Expression profiling of a spf deletion mutant suggests biological roles and mRNA targets for Spot 42 in the fish pathogen Aliivibrio salmonicida

Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role remained obscure until it was shown to cause discoordinate expression of the galactose operon (gal operon). Recently Spot 42 has also been implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria, which includes a number of serious pathogens of humans and animals, including the infamous Vibrio cholerae. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84 % identity to E. coli Spot 42 (spf). We generated a A. salmonicida spf deletion mutant. We then and used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose we found a surprisingly large number of ≥2× differentially expressed genes, and several major cellular processes were affected, such as carbohydrate metabolism and transport, motility and chemotaxis, iron homeostasis and quorum sensing. A gene encoding a pirin-like protein (VSAL_I1200) showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, in a global search we discovered a sRNA, named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is highly dependent on glucose. Our hypothesis is that this novel sRNA works in concert with Spot 42 to regulate carbohydrate metabolism and uptake. Two-condition experiment, wild type cells (control samples) vs. spf mutants grown in A. salmonicida-specific minimal media (ASMM) (stimulated samples). Samples were prepared by culturing the A. salmonicida wild type and a spf deletion mutant strains at 12 °C in ASMM without any added carbon source to OD 600nm 0.4. At this density glucose to 44.4 mM was added. Samples were collected 15 min after stimulation. Biological replicates for each sample: 3 wild type, 3 spf mutant, independently grown and harvested. One replicate per array.

Spot 42 于近40年前在大肠杆菌（Escherichia coli）中被发现，是一种丰度较高、分子量较小且稳定性较差的核糖核酸（RNA）。其生物学功能长期不明，直至研究证实它可引发半乳糖操纵子（galactose operon，gal操纵子）的表达失调。近年来的研究还表明，Spot 42在中央代谢与次级代谢中均具有更为广泛的调控作用。Spot 42由spf基因编码。该基因广泛分布于γ-变形菌门弧菌科（Vibrionaceae）家族中，该家族包含多种危及人类与动物的严重致病性病原菌，其中臭名昭著的霍乱弧菌（Vibrio cholerae）也隶属于此。该科的成员之一杀鲑弧菌（Aliivibrio salmonicida），可引发养殖大西洋鲑的冷水弧菌病。其基因组编码的Spot 42与大肠杆菌Spot 42（spf）的序列同源性达84%。本研究构建了杀鲑弧菌的spf基因缺失突变株，随后通过微阵列（microarray）与Northern印迹（Northern blot）分析对转录组的全局表达变化进行检测，以期阐明Spot 42在该细菌中的生物学功能。在葡萄糖存在的条件下，我们检测到数量可观的差异表达倍数≥2倍的基因，且多个关键细胞进程受到影响，包括碳水化合物代谢与转运、运动性与趋化性、铁稳态以及群体感应（quorum sensing）。一种编码类Pirin蛋白的基因（VSAL_I1200）在Spot 42存在与缺失的情况下呈现完全的“开/关”式表达模式，这提示Spot 42可通过调控发酵与呼吸途径的转换，在中央代谢中发挥关键调控作用。有趣的是，在全局转录组搜索中，我们发现了一个紧邻spf基因下游区域编码的小RNA（small RNA, sRNA），将其命名为VSsrna24。该新型sRNA的表达模式与Spot 42恰好相反，且其表达高度依赖葡萄糖的存在。我们提出假说：这一新型sRNA可与Spot 42协同调控碳水化合物的代谢与摄取。本实验为双条件对照设计：以在杀鲑弧菌特异性基础培养基（ASMM）中培养的野生型菌株为对照样本，同条件下培养的spf缺失突变株为处理样本。样本制备流程如下：将杀鲑弧菌野生型菌株与spf缺失突变株置于12℃、无外加碳源的ASMM培养基中培养，至光密度OD600nm为0.4时，向培养基中添加终浓度为44.4 mM的葡萄糖。于葡萄糖添加处理15分钟后收集样本。每个样本设置3次生物学重复，即野生型菌株与spf缺失突变株各3株独立培养并收获的样本，每块基因芯片阵列对应一个生物学重复。