Project description:[1] Transcription profiling of one Burkholderia cenocepacia clinical isolate, J2315, versus a soil isolate, HI2424, in conditions mimicking CF sputum [2] Transcription profiling of Burkholderia cenocepacia isolates J2315 and HI2424 in media mimicking CF sputum or the soil environment
Project description:Hfq proteins are RNA chaperones that play a critical role in post-transcription regulation of gene expression. Bacteria of the Burkholderia cepacia complex harbor two distinct and functional Hfq proteins, the Hfq and Hfq2. We have previously performed the functional analysis of Hfq and Hfq2 in the pathogen Burkholderia cenocepacia J2315. In order to examine the impacts of each RNA chaperone on the global transcriptome of B. cenocepacia J2315, we performed comparative transcriptome profile of mutants on the hfq and hfq2 genes, using as reference the wild-type strain.
Project description:Members of the genus Burkholderia are versatile bacteria capable of colonizing highly diverse environmental niches. In this study, we investigated the global response of the opportunistic pathogen Burkholderia cenocepacia H111 to nitrogen limitation at the transcript and protein expression level. In addition to a classical response to nitrogen starvation, including the activation of glutamine synthetase, PII proteins and the two component regulatory system ntrBC, B. cenocepacia H111 also up-regulated polyhydroxybutyrate (PHB) accumulation and exopolysaccharide (EPS) production in response to nitrogen shortage. A search for consensus sequences in promoter regions of nitrogen responsive genes identified a s54 consensus sequence. The mapping of the s54 regulon as well as the characterization of a s54 mutant suggests an important role of s54 not only in control of nitrogen metabolism, but also in virulence of this organism.
Project description:Burkholderia cenocepacia is a versatile opportunistic pathogen that survives in a wide variety of environments, which can be limited in nutrients such as nitrogen. We previously showed that B. cenocepacia sigma factor s54 played a major role in control of nitrogen assimilation and virulence. In this work, we investigated the role of the s54 enhancer binding protein NtrC in controlling the response to nitrogen limitation and virulence. RNA-Seq analyses and phenotypical analysis on a ntrC mutant strain showed that, in addition to orchestrating uptake of nitrogen sources, NtrC is also regulating exopolysaccharide (EPS) production and motility. A search for NtrC consensus sequences identified a potential binding sequence in the promoter region of gene clusters involved in EPS formation and flagellar rotation suggesting that NtrC directly controls the expression of these phenotypic traits in B. cenocepacia H111.
Project description:Burkholderia cenocepacia sequence type 32 (ST32) represents one of the most globally distributed strains from Bukrholderia cepacia complex (Bcc), which infected 30% of Czech cystic fibrosis (CF) patients. The aim of this study was to compare gene expression in two pairs of ST32 clinical isolates that were subjected to cultivation in two different conditions, characteristic for chronic B. cenocepacia infection in CF patients. ST32 strain is known to be a problematic epidemic strain, which caused a serious outbreak at the Prague CF centre.
Project description:BACKGROUND: We identified that a putative CzcRS-like two-component system (TCS) in Burkholderia cenocepacia K56-2 is required for heavy metal resistance and virulence. In an attempt to identify genes directly regulated by the CzcR response regulator, we performed ChIP-seq analysis to identify genomic regions bound by CzcR. METHODS. Sequence encoding a FLAG octapeptide (Sigma-Aldrich) was introduced to the 3’ end of the czcR gene at its native chromosomal position in wild-type B. cenocepacia K56-2. Wildtype K56-2 and the CzcR-FLAG strain were grown in 50 ml tryptone soya broth (TSB) in the presence or absence of 1.5 mM zinc chloride. After overnight incubation, anti-FLAG immunoprecipitation of CzcR-bound genomic DNA was performed. Wildtype K56-2 was processed in parallel, to serve as a control against which to assess for enrichment of genomic regions. Illumina sequencing libraries were prepared from the resulting DNA using the Nextflex ChIPseq protocol (Bioo Scientific) with indexed adapters. Libraries were amplified by 18 cycles PCR, purified using 0.8 volumes Ampure XP beads (Beckman Coulter) and quantified with a Bioanalyzer 7500 assay (Agilent). Libraries ranged in size from 150 bp to 800 bp with a average insert size of 310 bp. Libraries were pooled in equimolar concentrations, denatured and diluted to 6.5 pM, clustered on a flowcell using a cBOT (Illumina) and sequenced on a HiSeq2000 (Illumina). Paired-end reads were filtered using the fastq-mcf package from the ea-utils suite to remove reads with less than 90% Q20 scores or above and to trim off adaptor sequence. The reads were then aligned against the B. cenocepacia J2315 genome (NC_011001-NC_011003) using BWA (0.5.9) and converted to BAM format using Samtools. Potential PCR duplicates were removed using the samtools rmdup command. The MACS package (v1.4) was used to compare and contrast the control and sample data using the –call-subpeaks and –w options. RESULTS: Relative to wildtype B. cenocepacia K56-2, the only genomic region that was found to be enriched by the ChIP-seq analysis of the CzcR-FLAG strain mapped to nucleotide coordinates 787809-798988 on chromosome 2. This region includes the czcRS genes and those encoding the associated efflux pump (CzcCBA).