Gene regulation in Streptococcus pneumoniae - RePub - Erasmus ...

Reverse-transcriptase PCR
RNA isolation and cDNA synthesis was performed as described above, except that
aminoallyl-dUTP was replaced by dTTP during cDNA synthesis. To confirm the absence of
DNA contamination, reactions were also carried out without reverse transcriptase. 100 ng
cDNA was used for each PCR reaction, and after 20 amplification cycles (30 sec, 95ºC; 30
sec, 52ºC; 60 sec, 72ºC) with primers G6PDH4 and G6PDH5 reactions were analysed on 1%
agarose gels.
Adhesion assays
Nitrogen Metabolism in S. pneumoniae
Adhesion of pneumococci to epithelial cells was studied essentially as desribed
previously (21). Briefly, the human pharyngeal cell line Detroit 562 (ATCC CCL-138) was
cultured in RPMI 1640 without phenol red (Invitrogen) containing 1 mM sodium pyruvate
and 10% fetal calf serum (FCS). Aliquots of bacteria, grown to mid-exponential phase in
GM17 and stored till use at -80°C, were thawed rapidly, harvested by centrifugation, and
resuspended to 1x10 7 CFU/ml in RPMI 1640 medium without phenol red containing 1% FCS.
Monolayers of Detroit 562 in 24-well tissue culture plates were washed twice with 1 ml PBS,
after which 1 ml of bacterial suspension was allowed to adhere for 2h at 37°C in a 5% CO2
atmosphere. Subsequently, non-adherent bacteria were removed by three washes with 1 ml
PBS, and the epithelial cells were detached by treatment with 200 μl of 25% Trypsin, 1 mM
EDTA in PBS. Detroit 562 cells were lysed by the addition of 800 μl of ice-cold 0.025%
Triton X-100 in PBS, and appropriate dilutions were plated on blood agar plates to count the
number of adherent bacteria. This CFU count was first corrected mathematically to account
for small differences in count in the initial inoculum, after which data were normalized so that
the adhesion of the wild-type strain TK136 was expressed as 100%. Wild type and mutant
pneumococci grew comparably in RMPI medium without Detroit 562 cells. All experiments
were performed in triplicate and repeated at least three times. Significant differences between
wild-type and mutants were calculated by the Mann Whitney t-test (p