Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Sibold, Lionel; Pariot, Dominique; Bhatnagar, L.; Henriquet, Marc; Aubert, Jean-Paul

doi:10.1007/bf00383310

Cited by 46 publications

(13 citation statements)

References 39 publications

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“…The procedures to mobilize plasmid DNA from Escherichia coli to P. denitrificans were already described (8). M. ivanovii genomic DNA was purified as previously described (40). Nucleotide sequence was performed with Sequenase version 2.0 (United States Biochemical Corporation) with ot-35S-dATP (Amersham) on plasmid DNA as previously described (10) or on single-strand DNA according to the manufacturer's specifications and protocols.…”

Section: Methodsmentioning

confidence: 99%

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Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii

Blanche¹,

Robin²,

Couder³

et al. 1991

J Bacteriol

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An S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity has been identified in Methanobacterium ivanovii and was purified 4,500-fold to homogeneity with a 38% yield. The enzyme had an apparent molecular weight of 58,200 by gel filtration and consisted of two identical subunits of Mr 29,000, as estimated by gel electrophoresis under denaturing conditions. The Km value for uroporphyrinogen III was 52 nM. The enzyme catalyzed the two C-2 and C-7 methylation reactions converting uroporphyrinogen III into precorrin-2. Unlike Pseudomonas denitrificans SUMT, the only SUMT characterized to date (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet and B. Cameron, J. Bacteriol. 171:4222-4231, 1989), M. ivanovii SUMT did not show substrate inhibition at uroporphyrinogen Ill concentrations of up to 20 ,uM. Oligonucleotide probes from limited peptide sequence information were used to clone the corresponding gene. The encoded polypeptide showed more than 40% strict homology with P. denitrificans SUMT. The M. ivanovii SUMT structural gene is likely to be, as is P. denitrificans cobA, involved in corrinoid synthesis.Methanogenic bacteria synthesize particular types of corrinoids, mainly 5-hydroxybenzimidazolylcobamide (vitamin B12 factor III) (24,33,34,42,46) and Coa-t[-(7-adenyl)]cobamide (pseudovitamin B12) (24,43). Occurrence of cobina-

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Section: Methodsmentioning

confidence: 99%

“…The growth temperature was either 37°C for E. coli or 30°C for P. denitrificans. M. ivanovii was cultivated as previously reported (40) at 37°C in the complex medium described by Whitman et al (45). E. coli strains were grown anaerobically in jars using the BBL GasPak anaerobic system (Becton Dickinson and Co., Cockeysville, Md.…”

Section: Methodsmentioning

confidence: 99%

Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii

Blanche¹,

Robin²,

Couder³

et al. 1991

J Bacteriol

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“…vnf and anf are genes that have been positively identified as belonging to vanadium-or iron-type nitrogenases (6,13,15). Numerical designations follow the published convention where more than one nif gene is present in a species (11,(16)(17)(18)(19)(20)(21). maripaludis and sequenced on both strands with a combination of subcloning and walking primers by cycle sequencing with the Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems).…”

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Nitrogenase phylogeny and the molybdenum dependence of nitrogen fixation in Methanococcus maripaludis

1997

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We studied the effects of molybdenum, vanadium, and tungsten on the diazotrophic growth of Methanococcus maripaludis. Mo stimulated growth, with a maximal response at 4.0 M, while V had no effect at any concentration tested. W specifically inhibited diazotrophic growth in the presence of Mo. Coupling the results of our analysis and other known metal requirements with phylogenies derived from nifD and nifK genes revealed distinct clusters for Mo-, V-, and Fe-dinitrogenases and suggested that most methanogens also have molybdenum-type nitrogenases.Nitrogen fixation, the conversion of atmospheric N 2 to ammonia, is carried out by a variety of Bacteria and Archaea (23). Among the Bacteria, it occurs in at least four groups, the Proteobacteria, gram-positive bacteria, cyanobacteria, and green sulfur bacteria. Among Archaea, nitrogen fixation is found in all three orders of methanogens (10). Despite its wide phylogenetic distribution, the mechanism of nitrogen fixation is conserved. A typical dinitrogenase is an ␣ 2 ␤ 2 tetramer encoded by nifD and nifK and containing an iron-molybdenum cofactor, FeMoCo (22). Mo-independent dinitrogenases (2) have cofactors that coordinate vanadium in place of molybdenum (Vdinitrogenases) or that have neither molybdenum nor vanadium (Fe-dinitrogenases); these are encoded by the nifD and nifK homologs vnfD and vnfK and anfD and anfK, respectively. An additional small subunit (␦) found with the Mo-independent dinitrogenases is encoded by vnfG or anfG. Included in all nitrogenase complexes is dinitrogenase reductase, typically a homodimer in Bacteria, encoded by nifH, vnfH, or anfH.The discovery of nitrogen fixation in methanogens in 1984 redefined the phylogenetic extremity of nitrogen fixation and opened the door to a series of new questions regarding biological nitrogen fixation (1, 12). Many of the properties common to nitrogen fixation in the Bacteria hold true in the Archaea as well. Lobo and Zinder found the nitrogenase complex from Methanosarcina barkeri to resemble those from Bacteria in subunit composition (except that dinitrogenase reductase appeared to be a homotetramer), substrate range, and immunological cross-reactivity of dinitrogenase reductase (9). nif genes from a variety of methanogens proved to be homologous and similar in organization to those from Bacteria (10). Phylogenetic analysis of nifH, nifD, and nifK showed the methanogen genes to be related to certain groups of bacterial genes (discussed below).One question that has not been resolved is which metals are required for nitrogen fixation in methanogens and might therefore be incorporated into the dinitrogenase cofactor. Lobo and Zinder found that molybdenum but not vanadium stimulated nitrogen fixation in M. barkeri 227 (8). In contrast, Scherer reported stimulation by molybdenum and to a lesser extent by vanadium in two strains of M. barkeri, 227 and Fusaro (14). The issue is further complicated by the fact that M. barkeri 227 has two potentially active nitrogenases, as evidenced by the existence of two sets o...

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“…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).…”

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Nitrogenase in the archaebacterium Methanosarcina barkeri 227

Lobo

Zinder

1990

J Bacteriol

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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

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Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Cited by 46 publications

References 39 publications

Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii

Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii

Nitrogenase phylogeny and the molybdenum dependence of nitrogen fixation in Methanococcus maripaludis

Nitrogenase in the archaebacterium Methanosarcina barkeri 227

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