Defining the regulon of a CzcRS-like two-component system in Burkholderia cenocepacia K56-2 (ChIP-Seq)

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). Overall design: Genomic regions bound by the CzcR response regulator were identified by ChIP-seq analysis of a strain expressing a FLAG-tagged CzcR protein. Enriched genomic regions were determined by comparison to the wildtype (parental) strain which did not possess a FLAG tag. Sequencing was performed on an Illumina HiSeq2000.

研究背景：本研究证实，洋葱伯克霍尔德菌（Burkholderia cenocepacia）K56-2中一种推定的CzcRS样双组分系统（two-component system, TCS）参与重金属抗性与毒力调控。为鉴定受CzcR响应调控因子直接调控的基因，我们通过染色质免疫共沉淀测序（ChIP-seq）分析，鉴定CzcR结合的基因组区域。
实验方法：将编码FLAG八肽（Sigma-Aldrich）的序列插入野生型洋葱伯克霍尔德菌K56-2中czcR基因的3'末端，且插入位点位于该基因的天然染色体位置。将野生型K56-2与CzcR-FLAG菌株分别接种于50 ml胰蛋白胨大豆肉汤（tryptone soya broth, TSB）中，分别添加或不添加1.5 mM氯化锌。过夜培养后，采用抗FLAG抗体免疫沉淀结合了CzcR的基因组DNA；同时以野生型K56-2作为对照，用于评估基因组区域的富集情况。使用Nextflex ChIP-seq建库试剂盒（Bioo Scientific）搭配带索引的接头，从所得DNA中构建Illumina测序文库。通过18轮PCR扩增文库，使用0.8倍体积的Ampure XP磁珠（Beckman Coulter）纯化，并采用Agilent 7500生物分析仪进行定量。文库片段大小范围为150 bp至800 bp，平均插入片段大小为310 bp。将文库按等摩尔浓度混合，变性并稀释至6.5 pM，使用cBOT（Illumina）在流动槽上完成簇生成，随后在HiSeq2000（Illumina）测序平台上进行测序。使用ea-utils工具集的fastq-mcf软件包对双端读段进行过滤：去除Q20质量值占比低于90%的读段，并截除接头序列。随后使用BWA（0.5.9版本）将读段比对至洋葱伯克霍尔德菌J2315的基因组（NC_011001-NC_011003），并使用Samtools将比对结果转换为BAM格式。使用samtools rmdup命令去除潜在的PCR重复读段。使用MACS软件包（v1.4版本），通过–call-subpeaks和–w参数对对照与样本数据进行比较分析。
实验结果：相较于野生型洋葱伯克霍尔德菌K56-2，通过对CzcR-FLAG菌株的ChIP-seq分析，仅发现一个基因组区域存在富集，该区域定位于2号染色体的787809-798988核苷酸坐标区间。该区域包含czcRS基因以及编码相关外排泵（CzcCBA）的基因。
实验整体设计：通过对表达FLAG标签化CzcR蛋白的菌株进行ChIP-seq分析，鉴定结合CzcR响应调控因子的基因组区域；通过与未携带FLAG标签的野生型（亲本）菌株进行比对，确定富集的基因组区域；测序工作在Illumina HiSeq2000平台上完成。