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Genetics and Molecular Biology |
Centro de Investigaciones Biológicas, CSIC, Velázquez, 144, E-28006 Madrid, Spain1
Author for correspondence: Manuel Espinosa. Tel: +34 915611800. Fax: +34 915627518. e-mail: mespinosa{at}cib.csic.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Keywords: green fluorescent protein, pneumococci, chromosomal integration, maltose regulon, MalR repressor
Abbreviations: Ap, ampicillin; Cm, chloramphenicol; GFP, green fluorescent protein; Tc, tetracycline; TIR, translation initiation region
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Miller & Lindow, 1997 ). Among Gram-positive bacteria, the potential use of GFP as a reporter has also been documented (Geoffroy et al., 2000 ; Scott et al., 2000 ; Somkuti & Steinberg, 1999 ), although it has only been in Bacillus subtilis that GFP has been used to follow more complex processes such as DNA replication (Lemon & Grossman, 1998 ) and sporulation (Lewis & Errington, 1996 ). Measurement of GFP activity requires post-translational oxidation of the protein, and full activity is only achieved at pH values around 7·0 (reviewed by Sullivan & Kay, 1999 ). These features, although reversible, have hindered employment of this useful tool in some microaerophiles, such as the human pathogen Streptococcus pneumoniae.
We have been interested in the study of the utilization of maltosaccharides by S. pneumoniae (Puyet & Espinosa, 1993 ; Stassi et al., 1982 ). Genes involved in this process are organized as a regulon composed of three operons. Two of them are involved in maltosaccharide uptake (malXCD) and its utilization (malMP), and one (malAR) is involved in regulation (Puyet & Espinosa, 1993 ; Puyet et al., 1993 ). The two former operons are transcribed from two divergent promoters, termed PM (for the malMP operon) and PX (for the malXCD operon), which are negatively regulated by the product of malR (Nieto et al., 1997 ). MalR binds specifically to two operator sequences located in the intergenic region between operons malXCD and malMP (Nieto et al., 1997 ). The binding of MalR to its DNA target is reversed by the addition of maltose, pointing to inactivation of the repressor by the sugar (Nieto et al., 1997 ). In addition, the strong promoter PM has proved to support functional constitutive expression of gfp in Lactococcus lactis (Fernández de Palencia et al., 2000 ).
In this work, we used the recombinant plasmid pLS70 (Stassi et al., 1982 ), which harbours the pMV158 replicon (del Solar et al., 1998 ) and part of the pneumococcal mal regulon to clone a modified gfp gene (Miller & Lindow, 1997 ) from Aequorea victoria. In addition, by homologous recombination, we introduced the gfp gene into a single chromosomal copy. Expression of GFP was achieved in S. pneumoniae in both genetic conditions (multiple or single copies), as detected by direct measurement of the fluorescence of the cells. A combination of phase-contrast and fluorescence microscopy allowed us to distinguish between cells harbouring or not harbouring the gfp gene. Our results constitute a step forward in following directly the ability of pneumococcal cells to colonize epithelial cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). This strain, and S. pneumoniae R61 (Lacks, 1968 ), were also employed for expression and detection of GFP activity. Determination of the nucleotide sequence of the malR region of strain Rc19 showed that it harbours a deletion that removes four amino acids from the N-terminal region of MalR. This deletion is included within a region supposedly involved in the binding of MalR to its DNA target, leading to inactivation of MalR (C. Nieto, unpublished). The plasmid used was pLS70, a pMV158 derivative (Burdett, 1980 ) in which a fragment of the pneumococcal mal regulon was cloned (Stassi et al., 1982 ). The cloned region contains promoters PM and PX, the entire malM, and truncated malP and malX genes (Fig. 1). In addition, we used plasmid pGreenTIR (Miller & Lindow, 1997 ), a derivative of pUC18 in which a mutated gfp gene (containing a double F64L S65T amino acid change that increases GFP solubility and fluorescence) has been cloned. This plasmid also contains an improved translation initiation region (TIR) for prokaryotes, including the translational enhancer (ENH) and the Shine–Dalgarno (SD) regions of phage T7 gene 10, so that synthesis of GFP is enhanced. Finally, plasmid pJS3 (Ballester et al., 1990 ) is a pMV158-based replicon in which the cat gene, encoding resistance to chloramphenicol (Cm) of the staphylococcal plasmid pC194 (Horinouchi & Weisblum, 1982 ) has been placed under the control of the promoter for the tet gene of pMV158. E. coli DH5
cells were grown in TY medium (Sambrook et al., 1989 ), with selection for ampicillin (Ap) resistance (100 µg ml-1) for E. coli/pGreenTIR. S. pneumoniae cells were grown in AGCH medium (which contains casein hydrolysate, amino acids, vitamins, salts, albumin and catalase; Lacks, 1968 ), supplemented with sucrose, maltose or both sugars at a final concentration of 0·8%. To test whether gfp expression was limited to this medium, cells were also grown in Todd–Hewitt Broth (Difco), supplemented with 0·5% yeast extract and 0·8% sugars as above. Selection was applied for resistance to tetracycline (Tc) (1 µg ml-1) or Cm (5 µg ml-1).
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). Purified plasmid DNA from S. pneumoniae was prepared as reported by del Solar et al. (1987) . Restriction endonucleases, mung bean nuclease, the Klenow fragment of DNA polymerase I (PolIK) and DNA ligase were purchased from New England Biolabs or Boehringer Mannheim and were used as specified by the suppliers. Restriction fragments employed for cloning experiments were purified from agarose gels by phenol extraction (Sambrook et al., 1989 ). Determination of the nucleotide sequence of the desired DNA fragments of Rc19 and Rc19malM::gfp cat was done after PCR amplification by the dideoxynucleotide termination procedure on an automated sequencer (ABI 373A).
Measurement of GFP activity.
Pneumococcal cells harbouring plasmids or chromosomal inserts were grown in sugar-containing media, without selective pressure, to an OD650 of 0·7 (about 6x108 c.f.u. ml-1). In some cases, fluorescence was directly measured in the culture medium. In other cases, cells (1·5 ml from each culture) were sedimented by centrifugation and suspended in the same volume of PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 140 mM NaCl, 3 mM KCl, pH 7·2) prior to determination of their fluorescence. Cell lysates were prepared by sedimentation of the cells (1·5 ml), suspension in the same volume of lysis buffer (50 mM Tris/HCl pH 8·0, 10 mM EDTA, 0·1% sodium deoxycholate, 1% glucose) and incubation at 37 °C for 10 min. Aliquots (200 µl) of cells or cell lysates were used to measure the fluorescence on a LS-50B spectrophotometer (Perkin-Elmer) by excitation at a wavelength of 488 nm with a slit width of 2·5 or 5 nm for cuvettes or microtitre plates, respectively, and detection of emission at 510 nm with a slit width of 10 or 5 nm for cuvettes or microtitre plates, respectively. As a control to determine the background fluorescence, isogenic strains (plasmid-free or carrying pLS70) were used. Analysis of the culture media and of supernatants of cultures grown to different optical densities showed that their intrinsic fluorescence decreased as a function of cell growth (not shown), indicating that fluorescent compounds in the media were consumed by the cells.
Fluorescence microscopy.
Cultures of S. pneumoniae strains were grown in medium supplemented with sucrose plus maltose (0·8% each) to an OD650 of 0·4. Cells in growth medium were directly analysed by phase-contrast or epi-fluorescence microscopy with a Zeiss Axioplan Universal microscope using excitation standard FITC set D480/30 and emission TBP 460/530/610 filters.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The ligation mixture was used to transform competent pneumococcal cells, selecting for Tc resistance. Due to the high intrinsic fluorescence of the pneumococcal medium, direct selection for TcR and fluorescent colonies was not feasible on agar plates. Plasmid DNA from several clones was prepared and plasmids of an appropriate size were selected and analysed by restriction mapping. The resulting plasmid was termed pLS70GFP, and the integrity of the gfp gene cassette was checked by determination of its entire nucleotide sequence. This construction places the gfp gene under the control of promoter PM (Fig. 1).
