR product generated with oligonucleotides VL602 and Ery_3'_SalI using pJDC9 DNA (Chen & Morrison, 1988). The resulting plasmid pBAD-TOPO_
PGM was digested with MluI and used to transform S. gordonii competent cells. Correct pgm knockout of pgm : : erm, a randomly chosen erythromycin-resistant transformant, was verified by PCR (data not shown).
Transformation.
S. gordonii competent cells were prepared as described by Pozzi et al. (1990). Transformants were selected on Columbia agar (Difco) plates supplemented with 3 % human blood and 5 µg erythromycin ml–1.
Oligonucleotides.
The following oligonucleotides were used in this study:
Ery_3'_SalI, 5'-GTCGACCCTTGGAAGCTGTCAGTAGTATACCTA-3'; PGM_3'_B, 5'-AAAACGTTGAACATGGTTGC-3'; PGM_5', 5'-AAAGGAAGATGCCTTTTATACCAA-3'; VL602, 5'-AAAGCTTAAACGAAATGATACACCAATCAGTGCA-3'; VL837, 5'-TTGAGGTGGCGAACGGCGAATGAAAGTTCA-3'; VL838, 5'-TCGATAGCACGGATGTAAGTTGTCA-3'; VL840, 5'-CACTTCCACACCAACACGGTCAGCA-3'; VL851, 5'-GCCAAACCTTCAGTCGCTTGACGGACAACA-3'; VL852, 5'-CTGGAAGATCAGCGAAATCGA-3'; VL1008, 5'-GCATCAATATCTATGTTGTCCGTCA-3'. Underlined portions of the oligonucleotides represent engineered restriction sites: G/TCGAC, SalI; A/AGCTT, HindIII.
RNA extraction.
Total RNA was extracted by using the RNeasy Protect Bacteria Mini Kit (Qiagen). Briefly, 4 ml RNAprotect Bacteria Reagent (Qiagen) was added to a 2 ml aliquot of S. gordonii culture in BHI broth and vortexed for 5 s. After 5 min incubation at room temperature, the suspension was centrifuged at 3200 g for 10 min. The supernatant was discarded, and the pellet was resuspended in 100 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) containing 15 mg lysozyme ml–1 and incubated at room temperature for 15 min. The manufacturer's protocol was followed from this point.
Mapping of transcriptional start site.
The 5' end of the pgm transcript was mapped with the BD SMART RACE cDNA Amplification Kit (BD Biosciences), according to the manufacturer's protocol. Briefly, gene-specific first-strand cDNA, synthesized from 560 ng total RNA extracted at an OD600 of 0.6 with pgm-specific primers VL838 and VL840, was tailed with the BD SMART II A oligonucleotide. The products were then amplified with a nested gene-specific primer VL837 and the Universal Primer A Mix in a PCR involving 30 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s and extension at 72 °C for 3 min. The PCR product was purified with the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences), and a nested PCR was carried out under the conditions described above using Nested Universal Primer A and oligonucleotide VL851. The nested PCR product was sequenced using primer VL852 (Synergene Biotech).
Northern blot.
Total RNA was extracted from cells at an OD600 of 0.2. After incubation in 20 µl (total volume) of denaturation buffer (1 M glyoxal, 25 % DMSO, 10 mM sodium phosphate, pH 7.0) for 1 h at 50 °C, total RNA (10 µg) was separated by agarose electrophoresis and transferred onto a Hybond-N+ nylon membrane (Amersham Biosciences). RNA was blotted in 10x SSC with the VacuGene XL system (Amersham Biosciences) for 3.5 h under a vacuum of 50 mbar. After transfer, RNA was fixed to the membrane with a UV cross-linker (CX-2000, UVP) at a dose of 0.3 J cm–2. A 367 bp pgm probe labelled with digoxigenin was synthesized with a PCR DIG Labelling Mix (Roche Diagnostics) using oligonucleotide primers VL838 and VL1008. Hybridization and immunological detection were performed according to the supplier's instructions (Roche Diagnostics), using alkaline-phosphatase-conjugated anti-digoxigenin Fab fragments to probe the digoxigenin-labelled DNA, and disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD) as the reagent for chemiluminescent detection. Light emission was detected by using Hyperfilm ECL (Amersham Biosciences).
