Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism

Abstract

Small RNAs play a regulatory role in many central metabolic processes of bacteria, as well as in developmental processes such as biofilm formation. Small RNAs of Burkholderia cenocepacia, an opportunistic pathogenic beta-proteobacterium, are to date not well characterised. To address that, we performed genome-wide transcriptome structure analysis of biofilm grown B. cenocepacia J2315. 41 unannotated short transcripts were identified in intergenic regions of the B. cenocepacia genome. 15 of these short transcripts, highly abundant in biofilms, widely conserved in Burkholderia sp. and without known function, were selected for in-depth analysis. Expression profiling showed that most of these sRNAs are more abundant in biofilms than in planktonic cultures. Many are also highly abundant in cells grown in minimal media, suggesting they are involved in adaptation to nutrient limitation and growth arrest. Their computationally predicted targets include a high proportion of genes involved in carbon metabolism. Expression and target genes of one sRNA suggest a potential role in regulating iron homoeostasis. The strategy used for this study to detect sRNAs expressed in B. cenocepacia biofilms has successfully identified sRNAs with a regulatory function.

Low iron-induced small RNA BrrF regulates central metabolism and oxidative stress responses inBurkholderia cenocepacia

“…BrrF appeared to be cleaved from the hemP mRNA, as indicated by the strong depletion of the BrrF transcript by treatment with a 5’-monophosphate-dependent exonuclease (TEX, [7]). Its size was determined as 126 nt by dRNA-Seq (position 2548559 to 2548684 on replicon 1), confirmed by Northern blotting [8]. BrrF does not extend into the computationally predicted rho-independent terminator (position 2548709 to 2548732) downstream of hemP , instead it terminates a at succession of U-residues without a preceding stem loop (Fig.…”

“…BrrF was previously found to be more than 50-fold up-regulated under iron-depleted conditions, whereas other tested growth conditions such as biofilm growth, oxidative stress, stationary phase and starvation did not induce BrrF expression [8]. hemP was upregulated by iron depletion to approximately the same fold change as brrF .…”

“…A putative OxyR box was found upstream of hemP (Fig. 1), and hemP was upregulated 10-fold in H 2 O 2 -treated biofilms of B. cenocepacia J2315 [15] while in exponentially growing planktonic cells exposed to H 2 O 2 , neither hemP nor brrF change expression [8, 16]. However, in these studies biofilms were treated with 3% H 2 O 2 for 30 min, whereas planktonic cultures were treated with 0.05 or 0.15% H 2 O 2 for a shorter time period, which might account for the observed differences in hemP regulation under oxidative stress.…”

“…Functional enrichment analysis of predicted targets pointed to a relative over-representation of genes involved in aerobic respiration, iron- or heme-binding, and in the TCA cycle [8]. Furthermore, several genes involved in iron-sulfur cluster formation and in ROS detoxification, such as those encoding catalases and SOD, were predicted as targets (Table 1).…”

“…A small RNA highly upregulated under iron depletion was identified in B. cenocepacia J2315 by screening dRNA-Seq data for short transcripts [7, 8] and designated ncS63. Its computationally predicted targets included confirmed targets of E. coli RyhB and P. aeruginosa PrrF, such as sdhC and sodB [4, 5, 9, 10], yet ncS63 has no sequence similarity to any known Fur-regulated sRNAs.…”