Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, Ås, Norway.
Department of Chemistry, Biotechnology and Food Sciences, Norwegian University of Life Sciences, Ås, Norway.
Microbiology (Reading). 2012 Mar;158(Pt 3):826-834. doi: 10.1099/mic.0.054148-0. Epub 2011 Dec 15.
The reductases performing the four steps of denitrification are controlled by a network of transcriptional regulators and ancillary factors responding to intra- and extracellular signals, amongst which are oxygen and N oxides (NO and NO2(-)). Although many components of the regulatory network have been identified, there are gaps in our understanding of their role(s) in controlling the expression of the various reductases, in particular the environmentally important N(2)O reductase (N(2)OR). We investigated denitrification phenotypes of Paracoccus denitrificans mutants deficient in: (i) regulatory proteins (three FNR-type transcriptional regulators, NarR, NNR and FnrP, and NirI, which is involved in transcription activation of the structural nir cluster); (ii) functional enzymes (NO reductase and N(2)OR); or (iii) ancillary factors involved in N(2)O reduction (NirX and NosX). A robotized incubation system allowed us to closely monitor changes in concentrations of oxygen and all gaseous products during the transition from oxic to anoxic respiration. Strains deficient in NO reductase were able to grow during denitrification, despite reaching micromolar concentrations of NO, but were unable to return to oxic respiration. The FnrP mutant showed linear anoxic growth in a medium with nitrate as the sole NO(x), but exponential growth was restored by replacing nitrate with nitrite. We interpret this as nitrite limitation, suggesting dual transcriptional control of respiratory nitrate reductase (NAR) by FnrP and NarR. Mutations in either NirX or NosX did not affect the phenotype, but the double mutant lacked the potential to reduce N(2)O. Finally, we found that FnrP and NNR are alternative and equally effective inducers of N(2)OR.
执行反硝化的四个步骤的还原酶受转录调控网络和对细胞内和细胞外信号作出反应的辅助因子的控制,其中包括氧气和氮氧化物(NO 和 NO2(-))。尽管已经确定了调控网络的许多组成部分,但我们对它们在控制各种还原酶的表达方面的作用(特别是在环境中重要的 N2O 还原酶(N2OR)方面)的理解还存在差距。我们研究了缺失以下成分的脱氮假单胞菌突变体的反硝化表型:(i)调控蛋白(三种 FNR 型转录调控因子,NarR、NNR 和 FnrP,以及 NirI,其参与结构 nir 簇的转录激活);(ii)功能酶(NO 还原酶和 N2OR);或(iii)参与 N2O 还原的辅助因子(NirX 和 NosX)。一个自动化的孵育系统使我们能够在从需氧呼吸到缺氧呼吸的转变过程中密切监测氧气和所有气态产物浓度的变化。缺乏 NO 还原酶的菌株能够在反硝化过程中生长,尽管达到了微摩尔浓度的 NO,但无法恢复到需氧呼吸。FnrP 突变体在以硝酸盐为唯一 NO(x)的培养基中表现出线性缺氧生长,但通过用亚硝酸盐替代硝酸盐,指数生长得以恢复。我们将其解释为亚硝酸盐限制,表明 FnrP 和 NarR 对呼吸硝酸盐还原酶(NAR)进行双重转录控制。NirX 或 NosX 的突变均不影响表型,但双突变体缺乏还原 N2O 的潜力。最后,我们发现 FnrP 和 NNR 是 N2OR 的替代且同样有效的诱导物。
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