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Department of Molecular Genetics, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, the Netherlands
Correspondence
Oscar P. Kuipers
o.p.kuipers{at}rug.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.
A description of the chemically defined medium together with sequence data for plasmids pTK3 and pTK5 is available as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Current vaccines are directed against the polysaccharide capsule, but they do not cover the entire spectrum of different serotypes, and may offer only limited protection in children younger than 2 years (Bogaert et al., 2004). Furthermore, multi-antibiotic-resistant strains of S. pneumoniae have emerged in recent years, and are now a serious problem (Hancock, 2005). To be able to combat infections caused by S. pneumoniae in the future, new vaccines and antimicrobials are required. Therefore, a major effort is currently being made to dissect the molecular mechanisms underlying infection by S. pneumoniae.
Various molecular tools have been described to study the molecular biology of S. pneumoniae, such as inducible promoters, lacZ and green fluorescent protein (GFP) reporter vectors, and methods to construct mutant strains (Acebo et al., 2000; Chan et al., 2003; Claverys et al., 1995; Eichenbaum et al., 1998; Lee et al., 1998; Sung et al., 2001).
Here, we report the development and use of several additional molecular tools for S. pneumoniae, which were adapted from existing tools for the related bacterium Lactococcus lactis. These include an improved nisin-controlled gene expression system (NICE), a chemically defined medium (CDM), a plasmid-based lacZ reporter system, and a method to generate unmarked mutations in the chromosome. These tools extend the existing toolkit for the basic genetic manipulation of S. pneumoniae, and they are applicable in the sequenced strains R6 (D39) and TIGR4, and, most likely, in other strains as well.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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and were stored in 10 % (v/v) glycerol at –80 °C. S. pneumoniae was routinely grown as standing cultures in M17 broth (Terzaghi & Sandine, 1975) containing 0·25 % (w/v) glucose (GM17) or in Todd–Hewitt broth supplemented with 0·5 % yeast extract (THY) at 37 °C. For growth on plates, 1–5 % (v/v) defibrinated sheep blood (Johnny Rottier, Kloosterzande, The Netherlands) was added to GM17 agar. L. lactis was grown as standing cultures in M17 supplemented with 0·5 % (w/v) glucose at 30 °C. Escherichia coli EC1000 was grown in TY broth in a shaking incubator at 37 °C. Chloramphenicol and erythromycin were used at concentrations of 2·5 and 0·25 µg ml–1 for S. pneumoniae, and 5 and 4 µg ml–1 for L. lactis, respectively. Erythromycin was used at a concentration of 120 µg ml–1 for E. coli. Trimethoprim was used at a concentration of 15 µg ml–1 for S. pneumoniae. When appropriate, 0·006 % (w/v) X-Gal was used in plates. A nisin stock was obtained by extracting nisaplin (Aplin and Barrett, Danisco, Denmark) 1 : 1 (w/v) with 50 % ethanol, spinning the suspension for 1 min at 20 000 g in a table-top centrifuge and discarding the pellet. The supernatant contained approximately 20 µg nisin ml–1 and was stored at –20 °C. For induction of the nisA promoter, nisin was used at the concentrations indicated in the Results. Working stock solutions with the appropriate nisin concentration were freshly prepared in 50 % ethanol before each experiment.
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. Plasmid DNA was isolated using the plasmid isolation kit from Roche. DNA manipulation was done according to standard procedures. L. lactis and E. coli were transformed with plasmid DNA by electroporation (Holo & Nes, 1995). S. pneumoniae was transformed by growing the strain at 37 °C in GM17 to an OD600 of 0·1. The competence-stimulating peptide CSP-1 was used at 100 ng ml–1 to induce competence (Pozzi et al., 1996). Transformants were selected on GM17 agar with the appropriate antibiotic. Southern blotting was done as described by Chomczynski & Qasba (1984). Probe labelling, hybridization and detection were performed using the ECL direct nucleic acid labelling system according to the specifications of the manufacturer (Amersham Pharmacia Biotech).
Plasmid and strain construction.
