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MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia.

ABSTRACT: Burkholderia cenocepacia J2315 is a highly epidemic and transmissible clinical isolate of the Burkholderia cepacia complex (Bcc), a group of bacteria causing life-threatening respiratory infections among cystic fibrosis patients. This work describes the functional analysis of the 136-nucleotide (nt)-long MtvR small noncoding RNA (sRNA) from the Bcc member B. cenocepacia J2315, with homologues restricted to the genus Burkholderia. Bioinformatic target predictions revealed a total of 309 mRNAs to be putative MtvR targets. The mRNA levels corresponding to 17 of 19 selected genes were found to be affected when MtvR was either overexpressed or silenced. Analysis of the interaction between MtvR and the hfq mRNA, one of its targets, showed that the sRNA binds exclusively to the 5' untranslated region (UTR) of the hfq mRNA. This interaction resulted in decreased protein synthesis, suggesting a negative regulatory effect of MtvR on the RNA chaperone Hfq. Bacterial strains with MtvR silenced or overexpressed exhibited pleiotropic phenotypes related to growth and survival after several stresses, swimming and swarming motilities, biofilm formation, resistance to antibiotics, and ability to colonize and kill the nematode Caenorhabditis elegans. Together, the results indicate that the MtvR sRNA is a major posttranscriptional regulator in B. cenocepacia.

SUBMITTER: Ramos CG

PROVIDER: S-EPMC3754578 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia.

Ramos Christian G CG Grilo André M AM da Costa Paulo J P PJ Feliciano Joana R JR Leitão Jorge H JH

Journal of bacteriology 20130531 16

Burkholderia cenocepacia J2315 is a highly epidemic and transmissible clinical isolate of the Burkholderia cepacia complex (Bcc), a group of bacteria causing life-threatening respiratory infections among cystic fibrosis patients. This work describes the functional analysis of the 136-nucleotide (nt)-long MtvR small noncoding RNA (sRNA) from the Bcc member B. cenocepacia J2315, with homologues restricted to the genus Burkholderia. Bioinformatic target predictions revealed a total of 309 mRNAs to ...[more]

PMID: 23729649

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Project description:Burkholderia cenocepacia J2315 is a member of the B. cepacia complex. It has a large genome with three replicons and one plasmid; 7,261 genes code for annotated proteins, while 113 code for functional RNAs. Small regulatory RNAs of B. cenocepacia have not yet been functionally characterized. We investigated a small regulatory RNA, designated ncS35, that was discovered by differential RNA sequencing. Its expression under various conditions was quantified, and a deletion mutant, ΔncS35, was constructed. Compared to planktonic growth in a rich medium, the expression of ncS35 was elevated when B. cenocepacia J2315 was grown in biofilms and in minimal medium. Cells of the deletion mutant showed increased aggregation, higher metabolic activity, a higher growth rate, and an increased susceptibility to tobramycin. A transcriptomic analysis revealed upregulation of the phenylacetic acid and tryptophan degradation pathways in ΔncS35. Computational target prediction indicated that ncS35 likely interacts with the first gene of the tryptophan degradation pathway. Overall, we demonstrated that small RNA ncS35 is a noncoding RNA with an attenuating effect on the metabolic rate and growth. It is possible that slower growth protects B. cenocepacia J2315 against stressors acting on fast-dividing cells and enhances survival under unfavorable conditions. IMPORTANCE Small RNAs play an important role in the survival of bacteria in diverse environments. We explored the physiological role of ncS35, a small RNA expressed in B. cenocepacia J2315, an opportunistic pathogen in cystic fibrosis patients. In cystic fibrosis patients, infections can lead to "cepacia syndrome," a rapidly progressing and often fatal pneumonia. Infections with Burkholderia spp. are difficult to threat with antibiotics because of their high intrinsic resistance and ability to form biofilms. We show that ncS35 attenuates the growth and reduces the metabolic rate of B. cenocepacia and influences biofilm structure. This demonstrates that as-yet-uncharacterized small RNAs with regulatory function can influence physiological traits of B. cenocepacia that are relevant for infection.

Project description: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.

Project description:The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera.IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of "core essential genes" that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.

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MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia.

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MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia.

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