Abstract
Several lines of experimental analyses on the ploidy status of Azotobacter vinelandii genome lead to the conclusion that it contains more than 40 copies of its chromosome and therefore it is a polyploid organism. The genetic evidence argues against the existence of polyploidy in these cells since the segregation pattern of genetic markers under lack of selection pressure mimic that of haploids. However, when A. vinelandii was made Nif− by inserting a kanamycin resistance marker gene in the nifDK sequence and the cells were selected for kanamycin resistance and Nif+ phenotype, we were able to score colonies that are both kanamycin resistant and Nif+. Therefore, when the cells were subjected to forced double selection of the same locus, they behaved as if they carried at least two chromosomes, one carrying the kanamycin resistance marker in the nifDK genes and the other carrying the intact nifDK genes. These analyses suggested that at least a diploidy status can be induced in these cells under selection pressure.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith & K. Strul (ed) 1992. Current Protocols in Molecular Biology. Greene Publishing Wiley Interscience, New York, NY.
Contreras, A. & J. Casadesus, 1987. Tn10 mutagenesis in Azotobacter vinelandii. Mol. Gen. Genet. 209: 276–282.
Efuet, E.T., L. Pulakat & N. Gavini, 1996. Investigations on the cell volumes of Azotobacter vinelandii by Scanning Electron Microscopy. J. Basic Microbiol. 36: 229–234.
Gavini, N. & B.K. Burgess, 1992. FeMo cofactor synthesis by a nifH mutant with altered MgATP reactivity. J. Biol. Chem. 267: 21179–21186.
Gavini, N, B.S. Hausman, L. Pulakat, R.P. Schreiner & J.A. Williamson, 1997. Identification and mutational analysis of rfbG, the gene encoding CDP-D-Glucose-4,6-Dehydratase, isolated from free living soil bacterium Azotobacter vinelandii. Biochem. Biophys. Res. Commun. 240: 153–161.
Howard, J. & D.C. Rees, 2000. Structure of Nitrogenase Protein Components, pp. 43–54 in Prokaryotic Nitrogen Fixation: A Model System for Analysis of a Biological Process, edited by E. W. Triplett. Horizon Scientific Press, Wymondham, UK.
Jacobson, MR, K.E. Brigle, K.L.T. Bennet, R.A. Setterquist, M.S. Wilson, V.L. Cash, J. Beynon, W.E. Newton & D.R. Dean, 1989. Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J. Bacteriol. 219: 49–57.
Kennedy, C. & A. Toukdarian, 1987. Genetics of Azotobacter: Applications to nitrogen fixation and related aspects of nitrogen metabolism. Ann. Rev. Microbiol. 41: 227–258.
Kim, J. & D.C. Rees, 1992. Crystallographic structure and functional implications of the nitrogenase molybdenum-iron protein from Azotobacter vinelandii. Nature 257: 553–560.
Kim, J. & D.C. Rees, 1994. Nitrogenase and biological nitrogen fixation. Biochemistry 33: 389–397.
Maldonado, R, J. Jimenez & J. Casadeus, 1994. Changes of ploidy during the Azotobacter vinelandii growth cycle. J. Bacteriol. 176: 3911–3919.
Maldonado, R, A. Garzon, D.R. Dean & J. Casadeus, 1992. Gene dosage analysis in Azotobacter vinelandii. Genetics 132: 869–878.
Mullis, K.B. & F.A. Faloona, 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Meth. Enzymol. 155: 335–350.
Page, W.J. & M. Von Tigerstrom, 1979. Optimal conditions for transformation of Azotobacter vinelandii. J. Bacteriol. 139: 1058–1061.
Pulakat, L, E.T. Efuet & N. Gavini, 1998. Segregation pattern of kanamycin resistance marker in Azotobacter vinelandii did not show the constraints expected in a polyploid bacterium. FEMS Microbiol. Lett. 160: 247–252.
Pulakat, L, B.S. Hausman, S. Lei & N. Gavini, 1996. Nif- phenotype of Azotobacter vinelandii UW97: Characterization mutational analysis. J. Biol. Chem. 271: 1884–1889.
Punita, S. Jafri, M.A. Reddy & H.K. Das, 1989. Multiple chromosomes of Azotobacter vinelandii. J. Bacteriol. 171: 3133–3138.
Rangaraj, P., C. Rüttimann-Johnson, V.K. Shah & P.W. Ludden, 2000. Biosynthesis of the Iron-Molybdenum and Iron-Vanadium Cofactors of the nif-and vnf- encoded Nitrogenases, pp. 55–79 in Prokaryotic Nitrogen Fixation: A Model System for Analysis of a Biological Process, edited by E.W. Triplett. Horizon Scientific Press, Wymondham, UK.
Robson, R.L., J.A. Chesshyre, C. Wheeler, R. Jones, P.R. Woodley & J. Postgate, 1984. Genome size and complexity in Azotobacter chroococcum. J. Gen. Microbiol. 130: 1603–1612.
Sadoff, H.L., E. Berke & B. Loperfido, 1971. Physiological studies of encystment in Azotobacter vinelandii. J. Bacteriol. 105: 185–189.
Sadoff, H.L., B. Shimei & S. Ellis, 1979. Characterization of Azotobacter vinelandii deoxyribonucleic acid and folded chromosomes. J. Bacteriol. 138: 871–877.
Sambrook, J, E.F. Fritsch & T. Maniatis, 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Strandberg, G.W. & P.W. Wilson, 1968. Formation of the nitrogen fixing system in Azotobacter vinelandii. Can. J. Microbiol. 14: 25–31.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Suh, MH., Pulakat, L. & Gavini, N. Isolation and characterization of nif::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter. Genetica 110, 101–107 (2000). https://doi.org/10.1023/A:1017976920135
Issue Date:
DOI: https://doi.org/10.1023/A:1017976920135