REGULATION OF NAD METABOLISM IN SALMONELLA-TYPHIMURIUM - MOLECULAR SEQUENCE-ANALYSIS OF THE BIFUNCTIONAL NADR REGULATOR AND THE NADA-PNUC OPERON
REGULATION OF NAD METABOLISM IN SALMONELLA-TYPHIMURIUM - MOLECULAR SEQUENCE-ANALYSIS OF THE BIFUNCTIONAL NADR REGULATOR AND THE NADA-PNUC OPERON
复制标题
DOI：
10.1128/jb.172.8.4187-4196.1990
复制
发表时间：
1990-08-01
影响因子：
3.2
通讯作者：
SPECTOR, MP
中科院分区：
文献类型：
--
作者：
FOSTER, JW;PARK, YK;SPECTOR, MP
在鼠伤寒沙门氏菌中，NAD的从头合成通过nadA和nadB基因座的转录控制来调节。同样，吡啶核苷酸补救途径在pncB处受到控制。这三个位点的转录表达受nadR产物的协同调控。然而，有遗传学证据表明，NadR是双功能的，在监管和运输能力。nadR基因座中的一类突变赋予转运缺陷型PnuA-表型。这些突变体保留调节特性，但不能运输烟酰胺单核苷酸（NMN）完整的细胞膜。其他nadR突变体失去了调节和运输能力，而第三类只失去了调节能力。不寻常的NMN转运活性需要PnuC和NadR蛋白，其中pnuC基因座位于具有nadA的操纵子中。为了证明nadR编码单一蛋白质并深入了解调控靶基因座，对nadR和nadA pnuC基因座进行克隆和测序。发现补充调节和转运突变的DNA片段含有能够编码409个氨基酸蛋白（47，022道尔顿）的单个开放阅读框架，表明NadR确实是双功能的。通过对补充NadA和PnuC突变体表型的2.4-脱氢酶DNA片段的序列分析，获得了NadA和PnuC操纵子排列的确认。在大细胞中证实的nadA产物是365个氨基酸的蛋白质（40，750道尔顿），而pnuC编码322个氨基酸的蛋白质（36，930道尔顿）。PnuC蛋白的极疏水性（71%）表明它是一种完整的膜蛋白，与其在NMN跨细胞质膜运输中的核心作用一致。在这里和以前的研究中提出的结果表明一个假设的模型，其中NadR与PnuC在低的内部NAD水平下相互作用，允许NMN完整地运输到细胞中。随着细胞内NAD水平的增加，NadR对nadA、nadB和pncB操纵基因区的亲和力增加，从而抑制这些靶基因的转录。
In Salmonella typhimurium, de novo synthesis of NAD is regulated through the transcriptional control of the nadA and nadB loci. Likewise, the pyridine nucleotide salvage pathway is controlled at pncB. The transcriptional expression of these three loci is coordinately regulated by the product of nadR. However, there is genetic evidence suggesting that NadR is bifunctional, serving in both regulatory and transport capacities. One class of mutations in the nadR locus imparts a transport-defective PnuA- phenotype. These mutants retain regulation properties but are unable to transport nicotinamide mononucleotide (NMN) intact across the cell membrane. Other nadR mutants lose both regulatory and transport capabilities, while a third class loses only regulatory ability. The unusual NMN transport activity requires both the PnuC and NadR proteins, with the pnuC locus residing in an operon with nadA. To prove that nadR encoded a single protein and to gain insight into a regulatory target locus, the nadR and nadA pnuC loci were cloned and sequenced. A DNA fragment which complemented both regulatory and transport mutations was found to contain a single open reading frame capable of encoding a 409-amino-acid protein (47,022 daltons), indicating that NadR is indeed bifunctional. Confirmation of the operon arrangement for nadA and pnuC was obtained through the sequence analysis of a 2.4-kilobase DNA fragment which complemented both NadA and PnuC mutant phenotypes. The nadA product, confirmed in maxicells, was a 365-amino-acid protein (40,750 daltons), while pnuC encoded a 322-amino-acid protein (36,930 daltons). The extremely hydrophobic (71%) nature of the PnuC protein indicated that it was an integral membrane protein, consistent with its central role in the transport of NMN across the cytoplasmic membrane. The results presented here and in previous studies suggest a hypothetical model in which NadR interacts with PnuC at low internal NAD levels, permitting transport of NMN intact into the cell. As NAD levels increase within the cell, the affinity of NadR for the operator regions of nadA, nadB, and pncB increases, repressing the transcription of these target genes.