Dataset Information

Genome-wide transcription start site profiling in biofilm-grown Burkholderia cenocepacia J2315.

ABSTRACT: Burkholderia cenocepacia is a soil-dwelling Gram-negative Betaproteobacterium with an important role as opportunistic pathogen in humans. Infections with B. cenocepacia are very difficult to treat due to their high intrinsic resistance to most antibiotics. Biofilm formation further adds to their antibiotic resistance. B. cenocepacia harbours a large, multi-replicon genome with a high GC-content, the reference genome of strain J2315 includes 7374 annotated genes. This study aims to annotate transcription start sites and identify novel transcripts on a whole genome scale.RNA extracted from B. cenocepacia J2315 biofilms was analysed by differential RNA-sequencing and the resulting dataset compared to data derived from conventional, global RNA-sequencing. Transcription start sites were annotated and further analysed according to their position relative to annotated genes.Four thousand ten transcription start sites were mapped over the whole B. cenocepacia genome and the primary transcription start site of 2089 genes expressed in B. cenocepacia biofilms were defined. For 64 genes a start codon alternative to the annotated one was proposed. Substantial antisense transcription for 105 genes and two novel protein coding sequences were identified. The distribution of internal transcription start sites can be used to identify genomic islands in B. cenocepacia. A potassium pump strongly induced only under biofilm conditions was found and 15 non-coding small RNAs highly expressed in biofilms were discovered.Mapping transcription start sites across the B. cenocepacia genome added relevant information to the J2315 annotation. Genes and novel regulatory RNAs putatively involved in B. cenocepacia biofilm formation were identified. These findings will help in understanding regulation of B. cenocepacia biofilm formation.

SUBMITTER: Sass AM

PROVIDER: S-EPMC4603805 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Genome-wide transcription start site profiling in biofilm-grown Burkholderia cenocepacia J2315.

Sass Andrea M AM Van Acker Heleen H Förstner Konrad U KU Van Nieuwerburgh Filip F Deforce Dieter D Vogel Jörg J Coenye Tom T

BMC genomics 20151013

<h4>Background</h4>Burkholderia cenocepacia is a soil-dwelling Gram-negative Betaproteobacterium with an important role as opportunistic pathogen in humans. Infections with B. cenocepacia are very difficult to treat due to their high intrinsic resistance to most antibiotics. Biofilm formation further adds to their antibiotic resistance. B. cenocepacia harbours a large, multi-replicon genome with a high GC-content, the reference genome of strain J2315 includes 7374 annotated genes. This study aim ...[more]

PMID: 26462475

Similar Datasets

Project description:Burkholderia cenocepacia infections are difficult to treat due to resistance, biofilm formation and persistence. B. cenocepacia strain J2315 has a large multi-replicon genome (8.06 Mb) and the function of a large fraction of (conserved) hypothetical genes remains elusive. The goal of the present study is to elucidate the role of small proteins in B. cenocepacia, focusing on genes smaller than 300 base pairs of which the function is unknown. Almost 10% (572) of the B. cenocepacia J2315 genes are smaller than 300 base pairs and more than half of these are annotated as coding for hypothetical proteins. For 234 of them no similarity could be found with non-hypothetical genes in other bacteria using BLAST. Using available RNA sequencing data obtained from biofilms, a list of 27 highly expressed B. cenocepacia J2315 genes coding for small proteins was compiled. For nine of them expression in biofilms was also confirmed using LC-MS based proteomics and/or expression was confirmed using eGFP translational fusions. Overexpression of two of these genes negatively impacted growth, whereas for four others overexpression led to an increase in biofilm biomass. Overexpression did not have an influence on the MIC for tobramycin, ciprofloxacin or meropenem but for five small protein encoding genes, overexpression had an effect on the number of persister cells in biofilms. While there were no significant differences in adherence to and invasion of A549 epithelial cells between the overexpression mutants and the WT, significant differences were observed in intracellular growth/survival. Finally, the small protein BCAM0271 was identified as an antitoxin belonging to a toxin-antitoxin module. The toxin was found to encode a tRNA acetylase that inhibits translation. In conclusion, our results confirm that small proteins are present in the genome of B. cenocepacia J2315 and indicate that they are involved in various biological processes, including biofilm formation, persistence and intracellular growth.

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:Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of the cell cycle.

Dataset Information

Genome-wide transcription start site profiling in biofilm-grown Burkholderia cenocepacia J2315.

Publications

Genome-wide transcription start site profiling in biofilm-grown Burkholderia cenocepacia J2315.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets