Computational discovery and RT-PCR validation of novel Burkholderia conserved and Burkholderia pseudomallei unique sRNAs

Abstract

Background The sRNAs of bacterial pathogens are known to be involved in various cellular roles including environmental adaptation as well as regulation of virulence and pathogenicity. It is expected that sRNAs may also have similar functions for Burkholderia pseudomallei, a soil bacterium that can adapt to diverse environmental conditions, which causes the disease melioidosis and is also able to infect a wide variety of hosts. Results By integrating several proven sRNA prediction programs into a computational pipeline, available Burkholderia spp. genomes were screened to identify sRNA gene candidates. Orthologous sRNA candidates were then identified via comparative analysis. From the total prediction, 21 candidates were found to have Rfam homologs. RT-PCR and sequencing of candidate sRNA genes of unknown functions revealed six putative sRNAs which were highly conserved in Burkholderia spp. and two that were unique to B. pseudomallei present in a normal culture conditions transcriptome. The validated sRNAs include potential cis-acting elements associated with the modulation of methionine metabolism and one B. pseudomallei-specific sRNA that is expected to bind to the Hfq protein. Conclusions The use of the pipeline developed in this study and subsequent comparative analysis have successfully aided in the discovery and shortlisting of sRNA gene candidates for validation. This integrated approach identified 29 B. pseudomallei sRNA genes - of which 21 have Rfam homologs and 8 are novel.

Regulation of biofilm formation in Pseudomonas and Burkholderia species

“…Computational and experimental studies have revealed that sRNAs are abundant in Burkholderia species (Coenye et al, 2007;Yoder-Himes et al, 2009;Khoo et al, 2012;Ramos et al, 2013b), and that some of them are upregulated during growth in human sputum, indicating that sRNA plays an important role in survival of the bacteria in the human host (Coenye et al, 2007). However, functions in Burkholderia physiology were assigned only to the two sRNAs mtvR and h2cR.…”

In silico prediction and expression profile analysis of small non-coding RNAs in Herbaspirillum seropedicae SmR1

“…To confirm which of these sRNAs are expressed in H. seropedicae SmR1, we analysed RNA-seq data from three culture conditions (CRT, NAR and NIT) and verified the expression of 117 sRNAs, each expressed in at least one of the three conditions analysed. The number of sRNAs predicted by bioinformatics tools varies among species as already described for Streptomyces coelicolor (843 sRNAs) [44], Burkholderia pseudomallei (1306 sRNAs) [74] and B. cenocepacia J2315 (213 sRNAs) [75]. We can attribute this variation to the different methods of total RNA purification and to the sRNA prediction and validation methods employed.…”

Identification of novel sRNAs inBrucella abortus2308

“…NAPP is a program that searches the 'RNA-rich' clusters in query genomes, which employ the ab initio methods regardless of the sequence or structure similarity (Sridhar & Gunasekaran, 2013). According to a quantitative assessment of three different sRNA prediction methods (Rfam_scan, sRNAscanner, and SIPHT), the sensitivity of each method is < 30%, and the precision only 10% (Khoo et al, 2012). As the accuracy of these methods is so poor, many researchers combined several bioinformatic methods to increase the prediction accuracy.…”

“…As the accuracy of these methods is so poor, many researchers combined several bioinformatic methods to increase the prediction accuracy. In this way, many sRNAs have been identified in different bacterial species (Khoo et al, 2012;Tesorero et al, 2013).…”

“…Current strategies for identifying bacterial sRNAs often combine bioinformatics prediction and experimental validation (northern blot or reverse transcriptase PCR). One recent strategy involved bioinformatics programs (Rfam_scan, SIPHT, and sRNAscanner) and RT-PCR validation, resulting in 29 sRNAs identified in Burkholderia pseudomallei (Khoo et al, 2012).…”