RNase III participates in control of quorum sensing, pigmentation and oxidative stress resistance in Rhodobacter sphaeroides.

RNase III is a dsRNA-specific endoribonuclease, highly conserved in bacteria and eukarya. In this study, we analysed the effects of inactivation of RNase III on the transcriptome and the phenotype of the facultative phototrophic α-proteobacterium Rhodobacter sphaeroides. RNA-seq revealed an unexpectedly high amount of genes with increased expression located directly downstream to the rRNA operons. Chromosomal insertion of additional transcription terminators restored wild type-like expression of the downstream genes, indicating that RNase III may modulate the rRNA transcription termination in R. sphaeroides. Furthermore, we identified RNase III as a major regulator of quorum-sensing autoinducer synthesis in R. sphaeroides. It negatively controls the expression of the autoinducer synthase CerI by reducing cerI mRNA stability. In addition, RNase III inactivation caused altered resistance against oxidative stress and impaired formation of photosynthetically active pigment-protein complexes. We also observed an increase in the CcsR small RNAs that were previously shown to promote resistance to oxidative stress. Taken together, our data present interesting insights into RNase III-mediated regulation and expand the knowledge on the function of this important enzyme in bacteria.

Ribonuclease E strongly impacts bacterial adaptation to different growth conditions.

Adaptation of bacteria to changes in their environment is often accomplished by changes of the transcriptome. While we learned a lot on the impact of transcriptional regulation in bacterial adaptation over the last decades, much less is known on the role of ribonucleases. This study demonstrates an important function of the endoribonuclease RNase E in the adaptation to different growth conditions. It was shown previously that RNase E activity does not influence the doubling time of the facultative phototroph Rhodobacter sphaeroides during chemotrophic growth, however, it has a strong impact on phototrophic growth. To better understand the impact of RNase E on phototrophic growth, we now quantified gene expression by RNA-seq and mapped 5' ends during chemotrophic growth under high oxygen or low oxygen levels and during phototrophic growth in the wild type and a mutant expressing a thermosensitive RNase E. Based on the RNase E-dependent expression pattern, the RNAs could be grouped into different classes. A strong effect of RNase E on levels of RNAs for photosynthesis genes was observed, in agreement with poor growth under photosynthetic conditions. RNase E cleavage sites and 5' ends enriched in the rnets mutant were differently distributed among the gene classes. Furthermore, RNase E affects the level of RNAs for important transcription factors thus indirectly affecting the expression of their regulons. As a consequence, RNase E has an important role in the adaptation of R. sphaeroides to different growth conditions. [Figure: see text].

The Small RNA-Binding Protein CcaF1 Promotes Formation of Photosynthetic Complexes in Rhodobacter sphaeroides.

In natural habitats, bacteria frequently need to adapt to changing environmental conditions. Regulation of transcription plays an important role in this process. However, riboregulation also contributes substantially to adaptation. Riboregulation often acts at the level of mRNA stability, which is determined by sRNAs, RNases, and RNA-binding proteins. We previously identified the small RNA-binding protein CcaF1, which is involved in sRNA maturation and RNA turnover in Rhodobacter sphaeroides. Rhodobacter is a facultative phototroph that can perform aerobic and anaerobic respiration, fermentation, and anoxygenic photosynthesis. Oxygen concentration and light conditions decide the pathway for ATP production. Here, we show that CcaF1 promotes the formation of photosynthetic complexes by increasing levels of mRNAs for pigment synthesis and for some pigment-binding proteins. Levels of mRNAs for transcriptional regulators of photosynthesis genes are not affected by CcaF1. RIP-Seq analysis compares the binding of CcaF1 to RNAs during microaerobic and photosynthetic growth. The stability of the pufBA mRNA for proteins of the light-harvesting I complex is increased by CcaF1 during phototrophic growth but decreased during microaerobic growth. This research underlines the importance of RNA-binding proteins in adaptation to different environments and demonstrates that an RNA-binding protein can differentially affect its binding partners in dependence upon growth conditions.

The Functionality of IbpA from Acholeplasma laidlawii Is Governed by Dynamic Rearrangement of Its Globular-Fibrillar Quaternary Structure.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.

Interplay between formation of photosynthetic complexes and expression of genes for iron-sulfur cluster assembly in Rhodobacter sphaeroides?

Formation of photosynthetic complexes leads to a higher demand for Fe-S clusters. We hypothesized that in the facultative phototrophic alpha-proteobacterium Rhodobacter sphaeroides expression of the isc-suf operon for Fe-S cluster formation may be increased under conditions that promote formation of photosynthetic complexes and that, vice versa, lack of the IscR regulator may also affect photosynthesis gene expression. To test this hypothesis, we monitored the activities of the isc-suf sense and anti-sense promoters under different growth conditions and in mutants which are impaired in formation of photosynthetic complexes. We also tested expression of photosynthesis genes in a mutant lacking the IscR regulator. Our results are not in agreement with a co-regulation of the Isc-Suf system and the photosynthetic apparatus at level of transcription. We provide evidence that, coordination of the systems occurs at post-transcriptional levels. Increased levels of isc-suf mRNAs under conditions promoting formation of photosynthetic complexes are due to higher RNA stability.

Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus.

Bacteria use CRISPR Cas systems to defend against invading foreign nucleic acids, e.g., phage genomes, plasmids or mobile genetic elements. Some CRISPR Cas systems were reported to have physiological importance under a variety of abiotic stress conditions. We used physiological tests under different stress conditions and RNA-seq analyses to address the possible function of the RNA-targeting class 2 type VI CRISPR Cas system of the facultative phototrophic α-proteobacterium Rhodobacter capsulatus. Expression of the system was low under exponential non-stress conditions and high during oxidative stress, membrane stress and in stationary phase. Induction of the CRISPR Cas system in presence of a target protospacer RNA resulted in a growth arrest of R. capsulatus. RNA-seq revealed a strong alteration of the R. capsulatus transcriptome when cas13a was induced in presence of a target protospacer. RNA 5' end mapping indicated that the CRISPR Cas-dependent transcriptome remodeling is accompanied by fragmentation of cellular RNAs, e.g., for mRNAs originating from a genomic locus which encodes multiple ribosomal proteins and the RNA polymerase subunits RpoA, RpoB and RpoC. The data suggest a function of this CRISPR Cas system in regulated growth arrest, which may prevent the spread of phages within the population.