HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Guiral, S. Articles by Claverys, J.-P. Search for Related Content PubMed PubMed Citation Articles by Guiral, S. Articles by Claverys, J.-P. Agricola Articles by Guiral, S. Articles by Claverys, J.-P.

Construction and evaluation of a chromosomal expression platform (CEP) for ectopic, maltose-driven gene expression in Streptococcus pneumoniae

Maria-Halima Laaberki

Laboratoire de Microbiologie et Génétique Moléculaires, UMR 5100 CNRS-Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9, France

Correspondence
Jean-Pierre Claverys
claverys{at}ibcg.biotoul.fr

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
In this paper, the construction and evaluation of a chromosomal expression platform (CEP), which allows controlled gene expression following ectopic integration into the chromosome of Streptococcus pneumoniae, is described. CEP is based on the well-studied maltosaccharide-inducible system. To facilitate integration at CEP, a plasmid, pCEP, capable of replication in Escherichia coli, but not in S. pneumoniae, was assembled. This plasmid contains an expression/selection cassette flanked on each side by more than 2 kb of pneumococcal DNA. The cassette comprises a maltose-inducible promoter, P_M, separated from a kanamycin-resistance gene by NcoI and BamHI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of P_M can be obtained through direct transformation of an S. pneumoniae recipient with ligation products between that gene and NcoI/BamHI-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.

Present address: UMR1225 Interactions Hôtes-Agents Pathogènes, INRA-Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles BP 87614, 31076 Toulouse Cedex 3, France.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Streptococcus pneumoniae is widely used as a model Gram-positive pathogenic bacterium with low GC content, particularly since natural genetic transformation constitutes a convenient tool for molecular genetic studies of virulence. The involvement of many S. pneumoniae genes in virulence has been investigated through gene inactivation. An important step to validate inactivation data involves complementation of the inactivated function. While the amyE ectopic chromosomal platform is in routine use for complementation studies in Bacillus subtilis (Kong et al., 1993), no equivalent system has been developed so far for S. pneumoniae. Complementation has therefore been achieved in several different ways. The complementing gene can be cloned into a replicative plasmid and introduced by transformation (Balganesh & Lacks, 1985; Prats et al., 1985). However, multicopy complementation can lead to perturbations of cell physiology for various reasons, including the presence of the replicative plasmid itself. Thus, a recombinant plasmid carrying the nisRK two-component regulatory system and the gene of interest cloned under the control of the nisin-inducible nisA promoter has been reported to confer sensitivity to temperature (Luo et al., 2003).

To avoid copy-number effects, alternative strategies based on single-copy gene expression have therefore been developed over the years. One of these makes use of the ectopic integration of a recombinant plasmid (non-replicative in S. pneumoniae) in the chromosome through transformation (Vasseghi et al., 1981; Mannarelli & Lacks, 1984). A chromosome-targeting fragment is inserted into the recombinant plasmid to obtain ectopic integration. A 354 bp fragment from the ami operon of S. pneumoniae has thus been used to direct integration of a recombinant plasmid harbouring the firefly luciferase gene, luc, and luciferase activity is observed in pneumococcal cells (Stieger et al., 1999). Similarly, a qsrA fragment has been inserted into a new non-replicative plasmid, pEVP3qsrI, to obtain ectopic expression of the S. pneumoniae cbpD gene at the qsrA locus (Kausmally et al., 2005). In this example, the recombinant plasmid carries two regions of homology with the recipient chromosome, qsrA (886 bp) and cbpD (1377 bp), and can therefore integrate preferentially at cbpD. Integration at qsrA is obtained through the use of a recipient deleted for cbpD. It can thus be necessary for efficient ectopic integration to delete from the recipient chromosome the counterpart of the complementing gene. In any case, duplication of the chromosome-targeting fragment occurs during integration of the recombinant plasmid and creates the potential drawbacks that the plasmid is either amplified (hence is no longer in single copy) or is excised and lost (Vasseghi & Claverys, 1983).

