, 2006; Wen et al., 2006, 2010a, b). Previously, we reported that deficiency of BrpA (for biofilm regulatory protein A) in S. mutans caused major defects in the ability of the deficient mutants to tolerate acid and oxidative stresses and the ability to accumulate biofilms (Wen & Burne, 2002; Wen et al., 2006). The rex gene was found to be significantly decreased in the BrpA-deficient mutant, TW14D, during the early-exponential phase of growth (data not

shown), suggesting that rex expression is influenced by BrpA and that rex may be involved in the regulation of stress tolerance response and/or biofilm formation by S. mutans. To verify that rex is indeed a part of the BrpA-regulon, the expression of rex was analyzed using RealTime-PCR with total RNAs extracted from cultures grown in BHI and harvested during early (OD600 nm≅0.2), mid (OD600 nm≅0.4), and late (OD600 nm≅0.6) exponential Forskolin phase, respectively. The expression of rex in the wild-type strain was at its highest level during early-exponential phase, averaging 7.85E+07 copies μg−1 of total RNA, although the underlying mechanism governing the regulation remains unclear. Consistent with microarray data, rex expression in TW14D was decreased by more than sixfold during this period of growth, with an average of only 1.00E+07 copies μg−1 of total RNA

(P<0.001). However, no significant differences were observed in cells from mid- or late-exponential phase cultures (data not shown). To investigate whether Rex could be associated with phenotypes observed in BrpA-deficient mutants, an internal find more fragment (nucleotides 136–584 relative to the translational initiation site) of the rex gene was deleted and replaced with

a nonpolar kanamycin resistance element (Zeng et al., 2006). Rex-deficiency did not have a major impact on the morphology and growth rate in planktonic cultures in BHI (Fig. 1a). However, when biofilm formation in 96-well culture plates was analyzed (Loo et al., 2000; Wen & Burne, 2002), the Rex-deficient mutant, TW239, was shown to accumulate only a small fraction of the biofilms of the wild-type, UA159. Following staining with 0.1% crystal violet after 24 h, the OD575 nm of mutant biofilms was 3.5-fold (P<0.001) less than that of the wild-type strain when DNA ligase grown on glucose (Fig. 1b) and decreased by more than threefold (P<0.001) when sucrose was the carbohydrate source (Abstract, 87th IADR Annual Conference #2652). When grown on glass slides in BMGS (Nguyen et al., 2002; Wen et al., 2010a, b), the biofilms formed by TW239 after 3 days were about 6.2-fold less abundant than those formed by UA159, with an average of 1.82E7(±1.02E7) CFU for TW239 vs. 1.13E8(±2.88E7) (P<0.001) for UA159. Similar results were also observed with biofilms grown on hydroxylapatite discs, a commonly used in vitro tooth model. As compared with the wild-type, biofilms of the Rex-deficient mutant also had an altered structure.