Molecular genetic analysis of phosphite and hypophosphite oxidation by Pseudomonas stutzeri WM88
Molecular genetic analysis of phosphite and hypophosphite oxidation by Pseudomonas stutzeri WM88
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DOI：
10.1128/jb.180.21.5547-5558.1998
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发表时间：
1998-11-01
影响因子：
3.2
通讯作者：
Wolfe, RS
中科院分区：
文献类型：
--
作者：
Metcalf, WW;Wolfe, RS
首次报道了还原磷 (P) 化合物亚磷酸盐和次磷酸盐氧化生化途径的分子和遗传特征。该途径是在施氏假单胞菌 WM88 中发现的，根据其将次磷酸盐（+1 价）和亚磷酸盐（+3 价）氧化为磷酸盐（+5 价）的能力，从一组分离的生物体中选择该途径进行详细研究。将施氏疟原虫 WM88 氧化这两种化合物所需的基因克隆到单个约 1 上。通过筛选大肠杆菌和铜绿假单胞菌中的表达获得 30-kbp DNA 片段。两条证据表明次磷酸盐通过亚磷酸盐中间体氧化成磷酸盐。首先，容易获得在异源宿主上赋予亚磷酸盐氧化但不氧化次磷酸盐的质粒亚克隆。所有未能赋予亚磷酸盐氧化作用的质粒亚克隆也未能赋予次磷酸盐氧化作用。没有获得仅赋予次磷酸盐表达的亚克隆。其次，在体外制备了克隆基因的各种缺失衍生物，并将其重组到施氏疟原虫WM88的染色体上。个体突变体表现出两种表型。删除了编码亚磷酸盐氧化的区域的突变体（基于亚克隆结果）失去了氧化亚磷酸盐或次磷酸盐的能力。删除了编码次磷酸盐氧化的区域的突变体仅失去了氧化次磷酸盐的能力。这些突变体表现出的表型还表明，克隆的基因与原始施氏假单胞菌 WM88 分离株表现出的 P 氧化表型有关。确定了与每种化合物的氧化有关的最小区域的DNA序列。亚磷酸盐氧化成磷酸盐所需的区域推测编码：结合蛋白依赖性亚磷酸盐转运蛋白、NAD(+)依赖性亚磷酸盐脱氢酶和lysR家族的转录激活子。次磷酸盐氧化为亚磷酸盐所需的区域推测编码结合蛋白依赖性次磷酸盐转运蛋白和芳酮戊二酸依赖性次磷酸盐双加氧酶。致力于还原磷化合物氧化的基因的发现提供了进一步的证据，表明磷的氧化还原循环可能在这种必需且通常限制生长的营养素的代谢中很重要。
The first molecular and genetic characterization of a biochemical pathway for oxidation of the reduced phosphorus (P) compounds phosphite and hypophosphite is reported. The pathway was identified in Pseudomonas stutzeri WM88, which was chosen for detailed studies from a group of organisms isolated based on their ability to oxidize hypophosphite (+1 valence) and phosphite (+3 valence) to phosphate (+5 valence). The genes required for oxidation of both compounds by P: stutzeri WM88 were cloned on a single ca. 30-kbp DNA fragment by screening for expression in Escherichia coli and Pseudomonas aeruginosa. Two lines of evidence suggest that hypophosphite is oxidized to phosphate via a phosphite intermediate. First, plasmid subclones that conferred oxidation of phosphite, but not hypophosphite, upon heterologous hosts were readily obtained. All plasmid subclones that failed to confer phosphite oxidation also failed to confer hypophosphite oxidation. No subclones that conferred only hypophosphite expression were obtained. Second, various deletion derivatives of the cloned genes were made in vitro and recombined onto the chromosome of P. stutzeri WM88. Two phenotypes were displayed by individual mutants. Mutants with the region encoding phosphite oxidation deleted (based upon the subcloning results) lost the ability to oxidize either phosphite or hypophosphite. Mutants with the region encoding hypophosphite oxidation deleted lost only the ability to oxidize hypophosphite. The phenotypes displayed by these mutants also demonstrate that the cloned genes are responsible for the P oxidation phenotypes displayed by the original P. stutzeri WM88 isolate. The DNA sequences of the minimal regions implicated in oxidation of each compound were determined. The region required for oxidation of phosphite to phosphate putatively encodes: a binding-protein-dependent phosphite transporter, an NAD(+)-dependent phosphite dehydrogenase, and a transcriptional activator of the lysR family. The region required for oxidation of hypophosphite to phosphite putatively encodes a binding-protein-dependent hypophosphite transporter and an ar-ketoglutarate-dependent hypophosphite dioxygenase. The finding of genes dedicated to oxidation of reduced P compounds provides further evidence that a redox cycle for P may be important in the metabolism of this essential, and often growth-limiting, nutrient.