The Azotobacter vinelandii mutant strain UW45 contains a mutation in the nifB gene and produces an inactive dinitrogenase protein that can be activated by the addition of purified iron-molybdenum cofactor (FeMoco). This FeMoco-deficient dinitrogenase (Apo I) has now been purified 96-fold to greater than 95% purity and is FeMoco-activatable to 2200 nmol of C2H2 reduced/(min.mg of protein). The Apo I complex was found to contain two molecules of a 20-kDa protein, in addition to the alpha 2 beta 2 tetramer found for isolated holodinitrogenase (Holo I). The Apo I complex contained 15 +/- 2 mol of Fe per mole, but no Mo. While the presence of dinitrogenase reductase caused a 2-fold stimulation in the activation of the purified Apo I complex by FeMoco, this enhancement resulted from the stabilization of Apo I by dinitrogenase reductase to the denaturing effects of N-methylformamide. When the activation was performed in the absence of N-methylformamide, there was no enhancement by dinitrogenase reductase alone or by dinitrogenase reductase-Mg-ATP complex. The Apo I complex is more sensitive to O2 than Holo I, with a half-life in air of 6 min; however, the addition of dithiothreitol to Apo I during the exposure to air (or after exposure) resulted in a half-life very similar to that seen for Holo I. This suggests that sulfhydryl(s) is (are) important for the FeMoco-activation reaction.

Apodinitrogenase, which lacks the iron-molybdenum cofactor at its active site, is an oligomer that contains an additional protein not found in the active dinitrogenase tetramer. This associated protein in Kiebsiella pneumoniae is shown to be the product of the nifY gene. When apodinitrogenase is activated by the addition of the iron-molybdenum cofactor, NifY dissociates from the apodinitrogenase complex. The conditions for this dissociation are described. Finally, there are aspects of the dissociation and insertion process in K. pneumoniae that are different from that in Azotobacter vinelandii. (18).The active site of component I, the iron-molybdenum cofactor (FeMo-co), is synthesized by the nifgene products, including those of nifQ, -B, -V, -N, -E, and -H (18). Many mutations in nifB and nifNE result in strains that are unable to fix N2 (13) and accumulate a form of component I without the active site. Addition of purified FeMo-co to this apocomponent I (Apo I) in vitro yields an enzyme that is catalytically active (15).When Apo I was purified from a nifB Azotobacter vinelandii mutant, another protein of approximately 20 kDa copurified with it (11). A variety of efforts to dissociate this protein without destroying Apo I proved unsuccessful, demonstrating that the complex was very tight (11).Lacking either a good genetic or biochemical perspective on this associated protein from A. vinelandii, we examined the Apo I from Kiebsiella pneumoniae, reasoning that if it was important to the biochemistry of nitrogenase, a similar factor might be detected in that organism also. In fact, a previous publication describing the purification of Apo I from K pneumoniae had already reported the presence of a 20-kDa contaminant (5). Very recently, NifY has been detected in partially purified samples of Apo I from K pneumoniae, but the nature and role of this association were unclear (19). In this report, we show that the protein associated with pure Apo I from K pneumoniae is the product of nifY. We further characterize the nature and role of the proteins associated with Apo I from both K pneumoniae and A. vinelandii.The initial indication that the associated factor in K pneumoniae is NifY came from sequence analysis of the purified protein.To isolate the protein, we purified Apo I from K pneumoniae UN1217 (nifN4536) through the hy-* Corresponding author.droxylapatite column step as outlined previously (11), except that K pneumoniae Apo I was eluted from the DEAEcellulose column at 0.25 M NaCl. The reactivatible fractions were then analyzed on an alkyl superose fast protein liquid chromatography column and eluted at 0.63 M (NH4)2SO4 in 0.025 M MOPS (morpholinepropanesulfonic acid) (pH 7.4) containing 1.7 mM Na2S204. At this point, the Apo I protein is homogeneously pure. To isolate the associated protein from Apo I, the resulting protein preparation was separated on a sodium dodecyl sulfate-13% polyacrylamide gel electrophoresis (SDS-PAGE) gel prerun with 0.25% (wt/vol) thioglycolate in the buffer and transferred to an Immobil...

The niffl and -E gene products are involved in the synthesis of the iron-molybdenum cofactor of dinitrogenase, the enzyme responsible for the reduction of dinitrogen to ammonia. By using the in vitro iron-molybdenum cofactor biosynthesis assay, we have followed the purification of these gene products 450-fold to >95% purity. An overall recovery of 20% was obtained with the purified protein having a specific activity of 6900 units/mg of protein. The protein (hereafter referred to as NIFNE) was found to contain equimolar amounts of the nifN and -E gene products and have a native molecular mass of 200 ± 10 kDa, which indicates an a2.82 structure.NIFNE was oxygen labile with a half-life of 1 min in air. A UV-visible spectrum of the dye-oxidized protein showed an absorption maximum at 425 nm that could be bleached by reduction of NIFNE with sodium dithionite, suggesting the presence of an Fe center in NIFNE.

If we are to teach effectively, tools are needed to measure student learning. A widely used method for quickly measuring student understanding of core concepts in a discipline is the concept inventory (CI). Using the American Society for Microbiology Curriculum Guidelines (ASMCG) for microbiology, faculty from 11 academic institutions created and validated a new microbiology concept inventory (MCI). The MCI was developed in three phases. In phase one, learning outcomes and fundamental statements from the ASMCG were used to create T/F questions coupled with open responses. In phase two, the 743 responses to MCI 1.0 were examined to find the most common misconceptions, which were used to create distractors for multiple-choice questions. MCI 2.0 was then administered to 1,043 students. The responses of these students were used to create MCI 3.0, a 23-question CI that measures students’ understanding of all 27 fundamental statements. MCI 3.0 was found to be reliable, with a Cronbach’s alpha score of 0.705 and Ferguson’s delta of 0.97. Test item analysis demonstrated good validity and discriminatory power as judged by item difficulty, item discrimination, and point-biserial correlation coefficient. Comparison of pre- and posttest scores showed that microbiology students at 10 institutions showed an increase in understanding of concepts after instruction, except for questions probing metabolism (average normalized learning gain was 0.15). The MCI will enable quantitative analysis of student learning gains in understanding microbiology, help to identify misconceptions, and point toward areas where efforts should be made to develop teaching approaches to overcome them.

Azotobacter vinelandii DJ71, which contains a mutation in the nifV gene, was derepressed for nitrogenase in the presence of homocitrate. When dinitrogenase was isolated from this culture, it was found to be identical to the wild-type dinitrogenase. However, when the same NifV-strain was derepressed in the presence of erythrofluorohomocitrate, a homocitrate analog which produces a nitrogenase with wild-type properties in vitro, the isolated dinitrogenase was characteristic of the NifV-enzyme. These data show that homocitrate, but not fluorohomocitrate, is utilized by NifV-mutant cells. Fluorohomocitrate does not inhibit the uptake of homocitrate because the wild-type phenotype resulted when both compounds were added to the medium during nitrogenase derepression. Homocitrate lactone failed to cure the NifV-phenotype.