Methods: Various depth biofilm of S. mutans UA159 were generated in a constant-depth film fermenter (CDFF) and characterized by confocal laser scanning microscope (CLSM). The harvested cells from 100, 200 and 400-micron depth biofilms were subjected to the extraction of total RNA. An equal amount of purified RNA from each depth biofilms was used for cDNA synthesis. Probe labeling, hybridization and washing conditions were conducted according to http://www.tigr.org. The microarray data were normalized using global loess normalization. To identify differentially expressed genes, a parametric empirical Bayesian approach implemented in LIMMA was used.
Results: Direct comparison of the optical images of the CLSM consistently demonstrated that biofilm thickness is accompanied by significant shifts in cell viability. Analyses of microarray images indicated that expression of 20 genes was significantly changed in 400 vs. 100 microns and 49 genes in 200 vs. 100 microns biofilms. Only a small amount (8) of S. mutans genes showed significant differential expression in both 400 vs. 100-microns and 200 vs. 100-microns biofilms. Several of differentially expressed genes appear to modulate adherence and biofilm formation in S. mutans, such as SMU987, which encodes a cell wall-associated protein precursor WapA. SMU744 encoding to the membrane-associated receptor protein FtsY, involved in the signal recognition particle translocation pathway, was also found among those differentially expressed genes.
Conclusion: This study provides a genome-scale outline and adds important insights of gene expression in biofilm thickness process of S. mutans, which are highly associated in the pathogenesis of dental diseases.
Microarrays were provided by NIDCR through PFGRC at TIGR. This work was supported partially by the Norton-Ross Foundation of IADR.