Diazotrophy and Nitrogenase Activity in the Archaebacterium Methanosarcina barkeri 227

Abstract

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 N2-grown cultures than in NH4+-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 ,iM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 ,uM) 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-1 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 C2H4 formed h-1 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 C2H2 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.

Characterization of an H2-utilizing enrichment culture that reductively dechlorinates tetrachloroethene to vinyl chloride and ethene in the absence of methanogenesis and acetogenesis

“…When we added 5 mol of PCE to a 27-ml crimp-top tube containing 10 ml of growth medium, the nominal concentration was 0.5 mmol/liter, while the estimated aqueous concentration (15) was 0.16 mmol/liter. When methane and ETH were produced together, they were measured with a Varian 1400 GC using a Poropak R column as described previously (19). The detection limit for methane was ca.…”

Environmental Impact and Mechanisms of the Biological Clogging of Saturated Soils and Aquifer Materials

“…Several reasons seem to limit the activity of methanogens to the tail end of the successive redox reactions. First of all, they rely on the end-products of various types of fermentations for their supply of growth substrates, such as acetate, formate, methanol, CO, CO 2 , and H 2 (Zeikus, 1977;Blaut et al, 1985;Gottschalk, 1986, p. 252;Lobo and Zinder, 1988). Moreover, they are inhibited by O 2 , NO 3 − , and SO 4 2 − (Jakobsen et al, 1981).…”

Metal limitation of cyanobacterial N2fixation and implications for the Precambrian nitrogen cycle

“…Molybdenum has been proposed to limit modern N 2 fixation primarily through the competitive inhibition of molybdate uptake by high concentrations of sulfate in seawater (Howarth & Cole, 1985;Cole et al ., 1993;Marino et al ., 2003), but it is unclear how important this competition is in nature (Paerl et al ., 1987;Paulsen et al ., 1991). Molybdenum is directly involved in regulating the expression of the alternate nitrogenase enzymes in Azotobacter vinelandii (Premakumar et al ., 1984;Jacobson et al ., 1986;Joerger & Bishop, 1988;Jacobitz & Bishop, 1992) and Anabaena variabilis (Thiel, 1993), and has been shown to stimulate growth and N 2 fixation in methanogens (Lobo & Zinder, 1988;Kessler et al ., 1997). Azotobacter vinelandii can additionally extract Mo from silicates using a high-affinity ligand during diazotrophic growth under Mo-depleted conditions (Liermann et al ., 2005).…”