Metcalf W W, Wanner B L
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
Gene. 1993 Jul 15;129(1):27-32. doi: 10.1016/0378-1119(93)90692-v.
The Escherichia coli phn (psiD) locus consists of a large gene cluster encoding proteins necessary for the use of phosphonates (Pn) as a sole phosphorus source. On the basis of nucleotide (nt) sequence analysis, the phn locus contains a 12.6-kb operon of seventeen genes named, in alphabetical order, phnA to phnQ [Chen et al., J. Biol. Chem. 265 (1990) 4461-4471]. New Pn+ plasmids were made which are suitable for mutational analysis of this gene cluster. These plasmids contain the R6K origin for DNA replication, can be conjugatively transferred, contain the tetAR genes, and therefore provide a way for allele replacement. The construction of these plasmids showed that phnA and phnB have no role in Pn metabolism. Also, these plasmids were employed to introduce nonpolar phnD::lacZ and phnD::uidA fusions into the chromosome, which allowed us to show that phnD probably has a role in transport. In addition, it was shown that phnP is the most distal gene required for Pn use. This was done by testing the effect of phn::uidA insertions in or near the 3' end of phnP on Pn use. Altogether, these results show that all genes required for Pn use are in the 10.9-kb, fourteen-gene, phnCDEFGHIJKLMNOP locus.
大肠杆菌的phn（psiD）基因座由一个大的基因簇组成，该基因簇编码将膦酸盐（Pn）作为唯一磷源使用所需的蛋白质。根据核苷酸（nt）序列分析，phn基因座包含一个12.6 kb的十七个基因的操纵子，按字母顺序命名为phnA至phnQ [Chen等人，《生物化学杂志》265（1990）4461 - 4471]。构建了适用于该基因簇突变分析的新型Pn +质粒。这些质粒含有用于DNA复制的R6K起源，可以通过接合转移，含有tetAR基因，因此提供了一种等位基因替换的方法。这些质粒的构建表明phnA和phnB在Pn代谢中没有作用。此外，这些质粒被用于将非极性的phnD::lacZ和phnD::uidA融合基因导入染色体，这使我们能够表明phnD可能在转运中起作用。另外，通过测试phnP 3'端或其附近的phn::uidA插入对Pn使用的影响，表明phnP是Pn使用所需的最远端基因。总之，这些结果表明Pn使用所需的所有基因都位于10.9 kb的、包含十四个基因的phnCDEFGHIJKLMNOP基因座中。
