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Schematic diagram of the rex flanking region and transcriptional analysis of the rex operon.
(A) Schematic diagram of regions flanking rex, with the arrows indicating the direction of transcription and the numbers above indicating the sizes of the respective open reading frames in base pairs. Gene assignments and gene numbers above the diagram are based on Oralgen annotation. (B) RT-PCR analysis of rex operon. Following reverse transcription with iScriptase using primer GuaA-Rv, PCR amplification was performed with the primers Rex-Fw and GuaA-Rv, with RT products with and without iScriptase and genomic DNA as a positive control. Panel shows agarose gel electrophoresis of the PCR products. Lanes M, 1, 2, and 3 are MW marker, RT-PCR product, negative control without RT, and positive control with gDNA as a template. (C) EMSA analysis shows interaction of rex promoter with recombinant Rex. Inclusion of rRex resulted in mobility shift and such interaction was concentration-dependent. (D) Alignment of promoter regions of selected genes identified in TW239 that contain putative Rex binding sites. −10 and −35 regions as determined by BPROM program are in bold faces and putative Rex-binding sites are underlined or double-underlined in case where more than one site is identified. Positions of these elements were shown in numbers relative to start codon ATG of the respective genes. Consensus sequence was the conserved Rex-binding sites identified in B. subtilis and S. aureus. (E) Alignment of the putative and proven Rex-binding sites of the up-regulated genes in TW239. Promoters were scanned and aligned to the consensus Rex-binding site from S. aureus and B. subtilis. The indentified nucleotide sequences were then subjected to analysis using WebLogo (University of California, Berkeley) to generate the S. mutans consensus. Results showed that the Rex-binding site in S. mutans possesses more variability in the nucleotide composition than the other model organisms.