To test this hypothesis, DNA electrophoretic mobility shift assay were carried out. To do so, the His6-Rgg0182 protein was overproduced in E. coli C41(DE3), verified by SDS-PAGE and Western blot (data not shown). Immobilized Metal ion Affinity Chromatography (IMAC) purification of the His6-Rgg0182 protein was performed. The purity of the Rgg0182 protein was selleck products assessed by SDS-PAGE using Coomassie blue protein staining, i.e. only one band of the expected molecular mass (35.7 kDa) was revealed (data not shown). A 126 bp PCR amplified DNA fragment (Figure 1), including the entire 72 bp intergenic rgg 0182 -shp 0182 region and part of the 5′ end of the shp 0182 and rgg 0182 genes,

was incubated with the purified His6-Rgg0182 protein. As can be seen in Figure 4, the Rgg0182 protein retarded the shp 0182 promoter DNA fragment. The same experiment was realized with a 165 bp PCR amplified fragment, covering the entire

150 bp intergenic rgg 0182 -pep 0182 region including the pep 0182 promoter, and analogous results were obtained (Figure 4). The URMC-099 mw P ldh probe corresponding to the promoter region of the ldh gene was chosen as a negative control in EMSA experiments since its expression was not under the control of Rgg0182. Using P ldh as a probe, no DNA retardation was observed, demonstrating that Rgg0182 binds specifically to the promoter of its target genes. Thus, these results demonstrated conclusively that Rgg0182 activated the shp 0182 and pep 0182 genes transcription by binding to their promoter regions. Figure 4 Analysis of the Rgg 0182 binding to DNA. Electrophoretic mobility shift assay (EMSA) of the promoter regions of the two target genes (shp 0182 and pep 0182 ) of Rgg0182 in the absence or in the presence of the purified His6-Rgg0182 protein. DNA probes labelled with biotin (0.1 pmol each) were incubated with 2 pmol of Rgg0182. The P ldh probe is an ldh promoter fragment used as a negative control. Effects of the Rgg0182

protein on the transcription Thymidine kinase of genes encoding protease and chaperone proteins The impact of temperature on the rgg 0182 gene transcription suggested a role for the Rgg0182 protein on S. thermophilus LMG18311 adaptation to thermal changes. Thus, we hypothesized that Rgg0182 might control the transcription of genes encoding a set of heat- and cold-shock proteins including GSK458 cell line chaperones and proteases. Chaperones and ATP-dependent proteases play a major role for bacterial survival under conditions of heat stress where proteins tend to unfold and aggregate. Based upon the S. thermophilus LMG18311 genome sequence [26], genes predicted to encode the major chaperones and proteases involved in heat shock responses were selected for analysis: clpC, dnaK, dnaJ, hsp33, groES, groEL, clpP, clpX, clpE, clpL (Genbank Accession NC_006448, locus tags stu0077, stu0120-0121, stu0180, stu0203-0204, stu0356, stu0581, stu0602, stu1614, respectively).