Sundin G W, Shankar S, Chakrabarty A M
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612, USA.
J Bacteriol. 1996 Dec;178(24):7120-8. doi: 10.1128/jb.178.24.7120-7128.1996.
We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as critical to this process.
我们报道了利用定点诱变和随机诱变程序对铜绿假单胞菌核苷二磷酸激酶（ndk）编码基因进行操作，以研究Ndk在该生物体藻酸盐产生过程中的作用。在藻R2基因（8830R2::Cm）发生敲除突变的铜绿假单胞菌8830中，16 kDa形式的Ndk酶的细胞水平大幅降低；该菌株在胞外多糖藻酸盐的产生方面也存在缺陷。在本研究中，我们在ndk中分离出四个突变（丙氨酸-14→脯氨酸[Ala14Pro]、甘氨酸21→缬氨酸、组氨酸117→谷氨酰胺和丙氨酸125→精氨酸），这些突变导致Ndk生化活性丧失；这四个突变基因中的任何一个的过表达都未能使8830R2::Cm恢复藻酸盐的产生。我们还鉴定出另外六个氨基酸残基（丝氨酸-43、丙氨酸-56、丝氨酸-69、谷氨酸-80、甘氨酸-91和天冬氨酸-135），其改变导致Ndk无法补充藻酸盐的产生。在8830R2::Cm中过量表达后，确定这六个突变的Ndk中的每一个在生化上都是有活性的。然而，在四种情况下，Ndk的体内水平降低，这进而影响了8830R2::Cm在吐温20存在下的生长。两种不能补充8830R2::Cm中藻酸盐合成缺陷的突变Ndk蛋白在本研究中检测的任何特征方面均未受到影响。所有经鉴定仍具有生化活性的突变Ndk都与Pk形成膜复合物，从而导致GTP合成。所鉴定的四个Ndk活性突变体中的两个（组氨酸117→谷氨酰胺和丙氨酸125→精氨酸）能够被截短至12 kDa并与Pk形成膜复合物；然而，形成的复合物对GTP合成无活性。另外两个Ndk活性突变体可以被截短至12 kDa，但在膜组分中未检测到。这些结果进一步加深了我们对Ndk在藻酸盐合成中作用的理解，并鉴定出Ndk中以前未被研究认为对该过程至关重要的氨基酸残基。