To obtain a single copy of gfp, we used the procedure developed for S. pneumoniae based on recombination between the chromosomal DNA fragment present in pLS70GFP and the homologous chromosomal region in the host (López et al., 1982 ). As a selectable marker, we used the cat gene of plasmid pJS3, which is readily expressed in pneumococci because of the coupling of the tet promoter (Ptet) of plasmid pMV158 to the cat gene of the staphylococcal plasmid pC194 (Ballester et al., 1989 ). Plasmid pLS70GFP was partially digested with SacI, the linear forms were purified and the protruding ends were made blunt by treatment with mung bean nuclease and filling in with PolIK. Likewise, DNA from plasmid pJS3 was cleaved with Sau3AI and the 1·1 kb fragment containing the cat gene was purified and treated with PolIK. Both DNA fragments were ligated to generate gfp–cat or cat–gfp cassettes flanked by pneumococcal mal regions and the mixture was used to transform competent S. pneumoniae Rc19 cells. Since the donor DNA is taken up as linear single-stranded molecules by S. pneumoniae, chromosomal transformation is enhanced over plasmid establishment by a factor of 1000 when selection is applied for Cm resistance (López et al., 1982 ). Six transformants were selected and analysed for plasmid content and levels of fluorescence. None of them harboured plasmids, and three of them exhibited higher fluorescence than the parental strain (not shown). The chromosomal structure at the mal regulon of one of the fluorescent clones was checked by PCR amplification and determination of the nucleotide sequence. This clone, containing the desired construction with the insertion of the gfp–cat cassette within the malM gene (Fig. 1) was chosen and termed Rc19malM::gfp cat.
Expression of the gfp gene in S. pneumoniae
Detection of GFP depends upon the pH of the medium and the levels of oxygen (Sullivan & Kay, 1999 ). These features are reversible, but the use of GFP as a reporter in microaerophilic bacteria (such as pneumococci) usually demands exposure of cells to aerobic conditions, and neutralization and removal of fluorescent components of the culture media. These factors make it difficult to monitor the fluorescence of S. pneumoniae during cell growth. To test whether pneumococcal cells harbouring the gfp gene expressed GFP, we analysed the fluorescence in intact cells (either in the culture medium or after washing the cells and resuspension in PBS buffer) and in cell lysates. The observed total fluorescence was corrected for the intrinsic fluorescence of cells or lysates detected in the isogenic strains lacking gfp. The results (Table 1) showed that expression of GFP could be measured, although the levels were about twofold higher when the cells were suspended in PBS buffer as compared with the values obtained in growth medium. Similar levels were found in cell lysates and in cells suspended in PBS. The intensity of fluorescence measured in S. pneumoniae/pLS70GFP was fairly high and reproducible, being about 6–7 times higher than those obtained for the strain Rc19malM::gfp cat. These differences are consistent with the levels of amylomaltase (encoded by the malM gene) found in S. pneumoniae/pLS70 compared with those synthesized by a single chromosomal copy of malM (Stassi et al., 1982 ). Similar levels of gfp synthesis were detected when cells were grown in media other than AGCH (not shown). The above results allowed us to conclude that: (i) the gfp gene from plasmid pGreenTIR is expressed in S. pneumoniae; (ii) GFP synthesized is fully active in living pneumococcal cells; (iii) GFP can be detected in culture media, even in cells harbouring a single copy of the gene; and (iv) the levels of GFP are related to the gene dosage.
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) and fluorescence (Fig. 2b) of the cultures was monitored in the growth medium. The results showed that cell growth was slightly reduced when Rc19 cells harboured plasmids (pLS70 or pLS70GFP). The calculated doubling times were 40 min for plasmid-free cells and 45 min for plasmid-containing cells. However, strains harbouring gfp were not impaired in growth (Fig. 2a), they showed higher fluorescence than their isogenic strains lacking the gfp gene, and only the fluorescence of gfp+ strains increased with cell mass (not shown), indicating that the fluorescence is correlated with cell growth. Corrected fluorescence values for Rc19/pLS70GFP and Rc19malM::gfp cat strains (Fig. 2b) showed that, over the range of growth analysed, GFP expression increased linearly with cell mass and was related to gene dosage. Thus, the observed decrease in pH of the medium from 7·9 to 6·4 during growth of Rc19/pLS70 did not affect detection of GFP (top of Fig. 2b). In addition, alteration of the pH by addition of NaOH or HCl to the cultures revealed that full activity of GFP can be monitored within a pH range of 9·5–6·0 (not shown). We concluded that quantification of GFP in vivo can be achieved in a reliable manner during growth of S. pneumoniae in medium down to pH 6·0. Our results also showed that GFP can be used to monitor pneumococcal cells in real time.
