Microarray analysis of phosphate regulation in Synechococcus sp. WH8102

Primary productivity of open ocean environments, such as those inhabited by marine picocyanobacteria Synechococcus sp.WH8102, are often limited by low inorganic phosphate (P). To observe how this organism copes with P starvation, we constructed a full genome microarray and examined differences in gene expression under P-limited and P-replete growth conditions. To determine the temporal nature of the responses, comparisons were made for cells newly entered into P-stress (at a time point corresponding to the induction of extracellular alkaline phosphatase activity) and a later time point (late log phase). In almost all instances the P starvation response was transitory, with 36 genes showing significant upregulation (>log2 fold) while 23 genes were highly downregulated at the early time point; however, these changes in expression were maintained for only five of the upregulated genes. Knockout mutants were constructed for genes SYNW0947 or SYNW0948, comprising a two component regulator hypothesized to play a key role in regulating the response to P-limitation. A high degree of overlap in the sets of genes affected by P-limited conditions and in the knockout mutants supports this hypothesis; however there is some indication that other regulators may play a role in this response in Synechococcus sp. WH8102. Consistent with what has been observed in many other cyanobacteria, the Pho regulon of this strain is comprised largely of genes for alkaline phosphatases, P transport or P metabolism. Interestingly, however, the exact composition and arrangement of the Pho regulon appears highly variable in marine cyanobacteria. In this series four conditions have been analyzed. These are low phosphate stress during early log phase, low phosphate stress during late log phase, SYNW0947 mutation, and SYNW0948 mutation. There are six slides per condition, each with two biological replicates. There are three technical replicates for each biological replicate including one flip-dye comparison. The exception is the low phosphate stress during late log phase experiment which has a total of five slides, two biological replicates with three and two technical replicates, respectively, and one flip-dye comparison for each biological replicate. Each slide contains six replicate spots per gene.

开放海洋环境（如栖息着海洋微蓝细菌聚球藻（Synechococcus sp.）WH8102的海域）的初级生产力，常受低浓度无机磷（P）的限制。为探究该菌株如何应对磷饥饿胁迫，我们构建了全基因组微阵列，检测了磷限制与磷充足生长条件下的基因表达差异。为明确该应答的时序特征，我们分别对刚进入磷胁迫阶段（对应胞外碱性磷酸酶活性被诱导的时间节点）以及对数生长后期的细胞进行了比较分析。
在绝大多数情况下，磷饥饿应答具有瞬时性：在早期时间点，共有36个基因呈现显著上调（log₂倍变化>1），23个基因显著下调；但仅有5个上调基因的表达变化得以持续维持。我们针对SYNW0947和SYNW0948基因构建了敲除突变体，这两个基因属于双组分调控系统，被推测在磷限制应答调控中发挥关键作用。受磷限制条件影响的基因集与敲除突变体中的差异基因集存在高度重叠，这一结果支持了上述假说；但也有迹象表明，聚球藻WH8102中可能存在其他调控因子参与该应答过程。
与诸多其他蓝细菌的研究结果一致，该菌株的Pho调控子（Pho regulon）主要由编码碱性磷酸酶、磷转运蛋白以及磷代谢相关蛋白的基因构成。但值得注意的是，海洋蓝细菌中Pho调控子的具体组成与排布方式呈现出高度的多样性。本研究共分析了4组实验条件：分别为对数生长早期的低磷胁迫、对数生长后期的低磷胁迫、SYNW0947基因敲除突变体以及SYNW0948基因敲除突变体。
每组实验条件对应6张芯片玻片，每张玻片包含2个生物学重复；每个生物学重复设置3个技术重复，其中包含1次荧光反转（flip-dye）比较。唯有对数生长后期低磷胁迫实验组为例外：该组共计5张芯片玻片，包含2个生物学重复，分别对应3个和2个技术重复，且每个生物学重复均设置1次荧光反转比较。每张芯片玻片上每个基因设有6个重复点样。