A better system should thus allow stable single-copy integration at a neutral chromosomal site. Integration of an ectopic gene copy at the bgaA locus might fulfil this criterion (Robertson et al., 2002, 2003; Chan et al., 2003). However, although it has been reported that insertion mutations of various kinds into bgaA do not affect growth, the function(s) of BgaA is not fully understood. Taking into account the overall length of the gene (6·7 kb) and the surface location of the protein, it is not unlikely that BgaA has other function(s) in addition to the previously documented -galactosidase activity (Zähner & Hakenbeck, 2000). It is therefore difficult to consider bgaA as a neutral chromosomal site. Ectopic expression at the malM locus, which encodes amylomaltase (Stassi et al., 1982), has been used to demonstrate that the 5' to 3' exonuclease activity of DNA polymerase I is essential for S. pneumoniae (Díaz et al., 1992), but this system is not designed to allow easy further use. Ectopic expression of a gene inserted between aga, the gene encoding -galactosidase, and rafE, under the control of a raffinose-inducible promoter, has also been used (Luo et al., 2003). This insertion site is probably neutral with respect to the physiology of recipient cells, but this system also is not designed for easy repeated use. In view of these limitations, the construction of a new convenient single-copy gene-expression system, which is still lacking for S. pneumoniae, would be useful. In this paper, we describe the construction and evaluation of a versatile chromosomal expression platform (CEP), pre-assembled on a plasmid (pCEP) capable of replication in Escherichia coli, but not in S. pneumoniae. pCEP allows placement of the gene of interest under the control of a maltosaccharide-inducible promoter and provides flanking homology for its integration into a region of the pneumococcal chromosome originally devoid of expressed sequences.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial strains, growth conditions and transformation.
S. pneumoniae strains and plasmids used in this study are described in Table 1. Precultures were grown at 37 °C in CAT or in C+Y medium (Bergé et al., 2002) to OD₅₅₀ 0·4, then centrifuged, resuspended in fresh C+Y medium containing 15 % (v/v) glycerol and kept frozen at –70 °C. These precultures were used to initiate cultures in C+Y at 2x10⁶ cells ml^–1. Transformation was performed as described previously (Martin et al., 2000). Transformants were selected by plating in 10 ml CAT agar supplemented with 4 % horse blood, followed by challenge with a 10 ml overlay containing 0·1 µg ml^–1 erythromycin (Ery), 500 µg ml^–1 kanamycin (Kan) or 400 µg ml^–1 streptomycin (Sm), after phenotypic expression for 120 min at 37 °C.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Choice of an inducible system for controlled gene expression at CEP
The regulated systems previously used for controlled gene expression in S. pneumoniae depend on sugars, antibiotics, and antibacterial or regulatory peptides for induction. Maltose- (Nieto et al., 2000), raffinose- (Luo et al., 2003) or fucose-regulated promoters (Chan et al., 2003) have thus been used, as well as tetracycline- (Stieger et al., 1999), nisin- (Luo et al., 2003) or competence-stimulating peptide (CSP)1/bacteriocin inducing peptide (BIP)1-inducible promoters (Kausmally et al., 2005).

In choosing a regulatory system for inclusion at CEP, we were reluctant to use sublethal concentrations of antibiotic (tetracycline) or antibacterial peptide (nisin) as inducer. We also ruled out the use of CSP1, which induces the competence regulon (Dagkessamanskaia et al., 2004; Peterson et al., 2004), or BIP1, which induces the Blp regulon (de Saizieu et al., 2000), because they could not be considered to be neutral with respect to pneumococcal physiology. We therefore retained a sugar-inducible system and selected the maltosaccharide-inducible system because of the extensive studies conducted by Lacks, Espinosa and coworkers. P_M, the inducible promoter of the malMP operon (Stassi et al., 1982), has previously been used for the construction of a tightly regulated plasmid vector for S. pneumoniae (Nieto et al., 2000). P_M is regulated by the MalR repressor, the binding of MalR to P_M being relieved when pneumococcal cells are grown in maltose-containing media (Lacks, 1968; Nieto et al., 1997). A replicative plasmid containing the malR repressor gene (pAPM22; Puyet et al., 1993; Table 1) is available to increase the intracellular amount of MalR, thus ensuring high levels of repression.

