Premakumar R, Jacobitz S, Ricke S C, Bishop P E
USDA Agricultural Research Service, North Carolina State University, Raleigh 27695-7631, USA.
J Bacteriol. 1996 Feb;178(3):691-6. doi: 10.1128/jb.178.3.691-696.1996.
A tungsten-tolerant mutant strain (CA6) of Azotobacter vinelandii first described in 1980 (P. E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980) has been further characterized. Results from growth experiments suggest that both nitrogenases 1 and 3 are utilized when CA6 grows in N-free medium containing Na2MoO4. Strain CA6.1.71, which lacks both nitrogenases 2 and 3, grew as well as strain CA in N-free medium containing Na2MoO4 after an initial lag. This indicates that nitrogenase 1 is fully functional in strain CA6. nifH-lacZ and anfH-lacZ transcriptional fusions were expressed in CA6 in the presence of Na2MoO4. Thus, in contrast to wild-type strain CA, transcription of the anfHDGK gene cluster in strain CA6 is not repressed by Mo. Expression of the vnfD-lacZ fusion was the same in both strains CA and CA6. In agreement with the results obtained with lac fusions, subunits of both nitrogenases 1 and 3 were found in protein extracts of CA6 cells grown in N-free medium containing Na2MoO4. However, CA6 cells, cultured in the presence of Na2WO4, accumulated nitrogenase 3 proteins without detectable amounts of nitrogenase 1 proteins. This indicates that expression of Mo-independent nitrogenase 3 is the basis for the tungsten tolerance phenotype of strain CA6. A measure of Mo accumulation as a function of time showed that accumulation by strain CA6 was slower than that for strain CA. When Mo accumulation was studied as a function of Na2MoO4 concentration, the two strains accumulated similar amounts of Mo in the concentration range of 0 to 1 microM Na2MoO4 during a 2-h period. Within the range of 1 to 5 microM Na2MoO4, Mo accumulation by strain CA increased linearly with increasing concentration whereas no further increases were observed for strain CA6. These results are consistent with the possibility that the tungsten tolerance mutation carried by CA6 is in a Mo transport system.
1980年首次描述的维涅兰德固氮菌（Azotobacter vinelandii）的耐钨突变菌株（CA6）（P.E. Bishop、D.M.L. Jarlenski和D.R. Hetherington，《美国国家科学院院刊》77:7342 - 7346，1980年）已得到进一步表征。生长实验结果表明，当CA6在含有钼酸钠（Na2MoO4）的无氮培养基中生长时，固氮酶1和3都被利用。缺乏固氮酶2和3的菌株CA6.1.71，在含有钼酸钠的无氮培养基中经过初始延迟期后，生长情况与菌株CA相同。这表明固氮酶1在菌株CA6中功能完全正常。nifH - lacZ和anfH - lacZ转录融合体在有钼酸钠存在的情况下在CA6中表达。因此，与野生型菌株CA不同，菌株CA6中anfHDGK基因簇的转录不受钼的抑制。vnfD - lacZ融合体在菌株CA和CA6中的表达相同。与通过lac融合体获得的结果一致，在含有钼酸钠的无氮培养基中生长的CA6细胞的蛋白质提取物中发现了固氮酶1和3的亚基。然而，在钨酸钠（Na2WO4）存在下培养的CA6细胞积累了固氮酶3蛋白，而未检测到固氮酶1蛋白。这表明不依赖钼的固氮酶3的表达是菌株CA6耐钨表型的基础。对钼积累量随时间变化的测量表明，菌株CA6的钼积累速度比菌株CA慢。当研究钼积累量随钼酸钠浓度的变化时，在2小时内，在0至1微摩尔/升钼酸钠浓度范围内，这两种菌株积累的钼量相似。在1至5微摩尔/升钼酸钠范围内，菌株CA的钼积累量随浓度增加呈线性增加，而菌株CA6未观察到进一步增加。这些结果与CA6携带的耐钨突变位于钼转运系统中的可能性一致。
