JOURNAL OF BACTERIOLOGY, Apr. 1988, p. 1984-1985 Vol. 170, No. 4 0021-9193/88/041984-02$02.00/0 Copyright C 1988, American Society for Microbiology Plasmids of Azotobacter vinelandii MAURICIO MAIA,t JUAN M. SANCHEZ, AND G. R. VELA* Department ofBiological Sciences, North Texas State University, Denton, Texas 76203 Received 29 January 1987/Accepted 22 December 1987 Four laboratory strains and two isolates of Azotobacter vinelandii were found to contain plasmids. Twenty- five laboratory strains which could fix nitrogen did not have free, covalently closed circular plasmid DNA. The plasmids varied in size from 9 to 52 megadaltons, ahd each strain yielded only one plasmid. No discernible differences in ability to fix nitrogen were found between plasmid-bearing and cured cultures.

Since bacteria of the genus Azotobacter contain multiple (5). Nitrogen fixation was detected by measuring the conver- copies of their genome (4-6, 9), it seems reasonable to sion of acetylene to ethylene by gas chromatography, and all assume that, as a consequence, these bacteria may fail to other physiologic characteristics were determined by the mutate easily. Other workers (2, 3) have suggested that this methods of Thompson and Skerman (7). genetic redundancy may be due to multiple plasmids which Plasmids were found in 6 of the 32 cultures ofA. vinelandii carry many copies of each gene. In addition, Medhora et al. examined (Table 1). Strain UW-1, a non-nitrogen-fixing (2) propose that the nitrogen fixation genes which are easily mutant, contained a plasmid of 52 megadaltons. Since it has mutated may be located on single-copy plasmids, while other been suggested that nif genes are carried by azotobacter genes may exist in as many as 40 allelic copies either on plasmids (2, 3), it could be assumed that plasmid pUW-1 multicopy plasmids or on unique polyploid chromosomes. lacks such genes. Each of the six cultures of A. vinelandii The idea that nifgenes are carried on single-copy plasmids is had only one plasmid, while Robson et al. (5) found that each tenable only if all cultures of azotobacters which can grow strain of Azotobacter chroococcum they studied harbored on nitrogen-free media also possess plasmids either as a multiple plasmids. single-copy, free, double-stranded, circular DNA molecule Simultaneous comparison of cultures of each of the cured or as a single-copy plasmid integrated into the bacterial and plasmid-bearing strains of A. vinelandii with cultures genome. The data reported here show that the foregoing after curing of the plasmid showed no discernible differences theory is not tenable. The data also show that plasmids are in the ability to fix nitrogen (data not shown). not associated with nitrogen fixation in Azotobacter vinelan- Of 32 cultures capable of growing on nitrogen-free media, dii. only 6 contained double-stranded, covalently closed circular Thirty laboratory strains of A. vinelandii were obtained plasmid DNA. When the six strains that contained plasmids from 19 laboratories throughout the world, and two cultures were cured, nitrogen fixation was not affected; however, a were isolated from water. All cultures were grown and mutant called UW-1 contained a plasmid of 52 megadaltons maintained on Burk medium (8) supplemented with nutrient and yet was unable to fix nitrogen. These findings fail to broth powder at 4 g liter-1 and on nutrient agar. The same support the hypothesis that nif genes are on single-copy media were used for a non-nitrogen-fixing mutant, UW-1, plasmids. It is possible that other plasmids are integrated except that 8 g of ammonium acetate liter-' were added to into the chromosome and that among these are the ones Burk medium in lieu of glucose. Five