Cell extract preparation.
Bacteria were grown in 200 ml BHI medium at 37 °C. At different points during growth, cells were recovered by centrifugation at 7000 g and 4 °C. The pellets were rinsed with ice-cold distilled water and resuspended in 5 ml 100 mM triethanolamine, pH 7.4, containing 5 mM MgCl2 and 2.2 mM EDTA. The cells were disrupted by two passes through a One Shot cell disruptor (Constant Systems) at a pressure of 2.5 kbar (2.5 MPa). The cell debris was removed by centrifugation at 15 000 g for 15 min and the supernatant was frozen at –80 °C. The protein concentration was determined with the BCA Protein Assay Reagent (Pierce).
PGM assay.
PGM activity was assayed as previously described (Lazarevic et al., 2005). The reaction mixture contained cell extract (20 µg protein), 0.6 mM 
-NADP, 25 µM glucose 1,6-diphosphate and 1 unit glucose-6-phosphate dehydrogenase (Sigma), in 1 ml (total volume) of 100 mM triethanolamine, pH 7.4, containing 5 mM MgCl2 and 2.2 mM EDTA. The reaction was started by adding 1.5 mM -D-glucose 1-phosphate. Under the assay conditions used, one unit of enzyme activity corresponds to the conversion of 1 nmol substrate min–1 (mg cell protein)–1.
Phase-contrast microscopy.
Samples were fixed by addition of 0.1 vol. 20 % formaldehyde and applied to a slide. Phase-contrast images were captured with a x100 Plan-Neofluar objective on a Axioskop 2 microscope (Carl Zeiss) equipped with MetaVue imaging software (version 6.1r5; Molecular Devices).
Viable cell estimation.
The cell viability was estimated by using the LIVE/DEAD BacLight Bacterial Viability Kit for microscopy and quantitative assays (Molecular Probes). This kit differentially stains live or undamaged cells versus dead or damaged cells by using two nucleic acid stains. The Syto 9 green-fluorescent nucleic acid stain labels all cells whether living or dead, while the red-fluorescent nucleic acid stain propidium iodide enters only the cells with damaged membranes. The applicability of the kit to S. gordonii was verified with cells killed by incubation in 70 % 2-propanol for 1 h. Briefly, cells from samples of between 0.5 and 2 ml were harvested by centrifugation for 10 min at 10 000 g, washed with 1 ml 0.9 % NaCl and resuspended in 200 µl 0.9 % NaCl. Syto 9 (component A) and propidium iodide (component B) were mixed at a 1 : 1 ratio and 0.6 µl aliquots were added to each sample of cells. The suspensions were incubated for 15 min at room temperature in the dark. Bacteria were visualized with a DM IRE2 inverted laser scanning confocal microscope (Leica Microsystems) equipped with an HCX PL APO CS x100 1.4 oil objective and Leica Confocal Software version 2.5. Images were taken using 488 and 543 nm laser excitations and 503–523 nm and 595–631 nm band-pass filters, for the Syto 9 and propidium iodide stains, respectively. The cells were counted in at least eight random fields and the proportion of viable or undamaged (green-stained) cells was calculated as a percentage of all the cells.
Cell wall preparation.
Cell walls were purified from bacteria grown to an OD600 of 0.3 in BHI, as described by de Jonge et al. (1992).
Estimation of cell wall phosphate.
Lyophilized cell walls were mineralized according to Ames (1966). A 50 µl sample of a 1 mg ml–1 cell wall suspension was mixed with 50 µl 10 % Mg(NO3)2 (w/v in 96 % ethanol) in a borosilicate glass test tube and dried at 95 °C for 3–4 h. Samples were heated in a Bunsen flame for 2 min. After cooling, 300 µl 1 M HCl was added and tubes were heated for 15 min in a water bath at 100 °C. The phosphate concentration was determined according to Chen et al. (1956) against a standard curve generated using known concentrations of K2HPO4.
