Brucella spp are intracellular pathogens able to survive and establish themselves in the hostile environment of macrophages, and the etiological agent of brucellosis, the most prevalent worlwide zoonosis. Although there is an increasing knowledge of the virulence factors present in this genus, very little is known about the post-transcriptional regulation of these virulence factors at the RNA level. An hfq mutant in B. abortus is profoundly affected in its virulence and in several stress responses, suggesting that such a control exist. However the targets for Hfq, either mRNAs or small noncoding RNAs (sRNAs), are largely unknown for Brucella, and only two sRNAs regulated by Hfq have been published so far. With the aim of sRNA discovery and identification of new regulatory mechanisms, directional mRNA enriched libraries have been constructed from strain B. abortus 2308 and an hfq isogenic mutant derivative. The libraries were tagged and sequenced in an Illumina HiSeq2000 machine. Filtered reads were aligned to the reference B. abortus 2308 genome with bowtie2 and normalized read counts, expressed in RPKM's, was determined for specific features from the alignment sam or bam files with Artemis or Htseq. A strand specific plot of the reads starting at each genome position was also used to visualize the transcription along the genome. From this plot non annotated regions producing transcripts were selected as putative sRNAs. Secondary structure analysis with RNAfold, promotor and terminator searches and comparison with previous results, were used to filter the candidates and to elaborate a list containing 170 strong sRNA candidates. A subset of them have been selected for further biochemical characterization by Northern blot, RT-PCR and cloning of their 5' and 3' ends. The analysis performed also identified genes regulated by Hfq, and several prediction programs were used to determine the putative targets of the sRNAs. Comparison of the target list with the regulated genes allowed the identification of sRNAs-target pairs, some of which will be discussed.

Resumen del trabajo presentado al Brucellosis 2014 International Research Conference, celebrado en Berlín (Alemania) del 9 al 12 de septiembre de 2014.

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