Choice of CEP location
In our choice of a chromosomal location for CEP we looked for a transcriptionally silent site, at which integration of exogenous material would not perturb any known cellular function. We therefore chose to introduce CEP immediately downstream of the well-studied ami operon (Alloing et al., 1990), and designed it so that its integration would occur by replacement-recombination. Upon integration, CEP substitutes for a truncated IS1167 element (Fig. 1) between nucleotides 1 671 049 and 1 672 299 in the 2 038 615 bp R6 genome (Hoskins et al., 2001). When an empty CEP is integrated, DNA segments of similar size are exchanged. Two transcription terminators, which flank CEP on each side, are retained after integration (Fig. 1).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Organization of the chromosomal expression platform, CEP. (A) Primers used to construct pCEP and to generate CEPluc (Table 1, and Results and Discussion) are shown with horizontal arrows indicating primer orientation. Dotted lines (bordered by BM106 and BM111) indicate the IS1167 remnant sequences deleted and replaced by the PM–kan segment. The amiE and amiF genes belong to the oligopeptide permease ami operon (Alloing et al., 1990). treR and treP are part of a predicted trehalose utilization system. ter, transcription terminator; PM, maltose-inducible promoter; N and B, NcoI and BamHI restriction sites, respectively. (B) Sequence of the PM DNA fragment. The previously identified (Nieto et al., 1997) –35 and –10 regions are underlined and the MalR binding site is boxed. The residues flanking the ATG of malM (also boxed) have been modified in primer BM108M to introduce an NcoI site (Table 1). Note that our control sequencing revealed that the sequence of the PM fragment differed from that initially published by Stassi et al. (1982) at several positions (due to underestimations in the number of residues at runs of A or T). It also differs by a block of 10 residues (underlined italics) apparently mislocated (vertical arrow) in the sequence shown in Fig. 2 in Nieto et al. (1997). However, our sequence is 100 % identical to the sequence reported for the R6 strain (Hoskins et al., 2001). Note also that pCEP plasmid DNA generates no transformants resistant to methotrexate (a phenotype conferred by ami mutations) in a wild-type recipient strain, which strongly suggests that pCEP carries no mutation in the ami region. The tre region has not been sequenced.

The cassette is flanked on each side by more than 2 kb of pneumococcal DNA from the chromosomal region downstream of the ami operon (Fig. 1), a situation predicted to strongly favour cassette insertion over integration of the entire pCEP plasmid during transformation in S. pneumoniae (Pozzi & Guild, 1985). Transformants harbouring the appropriate construct can therefore be isolated directly following transformation of an S. pneumoniae strain with ligation products, with no need for an intermediate cloning step in E. coli. There are two main advantages to this strategy. First, the efficiency of pneumococcal transformation is much higher than that of E. coli. Second, it avoids potential cloning problems that arise in E. coli because of the previously documented frequent toxicity of cloned pneumococcal DNA fragments (Martin et al., 1989).

Construction of pCEP and integration of CEP into the pneumococcal chromosome
Plasmid pCEP carries a chromosomal fragment, bordered by primers BM105 and BM112, in which most of the IS1167 remnant sequences (1249 bp) are substituted by a 1140 bp segment containing P_M and the kan gene (Fig. 1). Construction of the engineered BM105–BM112 segment involved several PCR reactions with the AccuTaq LA DNA polymerase (Sigma), as follows (see primers with arrows in Fig. 1). The P_M and the Kan^R fragments were generated with primer pairs BM107M/BM108M and BM109/BM110.1 (Table 1) using R800 chromosomal DNA and plasmid pR410 DNA as template, respectively (Fig. 1). Fragments corresponding to the leftward and rightward flanking chromosomal regions were amplified from R800 chromosomal DNA with BM105/BM106 and BM111/BM112 primer pairs (Table 1), respectively (Fig. 1). After purification with a QIAquick nucleotide removal kit (Qiagen), fragments BM105–BM106 and BM107M–BM108M in one reaction, and BM109–BM110.1 and BM111–BM112 in another reaction, were mixed and used as template to generate, respectively, the BM105–BM108M and BM109–BM112 fragments (Fig. 1). The BM105–BM112 final PCR product was then amplified using the latter two PCR fragments as template. This fragment was digested with HindIII/XmaI and cloned into HindIII/XmaI-digested E. coli plasmid pJPB209 (Table 1) to generate plasmid pCEP.

