Transcription profiling of S. pneumoniae D39, a serotype 2 strain of pneumococcus, with that of an isogenic deletion mutant, (delta)luxS, time series

ABSTRACT: We were interested in determining if the LuxS signaling system contributed to the regulation of pneumococcal genes. To accomplish this, we compared the in vitro transcriptional profiles over time of S. pneumoniae D39, a serotype 2 strain of pneumococcus, with that of an isogenic deletion mutant, (delta)luxS, using a spotted S. pneumoniae- specific DNA microarray. For in vitro time course experiments, broth cultures of D39 and (delta)luxS were grown to low density (optical densities between ~0.01- 0.02), the cells were collected by centrifugation, washed 1x with sterile PBS, back diluted to ~3x105CFU/ ml into fresh broth, and immediately incubated at 37oC in a 5% CO2 chamber for 45 minutes. At this point, the first sample was collected. Subsequently, growth was monitored for approximately 8 generations by measuring both the optical density (OD600) and determining viable counts every 30 to 45 minutes. At these points samples were also harvested by centrifugation for RNA isolation and stored at -80oC until processing. For microarray probe synthesis, experimental samples were generated using 0.5ug total bacterial RNA isolated from either D39 or (delta)luxS strains at the various time points throughout the growth curve. These RNAs were used as templates for reverse transcription, and the resulting cDNAs were labelled with Cy5. The reference sample for each time course was generated from the D39 time zero RNA. Subsequent to reverse transcription, the resulting cDNAs were labelled with Cy3. The probes were mixed in hybridization buffer, heated to 99oC for at least 2 minutes, centrifuged briefly, and applied to the microarray. Hybridizations were carried out at 60oC for at least 24 hours.

ORGANISM(S): Streptococcus pneumoniae

SUBMITTER: Elizabeth Joyce 

PROVIDER: E-SMDB-2173 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress