The enzyme nitrogenase, which catalyzes nitrogen fixation in Azotobacter vinelandii, consists of two components, the Fe protein (NifH) and the MoFe protein (NifDK). NifK contains several highly conserved residues implicated in its functions throughout the protein. However, the carboxyl terminus of NifK is not implicated in any of the known functions of this protein. Therefore, the present study explored the role of carboxyl terminal region of NifK. The results of growth analysis showed that when the media was adjusted to be slightly acidic, the strain that expresses the mutated NifK yielded a lower growth compared to the wild type. These observations implied that the carboxyl terminus of the NifK contributes to the formation of a stable nitrogenase complex when A. vinelandii is grown in acidic environment. The proper interaction between NifH and NifK is essential for the nitrogenase conformation. To determine how the interaction is influenced by the characteristics of the amino acids available at position 112 of NifH, we introduced residue mutations to the codon encoding for Glu112. Growth analyses indicated that mutant strains are capable of propagation under nitrogen-deficit conditions although the growth rate is lower than that of wild type strain. Therefore the charge carried by the amino acid at position 112 of NifH plays a minor role in the interaction whereas; a more important factor is the length of the side chains. The research on hydrogenase expressed by bacteria shed light on the possibilities of utilizing this novel energy source. We endeavored to take advantage of the nature of A. vinelandii and construct an A. vinelandii mutant strain expressing Fe-hydrogenase. This ongoing research involves molecular manipulation of the enzyme-encoding gene hydA. The synthetic hydA was incorporated and expressed in A. vinelandii strain DJ54. At the same time, we screened several biomass materials for their capabilities in sustaining diazotrophic growth of A. vinelandii. The result indicated that the HydA protein can be expressed in A. vinelandii under certain conditions and a number of biomass substances can be supportive ingredients for putative biohydrogen media.

No description available.

No description available.

No description available.

The site of substrate binding and reduction for the nitrogenase complex is located on the iron molybdenum cofactor (FeMo-co) which is contained within the a-subunit of the molybdenum iron protein. FeMo co consists of a metal sulfur core composed of an FeS cluster bridged by three inorganic sulfides to a MoFeS cluster. An organic acid, homocitrate, is coordinated to the Mo atom through its 2-carboxy and 2-hydroxy groups. Homocitrate is formed by the condensation of acetyl-CoA and a-ketoglutarate, which is catalyzed by a homocitrate synthase encoded by nifV. By deleting the nifV gene from Azotobacter vinelandii we were able to study the role of homocitrate in nitrogenase catalysis. A poly-histidine tail was incorporated into the C-termini of the a-subunit permitting isolation of the homocitrateless MoFe protein by using metal affinity chromatography. We have found that the addition of a poly-histidine tag does not alter the catalytic behavior of the native enzyme. In NifV- strains of Klebsiella pneumoniae, citrate has been found to replace homocitrate as the organic constituent of FeMo-co. We have found no evidence this is so in A. vinelandii. Gas chromatography mass spectrophotometry studies indicate little or no organic acids are associated with FeMo-co. We examined the catalytic properties of the NifV- MoFe protein In the mutant, H2 evolution is inhibited by the addition of CO, unlike in the wild type. We have found that the NifV- MoFe protein from A. vinelandii is able to catalyze the reduction of acetylene to both ethylene and ethane. / Master of Science

The investigation presented here was accomplished using agglutination and agar gel immunodiffusion techniques to compare Azotobacter agilis s 3at, A. chroococcum Italy AC 16, A. macrocytogenes St. M and A. vinelandii ATCC 12837. It was found that the agglutination titers differed sufficiently to allow partial identification of the four species. The homologous and heterologous systems studied by agar gel immunodiffusion tests showed that each of the four Azoto bacter species differed sufficiently in their soluble antigens to give distinct, identifiable patterns of antigen-antibody reactions on Ouchterlony agar plates. These studies also showed several antigens common to the four species tested and the resultant antigen-antibody cross reactions. The results of these investigations suggest that this approach opens a new avenue for the identification of the organisms of genus Azotobacter and perhaps, by extension, the family Azotobacteraceae.

The work described in this thesis was undertaken to study the reasons why Azotobacter vinelandii ATCC 12837 after incubation in Burk's nitrogen-free liquid will not form as many colonies when plated on Difco Tryptic Soy Agar as when planted on Burk's nitrogen-free agar. The difference in growth of A. vinelandii on the two agars was established by performing viable cell-plate counts. The difference in growth was most apparent at 24-hours incubation of the Burk's liquid-media cultures. Phase contrast microscopic observations of Tryptic Soy media cultures of A. vinelandii disclosed the regular formation of fungoid cells at early stages of growth of the bacteria, 18 to 24 hours.

The purpose of this study is to determine if a strain of Azotobacter chroococcum isolated from the soil in northern Louisiana produces cysts which are as resistant to deleterious agents as those produced by previously reported strains os Azotobacter.

The purpose of the current study is to determine something of the nature of the heat resistance in Azotobacter, if in fact this is found to exist. An attempt is made to determine the specific physiological state associated with heat resistance as well as to resolve this resistance quantitatively.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 157-184). Also available via the Internet.