A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

Abstract

Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies.

Determination of the small RNA GcvB regulon in the Gram-negative bacterial pathogen Pasteurella multocida and identification of the GcvB seed binding region

“…In other bacteria, histidine production is regulated by multiple mechanisms including repression of transcription initiation and attenuation (Kulis-Horn et al 2014), but to our knowledge GcvB has not been previously linked with control of histidine biosynthesis. HisD encodes a histidinol dehydrogenase that has also been bioinformatically predicted to be a target of GcvB in the related Pasteurellaceae species A. pleuropneumoniae (Rossi et al 2016). Moreover, specific attenuator sequences that target histidine production have been identified in A. pleuropneumoniae (Rossi et al 2016).…”

“…HisD encodes a histidinol dehydrogenase that has also been bioinformatically predicted to be a target of GcvB in the related Pasteurellaceae species A. pleuropneumoniae (Rossi et al 2016). Moreover, specific attenuator sequences that target histidine production have been identified in A. pleuropneumoniae (Rossi et al 2016).…”

“…There is currently no information on the functional role of GcvB, its mRNA targets, or mRNA binding interactions in any organisms from the Pasteurellaceae family. A bioinformatics screen of multiple genomes identified a putative GcvB homolog in P. multocida, and a recent bioinformatics analysis of the related organism, Actinobacillus pleuropneumoniae, also identified a GcvB homolog and its expression was confirmed by northern blotting (Sharma et al 2007;Rossi et al 2016). Another study in Haemophilus influenzae showed expression of GcvB was high when grown in the presence of primary normal human bronchial epithelial cells using RNA-seq (Baddal et al 2015).…”

Non-Coding RNAs in Bacteria

“…The mechanism of post-transcriptional response of gene regulation involves a high degree of sequence complementarity, and this was considered an indication that interaction of the Hfq protein would not be required [23,45]. However, some researchers reported interference of Hfq in the cis-encoded ncRNA pairing [46][47][48]. In general, these ncRNAs act by complementing the mRNA ribosome-binding site, inhibiting, in turn, the translation [38,45].…”

Verificação Da Temperatura De Distribuição De Refeições Quentes Ofertadas Em Uma Instituição De Longa Permanência Para Idosos E a Correlação Com O Crescimento Microbiológico

“…SITTKA et al, 2008; LORENZ et al, 2010;ROSSI et al, 2016). Esse ncRNA contém 292 pb e regula negativamente a expressão do operon ureAB ao bloquear a tradução na porção 5' do ureB(FIGURA 4).…”