Analysis of the Haloferax volcanii Transcriptome. Haloferax volcanii

In this study the transcriptome of Haloferax volcanii DS70 (Wendoloski D, Ferrer C, Dyall-Smith ML. Microbiology. 2001, 147: 959-64), grown under of a variety of conditions, was mapped using a custom genome-wide tiled array consisting of 25 nt probes spaced in 35 nt windows. H. volcanii is a facultative aerobe capable of growth on simple carbon and nitrogen sources, it is amenable to genetic manipulations and has become a widely used model organism for studies on haloarchaeal physiology and global gene regulation. Of particular interest is the identification of genes encoding enzymes needed for growth on simple carbon and nitrogen sources and how these cells respond to limitations in these key nutrients. To address these questions we have compared the RNA populations of cells undergoing balanced growth in completely defined minimal medium (CDM) with succinate-glycerol, pyruvate or lactate as sole carbon sources with RNA from cells grown in complex rich medium (CX) containing yeast extract and tryptone. Data from these studies have identified a core group of genes expressing during balanced growth in either condition as well as specific genes associated with the biosynthesis of amino acids, nucleotides and other key building blocks. The response to nutrient limitation was also investigated. RNA populations from cells entering stationary phase from growth in CDM and CX media were compared to those undergoing balanced growth, and these RNAs were also compared with RNA populations of cells subjected to nitrogen or carbon starvation. Growth in the presence of histidine as the sole source of nitrogen in CDM medium also presented a model for growth with a “poor” nitrogen source. The results of these studies identified specific subpopulations of genes associated with nitrogen limitation and general nutrient limitation associated with the transition to stationary phase. Many of the responsive protein encoding genes were assigned general functions based on their similarity to known proteins; however, in each comparison, approximately half of the affected genes encode proteins classified as unknown or hypothetical proteins. The haloarchaea are also distinct among the Archaea in having multiple genes encoding the general transcription factor proteins TATA binding protein (TBP) and transcription factor IIB (TFB). We observed differential expression of these genes, providing further support for the proposal that alternative TBP-TFB pairings are associated with programmed changes in gene expression in the haloarchaea. Overall design: RNA was prepared from H. volcanii DS70 cells grown under a variety of conditions. These include: balanced growth in completely defined medium with succinate and glycerol, pyruvate, or lactate as sole carbon source; balanced growth in complex medium with yeast extract and tryptone; starvation for nitrogen or carbon by re-suspending cells, undergoing balanced growth, in media lacking these agents; balanced growth in the presence of low (10% w/v NaCl) and high (20 % or 25% w/v NaCl) salt, and cells entering stationary from growth in CDM or CX media. Four independent cultures were prepared for each growth variable and RNA was isolated from each culture. RNA samples were identified as acceptable for use in array analysis if clear 23S rRNA, 16S rRNA and tRNA-sized bands were present in agarose gel and capillary electrophoresis analysis of the samples. Three RNA samples were chosen for each growth variable and each was used as the source material for a separate chip experiment; these constituted three biological replicates for the growth variable. Data from 48 chip hybridizations, representing 16 growth conditions, were combined for the quantile scaling (Auer H, Newsom DL, Nowak NJ, McHugh KM, Singh S, Yu CY, Yang Y, Wenger GD, Gastier-Foster JM, Kornacker K. BMC Genomics. 2007, 8:111-124) presented in the data analyses. Two additional experiments are included where the RNAs of three independent biological samples were pooled into a single RNA population and used as the source material for an array experiment. These included RNA samples enriched for species less than 500 nt that were isolated from cells undergoing balanced growing, and entry into stationary phase, in CDM or CX media. A second pooled series were obtained from the H. volcanii DS70 mutant strains ASD40 (∆pyrF) and ASD41 (∆pyrF∆hutR) grown in CDM with ammonium ion or histidine as the sole source of nitrogen. Pooled samples are presented as quantile normalized values (Bolstad, B.M., Irizarry, R.A., Astrand, M. and Speed, T.P. Bioinformatics. 2003, 19: 185-193). Total genomic DNA from H. volcanii DS70 was also used as probe, in two independent array experiments, to measure the inherent hybridization to the probes. These data are presented as quantile scaled values (Auer et. al., 2007).