Muropeptide analysis.
WTAs were removed from the cell walls by gently mixing the cell wall suspension in hydrofluoric acid (49 %) for 48 h at 4 °C. The peptidoglycan was recovered, digested with mutanolysin (Sigma), and reduced and separated by HPLC, as described by de Jonge et al. (1992). The HPLC system (Hitachi) consisted of an L-7200 autosampler, an L-7100 gradient pump with low-pressure mixing and an L-7400 UV detector. Column temperature was maintained at 34 °C using the L-7360 column oven. Separation was performed by injection of a 100 ml sample, containing 100 µg of wall digest, onto a 250x4.6 mm reverse-phase column (ODS-Hypersil 3 µm; Keystone Scientific). The material was eluted at 0.5 ml min–1 using a linear gradient of 5 % (v/v) methanol in 100 mM sodium phosphate (pH 2.5) to 30 % (v/v) methanol in 100 mM sodium phosphate (pH 2.8) over 160 min. Eluted material was detected by its absorbance at 210 nm. The results were analysed using the D-7000 HPLC System Manager program (Hitachi). Principal peaks were collected and desalted using C18 ZipTips (Millipore), according to the manufacturer's protocol. The mass of desalted peaks was determined by reflector positive-ion mode MALDI-TOF MS (Autoflex; Bruker Daltonics) using a 5-chloro-2-mercaptobenzothiazole matrix in water, tetrahydrafuran and ethanol (1 : 1 : 1). Pure peaks were sequenced by post-source decay (PSD) analysis (Xu et al., 1997).
Autolysis of whole cells.
Cells were grown in BHI medium at 37 °C. At an OD600 of 0.2, cells from a 3.5 ml sample were recovered by centrifugation for 3 min at 10 000 g, washed with PBS buffer (0.68 % NaCl, 0.148 % Na2HPO4, 0.043 % KH2PO4, pH 7.2) and suspended in 1 ml 30 mM potassium phosphate (pH 7.0). The suspension was incubated at 37 °C and the OD600 was monitored. Results were expressed as percentages of the initial OD600.
Experimental endocarditis.
Sterile aortic vegetations were produced in rats, as described by Heraief et al. (1982). Groups of animals were inoculated by intravenous challenge with 106 c.f.u. exponential-phase streptococci. For the parent strain, this inoculum size produced endocarditis in >90 % of the rats, and thus it permitted a clear differentiation between virulent and putatively less virulent strains (Entenza et al., 1997; Stutzmann Meier et al., 2001). Rats were sacrificed 16 h after bacterial inoculation and viable colony counts in the vegetations and spleens were determined. The frequency of infection was compared by using Fisher's exact test. The median bacterial titres were compared by using a non-parametric Mann–Whitney unpaired test; P<0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and 48.8 % identity to the PGM of B. subtilis (Lazarevic et al., 2005). Analysis of the nucleotide sequence revealed that the 30 nt segment located 8 nt downstream of the S. gordonii pgm stop codon could form a stem–loop structure corresponding to a transcriptional terminator. The presence of a terminator-like sequence 193 nt upstream of the pgm start codon, followed by a functional promoter (Fig. 1a; see below), suggests that pgm forms a one-gene operon. The Northern hybridization experiments yielded a major product of the expected length of about 1.8 kb (Fig. 1b), confirming a monocistronic transcription of pgm. The minor band of about 1.5 kb was most probably an artefact caused by the partially degraded mRNA and the abundance of 16S rRNA.
