The use of chromatin immunoprecipitation to define PpsR binding activity in Rhodobacter sphaeroides 2.4.1

Abstract

The expression of genes involved in photosystem development in Rhodobacter sphaeroides is dependent upon three major regulatory networks: FnrL, the PrrBA (RegBA) two-component system, and the transcriptional repressor/antirepressor PpsR/AppA. Of the three regulators, PpsR appears to have the narrowest range of physiological effects, which are limited to effects on the structural and pigment biosynthetic activities involved in photosynthetic membrane function. Although a PrrA(-) mutant is unable to grow under photosynthetic conditions, when a ppsR mutation was present, photosynthetic growth occurred. An examination of the double mutant under anaerobic-dark-dimethyl sulfoxide conditions using microarray analysis revealed the existence of an "extended" PpsR regulon and new physiological roles. To characterize the PpsR regulon and to better ascertain the significance of degeneracy within the PpsR binding sequence in vivo, we adapted the chromatin immunoprecipitation technique to R. sphaeroides. We demonstrated that in vivo there was direct and significant binding by PpsR to newly identified genes involved in microaerobic respiration and periplasmic stress resistance, as well as to photosynthesis genes. The new members of the PpsR regulon are located outside the photosynthesis gene cluster and have degenerate PpsR binding sequences. The possible interaction under physiologic conditions with degenerate binding sequences in the presence of other biologically relevant molecules is discussed with respect to its importance in physiological processes and to the existence of complex phenotypes associated with regulatory mutants. This study further defines the DNA structure necessary for PpsR binding in situ.

FIG. 1.

Spectral analysis of photosynthetic complexes. Extracts from R. sphaeroides strains 2.4.1 (solid line) and PrrA⁻ PpsR⁻ (dashed line) containing equal amounts of protein and grown under anaerobic-dark-DMSO conditions were used.

FIG. 2.

Validation of microarray data by qRT-PCR. (A) Expression of the ppsR (RSP0282) gene measured by qRT-PCR. Experiments were performed with R. sphaeroides 2.4.1 (open bar) and 2.4.1(pPNs) (shaded bar) grown under anaerobic-dark-DMSO conditions. (B) Changes in expression of selected genes measured by qRT-PCR. Changes (expressed as relative changes on a logarithmic scale) were calculated using average values normalized with the RSP0154 gene from two independent experiments with standard deviations that did not exceed 15%. All strains were grown under anaerobic-dark-DMSO conditions. The R. sphaeroides PrrA⁻ strain was compared to the 2.4.1 strain (open bars), the R. sphaeroides PrrA⁻ PpsR⁻ strain was compared to strain 2.4.1 (bars with dots), the R. sphaeroides PrrA⁻ strain was compared to the PrrA⁻ PpsR⁻ strain (bars with horizontal stripes), and R. sphaeroides 2.4.1(pPNs) was compared to strain 2.4.1 (filled bars).

FIG. 3.

ChIP analysis of in vivo binding of PpsR inside the PGC. qRT-PCR was performed with immunoprecipitated samples of the wild-type (open bars) and PrrA⁻ PpsR⁻ double-mutant (bars with vertical stripes) strains of R. sphaeroides grown under aerobic (open bars) and anaerobic-dark-DMSO conditions (shaded bars). Enrichment (expressed as relative changes on a logarithmic scale) was calculated as described in Materials and Methods. The error bars indicate standard deviations. AB, antibody.

FIG. 4.

ChIP analysis of in vivo binding of PpsR outside the PGC. Selected DNA regions of newly identified members of the PpsR regulon were examined by performing qRT-PCR with immunoprecipitated samples of the wild-type (open bars) and PrrA⁻ PpsR⁻ double-mutant (bars with vertical stripes) strains of R. sphaeroides as the matrix. The strains were grown under aerobic (open bars) and anaerobic-dark-DMSO (shaded bars) conditions. Enrichment (expressed as relative changes on a logarithmic scale) was calculated as described in Materials and Methods. The error bars indicate standard deviations. AB, antibody.

FIG. 5.

PpsR binding site degeneration. The diagram was created after ChIP analysis using PpsR binding sequences shown in Table 3 and the WebLogo program (; http://weblogo.berkeley.edu/).