本研究针对在多种条件下培养的沃氏嗜盐古菌（Haloferax volcanii）DS70菌株（参考文献：Wendoloski D, Ferrer C, Dyall-Smith ML. Microbiology. 2001, 147: 959-64）的转录组进行了定位分析，所用工具为定制化全基因组平铺式微阵列（tiled array），该芯片由间隔35nt窗口排布的25nt探针组成。沃氏嗜盐古菌是一种兼性需氧菌，可利用简单碳源与氮源生长，易于开展遗传操作，现已成为研究嗜盐古菌生理学及全局基因调控的经典模式生物。本研究的核心关注点之一为鉴定以简单碳氮源生长所需的酶编码基因，以及此类细胞响应此类关键营养物质限制的分子机制。

为解答上述科学问题，本研究比较了两种培养条件下细胞的RNA群体：一是在完全限定基本培养基（completely defined minimal medium, CDM）中，分别以琥珀酸盐-甘油、丙酮酸或乳酸为唯一碳源进行平衡生长的细胞；二是在含有酵母提取物与胰蛋白胨的复合富培养基（complex rich medium, CX）中生长的细胞。本研究数据已鉴定出一组在两种培养条件下平衡生长过程中均表达的核心基因，以及与氨基酸、核苷酸及其他关键生物合成前体的生物合成相关的特异性基因。

本研究同时探究了细胞对营养限制的响应：将从CDM与CX培养基中生长进入稳定期的细胞的RNA群体，与处于平衡生长状态的细胞的RNA群体进行对比；同时还将此类RNA与经受氮饥饿或碳饥饿的细胞的RNA群体进行比较。在CDM培养基中以组氨酸作为唯一氮源进行培养的实验体系，也为利用"劣质"氮源生长的研究提供了模型。本研究结果鉴定出与氮限制以及与进入稳定期相关的一般性营养限制相关的特异性基因亚群。

诸多响应性蛋白编码基因可通过与已知蛋白的序列同源性被赋予一般功能；但在每一组比较分析中，约有半数受影响的基因编码的蛋白被归类为未知功能蛋白或假设蛋白。

嗜盐古菌在古菌域中具有独特性，其基因组携带多个编码通用转录因子的基因，包括TATA结合蛋白（TATA binding protein, TBP）与转录因子IIB（transcription factor IIB, TFB）。本研究观察到此类基因的差异表达，进一步支持了"替代性TBP-TFB配对与嗜盐古菌中基因表达的程序性变化密切相关"这一学术假说。

### 总体实验设计
从多种条件下培养的沃氏嗜盐古菌DS70细胞中提取制备RNA，所涵盖的培养条件包括：
1. 在以琥珀酸盐-甘油、丙酮酸或乳酸为唯一碳源的完全限定基本培养基中进行平衡生长；
2. 在含有酵母提取物与胰蛋白胨的复合富培养基中进行平衡生长；
3. 通过将处于平衡生长状态的细胞重悬于缺乏相应营养物质的培养基中，构建氮饥饿或碳饥饿模型；
4. 在低（10% w/v NaCl）与高（20%或25% w/v NaCl）盐浓度条件下的平衡生长；
5. 从CDM或CX培养基中生长进入稳定期的细胞。

每个培养变量设置4个独立重复培养物，并从每一个培养物中分离RNA。若琼脂糖凝胶电泳与毛细管电泳分析显示样本中存在清晰的23S rRNA、16S rRNA及tRNA特征条带，则认定该RNA样本可用于微阵列分析。每个培养变量选取3个符合要求的RNA样本，分别作为独立芯片实验的起始材料，以此构成该培养变量的3个生物学重复。

本数据分析采用的分位数缩放方法（quantile scaling，参考文献：Auer H, Newsom DL, Nowak NJ, McHugh KM, Singh S, Yu CY, Yang Y, Wenger GD, Gastier-Foster JM, Kornacker K. BMC Genomics. 2007, 8:111-124）整合了覆盖16种生长条件的48次芯片杂交数据。

本研究额外包含2项实验：将3个独立生物学样本的RNA混合为单个RNA群体，用作芯片实验的起始材料。其中一组混合样本为从CDM或CX培养基中平衡生长及进入稳定期的细胞中分离的、长度小于500nt的富集RNA片段；另一组混合样本来自沃氏嗜盐古菌DS70的突变菌株ASD40（∆pyrF）与ASD41（∆pyrF∆hutR），此类菌株在以铵离子或组氨酸为唯一氮源的CDM培养基中培养。混合样本以分位数归一化值（quantile normalized values，参考文献：Bolstad, B.M., Irizarry, R.A., Astrand, M. and Speed, T.P. Bioinformatics. 2003, 19: 185-193）呈现。

沃氏嗜盐古菌DS70的全基因组DNA也在2次独立芯片实验中用作探针，以检测探针的固有杂交信号。此类数据以分位数缩放值呈现（Auer等, 2007）。