The discovery of nitrogen fixation in the archaebacterium Methanosarcina barkeri 227 raises questions concerning the similarity of archaebacterial nitrogenases to Mo and alternative nitrogenases in eubacteria. A scheme for achieving a 20-to 40-fold partial purification of nitrogenase components from strain 227 was developed by using protamine sulfate precipitation, followed by using a fast protein liquid chromatography apparatus operated inside an anaerobic glove box. As in eubacteria, the nitrogenase activity was resolved into two components. by strain 227 cells. Antiserum against component 2 of Rhodospirillum rubrum nitrogenase was found to cross-react with component 2 from strain 227, and Western immunoblots using this antiserum showed no evidence for covalent modification of component 2. Also, extracts of strain 227 cells prepared before and after switch-off had virtually the same level of nitrogenase activity. In conclusion, the nitrogenase from strain 227 is similar in overall structure to the eubacterial nitrogenases and shows greatest similarity to alternative nitrogenases.In 1984 to 1985, the property of diazotrophy (nitrogen fixation) was reported in the methanogenic archaebacteria Methanosarcina barkeri 227 (19), Methanococcus thermolithotrophicus (2), and M. barkeri Fusaro (3). Sibold et al. (27) reported hybridization between eubacterial nifHDK probes and DNA from four different methanogenic species. These discoveries were the first confirmed examples of diazotrophy among members of the archaebacteria and have been followed by other descriptions of nitrogen-fixing methanogenic archaebacteria (1,9,13,18 There have been several recent studies on the physiology of diazotrophy in methanogens (1,3,16,18,25). In our previous study on M. barkeri 227 (16) we found similarities to the process in eubacteria, including significant decreases in growth yields and rates when N2 was the N source, stimulation of diazotrophic growth by Mo, and switch-off of C2H2-reducing activity in response to added NH4+. Scherer (25) has demonstrated stimulation of diazoatrophic growth in two strains of M. barkeri by V as well as Mo. We and others have found anomalously low in vivo and in vitro nitrogenase activity, as measured by rates of C2H2 reduction to C2H4 in all diazotrophic methanogens studied thus far (2, 3, 16, 18). The rates measured for methanogen cells and extracts are typically 100-to 500-fold lower than those of typical eubacterial diazotrophs with similar growth rates. One hypothesis considered (16) was that C2H2 was a poorer substrate than N2 for methanogen nitrogenases; another is that methanogen nitrogenases are switched off when assayed (18).We report here the partial purification of nitrogenase from strain 227, a description of its subunit composition, and measurements of its activity toward C2H2 and N2. We also describe more detailed studies on NH4' switch-off and its mechanism in strain 227. 6789on May 13, 2018 by guest
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.
Antibiotic resistance is a significant and growing public health problem. This work investigated the use of two different carbon nanomaterials, single-walled carbon nanotubes (SWNTs) and nanographene oxide (NGO), as a means of delivering the antibiotic tetracycline to a strain of Escherichia coli bacterium with an efflux pump resistance mechanism. Both SWNTs and NGO carrying tetracycline were found to inhibit the resistant strain of Escherichia coli, though the amount of tetracycline delivered was much lower than the minimum inhibitory concentration of free tetracycline. Attachment of the tetracycline to the nanomaterials was found to be necessary for the inhibition of bacterial growth, indicating that the nanomaterials were transporting the antibiotic into the cells and subverting the efflux pump. SWNTs were observed to have greater efficacy in delivering tetracycline than graphene oxide, which is attributed to the SWNTs’ needle-like shape. This work demonstrates both the use of carbon nanomaterials as antibiotic-delivery vehicles and the effect of nanomaterial shape on their efficacy. More importantly, it demonstrates that nanomaterials can successfully extend the life of existing antibiotics, making them an important tool for combatting antibiotic resistance mediated by an efflux pump mechanism.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.