Primers used in this study are listed in Table 2. The nisRK genes were introduced into the bgaA gene (spr0565) on the chromosome of S. pneumoniae as follows. Primers trpm-Fp and trmp-Rp were used to PCR-amplify the trmpR marker from pKOT. Using E. coli EC1000 as host, the PCR fragment was inserted into the HindIII site of pORI28 in the same orientation as the emR gene, yielding pTK1. The 5' and 3' ends of bgaA gene were amplified from D39 chromosomal DNA with primer pairs bgaA-1/bgaA-2 and bgaA-3/bgaA-4, and were cloned into the MluI/BamHI and KpnI/BglII sites of pTK1, respectively, resulting in pTK2. Finally, the nisRK genes, which were amplified from chromosomal DNA of L. lactis NZ9700 with primers NisRK-Fp-term/NisRK-Rp, were cloned into the PstI/BamHI sites of pTK2, resulting in pTK3. Both pTK2 and pTK3 were generated in L. lactis LL108.
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S. pneumoniae D39nisRK, harbouring the nisRK genes integrated via double cross-over in the bgaA locus, was constructed by transformation with a PCR product obtained with pTK3 as template using primers bgaA-1/bgaA-4 and selecting for trimethoprim-resistant clones. By analogy, a gene cassette was obtained from pTK5 by PCR with primers R6_bgaA-6/R6_bgaA-8, which was used to construct S. pneumoniae D39repA. The correctness of the mutations was verified by PCR and Southern blotting. In the same way, strains TIGR4nisRK, TIGR4repA, R6nisRK and R6repA were constructed.
Plasmids pTK6 and pJB1 containing glnA (spr0444) and spr0576 under the control of the nisin-inducible nisA promoter were constructed by amplifying the respective genes from chromosomal DNA of strain D39 using primer pairs glnA_R6-7/glnA_R6-8 and 659for/659rev, and cloning the resulting amplicons into the NcoI/XbaI sites of pNG8048E. The spr0576 gene was fused to a C-terminal Myc-tag. L. lactis NZ9000 was used as the cloning host.
To construct a transcriptional fusion of lacZ to the promoter of cpsA, the fragment was PCR-amplified from chromosomal DNA of S. pneumoniae D39 with primer pair cpsArev+50/cpsAfor-400 and cloned into the XmaI/PstI sites of pORI13, yielding pJB2. L. lactis LL108 was used as the cloning host.
pTK7, containing the deleted glnR gene (spr0443) surrounded by fragments of approximately 800 bp, was constructed by cloning the PCR products amplified from D39 chromosomal DNA with primer pairs glnR_R6-1/glnR_R6-2 and glnR-R6-3-ncoi/glnR_R6-4 in the XbaI/BamHI and NcoI/BglII sites of pORI280, using L. lactis LL108 as host.
Enzyme assays.
Cell-free extracts were made by resuspending cell pellets from 2 ml of culture in 20 mM Tris/HCl (pH 7·5) and disruption of cells by shaking them for 1 min with glass beads (75–150 µm) in a Biospec Mini-BeadBeater-8. Glutamine synthetase activity (transferase reaction) was determined as described by Fisher & Sonenshein (1984). -Galactosidase activity was determined in cell suspensions permeabilized by chloroform and SDS, as described by Israelsen et al. (1995).
Detection of the Spr576–Myc fusion protein by Western blotting.
Cells from 1 ml of cell culture grown in GM17 were harvested in 1 ml PBS and washed twice. Whole-cell samples were diluted in sample buffer (4 % SDS, 2 % 2-mercaptoethanol, 20 %, v/v, glycerol, 125 mM Tris/HCl, pH 6·8, 0·1 mg bromophenol blue ml–1), heated for 5 min at 100 °C, fractionated by 12·5 % SDS-PAGE and transferred to a nitrocellulose membrane. Spr576–Myc was detected with ECL (Amersham Biosciences) on immunoblots after incubation with a 1 : 5000 dilution of an anti-Myc monoclonal antibody (Gentaur, Brussels, Belgium), followed by a 1 : 10 000 dilution of anti-mouse IgG peroxidase (Amersham Biosciences).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Kleerebezem et al., 1997; Mierau & Kleerebezem, 2005). The core of NICE is formed by the NisRK two-component system, which in the presence of nisin activates expression of the nisA and nisP promoters in a dose-dependent manner (de Ruyter et al., 1996a; Kuipers et al., 1995). Nisin is a peptide bacteriocin belonging to the class of lantibiotics, and is produced by certain strains of L. lactis. An advantage of induction of expression with nisin is that it is metabolically inert at subinhibitory concentrations, unlike sugars and other metabolically active compounds that have been used in S. pneumoniae (Acebo et al., 2000; Chan et al., 2003).
