ABSTRACT: In consideration of the fact that BrpA expression is at its maximum during early-exponential phase as shown by Northern blotting and reporter gene fusion assays, we carried out a DNA microarray analysis using total RNA from early exponential phase cultures (OD600nm=0.3). It was found that 92 genes were up-regulated and 90 down-regulated in TW14D by a factor of at least 1.5-fold (p ≤ 0.001). At a level of P<0.01, 176 additional genes were found to be differentially expressed in TW14D, with 77 up- and 90 down-regulated. Based on the description and putative functions of the genes identified at the significance level of P<0.001, BrpA-deficiency affects almost every aspect of the cellular physiology as well as virulence properties, including amino acid biosynthesis, carbohydrates and energy metabolism, nucleic acids and DNA metabolism, transcriptional regulation, ABC transporters, molecular chaperones and other cellular processes, and hypothetical and conserved hypothetical proteins. The breadth of impact of BrpA-deficiency on the transcriptional profile of the deficient mutant is similar to what was observed previously with mid-exponential phase cells. However, comparison of the two transcriptional profiles revealed that only a small number of genes were consistently up- or down-regulated in both early- and mid-exponential phase cultures, which include recA (for recombinant protein RecA), gtfD (for glucosyltransferase D), wapA (for surface-associated protein WapA), groEL (for molecular chaperone GroEL) and sod (for Mn-dependent superoxide dismutase SOD). In addition, the magnitude of alterations in gene expression was also more dramatic in cells of the early-exponential phase, when compared to that in the mid-exponential phase cultures. Of the genes altered as a result of BrpA-deficiency in early-exponential phase cultures, several were found to encode proteins with potential roles in peptidoglycan biosynthesis. Among them are SMU.246 for a phospho-MurNAc-pentapeptide-transferase (RgpG), SMU.549 for an undecaprenyl-PP-MurNAc-pentapeptide-UDP-GlcNAc transferase (MurG), SMU.599 for a D-alanine-D-alanine ligase (DdlA), and SMU.1677 for a UDP-MurNAc-tripeptide synthetase (MurE). Besides, several genes encoding components of the Sec translocase were also found altered in TW14D. These included secA, secE and secY encoding the ATP-dependent motor of the translocation machinery, SecA, and the translocon pore components, SecE and SecY, respectively. Both secA and secE were down-regulated by more than 2-fold, while secY was up-regulated by more than 2-fold. Transcriptional profiling of S. mutans was done using a spotted array (version II) obtained from TIGR (JCVI). The wild-type UA159 and its BrpA-deficient mutant (TW14D) were grown in plain BHI broth until early phase OD=0.3. Four replicates each. A common reference (mid-exponential phase wild-type UA159) was used.
