Transcriptional regulation of two divergently transcribed genes from Streptococcus parasanguis

Two divergently transcribed loci of Streptococcus parasanguis FW213 have been previously cloned and sequenced. One gene, designated S. parasanguis pepO, encodes an enzyme with homology and activity similar to that of mammalian metallopeptidases, such as neutral endopeptidase and endothelin- converting enzyme 1. The locus that is upstream and divergently transcribed from pepO is the fimA operon which encodes an ATP-binding cassette transporter system. The coding regions of the two loci are separated by only 148 nucleotides. Objectives: To investigate the hypothesis that the promoter and regulatory regions of these two loci overlap and that the fimA operon and pepO may be coordinately regulated. Methods: The transcriptional start sites for both pepO and the fimA operon were mapped by primer extension analysis. Luciferase reporter analysis, as well as ELISA and immunoblot analysis, were used to investigate the effect of Mn²⁺ on transcriptional regulation and protein expression of genes encoded by these loci. Results: It was determined that the minimally defined promoter regions of pepO and the fimA operon do indeed overlap. It was also shown that the fimA operon transcript, a polycistronic message, has only one transcriptional start site, whereas the pepO message, which is monocistronic, has three transcriptional start sites. RNA folding analysis of the 5' untranslated region of pepO indicated that the two major transcriptional start sites occur on either side of a stem loop structure. This suggests that RNA stability rather than transcriptional regulation may determine which pepO transcript is most abundant. Luciferase reporter analysis revealed that the fimA operon promoter was regulated by Mn²⁺. Immunoblot and ELISA analysis indicated that FimA expression was repressed by Mn²⁺. In contrast, PepO expression appears to be constitutive. Conclusions: These data indicate that at least in terms of Mn²⁺ utilization the fimA operon and pepO are not coordinately regulated. This research was supported by NIH-NIDR grant R37DE11000.