Integration of CEP was readily obtained by transformation of the wild-type strain R800 with plasmid pCEP DNA and selection for Kan^R transformants. The frequency of Kan^R transformants was similar to that of Sm^R transformants obtained with the chromosomal marker str41 (data not shown). Since the Kan^R cassette is flanked on each side by more than 2 kb of pneumococcal DNA, integration of the engineered P_M–kan segment in place of the IS1167 remnant was expected to occur at a much higher frequency than insertion of the entire pCEP molecule through insertion-duplication mechanisms (Méjean et al., 1981; Pozzi & Guild, 1985). Analysis of a few Kan^R transformants confirmed the absence of pCEP vector sequences (data not shown). One of these transformants retained for further analysis, strain R1567, displayed growth and transformation properties indistinguishable from those of the parental strain (data not shown, but see Guiral et al., 2006), suggesting that modifications introduced downstream of the ami operon have no significant effect on pneumococcal physiology.

Insertion of the luc gene at CEP
To evaluate ectopic gene expression at CEP, we used the luc gene as transcriptional reporter. Its placement at CEP under the control of P_M was achieved as follows. A DNA fragment containing luc was amplified with the MP162/MP163 primer pair using plasmid pR414 DNA as template (Table 1). After digestion with NcoI and BamHI, the fragment (1657 bp) was ligated with NcoI/BamHI-digested plasmid pCEP, and strain R800 was transformed with the ligation mixture. Ten out of 10 Kan^R transformants analysed harboured the CEP^luc construct (Fig. 1). One of them was retained as strain R1474. Note that the sequence of the luc gene was not checked, but further experiments revealed that it encoded a functional luciferase (see below).

Evaluation of ectopic, P_M-driven luc expression
The effect of sugar concentrations on growth of pneumococcal cells was first evaluated (Fig. 2). In C+Y containing only 0·1 % sucrose, the growth rate was essentially unaffected, but cells stopped growing at an OD₄₉₂ value of 0·4, indicating that sucrose was limiting (Fig. 2A, C). Addition of 0·05 and 0·1 % maltose increased final OD values, indicating that maltose was metabolized and suggesting that its concentration declined progressively with growth. Cultures with 0·2 and 0·4 % maltose attained final OD values (0·8) similar to those of cultures in the presence of 0·3 % sucrose (Fig. 2, compare panels A and C with panels B and D). In C+Y containing 0·3 % sucrose, addition of 0·05–0·4 % maltose had no significant effect on growth rate and final OD values, indicating that sucrose was not limiting (Fig. 2B, D). Similar data were obtained when glucose (0·1 and 0·3 %) was substituted for sucrose (data not shown). A concentration of 0·3 % sucrose was employed in further expression studies.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Effect of sugar concentrations on growth of pneumococcal cells and on luc expression. Strains R1474 (CEPluc; panels A and B) and R1488 (CEPluc and the cloned malR gene, panels C and D) were grown in C+Y medium containing 0·1 % (panels A and C) or 0·3 % (panels B and D) sucrose (suc, open circles), or sucrose plus the following concentrations of maltose (filled symbols): 0·05 % (triangles), 0·1 % (squares), 0·2 % (diamonds) or 0·4 % (circles). Maltose was added at 60 min (+mal, black arrows). OD492 (grey lines) and luciferase activity (expressed in RLU, black lines in panels B and D) were recorded during incubation at 37 °C for 300 min.

