Methods: Biofilm growth of S. mutans UA159 was simulated on polystyrene multidishes in Brain Heart Infusion broth. Planktonically cultivated S. mutans in stationary phase and mature biofilm cells were harvested, washed and their RNA was isolated. The RNA quality and quantity were determined by agarose gel electrophoresis and spectrophotometrically. An equal amount of purified RNA from each condition culture was used for cDNA synthesis. S. mutans microarrays, based on the genome sequence of UA159 were used for comparative transcriptom analysis. We utilized the real-time reverse-transcription-PCR to further investigate differential gene expression. Specific primers for 14 selected genes were designed and for each set a standard amplification curve were plotted.
Results: By comparing expression under biofilm and planktonic growth conditions, we identified and statistically validated the mostly differentiated genes. Cellobiose-specificIIA component, gramicidine S synthetase, ABC transporter binding proteins were among the up-regulated genes associated with cellular transport. Other activated gene SMU.1037C encodes a putative histidine kinase in two component system. ComCDE and its upstream region genes: SMU.1910C–SMU.1914C demonstrated significant up-regulation as well. SorABCDG encode a PTS system transport and binding proteins, which were also activated in biofilm. The genes involved in protein synthesis showed reduced level of expression, which may indicate a reduced bacterial growth rate and/or limited metabolic activity in biofilms.
Conclusion: Relatively few S. mutans genes showed significant differential expression in biofilms compared with planktonic cultures. This study provides new insights of this bacterium gene expression under biofilm conditions.
Microarrays were provided by the NIDCR through a contract with the PFGRC at TIGR.