Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans.
Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans.
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DOI：
10.1042/bcj20170115
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发表时间：
2017-05-10
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影响因子：
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通讯作者：
Roldán MD
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作者：
Luque-Almagro VM;Manso I;Sullivan MJ;Rowley G;Ferguson SJ;Moreno-Vivián C;Richardson DJ;Gates AJ;Roldán MD
研究了脱氮拟球藻PD1222对硝酸盐依赖的合成代谢的转录适应。以硝酸盐为氮源，以氨氮为氮源，共诱导了74个基因，其中包括nasTSABGHC和ntrBC基因。NAST和NASS基因是共转录的，尽管NAST受硝酸盐的诱导比NASS更强。NasABGHC基因构成一个转录单位，其前面是一个非编码区，包含参与转录终止的发夹结构。以硝酸盐或谷氨酸为氮源时，NASTs和NasABGHC转录本的检测水平相似，但在氨基培养的细胞中未检测到NasABGHC转录本。用双向聚丙烯酰胺凝胶电泳法在硝酸盐细胞胞质组分中检测到亚硝酸还原酶NasG亚基，但以氨水或谷氨酸为氮源时均未检测到该亚基。Nast突变体缺乏nasABGHC转录本和依赖于NADH的硝酸还原酶活性。相反，NASS突变体表现出与野生型菌株相似的nasABGHC转录本水平，并且在除铵之外的所有氮源中都表现出NasG蛋白和NADH-硝酸还原酶活性。NASABGHC的铵抑制依赖于NTR系统。无论支持生长的氮源是什么，ntrBC和ntrYX基因都在低水平表达。对ntrBCyX基因的突变分析表明，虽然ntrBC基因是硝酸盐同化所必需的，但在缺乏ntrBC基因的情况下，ntrYX基因只能部分恢复硝酸盐的生长。提出了脱氮假单胞菌硝酸盐同化的调控机制，即硝酸盐的诱导在转录和翻译水平上均有作用。
Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS. The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH–nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed.