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

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.


Abstract 21
BrrF is a Fur-regulated small RNA highly upregulated in Burkholderia cenocepacia 22 under conditions of iron depletion. Its computationally predicted targets include iron-23 containing enzymes of the tricarboxylic acid (TCA) cycle such as aconitase and 24 succinate dehydrogenase, as well as iron-containing enzymes responsible for the 25 oxidative stress response, such as superoxide dismutase and catalase. Phenotypic Burkholderia cenocepacia J2315 is a member of the Burkholderia cepacia complex 50 (BCC), a group of aerobic Gram-negative beta-proteobacteria which mainly live in the 51 rhizosphere, but can also act as opportunistic pathogens, particularly in individuals 52 with cystic fibrosis [1]. 53 Iron is essential for living organisms, as part of the catalytic/active site of many 54 enzymes, yet it is mostly inaccessible for bacteria due to the low solubility under oxic The ferric uptake regulator (Fur) is important for iron homeostasis in many bacteria. 65 Its best known mechanism of action is the repression of genes involved in iron uptake 66 under iron replete conditions by iron-dependent binding to a specific sequence motif, 67 the Fur box [2]. In Escherichia coli and Pseudomonas aeruginosa Fur also positively 68 regulates SOD, the iron scavenger protein bacterioferritin and several enzymes of the 69 tricarboxylic acid cycle (TCA) [4,5]. This positive regulation was attributed to indirect 70 effects mediated by Fur-regulated small RNAs (sRNAs), RyhB in E. coli [5] and PrrF 71 in P. aeruginosa [4]. Under iron-depletion the Fur repression of these sRNAs is lifted, 72 they bind to the mRNA of their targets and the sRNA-mRNA hybrid is then rapidly 73 degraded, or the translation of targets is inhibited [6]. This reduces the demand for 74 iron in the cell, since many targets have iron or iron-sulfur clusters as cofactor.

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A small RNA highly upregulated under iron depletion was identified in B. cenocepacia 76 J2315 by screening dRNA-Seq data for short transcripts [7,8] and designated ncS63.

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Its computationally predicted targets included confirmed targets of E. coli RyhB and P. 78 aeruginosa PrrF, such as sdhC and sodB [4,5,9,10], yet ncS63 has no sequence 79 similarity to any known Fur-regulated sRNAs. Here we report on the full 80 characterisation of this low iron-induced sRNA. occurrences. Fur boxes were identified directly upstream of the TSS for hemP (Fig. 1), 118 and upstream of known iron transport-related genes such as orbS and fecI, but brrF 119 itself did not have a Fur box directly upstream of its 5' end. Expression of BrrF is 120 therefore probably under control of the Fur regulator via co-expression with hemP. In 121 contrast to that, the Fur-regulated sRNAs RyhB in E. coli and PrrFs in P. aeruginosa 122 are directly preceded by a Fur binding site.

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hemP is possibly also under the regulation of OxyR. A putative OxyR box was found 124 upstream of hemP (Fig. 1), and hemP was upregulated 10-fold in H2O2-treated biofilms 125 of B. cenocepacia J2315 [15] while in exponentially growing planktonic cells exposed 126 to H2O2, neither hemP nor brrF change expression [8,16]. However, in these studies 127 biofilms were treated with 3% H2O2 for 30 min, whereas planktonic cultures were 128 treated with 0.05 or 0.15% H2O2 for a shorter time period, which might account for the 129 observed differences in hemP regulation under oxidative stress.  Table S1). The categories with the largest number of genes with known 133 function were amino acid transport and metabolism (12.6%) and energy production 134 and conversion (10.0%). Most predicted interactions involved the 5' end of BrrF (Table   135 S1), notable exceptions were targets sdhC and sdhA (Fig. 2).

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Functional enrichment analysis of predicted targets pointed to a relative over-137 representation of genes involved in aerobic respiration, iron-or heme-binding, and in 138 the TCA cycle [8]. Furthermore, several genes involved in iron-sulfur cluster formation 139 and in ROS detoxification, such as those encoding catalases and SOD, were predicted 140 as targets (Table 1). These genes or functional categories are also targeted by RyhB 141 and/or PrrF (see Table 1 for references).  and it grew faster than WT on four of them (Fig. 3C, Fig. S3). This is consistent with a  (Table S2). In line with this, aconitase 211 activity was also reduced by BrrF overexpression (Fig. 6). This suggests that growth  (Table S2), although 219 the abundance of BrrF was similar (Fig. S4). This suggests that iron depletion is 220 necessary for the full function of BrrF.  Strains were routinely cultured in LB broth or agar (low-salt Lennox formulation: 10 g/L 278 tryptone, 5 g/L yeast extract, 5 g/L NaCl, 1.5% agar), supplemented with 600 µg/ml   All primer sequences are listed in Table S4.  per ml) were spread on low-nutrient LB agar plates (1 g/L tryptone, 0.5 g/L yeast 336 extract, 5 g/L NaCl, 1.5% agar) with a sterile cotton swab. Inoculated plates were pre-337 incubated for 1 hour to allow gene expression to start. Then, 10 µl of a 1% H2O2 338 (Sigma) solution were applied to 6 mm blank susceptibility testing discs (Oxoid) which   Table S4.         Complementation was dependent on the 5'end of BrrF for most genes except for sdhA and sdhC. Light grey: Expression compared to iron-replete condition. Dark grey: Expression compared to DbrrF-vector control. pBrrF-d1 and pBrrF-d2: plasmids overexpressing native BrrF with or without processing. pBrrF-d3: derivative of pBrrF-d2 with point mutations near the BrrF 5'end. Asterisks: Significant difference to the respective control, with p ≤ 0.05.

WT-pBrrF-d1
DbrrF-VC DbrrF-pBrrF-d1 DbrrF-pBrrF-d2 DbrrF-pBrrF-d3 Figure 6: BrrF-dependent reduction in aconitase activity. Cultures were grown under iron-replete conditions in LB broth to mid-log phase and rhamnose was added one hour before harvest and protein extraction. Cell-free protein extracts in a 20 mM citrate buffer were supplemented with 40 mM DL-isocitrate and increase in absorbance was measured at 240 nm in a spectrophotometer. Asterisks: Significant difference to the respective control, with p ≤ 0.05. VC: vector control.
mg cis-aconitate x min -1 x mg protein -1 * * * Figure 7: BrrF-dependent regulation of the oxidative stress response. A) SOD activity was determined in a cell-free protein extract from planktonic mid-log phase cells, indirectly via superoxide utilization of a xanthine oxidase. Results are presented as percent compared to control condition. B) Sensitivity to H 2 O 2 was determined on low-nutrient agar plates, via formation of a growth inhibition zone around a filter disk containing 1% H 2 O 2 . Plates were pre-incubated for one hour to induce gene expression changes before applying the filter disks. Light grey bars: Response to iron depletion in wild type (WT) and DbrrF. Dark grey bars: Overexpression of native BrrF with or without processing (pBrrF-d1 and pBrrF-d2) or of a derivative with point mutations near the BrrF 5'end (pBrrF-d3