Figure 1.
RpoHI and RpoHII accumulation following heat and singlet oxygen stresses.
Western blots illustrating the levels of RpoHI and RpoHII in wild-type R. sphaeroides (WT) at different times following (A) a shift of temperature from 30°C to 42°C (heat shock) or (B) addition of the photosensitizer methylene blue in the presence of oxygen (singlet oxygen stress). On the same western blots, the levels of FLAG-RpoHI and RpoHII obtained from ectopic expression vectors used in the expression profiling and ChIP-chip experiments under normal conditions. Note that because of the addition of the FLAG polypeptide, RpoHI-FLAG migrates slower than the wild-type RpoHI. The abundance of RpoHI and RpoHII in wild-type cells in the absence of added stress are shown in the first lane. As a gel loading control, the membranes were also subsequently treated polyclonal antibodies against the response regulator PrrA, a control transcription factor who's expression is not known to be dependent on either of the RpoH homologs. The experiment was designed to analyze changes in levels of RpoHI, RpoHII and PrrA before and after a stress, so the differences between panels reflect different exposure times used when developing the Western blots.
Figure 2.
Overlap between the RpoHI and RpoHII regulons.
Venn diagram representing the overlaps between genes that were significantly induced by the expression of RpoHI or RpoHII, and genes whose promoters were bound by RpoHI or RpoHII containing RNA polymerase holoenzyme in vivo. The total numbers of genes identified in each study are indicated in the parentheses. The RpoHI (solid outline) and RpoHII (dashed outline) regulons, as defined in this study, are identified by the emphasized outlines. The total numbers of genes contained in each regulon are indicated below the arrows.
Table 1.
Compositions of the RpoHI and RpoHII regulons.
Figure 3.
Conserved promoter sequences recognized by RpoHI and RpoHII.
The logos were constructed from promoter sequences alignments sorted into three categories according to their predicted specificity. The consensus sequence for σ32-dependent promoters in E. coli,as determined by Nomaka et al. [37], is shown as a reference. The heights of the letters represent the degree of conservation across sequences (information in bits, logos generated using WebLogo: http://weblogo.berkeley.edu/). The coordinates on the x-axes represent the positions relative to the predicted transcription start site. The numbers of promoter sequences used to create the logos are indicated in parentheses on the left of the logos. Below the logos are the sequence alignments of selected promoters that were used for direct experimental validation.
Figure 4.
Relative activities of selected RpoHI- and RpoHII-dependent promoters.
β-galactosidase activity of lacZ operon fusions with selected R. sphaeroides promoter regions monitored in tester strains expressing RpoHI (black), RpoHII (grey), or neither proteins. Genes are grouped according to the gene expression profiles displayed in the gene expression experiments: genes whose expressions were affected only by RpoHI, only by RpoHII, and by both RpoHI and RpoHII. Error bars represents the standard error of the mean from three independent replicates.
Figure 5.
Activities of selected mutant promoters when transcribed by RpoHI or RpoHII.
β-galactosidase activity of lacZ operon fusions with selected mutant E. coli (A) or R. sphaeroides (B) promoters monitored in an E. coli tester strain expressing R. sphaeroides RpoHI. The original promoter and specific base substitutions are indicated below the x-axis. (C) β-galatosidase activity of lacZ operon fusions integrated into the genome containing either the wild type of indicated mutant R. sphaeroides cycA P1 promoter in a tester strain expressing the indicated RpoH homolog. Most of the promoter mutations were made in the G-36T cycA P1 background, as this promoter had activity with RpoHI or RpoHII than its wild type (WT) counterpart. Base substitutions are indicated on the x-axis. Error bars represents the standard error of the mean from three independent replicates.