Two studies describe the application of NICE in S. pneumoniae (Eichenbaum et al., 1998; Luo et al., 2004). In the first study, two plasmids were used, one containing the nisin-inducible promoter, the other the nisRK genes. In this situation, transcription from the nisin-inducible promoter also occurred in the absence of nisin. Furthermore, nisin concentrations just below the growth inhibitory level resulted in only a tenfold overproduction (Eichenbaum et al., 1998). The second study used a single plasmid that contains both the nisin-inducible promoter and the nisRK genes, but in this case, nisin-induced expression was observed only at 30 °C (Luo et al., 2004).
To circumvent these problems, we have constructed a stable derivative of S. pneumoniae D39 (D39nisRK) that harbours nisRK with their native promoter in the bgaA locus (Fig. 1a), thereby abolishing endogenous -galactosidase activity (Fig. 3b).
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). Addition of nisin to the medium resulted in expression of glnA, which was dose-dependent (Fig. 1b). At nisin concentrations of 2–20 ng ml–1, which are far below the MIC value of 500 ng ml–1 (Kramer, 2005), a 20–50-fold higher glutamine synthetase activity was observed compared to the wild-type (Fig. 1b). Nisin-induced overexpression of spr0576 resulted in the appearance of two bands on the Western blot (Fig. 1c). The lower band probably represents the protein with the putative signal sequence cut off, and the upper band the unprocessed protein. The appearance of these two bands indicates that nisin induction leads to the production of large quantities of the Myc-tagged Spr0576. Furthermore, we also showed that nisin-induced expression occurs in both the early and the late exponential phase of growth (Fig. 1c). The nisRK integration cassette could be easily transferred to other strains via double cross-over and selection for resistance to the rarely used antiobiotic trimethoprim. In addition, nisin-dependent expression of GlnA and Spr0576 was equally functional in TIGR4 and R6 derivatives, which carry nisRK integrated in the bgaA locus (data not shown).
Although THY and brain heart infusion (BHI) are often used as growth media for S. pneumoniae, we carried out all experiments in GM17 medium, which is a commonly used medium for L. lactis. The growth rate and maximal OD595 of strains D39, R6 and TIGR4 were higher in GM17 than in THY or BHI (data not shown). Also, nisin-induced expression of glnA in THY seemed to be lower than that in GM17. As the stability and solubility of nisin are strongly diminished above pH 7 (Rollema et al., 1995), we speculated that this difference was due to the higher pH of THY, which is 7·8 versus 6·9 for GM17. To test this hypothesis, nisin induction was performed in GM17 medium at various pH values. In GM17 with a pH higher than 7·5, the level of induction was strongly decreased, while at lower pH values, the induction level was comparable to that obtained in standard GM17. As expected, lowering the pH of THY to 7·5 or below led to enhanced induction levels (data not shown). Thus, adjustment of the pH of the growth medium could be a good way to optimize NICE in S. pneumoniae.
Complementation of a glnA deletion mutant with NICE in chemically defined medium
Previously, complementation in S. pneumoniae has been accomplished with the fucose-inducible promoter (Ng et al., 2004). To validate the improved NICE system, we tested whether glnA, expressed under the control of the nisin-inducible nisA promoter in S. pneumoniae D39nisRK, could complement a chromosomal deletion in glnA. In order to do so, we first needed a CDM to ensure that the glnA mutant was auxotrophic for glutamine. Although chemically defined media for S. pneumoniae have been described (Adams & Roe, 1945; van de Rijn & Kessler, 1980; Willett & Morse, 1966), they are not commonly used, which could be due to their complexity. We adapted a CDM for L. lactis (Poolman & Konings, 1988) by supplementing it with pyruvate, adenine, uracil, choline chloride, aspartate and cysteine (supplementary data). This CDM is easy to prepare and supported growth of three S. pneumoniae strains tested, namely R6, D39 and TIGR4, to a level comparable to that in complex media, such as THY and GM17 (data not shown).
The glnA mutant grew well in CDM containing glutamine, but, unlike the wild-type, did not grow in CDM with glutamate instead of glutamine (Fig. 2a). In trans expression of glnA from the nisin-inducible promoter restored growth in CDM with glutamate instead of glutamine to the level of that in CDM containing glutamine (Fig. 2b), demonstrating the suitability of the NICE system we describe here for complementation experiments in S. pneumoniae.
