Electrophoretic mobility shift assays (EMSA) with selected upstream DNA fragments of PTS coding regions using the purified SugR protein

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> The physical maps of the extended fructose-PTS gene cluster as well as of the genes and are shown. Beneath the maps the fluorescently labelled PCR fragments are indicated which were used for EMSA studies. These studies were carried out with 15 pmol of purified SugR protein and 0.05 pmol of labeled PCR fragments. The results obtained for each PCR fragment are presented by agarose gel photos. In each picture, the left lane shows the shift in presence of the SugR protein, whereas the right lane shows the negative control without added SugR protein. Transcriptional terminators are denoted as stem loop structures

Comparison of experimentally determined and predicted SugR binding sites located upstream of the coding regions of , , , and and construction of a consensus sequence

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> A) Predicted binding motifs were determined by sequence comparisons of proven motifA and motifB to the DNA fragments showing positive SugR binding in the EMSA studies. Proven and predicted motifs are separated by a horizontal, dashed line. Boxed letters in the experimentally proven motifs A and B and the putative SugR binding sequences located upstream the coding regions of , , and denote identical nucleotides in all sequences. Distances to the according translation starts (TL) are indicated. B) A frequency plot of the deduced consensus sequence of all motifs is constructed by means of the WebLogo tool. The overall height of each stack of letters indicates the sequence conservation at each position of the 21-bp motif, whereas the height of each symbol within the stack reflects the relative frequency of the corresponding nucleotide at that position

Transcriptional regulation of the extended PTS cluster genes of cultures grown in liquid media containing glucose, fructose, or acetate

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> The strain RES167 was grown in liquid media containing glucose, fructose, or acetate. By RT-PCR the mRNA levels of the genes , , , , , , and of cultures grown in glucose (white bars) or fructose (grey bars) were compared to those of cultures grown in acetate containing media. Results are means of four measurements from two biological replicates. Standard deviations are indicated by error bars

Identification of effector substances inactivating the repressor protein SugR

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> Electrophoretic mobility shift analysis of 15 pmol purified SugR protein complexed with 0.05 pmol of the DNA fragment I2 in the presence of different putative effectors were conducted. Effectors applied to the SugR/I2-complex at varying concentrations as described were A) fructose-1-phosphate (F-1-P), B) fructose-1,6-bisphosphate (F-1,6-P), C) fructose-6-phosphate (F-6-P), and D) glucose-6-phosphate (G-6-P

Analyses of the transcriptomes of the mutant LG01 and its parent strain RES167 by microarray hybridizations

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> The strains were grown in liquid minimal media containing acetate as sole carbon source. Global gene expression of LG01 was compared to that of RES167 using microarray analyses performed on two biological and two technical replicates. Genes found to be significantly differentially expressed (m-value ≥ ± 1) are indicated by green (elevated expression) or red (decreased expression) dots, respectively. Gene names belonging to the fructose-PTS gene cluster are boxed, and the non clustered PTS genes are underlined

The transcriptional organization of the genes in the extended fructose-PTS cluster of

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> A) The extended fructose-PTS gene cluster as well as its transcriptional elements are depicted. Gene overlapping RT-PCR products are indicated by black bars and numbered. Transcriptional terminators predicted by TransTerm [26] are denoted as stem loop structures. Positions of the transcription start sites are indicated by arrows. B) The detailed sequences for the promoters P, P, P, and P. Transcription starts (+1), the distances to the translation start (in parentheses), and the start codons (underlined) are shown. The deduced-10 and -35 promoter regions are highlighted by dark and light grey boxes, respectively

The organization of the intergenic region between the genes and containing the binding motifs A and B

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> The double stranded sequence of the intergenic region between and is shown. The experimentally proven binding motifs A and B are also boxed. The transcriptional start sites for the two genes are indicated as Pand P. The predicted -10 and -35 promoter regions are shown as dark grey boxes, respectively. The translational start codons of and are underlined

Relative gene expressions of fructose-PTS cluster genes in dependence on the regulatory genes and

<p><b>Copyright information:</b></p><p>Taken from "The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in "</p><p>http://www.biomedcentral.com/1471-2199/8/104</p><p>BMC Molecular Biology 2007;8():104-104.</p><p>Published online 15 Nov 2007</p><p>PMCID:PMC2222622.</p><p></p> The strains LG01, LG02, and RES167 were grown in liquid media containing acetate as sole carbon source. By RT-PCR the mRNA levels of the fructose-PTS cluster genes , , , , and of the mutant strains LG01 and LG02 were compared to those of RES167. Due to the deletion of the coding region, the expression of the truncated gene in the mutant LG02 could not be determined. Results are means of four measurements from two biological replicates. Standard deviations are indicated by error bars