Hübner P, Masepohl B, Klipp W, Bickle T A
Department of Microbiology, Biozentrum der Universität Basel, Switzerland.
Mol Microbiol. 1993 Oct;10(1):123-32. doi: 10.1111/j.1365-2958.1993.tb00909.x.
The expression of nif genes in Rhodobacter capsulatus depends on the two regulatory genes, rpoN and nifA, encoding a nif-specific alternative sigma factor of RNA polymerase and a nif-specific transcriptional activator, respectively. The expression of the rpoN gene itself is also RPON/NIFA dependent. In order to better characterize the regulation of nif gene induction, chromosomal nifH-, rpoN-, nifA1- and nifA2- lacZ fusions were constructed and the expression of these different nif-lacZ fusions was determined under photoheterotrophic conditions at different starting ammonium concentrations. The two nifA genes were found to be induced first, followed by nifH and finally by rpoN upon weak, medium and strong nitrogen starvation, respectively. This induction profile and the correlation between the expression of the different nif genes suggested that nifA1 expression is the limiting factor for nif gene induction. This hypothesis was tested by construction of different nifA1 overexpressing mutants. Contrary to the current model of nif gene expression in R. capsulatus, which predicted constitutive nif gene expression in such mutants, a strong repression of nifH and rpoN was found at high ammonium concentration. The low nifH expression under these conditions is unaffected by nifA2 and is not increased in a ntrC mutant, ruling out any role of NTRC as a mediator of this repression. This finding implies an additional, so far unidentified, regulation by fixed nitrogen in R. capsulatus. Changing the expression level of rpoN indicated that low levels of RPON are already sufficient for full nifH induction. The nifA1 and rpoN expression mutants were also tested for diazotrophic growth. Similar generation times were determined for the mutants and for the wild type, but diazotrophic growth of the nifA1 over-expressing ntrC mutant RCM14 did not start until after a prolonged lag phase of two to three days.
荚膜红细菌中固氮基因（nif基因）的表达依赖于两个调控基因，即rpoN和nifA，它们分别编码RNA聚合酶的一种nif特异性替代σ因子和一种nif特异性转录激活因子。rpoN基因自身的表达也是RPON/NIFA依赖性的。为了更好地表征nif基因诱导的调控，构建了染色体nifH-、rpoN-、nifA1-和nifA2- lacZ融合体，并在不同起始铵浓度的光异养条件下测定了这些不同nif-lacZ融合体的表达。发现两个nifA基因分别在弱、中、强氮饥饿条件下首先被诱导，随后是nifH，最后是rpoN。这种诱导模式以及不同nif基因表达之间的相关性表明，nifA1的表达是nif基因诱导的限制因素。通过构建不同的nifA1过表达突变体对这一假设进行了验证。与目前荚膜红细菌中nif基因表达的模型相反，该模型预测此类突变体中nif基因组成型表达，但在高铵浓度下发现nifH和rpoN受到强烈抑制。在这些条件下低水平的nifH表达不受nifA2影响，并且在ntrC突变体中也不增加，排除了NTRC作为这种抑制作用介导因子的任何作用。这一发现意味着荚膜红细菌中存在一种额外的、迄今尚未明确的由固定氮介导的调控。改变rpoN的表达水平表明，低水平的RPON已经足以实现nifH的完全诱导。还对nifA1和rpoN表达突变体进行了固氮生长测试。测定了突变体和野生型的相似代时，但过表达nifA1的ntrC突变体RCM14的固氮生长直到两到三天的延长滞后期后才开始。