To investigate whether an increase in the intracellular level of the MalR repressor would lead to a better repression of the P_M promoter, luc expression was monitored in a derivative of strain R1474 containing a recombinant plasmid carrying the malR gene (strain R1488; Table 1). Luciferase activity in the control culture without maltose was reduced to 3·2±0·1 RLU (Fig. 2D), suggesting that repression of P_M was slightly reinforced by an increase in the intracellular level of MalR. In the presence of maltose (within the range 0·05–0·4 %), luciferase activity increased to 37·8±1·2 RLU (10·8±0·4-fold increase compared to the control culture; Fig. 2D).

Expression of the luc gene was then measured in cultures grown in the presence of concentrations of maltose below 0·05 % (Fig. 3). Luciferase activity was close to the maximum in the presence of 0·008 % maltose (48·9±0·6 compared to 53·9±0·2 RLU with 0·04 % maltose). While no significant induction of luc could be detected with 0·000064 % (0·64 µg ml^–1) maltose, intermediate expression values were obtained with 0·00032 % (3·2 µg ml^–1) and 0·0016 % maltose (17·5±0·4 and 38·5±1·3 RLU, respectively), indicating that maltose concentration can be adjusted to fine-tune gene expression at CEP.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Measurement of PM-driven expression of luc at CEP. Strain R1474 was grown in C+Y medium containing 0·3 % sucrose (suc, open circles) or 0·3 % sucrose plus 0·000064 % (0·64 µg ml–1; open squares), 0·00032 % (3·2 µg ml–1; open triangles), 0·0016 % (open diamonds), 0·008 % (filled squares) or 0·04 % (filled triangles) maltose. Maltose was added at 65 min (+mal, black arrow). Luciferase activity (black lines, expressed in RLU) and OD492 (grey lines) were recorded during incubation at 37 °C for 300 min. Note that maximum luciferase yields (grey arrows) occurred at higher OD values (indicated by horizontal lines) with increasing maltose concentration (OD492 0·165, 0·275 and 0·415 with 0·00032, 0·0016 and 0·008–0·04 % maltose, respectively), which suggests that maltose could be used for growth even in the presence of non-limiting sucrose.

	ACKNOWLEDGEMENTS

 
We thank Michael Chandler for critical reading of the manuscript and Paloma Lopez for kindly providing us with plasmid pAPM22. This research was financed in part by European Union Grant QLK2-CT-2000-00543.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Alloing, G., Trombe, M. C. & Claverys, J. P. (1990). The ami locus of the Gram-positive bacterium Streptococcus pneumoniae is similar to binding protein-dependent transport operons of Gram-negative bacteria. Mol Microbiol 4, 633–644.[CrossRef][Medline]

Balganesh, T. S. & Lacks, S. A. (1985). Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: cloning and expression of the hexA gene. J Bacteriol 162, 979–984.[Abstract/Free Full Text]

Bergé, M., Moscoso, M., Prudhomme, M., Martin, B. & Claverys, J. P. (2002). Uptake of transforming DNA in Gram-positive bacteria: a view from Streptococcus pneumoniae. Mol Microbiol 45, 411–421.[CrossRef][Medline]

Chan, P. F., O'Dwyer, K. M., Palmer, L. M. & 8 other authors (2003). Characterization of a novel fucose-regulated promoter (PfcsK) suitable for gene essentiality and antibacterial mode-of-action studies in Streptococcus pneumoniae. J Bacteriol 185, 2051–2058.[Abstract/Free Full Text]

Dagkessamanskaia, A., Moscoso, M., Hénard, V., Guiral, S., Overweg, K., Reuter, M., Martin, B., Wells, J. & Claverys, J. P. (2004). Interconnection of competence, stress and CiaR regulons in Streptococcus pneumoniae: competence triggers stationary phase autolysis of ciaR mutant cells. Mol Microbiol 51, 1071–1086.[CrossRef][Medline]

de Saizieu, A., Gardes, C., Flint, N., Wagner, C., Kamber, M., Mitchell, T. J., Keck, W., Amrein, K. E. & Lange, R. (2000). Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide. J Bacteriol 182, 4696–4703.[Abstract/Free Full Text]

