Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ

Abstract

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

Fig. 1.

Involvement of PcrZ in the regulatory network controlling photosynthesis gene expression. At low oxygen tension PrrA and FnrL activate expression of photosynthesis (PS) genes and at the same time expression of PcrZ. PcrZ counteracts the activation by PrrA and FnrL and represses photosynthesis genes, indicating an incoherent feed-forward loop. Also the appA gene is repressed by PcrZ, reducing the amount of AppA, which further leads to a stronger repression of photosynthesis genes by PpsR.

Fig. 2.

PrrA regulates oxygen-dependent expression of PcrZ. (A) Genetic context of the pcrZ gene. The gene RSP_0819 encoding a DEAD/DEAH box helicase ends 91 nt upstream of PcrZ and is preceded by a PrrA consensus promoter. The hypothetical protein RSP_6134 starts 134 nt downstream and is preceded by a FnrL consensus promoter. (B) PrrA-dependent expression of PcrZ shown by Northern blot analysis of total RNA, isolated from R. sphaeroides WT 2.4.1 and PrrA2 (prrA⁻). Cultures were grown under high-oxygen conditions (HO) and shifted to low-oxygen conditions (LO) at t₀. The oxygen tension dropped from 8 to 0.5 mg/L during the first 30 min. A PcrZ-specific oligonucleotide was used as a probe for hybridization. Detection of PcrZ primary transcript (136 nt) and processed fragments (127 nt, 73 nt, and 51–56 nt) is indicated. 5S rRNA served as a loading control.

Fig. 3.

The stable processing product of PcrZ stems from its 5′ end. (A) Predicted secondary structure of PcrZ by the Mfold program (25). Solid stars indicate the five different 3′ ends, identified by 3′ RACE. Hybridization sites of different oligonucleotide probes used for Northern blot analysis are displayed (5′ probe in gray and 3′ probe in black). (B) Northern blot analysis of total RNA, isolated from R. sphaeroides 2.4.1pRK4352 and 2.4.1pRKPcrZ. Cultures were grown under low-oxygen conditions to an OD₆₆₀ of 0.8. Two different oligonucleotides, binding near the 5′ and 3′ ends of PcrZ, were used for hybridization. Detection of PcrZ primary transcript (136 nt) and processed fragments (127 nt, 73 nt, and 51–56 nt) is indicated. tmRNA (coding piece), 5S rRNA, and tRNA-Ala oligonucleotides served as internal size markers. (C) Quantification of Northern blot signals from strains 2.4.1pRK4352 (solid bars) and 2.4.1pRKPcrZ (open bars). RNA levels were calculated after normalizing PcrZ signal intensities to 5S rRNA signal intensities (B, 5′ probe). 2.4.1pRK4352 intensities were set to 1 and fold changes of 2.4.1pRKPcrZ were calculated relative to 2.4.1pRK4352.

Fig. 4.

PcrZ reduces levels of photosynthetic complexes. (A) Absorption spectra of R. sphaeroides whole-cell extracts. Three independent cultures of each strain were grown under low-oxygen conditions to an OD₆₆₀ of 0.8. The absorbance was measured from 500 nm to 900 nm. One representative spectrum of 2.4.1pRK4352 (solid dashed line), 2.4.1pRKPcrZ (shaded dashed line), and 2.4.1pRKPcrZ-51nt (shaded line) is shown. Peaks at 800 and 850 nm correspond to the light-harvesting complex II (B800–850) and the light-harvesting complex I (B870) of the photosynthetic apparatus. (B) Relative bacteriochlorophyll content of R. sphaeroides strains 2.4.1pRK4352 (solid bar), 2.4.1pRKPcrZ-51nt (shaded bar), and 2.4.1pRKPcrZ (open bar) grown under low-oxygen conditions to an OD₆₆₀ of 0.8. The relative bacteriochlorophyll content was calculated on the basis of the absorbance at 770 nm after acetone-methanol (7:2) extraction of 4 mL cells, normalized to the OD₆₆₀. Results from three independent experiments are shown with error bars depicting the SE of mean.

Fig. 5.

PcrZ directly targets bchN and puc2A. (A) Predicted PcrZ-bchN duplex structure. Single- and triple-nucleotide exchanges of PcrZ and bchN are indicated by shaded arrows. Position +1 of the mRNA refers to the A of the translational start codon (shaded). For PcrZ position 1 refers to the 5′ nucleotide. (B) Predicted PcrZ-puc2A duplex structure. (C) Schematic picture of mRNA::lacZ fusions. 16S rRNA promoter is depicted as P₄₃₅₂. “+1” indicates the first nucleotide of the corresponding start codon. (D) Relative β-galactosidase activity of the lacZ-based in vivo reporter system. Activity in the control strains containing the bchN::lacZ, bchN-M1::lacZ, or bchN-M3::lacZ reporter fusions and plasmid pBBR4352 is set to 100% (open bar). Activities for all other combinations are calculated in relation to their respective control. Activity in the strain containing the reporter fusion and plasmid pBBRPrcZ is indicated by a solid bar. Bars with light shading and bars with dark shading represent strains overexpressing PrcZ with single (M1) or triple (M3) mutations, respectively. Compensatory mutations of bchN in the reporter plasmid are represented by dashed bars. (E) Relative β-galactosidase activity of the lacZ-based in vivo reporter system. Activity in the control strains containing the reporter fusion and plasmid pBBR4352 is set to 100% (open bar). Activity of strains with plasmid pBBRPcrZ and the RSP_0557::lacZ reporter (bar with dark shading), the puc2A::lacZ reporter (solid bar), and the appA::lacZ reporter (bar with light shading) is shown. For each strain, three independent biological experiments with technical duplicates were performed. SDs are depicted by error bars. β-Galactosidase assays were performed as described previously (42).