Abstract
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.
Highlights
Burkholderia cenocepacia J2315 is a member of the B. cepacia complex
transcription start sites (TSS) in an intergenic region on the second largest chromosome of B. cenocepacia J2315 [7, 8]. This TSS is located on the opposite strand of its adjacent genes, BCAM2068 and BCAM2069 (Fig. 1), indicating an independently transcribed sRNA, which was designated ncS35. dRNA-seq data further indicated a processing site 29 nt downstream of the TSS, revealed by a coverage peak depleted in the Terminator RNA exonuclease (TEX)-treated subsample (Fig. 1A). 5= rapid amplification of cDNA ends (RACE) confirmed the TSS as the beginning of the transcript, as well as the processing site. dRNA-seq data showed an abrupt decrease in coverage at a distinct location, and 3= RACE confirmed this as the end of the transcript
Known regulatory sRNAs of other bacterial species tend to have a low Z score, which serves as an indicator of their structural significance [16], as it confirms that the actual RNA sequence has a significantly lower minimum free energy (MFE) than sequences with the same length and nucleotide composition
Summary
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. 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. The small RNA ncS35 affects metabolic rate, growth, and antimicrobial susceptibility in the opportunistic cystic fibrosis pathogen Burkholderia cenocepacia msphere.asm.org 1 of the high innate resistance to most antibiotics and because of their ability to form biofilms, eradication of Bcc bacteria is difficult [4, 5]. SRNAs are small noncoding RNA molecules in bacteria that possess regulatory functions on the posttranscriptional level [11] They are typically relatively short (40 to 500 nucleotides [nt]) and act mostly by base pairing with mRNA. MRNA stability can be directly affected by sRNAs, as binding within the coding sequence (CDS) of the mRNA target can result in increased degradation of the sRNA-mRNA duplex [13, 14]
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