Díaz, A., Lacks, S. A. & López, P. (1992). The 5' to 3' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae. Mol Microbiol 6, 3009–3019.[CrossRef][Medline]

Guiral, S., Hénard, V., Granadel, C., Martin, B. & Claverys, J. P. (2006). Inhibition of competence development in Streptococcus pneumoniae by increased basal-level expression of the ComDE two-component regulatory system. Microbiology, 152, 323–331.[Abstract/Free Full Text]

Hoskins, J., Alborn, W. E., Jr, Arnold, J. & 40 other authors (2001). Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol 183, 5709–5717.[Abstract/Free Full Text]

Kausmally, L., Johnsborg, O., Lunde, M., Knutsen, E. & Håvarstein, L. S. (2005). Choline-binding protein D (CbpD) in Streptococcus pneumoniae is essential for competence-induced cell lysis. J Bacteriol 187, 4338–4345.[Abstract/Free Full Text]

Kong, L., Siranosian, K. J., Grossman, A. D. & Dubnau, D. (1993). Sequence and properties of mecA, a negative regulator of genetic competence in Bacillus subtilis. Mol Microbiol 9, 365–373.[CrossRef][Medline]

Lacks, S. A. (1968). Genetic regulation of maltosaccharide utilization in pneumococcus. Genetics 60, 685–706.[Free Full Text]

Luo, P., Li, H. & Morrison, D. A. (2003). ComX is a unique link between multiple quorum sensing outputs and competence in Streptococcus pneumoniae. Mol Microbiol 50, 623–633.[CrossRef][Medline]

Mannarelli, B. M. & Lacks, S. A. (1984). Ectopic integration of chromosomal genes in Streptococcus pneumoniae. J Bacteriol 160, 867–873.[Abstract/Free Full Text]

Martin, B., Prats, H. & Claverys, J. P. (1985). Cloning of the hexA mismatch repair of Streptococcus pneumoniae and identification of the product. Gene 34, 293–303.[CrossRef][Medline]

Martin, B., Alloing, G., Boucraut, C. & Claverys, J. P. (1989). The difficulty of cloning Streptococcus pneumoniae mal and ami loci in Escherichia coli: toxicity of malX and amiA gene products. Gene 80, 227–237.[CrossRef][Medline]

Martin, B., Prudhomme, M., Alloing, G., Granadel, C. & Claverys, J. P. (2000). Cross-regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae. Mol Microbiol 38, 867–878.[CrossRef][Medline]

Méjean, V., Claverys, J. P., Vasseghi, H. & Sicard, A. M. (1981). Rapid cloning of specific DNA fragments of Streptococcus pneumoniae by vector integration into the chromosome followed by endonucleolytic excision. Gene 15, 289–293.[CrossRef][Medline]

Mortier-Barrière, I., de Saizieu, A., Claverys, J. P. & Martin, B. (1998). Competence-specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae. Mol Microbiol 27, 159–170.[CrossRef][Medline]

Nieto, C., Espinosa, M. & Puyet, A. (1997). The maltose/maltodextrin regulon of Streptococcus pneumoniae. J Biol Chem 272, 30860–30865.[Abstract/Free Full Text]

Nieto, C., Fernández de Palencia, P., López, P. & Espinosa, M. (2000). Construction of a tightly regulated plasmid vector for Streptococcus pneumoniae: controlled expression of the green fluorescent protein. Plasmid 43, 205–213.[CrossRef][Medline]

Peterson, S., Sung, C. K., Cline, R. & 13 other authors (2004). Identification of competence pheromone responsive genes in Streptococcus pneumoniae. Mol Microbiol 51, 1051–1070.[CrossRef][Medline]

Pichoff, S., Vollrath, B., Touriol, C. & Bouché, J. P. (1995). Deletion analysis of gene minE which encodes the topological specificity factor of cell division in Escherichia coli. Mol Microbiol 18, 321–329.[CrossRef][Medline]