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). This would not be the case for strain Rc19 lacking a functional MalR, in which expression of gfp from PM should be constitutive and independent of the carbon source. To compare the levels of GFP synthesized in strain Rc19 with those in a wild-type background (i.e. one intact copy of the malR gene), both the chromosomal gfp–cat gene cassette and plasmid pLS70GFP were independently transferred by transformation into the wild-type S. pneumoniae R61. After checking the appropriateness of the strains harbouring single or multiple copies of gfp, we tested whether a possible correlation between biomass and expression of GFP existed when cells were grown in media containing sucrose, maltose or a mixture of both sugars. Since amylomaltase is required for utilization of maltose as a carbon source by pneumococci, strains harbouring the malM::gfp cat insertion could not be grown in medium containing only maltose. Growth of the cultures was monitored by spectrophotometry, and the levels of GFP were measured by fluorescence spectroscopy in growth medium at different stages of cell growth (Fig. 3). In the case of strains Rc19 harbouring gfp, the fluorescence of the cultures increased with cell mass, independently of the carbon source (Fig. 3a, c), as expected for a constitutive strain. There was a clear relationship between the levels of GFP synthesized and the dosage of the gfp gene, GFP being about 6–7 times higher in Rc19/pLS70GFP than in the strain harbouring one chromosomal copy of gfp. These differences agree with previous measurements of amylomaltase activity when tested in a single chromosomal copy or in plasmid pLS70 (Stassi et al., 1982 ). Consequently, the defective MalR synthesized by Rc19 does not repress promoter PM and gfp expression is constitutive.
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). In the medium containing both sugars, higher levels of GFP were detected when the concentration of maltose was increased, until the values measured were the same as those obtained for cultures grown only in maltose (not shown). When the gfp gene was provided in a single chromosomal copy (Fig. 3d), a similar result was found for cultures grown in sucrose plus maltose. In these cases, the GFP levels found in maltose-grown cultures were comparable to those obtained in the isogenic Rc19 strains (compare Fig. 3a with 3b, and 3c with 3d), and are consistent with the amylomaltase levels previously measured (Stassi et al., 1982 ). However, when the cultures were grown in sucrose (Figs 3b, d), low but significant amounts of GFP were detectable, indicating that repression by MalR was not enough to shut off gfp transcription from promoter PM. This would be expected when gfp was present in multiple copies but not when a single copy was assayed, a result for which we have no explanation at present. We conclude that over the range of growth analysed, the expression of GFP can be reliably quantified and, in consequence, GFP can be used to monitor pneumococcal cells.
Detection of GFP-expressing pneumococcal cells by fluorescence microscopy
To test whether S. pneumoniae Rc19 cells harbouring single or multiple copies of gfp could be visualized by fluorescence microscopy, the strains were grown and directly analysed in growth medium (without fixing) by phase-contrast or by fluorescence microscopy. The analysis was also performed in mixed cultures of Rc19 expressing or not expressing GFP. All cells harbouring a single gfp copy or pLS70GFP were fluorescently labelled (not shown). Employment of mixed cultures of cells expressing or not expressing GFP allowed us to distinguish the two isogenic strains, and expression of GFP in monocopy (Fig. 4a) or multicopy (Fig. 4b) did not affect cell morphology. We believe that this is a fundamental step toward monitoring the adherence of S. pneumoniae to human cells, since one copy of the gfp gene, integrated into the bacterial chromosome, is enough to follow their fate upon invasion. Adhesion can be thus monitored directly without the need for tedious analyses of binding of S. pneumoniae to human cell lines.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Ballester, S., Alonso, J. C., López, P. & Espinosa, M. (1990). Comparative expression of the pC194 cat gene in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli.Gene 86, 71-79.[Medline]
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Horinouchi, S. & Weisblum, B. (1982). Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance.J Bacteriol 150, 815-825.
Lacks, S. A. (1968). Genetic regulation of maltosaccharide utilization in Pneumococcus.Genetics 60, 685-706.
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Somkuti, G. A. & Steinberg, D. H. (1999). Promoter activity of the pER341-borne ST(Phsp) in heterologous gene expression in Escherichia coli and Streptococcus thermophilus.FEMS Microbiol Lett 179, 431-436.[Medline]
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Received 1 December 1999;
revised 21 February 2000;
accepted 20 March 2000.
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), Rc19malM::gfp cat (
), Rc19/pLS70 (
) and Rc19/pLS70GFP (
). The fluorescence due to GFP in strains Rc19/pLS70GFP and Rc19malM::gfp cat was plotted after subtraction of the fluorescence background detected in Rc19/pLS70 and Rc19, respectively. The pH of the medium during growth of Rc19/pLS70GFP was monitored (shown at the top of b).
). Strains used were Rc19/pLS70GFP (a), R61/pLS70GFP (b), Rc19malM::gfp cat (c) and R61malM::gfp cat (d). Fluorescence due to GFP was calculated as in Fig. 2