Abstract
Regulatory small RNAs play an essential role in maintaining cell homeostasis in bacteria in response to environmental stresses such as iron starvation. Prokaryotes generally encode a large number of RNA regulators, yet their identification and characterisation is still in its infancy for most bacterial species. Burkholderia cenocepacia is an opportunistic pathogen with high innate antimicrobial resistance, which can cause the often fatal cepacia syndrome in individuals with cystic fibrosis. In this study we characterise a small RNA which is involved in the response to iron starvation, a condition that pathogenic bacteria are likely to encounter in the host. BrrF is a small RNA highly upregulated in Burkholderia cenocepacia under conditions of iron depletion and with a genome context consistent with Fur regulation. Its computationally predicted targets include iron-containing enzymes of the tricarboxylic acid (TCA) cycle such as aconitase and succinate dehydrogenase, as well as iron-containing enzymes responsible for the oxidative stress response, such as superoxide dismutase and catalase. Phenotypic and gene expression analysis of BrrF deletion and overexpression mutants show that the regulation of these genes is BrrF-dependent. Expression of acnA, fumA, sdhA and sdhC was downregulated during iron depletion in the wild type strain, but not in a BrrF deletion mutant. TCA cycle genes not predicted as target for BrrF were not affected in the same manner by iron depletion. Likewise, expression of sodB and katB was dowregulated during iron depletion in the wild type strain, but not in a BrrF deletion mutant. BrrF overexpression reduced aconitase and superoxide dismutase activities and increased sensitivity to hydrogen peroxide. All phenotypes and gene expression changes of the BrrF deletion mutant could be complemented by overexpressing BrrF in trans. Overall, BrrF acts as a regulator of central metabolism and oxidative stress response, possibly as an iron-sparing measure to maintain iron homeostasis under conditions of iron starvation.
Highlights
As our results suggest that B. cenocepacia ncS63 shows analogy to RyhB and PrrF, we suggest the name BrrF (Burkholderia regulatory RNA involving iron, Fe), analogous to P. aeruginosa PrrF [5] and Neisseria meningitidis NrrF [12]
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, [8])
Our results demonstrate that small RNAs (sRNAs) BrrF is involved in downregulating tricarboxylic acid (TCA) and oxidative stress response in B. cenocepacia J2315
Summary
Burkholderia cenocepacia J2315 is a member of the Burkholderia cepacia complex (BCC), a group of aerobic Gram-negative beta-proteobacteria which mainly live in the rhizosphere, but can act as opportunistic pathogens, in individuals with cystic fibrosis [1]. In Escherichia coli and Pseudomonas aeruginosa Fur positively regulates SOD, the iron scavenger protein bacterioferritin and several enzymes of the tricarboxylic acid cycle (TCA) [5, 6]. This positive regulation was attributed to indirect effects mediated by Fur-regulated small RNAs (sRNAs), RyhB in E. coli [6] and PrrF in P. aeruginosa [5]. Under iron-depletion the Fur repression of these sRNAs is lifted, they bind to the mRNA of their targets and the sRNA-mRNA hybrid is rapidly degraded, or the translation of targets is inhibited [7]. As our results suggest that B. cenocepacia ncS63 shows analogy to RyhB and PrrF, we suggest the name BrrF (Burkholderia regulatory RNA involving iron, Fe), analogous to P. aeruginosa PrrF [5] and Neisseria meningitidis NrrF [12]
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