Characterization of the gene for the Fe-protein of the vanadium dependent alternative nitrogenase of Azotobacter vinelandii and construction of a Tn5 mutant

1988 ◽  
Vol 214 (1) ◽  
pp. 121-127 ◽  
Author(s):  
Ramesh Raina ◽  
M. Amarender Reddy ◽  
Debabrota Ghosal ◽  
H. K. Das
Keyword(s):  
Azotobacter Vinelandii ◽  
Fe Protein ◽  
Alternative Nitrogenase ◽  
Tn5 Mutant

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

Characterization of transcripts expressed from nitrogenase-3 structural genes of Azotobacter vinelandii

1992 ◽  
Vol 38 (9) ◽  
pp. 929-936 ◽  
Author(s):  
R. Premakumar ◽  
Marty R. Jacobson ◽  
Telisa M. Loveless ◽  
Paul E. Bishop
Keyword(s):  
Azotobacter Vinelandii ◽  
Coding Region ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Mrna Species ◽  
Mutant Strains ◽  
S1 Nuclease Mapping ◽  
Alternative Nitrogenase ◽  
Nuclease Mapping

Five major anfH-hybridizing mRNA species accumulated in Azotobacter vinelandii cells derepressed for nitrogenase-3 (an alternative nitrogenase, which appears to lack Mo and V). Using anfH-, anfD-, anfG-, anfK-, and orf1orf2-specific probes and mutant strains of A. vinelandii these mRNA species have been identified as encoding anfHDGKorf1orf2 (6.0 kb), anfHDGK (4.3 kb), anfHD (2.6 kb), vnfHorfFd (1.3 kb), and vnfH and (or) anfH(1.0 kb). A 0.6-kb mRNA species, which hybridized only to the orf1orf2-specific probe, and a 3.5-kb mRNA species, which hybridized to anfD or anfK, also accumulated under these conditions. Northern blot analysis and S1 nuclease mapping indicate that transcription of the anf structural gene cluster initiates at a unique nif consensus promoter situated 127 base pairs upstream from the anfH coding region. Observation of anfH-hybridizing mRNA species that accumulate in strains derepressed for nitrogen fixation demonstrates that transcription of the anfHDGKorf1orf2 cluster is normally repressed by Mo, V, and NH4+, whereas transcription of the vnfHorfFd cluster does not require the presence of V and is repressed only by Mo, but not NH4+. Analysis of the accumulation of mRNAs in a tungsten-tolerant strain revealed that Mo and V repression of anf transcription must occur by different mechanisms. Key words: Azotobacter vinelandii, nitrogenase-3, transcripts, regulation, molybdenum, vanadium.

scholarly journals Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor

1993 ◽  
Vol 293 (1) ◽  
pp. 101-107 ◽  
Author(s):  
R N Pau ◽  
M E Eldridge ◽  
D J Lowe ◽  
L A Mitchenall ◽  
R R Eady
Keyword(s):  
Azotobacter Vinelandii ◽  
H2 Evolution ◽  
Titration Curves ◽  
Sds Page ◽  
Metal Requirement ◽  
Alternative Nitrogenase ◽  
Terminal Amino ◽  
Iron Molybdenum Cofactor ◽  
A New Species

Nitrogenase-3 of Azotobacter vinelandii is synthesized under conditions of molybdenum and vanadium deficiency. The minimal metal requirement for its synthesis, and its metal content, indicated that the only transition metal in nitrogenase-3 was iron [Chisnell, Premakumar and Bishop (1988) J. Bacteriol. 170, 27-33; Pau, Mitchenall and Robson (1989) J. Bacteriol. 171, 124-129]. A new species of nitrogenase-3 has been purified from a strain of A. vinelandii (RP306) lacking structural genes for the Mo- and V-nitrogenases and containing a mutation which enables nitrogenase-3 to be synthesized in the presence of molybdenum. SDS/PAGE showed that component 1 contained a 15 kDa polypeptide which N-terminal amino acid sequence determination showed to be encoded by anfG. This confirms that nitrogenase-3, like V-nitrogenase, comprises three subunits. Preparations of the nitrogenase-3 from strain RP306 contained 24 Fe atoms and 1 Mo atom per molecule. Characterization of the cofactor centre of the enzyme by e.p.r. spectroscopy and an enzymic cofactor assay, together with stimulation of the growth of strain RP306 by Mo, showed that nitrogenase-3 can incorporate the Mo-nitrogenase cofactor (FeMoco) to form a functional enzyme. The specific activities (nmol of product produced/min per mg of protein) determined from activity titration curves were: under N2, NH3 formation 110, with concomitant H2 evolution of 220; under argon, H2 evolution 350; under 10% acetylene (C2H2) in argon, ethylene (C2H4) 58, ethane (C2H6) 26, and concomitant H2 evolution 226. The rate of formation of C2H6 was non-linear, and the C2H6/C2H4 ratio strongly dependent on the ratio of nitrogenase components.

scholarly journals Mössbauer, EPR, and magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]1+ cluster with spin S = 3/2.

1985 ◽  
Vol 260 (20) ◽  
pp. 11160-11173 ◽  
Author(s):  
P A Lindahl ◽  
E P Day ◽  
T A Kent ◽  
W H Orme-Johnson ◽  
E Münck
Keyword(s):  
Azotobacter Vinelandii ◽  
Fe Protein

scholarly journals The molybdenum–iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ‘mapping’

1976 ◽  
Vol 155 (2) ◽  
pp. 383-389 ◽  
Author(s):  
C Kennedy ◽  
R R. Eady ◽  
E Kondorosi ◽  
D K Rekosh
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Sodium Dodecyl Sulphate ◽  
Azotobacter Vinelandii ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Rhizobium Japonicum ◽  
Fe Protein

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide ‘maps’ after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.

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