Dombrecht Bruno, Marchal Kathleen, Vanderleyden Jos, Michiels Jan
Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium.
Genome Biol. 2002;3(12):RESEARCH0076. doi: 10.1186/gb-2002-3-12-research0076. Epub 2002 Nov 26.
In the rhizobia, a group of symbiotic Gram-negative soil bacteria, RpoN (sigma54, sigmaN, NtrA) is best known as the sigma factor enabling transcription of the nitrogen fixation genes. Recent reports, however, demonstrate the involvement of RpoN in other symbiotic functions, although no large-scale effort has yet been undertaken to unravel the RpoN-regulon in rhizobia. We screened two complete rhizobial genomes (Mesorhizobium loti, Sinorhizobium meliloti) and four symbiotic regions (Rhizobium etli, Rhizobium sp. NGR234, Bradyrhizobium japonicum, M. loti) for the presence of the highly conserved RpoN-binding sites. A comparison was also made with two closely related non-symbiotic members of the Rhizobiales (Agrobacterium tumefaciens, Brucella melitensis).
A highly specific weight-matrix-based screening method was applied to predict members of the RpoN-regulon, which were stored in a highly annotated and manually curated dataset. Possible enhancer-binding proteins (EBPs) controlling the expression of RpoN-dependent genes were predicted with a profile hidden Markov model.
The methodology used to predict RpoN-binding sites proved highly effective as nearly all known RpoN-controlled genes were identified. In addition, many new RpoN-dependent functions were found. The dependency of several of these diverse functions on RpoN seems species-specific. Around 30% of the identified genes are hypothetical. Rhizobia appear to have recruited RpoN for symbiotic processes, whereas the role of RpoN in A. tumefaciens and B. melitensis remains largely to be elucidated. All species screened possess at least one uncharacterized EBP as well as the usual ones. Lastly, RpoN could significantly broaden its working range by direct interfering with the binding of regulatory proteins to the promoter DNA.
在根瘤菌（一类共生的革兰氏阴性土壤细菌）中，RpoN（σ54、σN、NtrA）作为能够使固氮基因转录的σ因子最为人所知。然而，最近的报告表明RpoN参与了其他共生功能，尽管尚未开展大规模工作来解析根瘤菌中的RpoN调控子。我们在两个完整的根瘤菌基因组（百脉根中生根瘤菌、苜蓿中华根瘤菌）和四个共生区域（菜豆根瘤菌、根瘤菌属NGR234、大豆慢生根瘤菌、百脉根中生根瘤菌）中筛选了高度保守的RpoN结合位点的存在情况。还与根瘤菌目中两个密切相关的非共生成员（根癌农杆菌、羊布鲁氏菌）进行了比较。
应用了一种基于高度特异性权重矩阵的筛选方法来预测RpoN调控子的成员，这些成员存储在一个经过高度注释和人工整理的数据集中。使用轮廓隐马尔可夫模型预测了控制RpoN依赖性基因表达的可能的增强子结合蛋白（EBP）。
用于预测RpoN结合位点的方法被证明非常有效，因为几乎所有已知的受RpoN控制的基因都被识别出来了。此外，还发现了许多新的RpoN依赖性功能。这些不同功能中的几种对RpoN的依赖性似乎具有物种特异性。大约30%的已识别基因是假设性的。根瘤菌似乎已将RpoN用于共生过程，而RpoN在根癌农杆菌和羊布鲁氏菌中的作用在很大程度上仍有待阐明。所有筛选的物种都至少拥有一种未表征的EBP以及常见的EBP。最后，RpoN可以通过直接干扰调节蛋白与启动子DNA的结合来显著扩大其工作范围。
