MATERIALS AND METHODS
Bacterial strains and growth conditions
All bacterial strains and plasmids used in this work are listed in Table S1. The
pneumococcal strains were grown in casein tryptone (CAT) medium containing (per liter)
167 mmol of K2HPO4, 5 mg of choline chloride, 5 g of tryptone, 10 g of enzymatic casein
hydrolysate, 1 g of yeast extract, and 5 g of NaCl (5). After sterilization, glucose was
added to a concentration of 0.2%. S. pneumoniae was grown at 37C, and growth was
monitored by measuring OD at 550 nm or 492 nm. E. coli was grown in Luria-Bertani
(LB) medium (7) at 37C. When appropriate, S. pneumoniae strains were grown in the
presence of 2.5 g ml-1 chloramphenicol, 200 g ml-1 spectinomycin or 2 g ml-1
Construction of transcriptional fusions and modifications of boxAB2C at the qsrAB
To modify the boxAB2C element at the qsrAB locus, we first amplified a 2700 bp DNA
fragment containing this BOX element using the primers 4144 and Eiv2 (Table S2). This
fragment was used as template in a new PCR reaction with the primers Box1 and Box2,
containing BamHI sites in their 5’-ends, resulting in specific amplification of the qsr
boxAB2C element. This fragment was ligated into pCR2.1-TOPO vector (Invitrogen)
giving rise to plasmid pCRAB2C. pCRAB2C derivatives containing deleted variants of
the AB2C motif were then generated using a PCR-restriction protocol as follows. To
remove any of the four boxes in the AB2C motif, primer pairs that annealed to DNA
regions flanking the box modules to be deleted were first designed. The oligonucleotide
primers, each complementary to opposite strands of the pCRAB2C plasmid, were used to
replicate both plasmid strands by PCR, thereby generating a plasmid with staggered
nicks. Since the primer pairs were designed to contain a unique restriction site at their 5’
ends, subsequent treatment of the PCR product with the appropriate restriction enzyme
efficiently removed the box module(s) lying between the 5’ ends of the two primers used
in the PCR reaction. Next, the PCR product was treated with DpnI to remove template
DNA, and purified by agarose gel electrophoresis. Finally, the plasmid was religated and
transformed into E. coli Top 10 competent cells (Invitrogen). This PCR-restriction
strategy was first applied to construct pCRAB2C derivatives containing AB2 (pCRAB2),
B2C (pCRB2C) and AC (pCRAC) box motifs, using the primer pairs Fjcf/Fjcr (contains a
StuI 5’ restriction site), Fjaf/Fjar (contains a NdeI 5’ restriction site) and Fjbf/Fjbr
(contains a StuI 5’ restriction site) respectively. To construct a derivative containing a
boxB2 motif (pCRB2), we used the pCRAB2 plasmid as a template in a PCR together with
the primer pair Fjaf/Fjar. The various box combinations were isolated from their
respective recombinant plasmid by digestion with BamHI, and ligated in either
orientation into the BamHI site downstream of the PE promoter in pOE4144 (for details
see Knutsen et al. (2)). A pOE4144 derivative containing a boxAB7C motif was
constructed by PCR amplification of a boxAB7C element located upstream of the spr1604
locus in the S. pneumoniae R6 genome. First, a 600 bp DNA fragment encompassing the
AB7C motif was amplified using the primers Box7.1 and Box7.2. This DNA fragment
was subsequently used as a template for specific amplification of the boxAB7C element
using primers (Box7.3 and Box7.4) containing BamHI sites in their 5’-ends. The
fragment was treated with BamHI, and ligated in either orientation into the BamHI site of
pOE4144. All the pOE4144 derivatives constructed in this work were integrated into the
genome of S. pneumoniae strain EK100 by homologous recombination in the region
upstream of the qrsAB promoter.
Construction of transcriptional fusions and modifications of boxABC at the comAB
The OE4151 strain, which contains a promoterless lacZ gene inserted into the
pneumococcal genome behind a comAB promoter lacking boxABC, and the positive
control strain OE4150, which is identical to OE4151 except that boxABC is left intact,
were constructed as follows. A 1302 bp DNA fragment from the comAB locus including
the first fifty-eight 5’-nucleotides of comA together with the complete comAB promoter
and its upstream region was amplified from the CP1200 genome using the primers
ComA7 and ComA8. This fragment was restricted with NsiI and BamHI, and ligated into
the corresponding sites of the vector Litmus 28 (New England Biolabs), giving rise to the
recombinant plasmid pLicom1. To remove the boxABC element, pLicom1 was
subsequently used as a template in a PCR reaction with the primer pair ComA3/ComA4
(contains a NheI 5’ restriction site) using the same technique as described above. The
PCR products were digested with DpnI to remove template DNA, and NheI to remove the
ABC box motif. Agarose gel purified products were next religated, and transformed into
E. coli TOP10 cells, giving rise to pLicom2. The inserts in pLicom1 and pLicom2 were
isolated by digesting the plasmids with HindIII and BamHI, and purified by agarose gel
electrophoresis. Next, the fragments were ligated into the corresponding restriction sites
upstream of the promoterless lacZ gene in the nonreplicating pEVP3 vector, resulting in
the constructs pEVP3-4150 and pEVP3-4151, respectively. Finally, natural
transformation was used to integrate pEVP3-4150 and pEVP3-4151 into the chromosome
of the S. pneumoniae EK100 strain by homologous recombination, giving rise to the
mutant strains OE4150 and OE4151.