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-galactosidase activity, the strain can be employed for promoter-expression studies using the E. coli lacZ gene as a reporter, either in combination or not in combination with nisin-controlled expression of the protein of interest.
Plasmid-based lacZ expression system for studying promoter function in S. pneumoniae
A plasmid-based GFP reporter system has been applied in S. pneumoniae (Bartilson et al., 2001; Marra et al., 2002), but the lacZ reporter systems described to date are based on integrative plasmids (Claverys et al., 1995; Pestova & Morrison, 1998). The efficiency of integration into the chromosome via insertion-duplication, however, strongly depends on the length of the targeting fragment, which could give problems when a small insert is required (Lee et al., 1998). In addition, an integration plasmid is less suitable for the detailed dissection of promoter function, for example by point mutations or deletions.
We developed a plasmid-based lacZ reporter system using the lactococcal pWV01 replicon (Leenhouts et al., 1991). This replicon has been exploited to construct various molecular tools in L. lactis, and it functions in both Gram-positive and Gram-negative bacteria (Kok et al., 1984). Replication of pORI vectors, which are derivatives of pWV01, is dependent on an in trans copy of the pWV01 replication initiation gene repA (Leenhouts et al., 1991).
To maintain the pORI vectors in S. pneumoniae, strain D39repA was constructed, which carries the repA gene, driven by a constitutive promoter, in the bgaA locus (Fig. 3a). Like strain D39nisRK, D39repA does not display endogenous -galactosidase activity (Fig. 3b). Subsequently, we tested whether the RepA-dependent plasmid pORI13 (Sanders et al., 1998), which contains a promoterless E. coli lacZ for transcriptional fusions preceded by a lactococcal ribosome-binding site, a multiple cloning site and stop codons in all three reading frames, could be employed as an integrative and replicative lacZ reporter vector in S. pneumoniae strains D39nisRK and D39repA, respectively. To this end, the promoter of cpsA, the first gene of the locus, which encodes enzymes necessary for capsule production, was cloned upstream of the lacZ of pORI13. The resulting construct was either integrated into the chromosome of strain D39nisRK or introduced into strain D39repA. The plasmid could be isolated from D39repA but not from D39nisRK (data not shown). Moreover, D39repA could be transformed easily with empty pORI13, while wild-type D39 could not, showing that pORI13 is maintained as an autonomous plasmid in D39repA. The cpsA promoter is active in GM17 medium, and lacZ expression is approximately threefold higher in strain D39repA than in strain D39nisRK (Fig. 3b), indicating that pORI13 is present as a low-copy-number plasmid in the former, giving -galactosidase activity close to that of a single-copy situation in the chromosome. Likewise, TIGR4 and R6 derivatives harbouring repA in the bgaA locus were able to replicate pORI13 (data not shown).
Thus, this system enables the use of the lacZ expression plasmid pORI13 both for integration and as a low-copy-number plasmid, allowing detailed investigation of promoter function. Another advantage of having a RepA+ derivative of S. pneumoniae is that there is a range of different pORI vectors, with various antibiotic-resistance genes and extensive multiple-cloning sites (Leenhouts et al., 1996, 1998b), which can be maintained as replicative plasmids in D39repA as well (data not shown). In addition, pORI13 can be combined with the NICE system described above, since plasmid pNG8048E, containing the nisin-inducible promoter, provides the pWV01 repA gene required for the replication of pORI13 (data not shown).
Construction of unmarked chromosomal mutations with pORI280 in S. pneumoniae
Generally, mutant S. pneumoniae strains are constructed by substitution of a target gene with an antibiotic-resistance gene via a double cross-over event. This method has several disadvantages. First, it could cause polar effects on the expression of downstream genes, especially when the target gene is one of the first genes in an operon. Second, the introduction of point mutations or small in-frame deletions in a wild-type gene on the chromosome is not possible. Third, construction of strains containing multiple mutations could be hampered by the lack of sufficient antibiotic markers. The introduction of unmarked mutations into the chromosome would circumvent these drawbacks.
Previously, rpsL has successfully been applied as a counter-selectable marker in S. pneumoniae to construct unmarked mutations (Sung et al., 2001). However, this system requires the presence of a mutation in the wild-type chromosomal copy of rpsL. Furthermore, the mutated rpsL allele spontaneously converts with low frequency to wild-type, giving rise to false positives (Sung et al., 2001). Unmarked mutants have also been constructed in S. pneumoniae by overlap-extension PCR mutagenesis (Standish et al., 2005) and by direct transformation with a PCR fragment containing the desired mutation (Iannelli & Pozzi, 2004). With these methods, selection for mutant clones is done by checking for antibiotic sensitivity and by colony PCR.