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). The transcriptional start located 75 nt upstream of the putative pgm start codon was preceded at a distance of 6 nt by the sequence ATATGCTACAAT (Fig. 1a
). This sequence exhibited a strong (10/11) adherence to the so-called extended –10 consensus sequence (RTRTGNTATAAT) of B. subtilis and S. pneumoniae A-type promoters (Helmann, 1995; Sabelnikov et al., 1995). Only a poorly matching (3/6) –35 region was found at the usual distance of 11 nt upstream of the extended –10. Therefore, it seems likely that, as in B. subtilis (Helmann, 1995) and S. pneumoniae (Sabelnikov et al., 1995), the extended –10 site may function without a recognizable –35 region. Moreover, in S. gordonii, transcription initiation from such promoters seems to be relatively frequent (M. Haenni and V. Lazarevic, unpublished).
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), no detectable PGM activity was found in the pgm : : erm mutant, strongly suggesting that pgm encodes a unique PGM in S. gordonii. In the wild-type strain, after growth from an OD600 of 0.03 to a final reading of 1.39, PGM activity increased by a factor of 2.3.
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Microscopic examination of the pgm : : erm mutant grown in BHI liquid medium revealed an enlarged diameter compared with that of wild-type cells (Fig. 4). The cell size varied within the same chain. This aberrant cell morphology, which is in accordance with the observations recently made in S. iniae (Buchanan et al., 2005), was not reversible by addition of Mg2+, as shown for B. subtilis PGM-deficient mutants (Lazarevic et al., 2005).
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pgm : : erm mutant was investigated using the LIVE/DEAD BacLight Bacterial Viability Kit, which includes stains to determine whether cells have an intact or damaged membrane. In the exponential phase, at an OD600 of 0.3, 99 and 93 % of the wild-type and pgm-deficient cells, respectively, were stained as viable (Table 1). In contrast, a striking difference in viability was observed after 24 h batch cultivation. Wild-type cells showed only a 2 % decline in the viable count, whereas the pgm-deficient mutant exhibited >30 % loss of viability under the same conditions.
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. Whereas the parent strain infected 100 % of both vegetations and spleens, the pgm deletion mutant infected only 54 % of these tissues (P<0.05). Moreover, the rats inoculated with the parent strain had significantly higher vegetation and spleen bacterial densities than those inoculated with the pgm : : erm mutant (P<0.05). Thus, in agreement with previous findings demonstrating that mutations in PGM had a significant effect on the virulence of S. pneumoniae and S. iniae in different animal models (Buchanan et al., 2005; Hardy et al., 2001), our results showed that the S. gordonii pgm : : erm mutant was also less virulent in experimental endocarditis.
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). The increased autolysis might be due to an altered cell wall structure that allows hydrolytic enzymes increased access to the peptidoglycan.
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). The composition of the components present in individual peaks was determined (Table 3). Identical structures were present in both the parent and the mutant, but the relative amounts varied between strains (Fig. 6, Table 4). These differences were quantified by calculating the relative amounts of the different peaks labelled in Fig. 6. Peaks 1, 2, 5b and 7 were significantly increased in the pgm : : erm strain compared with the wild-type, whereas peaks 3, 4, 5a and 6 were significantly decreased. No significant difference was observed in the amounts of peak 5c found in either strain (Table 4). Peaks 1, 2, 3, 4, 6 and 7 were found to be pure by MALDI-TOF MS and were identified by PSD analysis (Table 3). Peaks 5a, 5b and 5c contained several components and were not further analysed in this study. The fact that peak 2 was more abundant in the mutant than in the parent, and that it was a simple monomer, suggests that the wall of the mutant might be simpler than that of the parent.
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pgm : : erm and the wild-type strains. The phosphate content of the wild-type (Table 1) was very close to 0.23 µmol phosphate (mg cell wall) –1, a value that has been reported for S. gordonii NCTC 7865 (Rose et al., 1997). Phosphate measurements revealed about 30 % reduction in the cell wall of the PGM-deficient mutant (Table 1). This decreased value is to be expected for walls impoverished in sugar phosphate polymers. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Other studies have shown that at least part of the virulence attenuation observed in PGM-deficient S. iniae and S. pneumoniae is likely to be due to a diminished capsule expression (Buchanan et al., 2005; Hardy et al., 2001). Interestingly, S. gordonii appears to be an unencapsulated bacterium. Therefore, the reduced in vivo and in vitro fitness in the PGM-deficient mutant may be caused by changes in other cell envelope components.
