The world of small RNAs in Alphaproteobacteria

Sudden changes in the environment entail the need for sophisticated regulation of cellular responses in order to adapt and survive. Bacteria regulate their gene expression on both transcriptional and post-transcriptional level. Besides intrinsic regulatory elements of mRNAs, like riboswitches, small regulatory RNAs (sRNAs) are at the forefront of post-transcriptional regulation. Typical trans-encoded sRNAs influence their target mRNAs by means of stability and/or translation initiation. They are commonly induced upon stress and involved in regulatory fine-tuning and feedback regulations.

In the group of Alphaproteobacteria metabolic diversity is enormous and even in a single species high metabolic flexibility can exist. Environmental cues directly determine the bacterial life-style/metabolism and the question of how these bacteria regulate their gene expression is of certain interest.

Small RNAs in Rhodobacter sphaeroides

The purple bacterium Rhodobacter sphaeroides is a well-studied model organism with regard to the regulation of photosynthesis gene expression and the response to oxidative and photooxidative stress. The search for sRNAs using a differential RNA-sequencing approach (dRNA-seq) revealed that many of these small regulators exist in R. sphaeroides and implied their regulatory relevance (Figure 1). The functional characterization of these sRNAs is currently the main focus. We found one sRNA (RSs2430) that is controlled by the oxygen-dependent two component system PrrB/PrrA and is involved in regulation under oxygen depletion. The RSs0019 sRNA specifically responds to photooxidative stress caused by singlet oxygen in an RpoE-dependent manner and may fine-tune sulfur metabolism. Interestingly, RSs0019 contains a small open reading frame (ORF) and might therefore represent a dual-function RNA with a regulatory and a coding function. Three additional sRNAs (RSs0680a, RSs1543 and RSs2461) belong to the RpoH_I/RpoH_II regulon and most likely depict RNA regulators with a role under more general stress conditions. Last but not least, the strongly abundant sRNA RSs0682 undergoes a singlet oxygen-specific processing which depends on the important RNA-binding protein Hfq. We successfully over-expressed several of these sRNAs, resulting in distinct phenotypes and altered global expression patterns as analyzed by microarray and proteome studies. The bioinformatic prediction of interaction sites of sRNA/mRNA pairs confines the set of direct mRNA targets, which are subsequently tested by in vivo and in vitro studies. In addition, the important RNA-binding protein Hfq was demonstrated to have a global regulatory function in R. sphaeroides and to interact with several of the above mentioned sRNAs (Figure 1). We therefore study the role of Hfq in sRNA function in R. sphaeroides.

Figure 1: Model of sRNAs and Hfq in Rhodobacter sphaeroides.

Role of an sRNA repeat in the general stress response of Alphaproteobacteria

In R. sphaeroides a repeat of four homologous sRNAs was identified by dRNA-seq. The sRNAs RSs0680a-d are induced from an RpoH_I/RpoH_II dependent promoter and co-transcribed with RSP_6037, encoding a hypothetical protein with a domain of unknown function (DUF) 1127 (Figure 2A). The sRNAs fold into a typical secondary structure containing two stem-loops with the diagnostic CCUCCUCCC motif within the loops (Figure 2B). We are interested in the regulatory mechanisms and the role in the general stress response of these sRNAs. They are conserved among Alphaproteobacteria and regularly associated with DUF 1127 proteins. We therefore investigate this set of sRNAs in R. sphaeroides and related Alphaproteobacteria, as e.g. rhizobia.

Figure 2: The homologous RSs0680 sRNAs. (A) Genetic localization of RSP_6037 and RSs0680a-d. (B) Consensus secondary structure of RSs0680-type sRNAs containing the CCUCCUCCC motives.

Small RNAs in rhizobia

Using a comparative genomics approach, candidates for small RNAs were predicted and verified in the plant symbionts Sinorhizobium meliloti and Bradyrhizobium japonicum. We analyzed several growth phase dependent small RNAs of S. meliloti, the nitrogen fixing symbiont of alfalfa. These sRNAs are conserved and expressed in representatives of the genera Sinorhizobium and Rhizobium. Two of them are Hfq-dependent: Hfq is important for their stability and degradation pathway in S. meliloti. The expression of sRNAs in B. japonicum, the symbiont of soyben plants, was studied under free-living conditions and in root nodules. Northern blots, microarrays and deep sequencing analyses revealed differential expression of several sRNAs under the two physiological conditions. Based on the deep sequencing data, we are currently mapping sRNAs, their transcriptional start sites and putative promoters. Future analyses aim to unravel the role of sRNAs in the interaction between bacteria and plants. The projects are performed in cooperation with the research groups of A. Becker (Marburg), M. Gelfand (Moskau), H.-M. Fischer (ETH Zürich), W.R. Hess (Freiburg), J. Vogel and C. Sharma (Würzburg).

Methods

Cultivation of Alphaproteobacteria; Cloning procedures; RNA isolation; Northern blotting; real time RT-PCR; Microarray; RNA 5’ and 3’ end detection (primer extension, RNA protection, 5’ and 3’ RACE); EMSA; In vitro transcription and in vitro RNA degradation; Isolation and characterization of recombinant proteins; Western blotting; 2D gel electrophoresis; in vivo reporter studies

Reference

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