The OE4061 strain, which lacks the BOX-element upstream of comAB, and the
positive control strain OE4060, which is identical to OE4061 except that boxABC is left
intact, were derived from strain CP1200 as follows. A 1 kb DNA fragment containing the
complete comAB promoter and the 5’-half of the comA gene was amplified using the
primers comA1 and comA2. The fragment was digested with NsiI and ligated into
pLitmus 28 (New England Biolabs), resulting in the plasmid pLicom3. To construct a
pLicom3 derivative (pLicom4) harboring a comAB promoter region lacking boxABC,
pLitcom 3 was used as template in a PCR-deletion protocol identical to the one used for
generating pLitcom2. The inserts in pLicom3 and pLicom4 were next isolated by
digesting the plasmids with NsiI, purified by agarose gel electrophoresis, and ligated into
the corresponding restriction site in the vector pEVP3, resulting in the derivatives
pEVP3-4160 and pEVP3-4161, respectively. These plasmids were integrated into the
chromosome of S. pneumoniae CP1200 by natural transformation, giving rise to the
mutant strains OE4160 and OE4161. Upon integration of pEVP3-4160 and pEVP3-4161
into the CP1200 genome in the comA gene, the promoter regions originating from
pEVP3-4160 and pEVP3-4161 were inserted in front of fully functional comAB genes.
To monitor spontaneous competence development in the OE4160 and OE4161
strains, a competence inducible luciferase reporter gene was next integrated into the
chromosome of both mutants. A derivative of the pR424 recombinant plasmid (1, 6) was
constructed for this purpose (see Table S1). pR424 contains the Photinus pyralis luc gene
placed under control of the competence inducible promoter of the late com gene ssbB.
The chloramphenicol resistance gene in pR424 was exchanged with the spectinomycin
(spc) resistance gene from pR412 (3). First, the spc gene was amplified from pR412
using the primers Spec1 and Spec2. The PCR products were subsequently digested with
PstI and HindIII, and ligated into the corresponding sites of pR424. The resulting
plasmid, pR459, was then transformed into the OE4160, OE4161, and CP1200 strains.
Homology-dependent integration of pR459 into the ssbB locus resulted in strains
OE4170, OE4171, and OE4180, respectively (Table S1). All mutants constructed in this
work were verified by DNA sequencing.
Transformation of S. pneumoniae
An overnight culture of the pneumococcal strain to be transformed was diluted to an
OD550 of 0.05 in prewarmed (37°C) CAT broth. Before samples were withdrawn for
transformation, reconstitution of growth was allowed by incubating 1 ml samples at 37°C
for 30 min. Genomic or plasmid DNA was added to a final concentration of 2 to 5 µg ml–
together with 250 ng of CSP-1 ml–1. Cells were incubated for 2.5 h before plating on
CAT-agar plates containing the appropriate antibiotics for selection.
β-galactosidase assays were carried out as described previously (2). Hydrolysis of ONPG
was recorded in a spectrophotometer at 420 nm, and enzyme activity was calculated
according to the method of Miller (4).
Strains used for luciferase assays were grown to OD550 = 0.4, aliquoted, and
stored as glycerol stocks at -80C. Prior to freezing, cells were washed 10 times in cold
CAT medium to remove any endogenously produced CSP from the culture supernatant.
Detection of luciferase activity was essentially performed as described previously (1, 6).
After thawing glycerol stocks, 100 l of stored cells were pipetted into Eppendorf tubes,
pelleted by centrifugation, and resuspended in 1 ml fresh CAT medium supplemented
with 0.2% (w/v) bovine serum albumin. For each test sample, 280 µl diluted culture was
mixed with 20 µl of firefly D-luciferin (10 mM solution in growth medium) and
transferred into a 96-well Corning NBS plate with clear bottom. The plate was incubated
at 37°C in an Anthos Lucy 1 luminometer for 7.5 hours. Optical density (OD492) and
luminescence were measured automatically by the luminometer at 10-minute intervals.
1. Chastanet, A., M. Prudhomme, J.P. Claverys, and T. Msadek. 2001. Regulation of
Streptococcus pneumoniae clp genes and their role in competence development and stress
survival. J. Bacteriol. 183: 7295-7307.
2. Knutsen, E., O. Ween, and L.S. Håvarstein. 2004. Two separate quorum-sensing
systems upregulate transcription of the same ABC transporter in Streptococcus
pneumoniae. J. Bacteriol. 186: 3078-3085.
3. Martin, B., M. Prudhomme, G. Alloing, C. Granadel, and J.P. Claverys. 2000.
Cross-regulation of competence pheromone production and export in the early control of
transformation in Streptococcus pneumoniae. Mol. Microbiol. 38: 867-878.
4. Miller, J.H. 1972. Experiments in molecular genetics. Cold Spring Harbor Press, Cold
Spring Harbor, N.Y.
5. Morrison, D.A., S.A. Lacks, W.R. Guild, and J.M. Hageman. 1983. Isolation and
characterization of three new classes of transformation-deficient mutants of
Streptococcus pneumoniae that are defective in DNA transport and genetic
recombination. J. Bacteriol. 156:281-290.
6. Prudhomme, M. and J. P. Claverys. 2006. There will be a light: the use of luc
transcriptional fusions in living pneumococcal cells, p. in press. In R. Hakenbeck and G.
S. Chhatwal (ed.), The Molecular Biology of Streptococci. Horizon Scientific Press.
7. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning, vol. 3. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.