Minerdi D, Fani R, Gallo R, Boarino A, Bonfante P
Dipartimento di Biologia Vegetale dell'Università di Torino and Centro di Studio sulla Micologia del Terreno del Consiglio Nazionale delle Ricerche, 10125 Turin, Italy.
Appl Environ Microbiol. 2001 Feb;67(2):725-32. doi: 10.1128/AEM.67.2.725-732.2001.
In this paper we report the identification and characterization of a DNA region containing putative nif genes and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome. Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6,413-bp region. Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs. PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G. margarita endosymbiont. They offer, therefore, the first sequence for the nif operon described for Burkholderia. Reverse transcriptase PCR experiments with primers designed on the Burkholderia nifH and nifD genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase. A phylogenetic analysis performed on the available nifK sequences placed the endosymbiotic Burkholderia close to A. brasilense.
在本文中，我们报告了一个DNA区域的鉴定和特征，该区域包含假定的固氮基因，属于丛枝菌根真菌珠状巨孢囊霉的伯克霍尔德氏菌内共生体。从真菌孢子中提取的总DNA基因组文库也代表了细菌基因组，并用于研究原核基因组。以巴西固氮螺菌的nifHDK基因作为原核探针筛选该文库，鉴定出一个6413 bp的区域。分析显示有三个开放阅读框，编码的假定蛋白质与来自不同固氮菌的固氮酶组分I的两个亚基（NifD和NifK）以及组分II（NifH）具有非常高的序列相似性。这三个基因排列成一个操纵子，类似于大多数古菌和细菌固氮菌所显示的操纵子。用基于伯克霍尔德氏菌nifHDK基因设计的引物进行的PCR实验和Southern印迹分析表明，它们实际上属于珠状巨孢囊霉内共生体的基因组。因此，它们提供了伯克霍尔德氏菌中描述的nif操纵子的第一个序列。用基于伯克霍尔德氏菌nifH和nifD基因设计的引物对从孢子中提取的总RNA进行逆转录酶PCR实验，结果表明基因表达仅限于萌发阶段。对可用的nifK序列进行的系统发育分析将内共生伯克霍尔德氏菌置于靠近巴西固氮螺菌的位置。
