Institute of Microbiology, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
Microbiology (Reading). 2010 Aug;156(Pt 8):2575-2586. doi: 10.1099/mic.0.038570-0. Epub 2010 May 6.
Methanol dehydrogenase-like protein XoxF of Methylobacterium extorquens AM1 exhibits a sequence identity of 50 % to the catalytic subunit MxaF of periplasmic methanol dehydrogenase in the same organism. The latter has been characterized in detail, identified as a pyrroloquinoline quinone (PQQ)-dependent protein, and shown to be essential for growth in the presence of methanol in this methylotrophic model bacterium. In contrast, the function of XoxF in M. extorquens AM1 has not yet been elucidated, and a phenotype remained to be described for a xoxF mutant. Here, we found that a xoxF mutant is less competitive than the wild-type during colonization of the phyllosphere of Arabidopsis thaliana, indicating a function for XoxF during plant colonization. A comparison of the growth parameters of the M. extorquens AM1 xoxF mutant with those of the wild-type during exponential growth revealed a reduced methanol uptake rate and a reduced growth rate for the xoxF mutant of about 30 %. Experiments with cells starved for carbon revealed that methanol oxidation in the xoxF mutant occurs less rapidly compared with the wild-type, especially in the first minutes after methanol addition. A distinct phenotype for the xoxF mutant was also observed when formate and CO(2) production were measured after the addition of methanol or formaldehyde to starved cells. The wild-type, but not the xoxF mutant, accumulated formate upon substrate addition and had a 1 h lag in CO(2) production under the experimental conditions. Determination of the kinetic properties of the purified enzyme showed a conversion capacity for both formaldehyde and methanol. The results suggest that XoxF is involved in one-carbon metabolism in M. extorquens AM1.
甲基杆菌 AM1 的甲醇脱氢酶样蛋白 XoxF 与同一生物体中周质甲醇脱氢酶的催化亚基 MxaF 的序列同一性为 50%。后者已被详细表征，被鉴定为吡咯喹啉醌（PQQ）依赖性蛋白，并被证明是该甲基营养型模式细菌在甲醇存在下生长所必需的。相比之下，XoxF 在甲基杆菌 AM1 中的功能尚未阐明，并且对于 xoxF 突变体仍有待描述表型。在这里，我们发现 xoxF 突变体在拟南芥叶片定殖过程中的竞争力低于野生型，表明 XoxF 在植物定殖过程中具有功能。比较 M. extorquens AM1 的 xoxF 突变体和野生型在指数生长期的生长参数时，发现 xoxF 突变体的甲醇摄取率降低，生长速度降低约 30%。用饥饿细胞进行的实验表明，与野生型相比，xoxF 突变体中的甲醇氧化发生得更慢，尤其是在添加甲醇后的最初几分钟内。当向饥饿细胞中添加甲醇或甲醛后测量甲酸和 CO2 的产生时，也观察到 xoxF 突变体的明显表型。野生型，但不是 xoxF 突变体，在底物添加后积累甲酸，并且在实验条件下 CO2 产生有 1 小时的滞后。纯化酶的动力学特性测定表明其具有甲醛和甲醇的转化能力。结果表明，XoxF 参与了甲基杆菌 AM1 的一碳代谢。