The cell wall of most Gram-positive bacteria is composed of peptidoglycan, which is a structurally defining cell wall polymer that contains covalently attached anionic polymers, such as WTA and teichuronic acid. The repeating units of most WTAs contain substituted polyol phosphate. WTAs contribute to the cell wall electrolyte properties, modify the activity of peptidoglycan hydrolases and maintain cation homeostasis (Neuhaus & Baddiley, 2003). In the rod-shaped bacterium B. subtilis, as well as in the spherical Streptococcus oralis, WTA deficiency correlates with abnormal shape and cell growth (Horne & Tomasz, 1993; Soldo et al., 2002). Since most species of viridans streptococci do not contain polyol-WTA (Rose et al., 1997), their sugar-phosphate polymers probably perform some of the roles carried out by WTA. Moreover, Hancock & Baddiley (1985) proposed that these so-called sugar 1-phosphate polymers should be recognized as WTAs. Although the structure of S. gordonii Challis sugar-phosphate polymer has not been determined, it may be similar to those found in the viridans group of streptococcal strains whose repeated unit is a glucose- and phosphate-containing hexasaccharide or heptasaccharide (Cisar et al., 1997). Therefore, the PGM deficiency that prevents the synthesis of UDP-glucose, which is an important glucosyl donor for the synthesis of a variety of cell wall polymers, may lead to the abnormality in sugar-phosphate polymer metabolism and, as a consequence, altered cell morphology.
Since PGM does not appear to be involved in the muropeptide biosynthetic pathway, PGM deficiency does not provide a straightforward explanation for changes in the muropeptide pattern of the pgm : : erm strain. Moreover, we are not aware of any other study linking PGM deficiency to a change in the muropeptide pattern in other bacterial species. One possibility is that changes in WTA metabolism may influence the activity of peptidoglycan-hydrolytic or -biosynthetic enzymes, resulting in an altered muropeptide composition and an increased autolysis rate.
It has been hypothesized that the altered cell morphology in a PGM-deficient B. subtilis strain might be due to deficiencies in glucose 1-phosphate metabolites, monoglucosyldiacylglycerol and/or diglucosyldiacylglycerol (Lazarevic et al., 2005). In B. subtilis, synthesis of these glycolipids requires YpfP, a processive enzyme that catalyses successive addition of glucosyl residues from UDP-glucose to diacylglycerol (Jorasch et al., 1998). Interestingly, sequence similarity searches failed to reveal orthologues of B. subtilis ypfP in S. gordonii and other streptococci. Therefore, the synthesis of membrane glycolipids in streptococci seems to involve a family of enzymes that are evolutionarily distinct from those operating in bacilli and staphylococci. Nevertheless, a deficiency in membrane glycolipids may be due to an inability to synthesize the putative glycosyl donor UDP-glucose.
S. gordonii is a commensal oral bacterium that has been considered as a potential live vaccine vector in humans (Pozzi et al., 1992; Kotloff et al., 2005). It may be theoretically advantageous to use a pgm-deficient S. gordonii as a vector in cases where only transient antigen exposure is required. An attenuated strain would be naturally outcompeted by the resident microflora, preventing long-term antigen stimulation. The use of such a strain may also reduce the risk of infective endocarditis after accidental entry of the bacterium into the bloodstream. Whether or not the diminished virulence properties conferred by pgm deficiency may offer an advantage for the use of S. gordonii as a potential vaccine vector in humans warrants further investigation.
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ACKNOWLEDGEMENTS |
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Edited by: J. C. Paton
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 27 June 2006;
revised 16 October 2006;
accepted 25 October 2006.
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) and pgm-deficient strains (
) was monitored. (b) PGM activity [nmol min–1 (mg cell protein)–1] and error bars (SD) are indicated for the wild-type strain only, since activity was not detected in the 
