The Small RNA ncS35 Regulates Growth in Burkholderia cenocepacia J2315

Kiekens, Sanne; Saß, Andrea; Nieuwerburgh, Filip Van; Deforce, Dieter; Coenye, Tom

doi:10.1128/msphere.00579-17

Cited by 19 publications

(31 citation statements)

References 46 publications

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“…katB and BCAM0931 are predicted as targets for BrrF. katA did not change expression upon iron depletion (Table S2) and BCAS0635 is not expressed under iron-replete conditions [22]. Taken together, this suggest the increased sensitivity to H 2 O 2 can be linked to downregulation of katB .…”

Section: Resultsmentioning

confidence: 99%

“…Our results demonstrate that sRNA BrrF is involved in downregulating TCA and oxidative stress response in B. cenocepacia J2315. This sRNA can be added to the growing list of sRNA involved in regulating metabolism, growth and stress responses in the versatile opportunistic pathogen B. cenocepacia [22, 26].…”

Section: Resultsmentioning

confidence: 99%

“…For overexpressing BrrF, a region ranging from 80 nt upstream of the putative processing site to 49 nt downstream of the computationally predicted terminator sequence was amplified and cloned into an overexpression vector with a rhamnose-inducible promoter [30], yielding pBrrF-d1. The overexpression vector had previously been modified to remove the ribosome binding site (RBS) and start codons [22].…”

Section: Methodsmentioning

confidence: 99%

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

Saß

Coenye

2019

Preprint

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6 7 Running title: Role of Burkholderia cenocepacia regulatory sRNA BrrF 8 9 # email corresponding author: tom.coenye@ugent.be 10 11 12 13 14 15 16 17 Word count abstract and importance: 199 and 99 Abstract 21BrrF 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 26 and gene expression analysis of BrrF deletion and overexpression mutants show that 27 the regulation of these genes is BrrF-dependent. Expression of acnA, fumA, sdhA and 28 sdhC was downregulated during iron depletion in the wild type strain, but not in a BrrF 29 deletion mutant. TCA cycle genes not predicted as target for BrrF were not affected in 30 the same manner by iron depletion. Likewise, expression of sodB and katB was 31 dowregulated during iron depletion in the wild type strain, but not in a BrrF deletion 32 mutant. BrrF overexpression reduced aconitase and superoxide dismutase activities 33 and increased sensitivity to hydrogen peroxide. All phenotypes and gene expression 34 changes of the BrrF deletion mutant could be complemented by overexpressing BrrF 35 in trans. Overall, BrrF acts as a regulator of central metabolism and oxidative stress 36 response, possibly as an iron-sparing measure to maintain iron homeostasis under 37 conditions of iron starvation. 38 Importance 39 Regulatory small RNAs play an essential role in maintaining cell homeostasis in 40 bacteria in response to environmental stresses such as iron starvation. Prokaryotes 41 generally encode a large number of RNA regulators, yet their identification and 42 characterisation is still in its infancy for most bacterial species. Burkholderia 43 cenocepacia is an opportunistic pathogen with high innate antimicrobial resistance, 44 which can cause the often fatal cepacia syndrome in individuals with cystic fibrosis. In 45 3this study we characterise a small RNA which is involved in the response to iron 46 starvation, a condition that pathogenic bacteria are likely to encounter in the host. 47 48 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. 75A 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. 77Its 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. As our results suggest that B. 81 cenocepacia ncS63 shows analogy to RyhB and PrrF, we s...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Saß

Coenye

2019

Preprint

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“…qPCR was performed in a CFX96 Real-Time System C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) using GoTaq qPCR Master Mix (Promega, Madison, WI, USA). Cq values were normalised against a previously-validated control gene ( rpo D, BCAM0918) (51, 52). Fold changes were calculated compared to a standard (mix of all cDNAs in experiment) and log-transformed.…”

Section: Methodsmentioning

confidence: 99%

Various evolutionary trajectories lead to loss of the tobramycin-potentiating activity of the quorum sensing inhibitor baicalin hydrate inBurkholderia cenocepaciabiofilms

Saß

Slachmuylders

Acker

et al. 2018

Preprint

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Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant and -tolerant bacteria. As these potentiators typically do not interfere with essential processes of bacteria, it has been hypothesized that they are less likely to induce resistance than conventional antibiotics. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop resistance towards one such adjuvant, baicalin hydrate (BH), a quorum sensing inhibitor known to increase antibiotic-induced oxidative stress. Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After each cycle of treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to treatments with TOB and TOB+BH, compared to the start population, and the potentiating effect of BH towards TOB was lost. Whole genome sequencing was performed to probe which changes were involved in the reduced effect of BH and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter), as well as a partial deletion of two larger regions. No changes in the minimal inhibitory or minimal bactericidal concentration of TOB or changes in the number of persister cells were observed in the evolved populations. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that resistance towards antibiotic-potentiating activity can develop rapidly in B. cenocepacia J2315 biofilms and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as potential mechanisms.ImportanceBacteria show a markedly reduced susceptibility to antibiotics when growing in a biofilm, which hampers effective treatment of biofilm-related infections. The use of potentiators that increase the activity of antibiotics against biofilms has been proposed as a solution to this problem, but it is unclear whether resistance to these potentiators could develop. Using an experimental evolution approach, we convincingly demonstrate that Burkholderia cenocepacia biofilms rapidly develop resistance towards the tobramycin-potentiating activity of baicalin hydrate. Whole genome sequencing revealed that there are different mechanisms that lead to this resistance, including mutations resulting in metabolic changes, changes in production of intracellular levels of reactive oxygen species, and differences in transporter-mediated tobramycin uptake. Our study suggests that this form of combination therapy is not ‘evolution-proof’ and highlights the usefulness of experimental evolution to identify mechanisms of resistance and tolerance in biofilm-grown bacteria.

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“…Originally, CopraRNA was successfully tested on 18 well-known Enterobacteriaceae sRNAs and on a small number of sRNAs from different bacteria revealing the previously identified mRNA targets as well as novel base pairing interactions (Wright et al, 2013). In the following, multiple studies confirmed the power of such approach in diverse bacterial groups including, for example, Cyanobacteria (Synechocystis: PsrR1 , NsiR4 (Klähn et al, 2015), IsaR1 (Georg et al, 2017); Anabaena: NsrR1 (Álvarez-Escribano, Vioque, & Muro-Pastor, 2018, p. 1)), Firmicutes (Bacillus: RsaE (Durand et al, 2015)), γ-Proteobacteria (Pseudomonas: RgsA (Lu et al, 2016)), α-Proteobacteria (Agrobacterium: AbcR1 (Overlöper et al, 2014); Sinorhizobium: EcpR1, GspR (Robledo et al, 2018)), or β-Proteobacteria (Burkholderia: ncS35, Neisseria: FnrS (Kiekens, Sass, Van Nieuwerburgh, Deforce, & Coenye, 2018;Wright et al, 2013)).…”

Section: Advan Ced Algorithmic Approache S In the Identifi C Ati Onmentioning

confidence: 99%

The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs

Georg

Lalaouna

Hou

et al. 2019

Molecular Microbiology

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Trans‐acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic microRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post‐transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNAs targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB, and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more details. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA‐dependent regulatory networks is now attainable.

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The Small RNA ncS35 Regulates Growth in Burkholderia cenocepacia J2315

Cited by 19 publications

References 46 publications

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

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

Various evolutionary trajectories lead to loss of the tobramycin-potentiating activity of the quorum sensing inhibitor baicalin hydrate inBurkholderia cenocepaciabiofilms

The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs

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