Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows exclusively in the xylem of several plants, causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time-course experiment (2, 8 and 12 hours) revealed many differentially expressed genes under nitrogen starvation, such as genes related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding a glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter and contains a predicted NtrC binding site. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon. For time-course studies, cells cultivated at late-exponential phase in PWG medium were used to inoculate a culture in 100 ml XDM2 medium to an optical density at 600 nm (OD600 nm) of 0.1. Cells were grown during 12 days in the XDM2 medium (mid-log phase) and harvested by centrifugation. Then, the culture was divided into two 6 portions and cells were washed with XDM2 medium (zero time) or XDM2 medium lacking all nitrogen sources (XDM0), respectively. The cultivation was continued for 2h, 8h and 12h in XDM0 to establish nitrogen starvation conditions. For each time point, cells in a 25-ml culture were collected by centrifugation and rapidly frozen in dry ice, until RNA isolation. Three RNA samples isolated from independently grown cultures of the cells at each starvation period (2h, 8h and 12h) were examined, and each preparation was subjected to microarray analysis. As the genes were spotted at least in duplicate, we obtained six replicates for each gene from three independent data sets per gene per starvation period. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a σ54-dependent manner. To determine the effect of rpoN inactivation on gene expression after nitrogen starvation, the transcriptomes of the wild type and the rpoN strains were compared using DNA microarrays, with both strains grown on XDM2 medium and submitted to nitrogen starvation (XDM0) during 2 hours. Three RNA samples isolated from independently grown cultures of the cells were examined. Due to the platform design, each microarray slide was divided into "LEFT" and "RIGHT", allowing the probing of two technical replicates per slide.

**背景**：苛养革兰氏阴性菌快速约翰逊氏菌（Xylella fastidiosa）仅能在多种植物的木质部中定植繁殖，可引发柑橘杂色褪绿病等病害。由于木质部汁液中氨基酸及其他化合物浓度较低，该菌在自然生境中需应对氮限制压力。
**结果**：本研究对快速约翰逊氏菌的氮饥饿响应开展了全基因组微阵列分析（whole-genome microarray analysis）。通过2小时、8小时及12小时的时间进程实验，发现氮饥饿条件下存在大量差异表达基因，包括与物质转运、氮同化、氨基酸生物合成、转录调控相关的基因，以及大量编码假定蛋白的基因。此外，本研究还观察到参与碳代谢与能量生成通路的诸多基因的表达水平出现下调。通过比较野生型菌株与rpoN基因缺失突变株的基因表达谱，鉴定出以σ54依赖方式受氮饥饿诱导的基因。结合转录组数据与基于位置权重矩阵（position weight matrix）的计算机模拟（in silico）σ54依赖型启动子预测，进一步完善了σ54调节子的调控网络。本研究通过引物延伸实验（primer extension assays）验证了位于编码谷氨酰胺合成酶的glnA基因上游的一个σ54预测结合位点，证实该基因确实拥有σ54依赖型启动子，并包含预测的NtrC结合位点。
**结论**：综上，本研究结果表明氮饥饿会引发快速约翰逊氏菌转录组的剧烈变化，其中部分差异表达基因属于σ54调节子。
对于时间进程实验，本研究将在PWG培养基中培养至晚期指数生长期的菌体，接种至100 mL XDM2培养基中，使初始600 nm光密度（OD600 nm）达到0.1。将菌体在XDM2培养基中培养12天至中期对数生长期后，通过离心收集菌体。随后将培养物均分为两份，分别用XDM2培养基（零时间点）与不含任何氮源的XDM2培养基（XDM0）重悬洗涤菌体。将菌体在XDM0培养基中继续培养2小时、8小时及12小时以建立氮饥饿模型。在每个时间点，取25 mL培养物通过离心收集菌体，并用干冰快速冷冻，直至进行RNA提取。针对每个饥饿处理时长（2小时、8小时、12小时），本研究均使用3份来自独立培养物的RNA样本进行检测，且每份样本均开展微阵列分析。由于基因探针至少以重复点样形式排布，每个饥饿处理时长下每个基因均可从3组独立数据集获得6次技术重复。
通过比较野生型菌株与rpoN基因缺失突变株的基因表达谱，可鉴定出以σ54依赖方式受氮饥饿诱导的基因。为确定rpoN基因失活对氮饥饿后基因表达的影响，本研究利用DNA微阵列比较野生型菌株与rpoN突变株的转录组，两组菌株均在XDM2培养基中培养并经2小时XDM0氮饥饿处理。本研究同样使用3份来自独立培养物的RNA样本进行检测。
由于芯片平台的设计，每块微阵列玻片分为"LEFT"与"RIGHT"两个区域，可在单张玻片上完成2次技术重复检测。