We investigated whether the lactococcal plasmid pORI280, which contains a constitutively expressed lacZ and an erythromycin-resistance gene, can be used for the introduction of unmarked mutations in S. pneumoniae. pORI280 was originally developed to generate unmarked mutations in the chromosome of L. lactis independent of the genotype of the host, and it allows the screening of possible mutant clones on plates by visual blue/white selection (Leenhouts et al., 1996). We used gene spr0443 (glnR) to test the system in S. pneumoniae. To this end, chromosomal fragments of 800 bp surrounding glnR were inserted into pORI280, and the construct (pTK7) was used to transform S. pneumoniae D39 with selection for erythromycin resistance (see Fig. 4 for a schematic representation of the procedure). As replication of pORI280 is dependent on RepA, the transformation leads to single cross-over integration of the construct into the chromosome. After growing several erythromycin-resistant, LacZ-positive integrants as separate cultures for 30–50 generations (culturing two to four times until stationary phase) without antibiotic selection, cells were plated on X-Gal medium to screen for clones that had lost the plasmid as the result of a second recombination event. Of the colonies, 0·5 % were both white and erythromycin sensitive, indicating excision of the plasmid from the chromosome. Of these white erythromycin-sensitive colonies, 80 % contained the desired mutation, as verified by PCR, Southern blotting and nucleotide sequencing (data not shown). In the same way, we successfully introduced two point mutations in the chromosomal copy of glnR of S. pneumoniae D39. Both mutant strains will be described in detail elsewhere. An advantage of the use of pORI280 compared to the methods mentioned above is that possible mutants can be easily selected on the basis of their white colour on plates with X-Gal. Thus, this system provides an efficient way to obtain unlabelled mutants in S. pneumoniae.
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ACKNOWLEDGEMENTS |
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Received 14 September 2005;
revised 8 November 2005;
accepted 8 November 2005.
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  I. R. Monk, C. G. M. Gahan, and C. Hill Tools for Functional Postgenomic Analysis of Listeria monocytogenes Appl. Envir. Microbiol., July 1, 2008; 74(13): 3921 - 3934. [Abstract] [Full Text] [PDF]
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 P. Burghout, H. J. Bootsma, T. G. Kloosterman, J. J. E. Bijlsma, C. E. de Jongh, O. P. Kuipers, and P. W. M. Hermans Search for Genes Essential for Pneumococcal Transformation: the RadA DNA Repair Protein Plays a Role in Genomic Recombination of Donor DNA J. Bacteriol., September 15, 2007; 189(18): 6540 - 6550. [Abstract] [Full Text] [PDF]
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U. Wegmann, M. O'Connell-Motherway, A. Zomer, G. Buist, C. Shearman, C. Canchaya, M. Ventura, A. Goesmann, M. J. Gasson, O. P. Kuipers, et al. Complete Genome Sequence of the Prototype Lactic Acid Bacterium Lactococcus lactis subsp. cremoris MG1363 J. Bacteriol., April 15, 2007; 189(8): 3256 - 3270. [Abstract] [Full Text] [PDF]
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J. J. E. Bijlsma, P. Burghout, T. G. Kloosterman, H. J. Bootsma, A. de Jong, P. W. M. Hermans, and O. P. Kuipers Development of Genomic Array Footprinting for Identification of Conditionally Essential Genes in Streptococcus pneumoniae Appl. Envir. Microbiol., March 1, 2007; 73(5): 1514 - 1524. [Abstract] [Full Text] [PDF]
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 T. G. Kloosterman, W. T. Hendriksen, J. J. E. Bijlsma, H. J. Bootsma, S. A. F. T. van Hijum, J. Kok, P. W. M. Hermans, and O. P. Kuipers Regulation of Glutamine and Glutamate Metabolism by GlnR and GlnA in Streptococcus pneumoniae J. Biol. Chem., September 1, 2006; 281(35): 25097 - 25109. [Abstract] [Full Text] [PDF]
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 A. Finn and H. F. Jenkinson The pneumococcus: 'old man's friend' and children's foe Microbiology, February 1, 2006; 152(2): 281 - 283. [Full Text] [PDF]
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