MM

					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 37C, and growth was

monitored by measuring OD at 550 nm or 492 nm. E. coli was grown in Luria-Bertani

(LB) medium (7) at 37C. 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

erythromycin.



Construction of transcriptional fusions and modifications of boxAB2C at the qsrAB

locus

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

locus

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–
1
    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.



Reporter assays

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 -80C. 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.


REFERENCES

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.

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:3
posted:11/23/2012
language:English
pages:6