Department of Microbiology, 410 Stocking Hall, Cornell University, Ithaca, New York 14853.
Appl Environ Microbiol. 1988 Jul;54(7):1656-61. doi: 10.1128/aem.54.7.1656-1661.1988.
Nitrogen fixation (diazotrophy) has recently been demonstrated in several methanogenic archaebacteria. To compare the process in an archaebacterium with that in eubacteria, we examined the properties of diazotrophic growth and nitrogenase activity in Methanosarcina barkeri 227. Growth yields with methanol or acetate as a growth substrate were significantly lower in N(2)-grown cultures than in NH(4)-grown cultures, and the culture doubling times were increased, indicating that diazotrophy was energetically costly, as it is in eubacteria. Growth of nitrogen-fixing cells was inhibited when molybdenum was omitted from the medium; addition of 10 nM molybdate stimulated growth, while 1 muM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 muM) strongly inhibited growth of molybdenum-deficient diazotrophic cells, while ammonia-grown cells were unaffected. The addition of 100 nM vanadate or chromate did not stimulate diazotrophic growth of molybdenum-starved cells. These results are consistent with the presence of a molybdenum-containing nitrogenase in M. barkeri. Acetylene, the usual substrate for assaying nitrogenase activity, inhibited methanogenesis by M. barkeri and consequently needed to be used at a low partial pressure (0.3% of the headspace) when acetylene reduction by whole cells was assayed. Whole cells reduced 0.3% acetylene to ethylene at a very low rate (1 to 2 nmol h mg of protein), and they "switched off" acetylene reduction in response to added ammonia or glutamine. Crude extracts from diazotrophic cells reduced 10% acetylene at a rate of 4 to 5 nmol of C(2)H(4) formed h mg of protein when supplied with ATP and reducing power, while extracts of Klebsiella pneumoniae prepared by the same procedures had rates 100-fold higher. Acetylene reduction by extracts required ATP and was completely inhibited by 1 mM ADP in the presence of 5 mM ATP. The low rates of C(2)H(2) reduction could be due to improper assay conditions, to switched-off enzyme, or to the nitrogenase's having lower activity towards acetylene than towards dinitrogen.
氮固定（固氮作用）最近已在几种产甲烷古菌中得到证实。为了比较古菌和真细菌中的这一过程，我们研究了 Methanosarcina barkeri 227 的固氮生长和固氮酶活性的特性。以甲醇或乙酸作为生长基质时，与 NH4+ 生长培养相比，N2 生长培养中的生长产率显著降低，培养倍增时间延长，表明固氮作用在能量上是昂贵的，就像在真细菌中一样。从培养基中省略钼会抑制固氮细胞的生长；添加 10 nM 钼酸盐会刺激生长，而 1 μM 钼酸盐则恢复最大固氮生长。从培养基中省略钼不会抑制氨生长细胞的生长。钨酸盐（100 μM）强烈抑制缺钼固氮细胞的生长，而对氨生长细胞没有影响。添加 100 nM 钒酸盐或铬酸盐不会刺激缺钼固氮细胞的固氮生长。这些结果与 M. barkeri 中存在钼含氮酶一致。乙炔是测定固氮酶活性的常用底物，它抑制了 M. barkeri 的甲烷生成，因此当测定整个细胞的乙炔还原时，需要在低分压（气相的 0.3%）下使用。整个细胞以非常低的速率（1 至 2 nmol h mg 的蛋白质）将 0.3%的乙炔还原为乙烯，并且它们对添加的氨或谷氨酰胺做出反应而“关闭”乙炔还原。从固氮细胞中提取的粗提取物在供应 ATP 和还原力时，以 4 至 5 nmol 的 C2H4 形成 h mg 的蛋白质的速率还原 10%的乙炔，而用相同程序制备的 Klebsiella pneumoniae 提取物的速率高 100 倍。提取物的乙炔还原需要 ATP，并且在存在 1 mM ADP 的情况下，5 mM ATP 可完全抑制 ADP。C2H2 还原的低速率可能是由于不正确的测定条件、关闭的酶或氮酶对乙炔的活性比对二氮的活性低所致。
