Unraveling small RNA function in alpha-proteobacteria: Design and validation of a novel synthetic system for a tunable expression of sRNAs

LAGARES ANTONIO; SERRANIA, JAVIER; BECKER, ANKE; VALVERDE, CLAUDIO

Würzburg

Congreso; 3rd International Conference on regulating with RNA in bacteria; 2013

Institute for Molecular Infection Biology - Würzburg Universität

INTRODUCTION Small non-coding RNAs (sRNAs) constitute a major class of post-transcriptional regulators of gene expression in prokaryotes. Over the last decade, the emergence of high throughput RNA-sequencing methods allowed the systematic detection of a large number of short non-coding RNAs in many alpha-proteobacterial species. However, little is known yet about their biological functions. Reverse genetics approaches have been widely used to study the function of sRNAs in enterobacterial models. In order to identify direct targets of sRNA regulation it is mandatory to detect those changes in gene expression that occur shortly after modifying the sRNA activity inside the cell. Since Hfq availability is a limiting factor for the sRNA-mediated riboregulatory networks, strong over-expression must be avoided to prevent Hfq titration and indirect impairment of other Hfq-dependent regulatory pathways. OBJECTIVES 1- To design and construct a novel synthetic system that allows an adjustable expression of sRNA genes. 2- To validate its application in the alfalfa root-symbiont soil model bacterium Sinorhizobium meliloti 2011 (Sm). RESULTS The synthetic system consists of an expression module in which blunt-end DNA fragments encoding sRNA genes can be cloned precisely at the transcriptional start site (TSS) of the Escherichia coli Tryptophan promoter (pTrp), and a regulatory module from which the negative regulator TrpR is constitutively expressed under the control of a moderately active promoter. Both pTrp and TrpR are absent in alpha-proteobacteria. In the presence of Tryptophan in the culture medium, expression downstream to pTrp is repressed by TrpR binding to the promoter region. After the addition of the synthetic inducer β-indoleacrylic acid (IAA), the repression is released thus starting sRNA transcription from the pTrp. The system was tested by cloning the sm8 sRNA gene of Sm at the pTrp TSS position, and then it was introduced as a single chromosomal copy in a neutral intergenic region of the Sm sm8- mutant strain. The engineered strain was cultured to the exponential phase of growth in defined medium supplemented with Tryptophan. Sm8 expression levels from pTrp showed a positive correlation with the inducer concentration, as determined by RT-qPCR assays. Within ten minutes after IAA addition, Sm8 concentration increased between 5-fold and 120-fold for IAA ranging 10-100 μg mL-1, compared to the basal Sm8 levels measured before induction. Longer incubation times did not increase the sRNA levels. A transcriptomic profile monitoring during a short pulse of Sm8 expression in Sm allowed us to detect significant changes of transcript abundance of a comprised group of genes that were previously found as deregulated in the sm8- Sm mutant when it was compared to the wild type strain. CONCLUSION Altogether, these results support the use of the pTrp-based expression system to tightly regulate the expression of potentially any RNA transcript in Sm. It provides a valuable alternative tool that allows to take sensitive control on the expression of RNA transcripts in which it is critical that the 5?-end is precisely defined, as in the case of small RNAs, being also feasible to extend its application to other alpha-proteobacteria species than Sm. Searching for Sm8 targets following its induction from the expression system helped to sharply demarcate the extent of its regulon.