RNase E cleavage shapes the transcriptome of Rhodobacter sphaeroides and strongly impacts phototrophic growth.

Bacteria adapt to changing environmental conditions by rapid changes in their transcriptome. This is achieved not only by adjusting rates of transcription but also by processing and degradation of RNAs. We applied TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) for the transcriptome-wide identification of RNase E cleavage sites and of 5' RNA ends, which are enriched when RNase E activity is reduced in Rhodobacter sphaeroides. These results reveal the importance of RNase E for the maturation and turnover of mRNAs, rRNAs, and sRNAs in this guanine-cytosine-rich α-proteobacterium, some of the latter have well-described functions in the oxidative stress response. In agreement with this, a role of RNase E in the oxidative stress response is demonstrated. A remarkably strong phenotype of a mutant with reduced RNase E activity was observed regarding the formation of photosynthetic complexes and phototrophic growth, whereas there was no effect on chemotrophic growth.

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

Bacterial regulatory networks include direct contact of response regulator proteins: interaction of RegA and NtrX in Rhodobacter capsulatus.

The formation of photosynthetic complexes in facultatively photosynthetic bacteria is controlled by the oxygen tension in the environment. In Rhodobacter capsulatus the two-component system RegB/RegA plays a major role in the redox control of photosynthesis genes but also controls other redox-dependent systems. The response regulator RegA is phosphorylated under low oxygen tension and activates the puf and puc operons, which encode pigment binding proteins, by binding to their promoter regions. Data from a yeast two-hybrid analysis as well as an in vitroanalysis indicate that RegA interacts with the NtrX protein, the response regulator of the NtrY/NtrX two-component system which is believed to be involved in regulation of nitrogen fixation genes. Our further analysis revealed that NtrX is indeed involved in the regulation of the puf and puc operons. Furthermore, we showed that an altered NtrX protein, which is predicted to adopt the conformation of phosphorylated NtrX protein, binds within the puf promoter region close to the RegA binding sites. We conclude that a direct interaction of two response regulators connects the regulatory systems for redox control and nitrogen control.

The AppA and PpsR proteins from Rhodobacter sphaeroides can establish a redox-dependent signal chain but fail to transmit blue-light signals in other bacteria.

The AppA protein of Rhodobacter sphaeroides has the unique ability to sense and transmit redox and light signals. In response to decreasing oxygen tension, AppA antagonizes the transcriptional regulator PpsR, which represses the expression of photosynthesis genes, including the puc operon. This mechanism, which is based on direct protein-protein interaction, is prevented by blue-light absorption of the BLUF domain located in the N-terminal part of AppA. In order to test whether AppA and PpsR are sufficient to transmit redox and light signals, we expressed these proteins in three different bacterial species and monitored oxygen- and blue-light-dependent puc expression either directly or by using a luciferase-based reporter construct. The AppA/PpsR system could mediate redox-dependent gene expression in the alphaproteobacteria Rhodobacter capsulatus and Paracoccus denitrificans but not in the gammaproteobacterium Escherichia coli. Analysis of a prrA mutant strain of R. sphaeroides strongly suggests that light-dependent gene expression requires a balanced interplay of the AppA/PpsR system with the PrrA response regulator. Therefore, the AppA/PpsR system was unable to establish light signaling in other bacteria. Based on our data, we present a model for the interdependence of AppA/PpsR signaling and the PrrA transcriptional activator.

Thioredoxin can influence gene expression by affecting gyrase activity.

The expression of many genes of facultatively photosynthetic bacteria of the genus Rhodobacter is controlled by the oxygen tension. Among these are the genes of the puf and puc operons, which encode proteins of the photosynthetic apparatus. Previous results revealed that thioredoxins are involved in the regulated expression of these operons, but it remained unsolved as to the mechanisms by which thioredoxins affect puf and puc expression. Here we show that reduced TrxA of Rhodobacter capsulatus and Rhodobacter sphaeroides and oxidized TrxC of R.capsulatus interact with DNA gyrase and alter its DNA supercoiling activity. While TrxA enhances supercoiling, TrxC exerts a negative effect on this activity. Furthermore, inhibition of gyrase activity strongly reduces puf and puc expression. Our results reveal a new signaling pathway by which oxygen can affect the expression of bacterial genes.