Pozzi, G. & Guild, W. R. (1985). Modes of integration of heterologous plasmid DNA into the chromosome of Streptococcus pneumoniae. J Bacteriol 161, 909–912.[Abstract/Free Full Text]

Prats, H., Martin, B. & Claverys, J. P. (1985). The hexB mismatch repair gene of Streptococcus pneumoniae: characterization, cloning and identification of the product. Mol Gen Genet 200, 482–489.[CrossRef][Medline]

Prudhomme, M. & Claverys, J. P. (2006). There will be a light: the use of luc transcriptional fusions in living pneumococcal cells. In The Molecular Biology of Streptococci. Edited by R. Hakenbeck & G. S. Chhatwal. Norwich, UK: Horizon Scientific Press.

Puyet, A., Ibáñez, A. M. & Espinosa, M. (1993). Characterization of the Streptococcus pneumoniae maltosaccharide regulator MalR, a member of the LacI-GalR family of repressors displaying distinctive genetic features. J Biol Chem 268, 25402–25408.[Abstract/Free Full Text]

Robertson, G. T., Ng, W. L., Foley, J., Gilmour, R. & Winkler, M. E. (2002). Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and virulence. J Bacteriol 184, 3508–3520.[Abstract/Free Full Text]

Robertson, G. T., Ng, W. L., Gilmour, R. & Winkler, M. E. (2003). Essentiality of clpX, but not clpP, clpL, clpC, or clpE, in Streptococcus pneumoniae R6. J Bacteriol 185, 2961–2966.[Abstract/Free Full Text]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Stassi, D. L., Dunn, J. J. & Lacks, S. A. (1982). Nucleotide sequence of DNA controlling expression of genes for maltosaccharide utilization in Streptococcus pneumoniae. Gene 20, 359–366.[CrossRef][Medline]

Stieger, M., Wohlgensinger, B., Kamber, M., Lutz, R. & Keck, W. (1999). Integrational plasmids for the tetracycline-regulated expression of genes in Streptococcus pneumoniae. Gene 226, 243–251.[CrossRef][Medline]

Sung, C. K., Li, H., Claverys, J. P. & Morrison, D. A. (2001). An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ Microbiol 67, 5190–5196.[Abstract/Free Full Text]

Vasseghi, H. & Claverys, J. P. (1983). Amplification of a chimeric plasmid carrying an erythromycin-resistance determinant introduced into the genome of Streptococcus pneumoniae. Gene 21, 285–292.[CrossRef][Medline]

Vasseghi, H., Claverys, J. P. & Sicard, A. M. (1981). Mechanism of integrating foreign DNA during transformation of Streptococcus pneumoniae. In Transformation-1980, pp. 137–154. Edited by M. Polsinelli & G. Mazza. Oxford: Cotswold Press.

Zähner, D. & Hakenbeck, R. (2000). The Streptococcus pneumoniae beta-galactosidase is a surface protein. J Bacteriol 182, 5919–5921.[Abstract/Free Full Text]

Received 11 August 2005; revised 20 September 2005; accepted 21 September 2005.

This article has been cited by other articles:

				A. Le Gouellec, L. Roux, D. Fadda, O. Massidda, T. Vernet, and A. Zapun Roles of Pneumococcal DivIB in Cell Division J. Bacteriol., July 1, 2008; 190(13): 4501 - 4511. [Abstract] [Full Text] [PDF]

				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]

				S. Guiral, V. Henard, C. Granadel, B. Martin, and J.-P. Claverys Inhibition of competence development in Streptococcus pneumoniae by increased basal-level expression of the ComDE two-component regulatory system Microbiology, February 1, 2006; 152(2): 323 - 331. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Guiral, S. Articles by Claverys, J.-P. Search for Related Content PubMed PubMed Citation Articles by Guiral, S. Articles by Claverys, J.-P. Agricola Articles by Guiral, S. Articles by Claverys, J.-P.