strains of Escherichia which bear nifgenes, but there is also the problem of how to coli were used as references for plasmid DNA of known molecular weight. The plasmids ranged in size from 5.5 to 96 megadaltons, and each conferred resistance to one or more TABLE 1. Strains of A. vinelandii containing plasmids antibiotics. All E. coli were grown on tryptone salt-yeast extract medium, and the presence of plasmids was confirmed Strain (MDa)a Source Plasmid by adding the appropriate antibiotics. Antibiotic susceptibil- UW-1 52 H. J. Sadoff, Michigan pUW-1 ity was used to indicate curing of plasmids after treatment State University with ethidium bromide. A. vinelandii and E. coli were grown Soil isolate 48 J. M. Sanchez, North pNT5S in 200-ml batch cultures on a reciprocal shaker at 30 and Texas State 37°C, respectively, to late-logarithmic-growth phase (ap- University proximately 108 cells ml-'). The method of Kado and Liu (1) UW 43 V. Shaw, University pUWW was used for extracting and isolating plasmid DNA. SeaKem of Wisconsin ME agarose gel electrophoresis was used to resolve plasmid UW 12 W. J. Page, University pUWA DNA and to estimate molecular weights. For curing, the of Alberta azotobacter cultures were grown in Burk medium with Water isolate 3 11 R. Peters, University pWM3 various quantities of ethidium bromide, sodium dodecyl of Chihuahua Water isolate 16 10 R. Peters, University pWM16 sulfate, or acridine orange by the method of Robson et al. of Chihuahua 2489 9 J. Moreno, University p2489 * Corresponding author. of Granada t Present address: Biology Department, University of California a The sizes of strains were determined by comparison with E. coli plasmids at Davis, Davis, CA 95616. of known molecular weight. MDa, Megadaltons. 1984 VOL. 170, 1988 NOTES 1985 distinguish between that type of plasmid and chromosomal F. C. Cannon. 1979. Evidence for nitrogen fixation (nif) genes on genes. indigenous Rhizobium plasmids. Nature (London) 282:533-535. 4. Roberts, G. P., and W. J. Brill. 1981. Genetics and regulation of This work was supported by grant PCM 8213951 from the Na- nitrogen fixation. Annu. Rev. Microbiol. 35:207-235. tional Science Foundation. 5. Robson, R. L., J. A. Chesshyre, C. Wheeler, R. Jones, P. R. The work of Maria Luisa Salaiz and Betzabet Quintanilla, Uni- Woodley, and J. R. Postgate. 1984. Genome size and complexity versity of Chihuahua, is gratefully acknowledged. We also thank the in Azotobacter chroococcum. J. Gen. Microbiol. 130:1603-1612. individuals listed in Table 1 for kindly providing us with their 6. Sadoff, H. L., B. Shimei, and S. Ellis. 1979. Characterization of cultures of A. vinelandii. Azotobacter vinelandii deoxyribonucleic acid and folded chro- mosomes. J. Bacteriol. 138:871-877. 7. Thompson, J. P., and V. B. D. Skerman. 1979. Azotobacteraceae: LITERATURE CITED the taxonomy and ecology of aerobic nitrogen-fixing bacteria. Academic Press, Inc. (London), Ltd., London. 1. Kado, C. I., and S.-T. Liu. 1981. Rapid procedure for detection 8. Vela, G. R., and R. S. Rosenthal. 1972. Effect of peptone on and isolation of large and small plasmids. J. Bacteriol. 145:1365- Azotobacter morphology. J. Bacteriol. 111:260-266. 1373. 9. Yano, K., M. Anazawa, F. Murai, and M. Fukuda. 1984. pMYL, 2. Medhora, M., S. H. Phandir, and K. S. Das. 1983. Construction a large plasmid of Azotobacter vinelandii AVY5, has DNA of a gene library from the nitrogen-fixing aerobe Azotobacter sequences hybridizable to Kpnifand PpXyl, p. 751. In C. Veeger vinelandii. Gene 25:355-360. and W. E. Newton (ed.), Advances in nitrogen fixation research. 3. Nuti, M. P., A. A. Lepidi, R. K. Prakash, R. A. Schilperoort, and Kluwer-Nijhoff Publishing, Waggeningen, The Netherlands.