Teixeira R L F, von der Weid I, Seldin L, Rosado A S
Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brasil.
Lett Appl Microbiol. 2008 Mar;46(3):344-9. doi: 10.1111/j.1472-765X.2008.02322.x. Epub 2008 Feb 7.
The aim of this study was to develop an approach based on a reverse transcriptase (RT)-PCR/denaturing gradient gel electrophoresis (DGGE) for the detection of the functional genes nifH and anfH in Paenibacillus durus.
Two sets of primers were employed to study the expression of the nitrogen fixation genes in a pure-culture system of P. durus grown in media with increasing concentrations of ammonium (NH(4)(+)), tungsten (W) or molybdenum (Mo). The results obtained indicate that the expression of nitrogenase genes from P. durus can take place in the presence of relatively high levels of fixed nitrogen. It was also observed that the addition of 20 micromol l(-1) molybdenum and 2 mmol l(-1) tungstate did not interfere in the mRNA levels of nifH and anfH genes.
Our results demonstrate the presence and transcription of nifH and anfH in P. durus under a variety of growth conditions. A specific set of primers was designed for the detection of the alternative system for nitrogen fixation in P. durus.
The RT-PCR/DGGE system enables the rapid gathering of incremental data about the regulation of conventional and alternative nitrogenase genes in P. durus strains.
本研究的目的是开发一种基于逆转录酶（RT）-聚合酶链反应（PCR）/变性梯度凝胶电泳（DGGE）的方法，用于检测耐久芽孢杆菌中的功能基因nifH和anfH。
使用两组引物研究了在添加浓度递增的铵（NH₄⁺）、钨（W）或钼（Mo）的培养基中生长的耐久芽孢杆菌纯培养系统中固氮基因的表达。所得结果表明，在相对高水平的固定氮存在下，耐久芽孢杆菌的固氮酶基因仍可表达。还观察到添加20 μmol l⁻¹钼和2 mmol l⁻¹钨酸盐不会干扰nifH和anfH基因的mRNA水平。
我们的结果证明了在各种生长条件下，耐久芽孢杆菌中存在nifH和anfH并能进行转录。设计了一组特异性引物用于检测耐久芽孢杆菌中固氮的替代系统。
RT-PCR/DGGE系统能够快速收集关于耐久芽孢杆菌菌株中传统和替代固氮酶基因调控的增量数据。
