Comparative transcriptomics of wild-type (UCD 153) and patN-deletion strains (UCD 524) of Nostoc punctiforme ATCC 29133. Nostoc punctiforme PCC 73102

Heterocysts, cells specialized for nitrogen fixation in certain filamentous cyanobacteria, appear singly in a nonrandom spacing pattern along the chain of vegetative cells. A two-stage, biased initiation and competitive resolution model has been proposed to explain the establishment of this spacing pattern. There is substantial evidence that competitive resolution of a subset of cells initiating differentiation occurs by interactions between a self enhancing activator, HetR, and a diffusible inhibitor PatS-5 (RGSGR). Results presented here show that the absence of a unique membrane protein, PatN, in Nostoc punctiforme strain ATCC 29133 leads to a threefold increase in heterocyst frequency and a fourfold decrease in the vegetative cell interval between heterocysts. A PatN-GFP translational fusion shows a pattern of biased inheritance in daughter vegetative cells of ammonium-grown cultures. Inactivation of another heterocyst patterning gene, patA, is epistatic to inactivation of patN, and transcription of patA increases in a patN- deletion strain, implying that patN may function by modulating levels of patA. The presence of PatN is hypothesized to decrease the competency of a vegetative cell to initiate heterocyst differentiation, and the cellular concentration of PatN is dependent on cell division that results in cells transiently depleted of PatN. We suggest that biased inheritance of cell-fate determinants is a phylogenetic domain- spanning paradigm in the development of biological patterns. Overall design: Change in gene expression for a wild-type (UCD 153) and patN-deletion strain, (UCD 524) of Nostoc punctiforme ATCC 29133 over the time course of heterocyst developments. Total RNA from 3 biological replicates at each time point from 0 to 120 hours after removal of combined nitrogen (Nitrogen step-down) was converted to cDNA, dye-labled and hybridized to nimblegen 12x135k array slides.

异形胞（Heterocysts）是某些丝状蓝细菌中特化用于固氮作用（nitrogen fixation）的细胞，它们沿营养细胞（vegetative cells）链以非随机间距模式单个排布。已有研究提出两阶段偏倚起始与竞争性解析模型（two-stage, biased initiation and competitive resolution model），用以阐释该间距模式的建立机制。现有大量证据表明，分化起始细胞亚群的竞争性解析过程，是通过自我增强激活因子HetR与可扩散抑制因子PatS-5（RGSGR）之间的相互作用实现的。
本研究结果显示，在点状念珠藻ATCC 29133菌株（Nostoc punctiforme strain ATCC 29133）中，特异性膜蛋白PatN的缺失会使异形胞频率升高三倍，且异形胞之间的营养细胞间隔缩短四倍。PatN-GFP翻译融合蛋白（PatN-GFP translational fusion）实验表明，在铵盐培养的菌体中，该融合蛋白在子代营养细胞内呈现偏倚遗传模式。另一异形胞模式形成基因patA的失活对patN的失活具有上位性；且在patN缺失菌株中，patA的转录水平显著升高，这提示patN可能通过调控patA的表达水平发挥功能。我们推测，PatN的存在会降低营养细胞启动异形胞分化的感受态，而PatN的细胞浓度依赖于细胞分裂过程，这会使得细胞暂时出现PatN耗竭的状态。本研究提出，细胞命运决定因子（cell-fate determinants）的偏倚遗传是一种跨系统发育域的通用范式，广泛存在于生物模式发育进程中。
整体实验设计：本研究针对点状念珠藻ATCC 29133的野生型菌株（UCD 153）与patN缺失菌株（UCD 524），在异形胞发育的时间进程中检测基因表达变化。在去除结合态氮（即氮素降阶处理，Nitrogen step-down）后的0至120小时的每个时间点，分别收集3次生物学重复的总RNA，将其反转录为cDNA并进行荧光染料标记，随后与NimbleGen 12x135k基因芯片玻片进行杂交。