Plasmids of Azotobacter vinelandii Page: 1,984
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Vol. 170, No. 4
JOURNAL OF BACTERIOLOGY, Apr. 1988, p. 1984-1985
0021-9193/88/041984-02$02.00/0
Copyright 1988, American Society for MicrobiologyPlasmids of Azotobacter vinelandii
MAURICIO MAIA,t JUAN M. SANCHEZ, AND G. R. VELA*
Department of Biological 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, and 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
copies of their genome (4-6, 9), it seems reasonable to
assume that, as a consequence, these bacteria may fail to
mutate easily. Other workers (2, 3) have suggested that this
genetic redundancy may be due to multiple plasmids which
carry many copies of each gene. In addition, Medhora et al.
(2) propose that the nitrogen fixation genes which are easily
mutated may be located on single-copy plasmids, while other
genes may exist in as many as 40 allelic copies either on
multicopy plasmids or on unique polyploid chromosomes.
The idea that nifgenes are carried on single-copy plasmids is
tenable only if all cultures of azotobacters which can grow
on nitrogen-free media also possess plasmids either as a
single-copy, free, double-stranded, circular DNA molecule
or as a single-copy plasmid integrated into the bacterial
genome. The data reported here show that the foregoing
theory is not tenable. The data also show that plasmids are
not associated with nitrogen fixation in Azotobacter vinelan-
dii.
Thirty laboratory strains of A. vinelandii were obtained
from 19 laboratories throughout the world, and two cultures
were isolated from water. All cultures were grown and
maintained on Burk medium (8) supplemented with nutrient
broth powder at 4 g liter-1 and on nutrient agar. The same
media were used for a non-nitrogen-fixing mutant, UW-1,
except that 8 g of ammonium acetate liter-' were added to
Burk medium in lieu of glucose. Five strains of Escherichia
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
antibiotics. All E. coli were grown on tryptone salt-yeast
extract medium, and the presence of plasmids was confirmed
by adding the appropriate antibiotics. Antibiotic susceptibil-
ity was used to indicate curing of plasmids after treatment
with ethidium bromide. A. vinelandii and E. coli were grown
in 200-ml batch cultures on a reciprocal shaker at 30 and
37C, respectively, to late-logarithmic-growth phase (ap-
proximately 108 cells mi-'). The method of Kado and Liu (1)
was used for extracting and isolating plasmid DNA. SeaKem
ME agarose gel electrophoresis was used to resolve plasmid
DNA and to estimate molecular weights. For curing, the
azotobacter cultures were grown in Burk medium with
various quantities of ethidium bromide, sodium dodecyl
sulfate, or acridine orange by the method of Robson et al.
* Corresponding author.
t Present address: Biology Department, University of California
at Davis, Davis, CA 95616.(5). Nitrogen fixation was detected by measuring the conver-
sion of acetylene to ethylene by gas chromatography, and all
other physiologic characteristics were determined by the
methods of Thompson and Skerman (7).
Plasmids were found in 6 of the 32 cultures of A. vinelandii
examined (Table 1). Strain UW-1, a non-nitrogen-fixing
mutant, contained a plasmid of 52 megadaltons. Since it has
been suggested that nif genes are carried by azotobacter
plasmids (2, 3), it could be assumed that plasmid pUW-1
lacks such genes. Each of the six cultures of A. vinelandii
had only one plasmid, while Robson et al. (5) found that each
strain of Azotobacter chroococcum they studied harbored
multiple plasmids.
Simultaneous comparison of cultures of each of the cured
and plasmid-bearing strains of A. vinelandii with cultures
after curing of the plasmid showed no discernible differences
in the ability to fix nitrogen (data not shown).
Of 32 cultures capable of growing on nitrogen-free media,
only 6 contained double-stranded, covalently closed circular
plasmid DNA. When the six strains that contained plasmids
were cured, nitrogen fixation was not affected; however, a
mutant called UW-1 contained a plasmid of 52 megadaltons
and yet was unable to fix nitrogen. These findings fail to
support the hypothesis that nif genes are on single-copy
plasmids. It is possible that other plasmids are integrated
into the chromosome and that among these are the ones
which bear nifgenes, but there is also the problem of how to
TABLE 1. Strains of A. vinelandii containing plasmids
Mol wt
Strain (MDa)a Source Plasmid
(MDa)"
UW-1 52 H. J. Sadoff, Michigan pUW-1
State University
Soil isolate 48 J. M. Sanchez, North pNT5S
Texas State
University
UW 43 V. Shaw, University pUWW
of Wisconsin
UW 12 W. J. Page, University pUWA
of Alberta
Water isolate 3 11 R. Peters, University pWM3
of Chihuahua
Water isolate 16 10 R. Peters, University pWM16
of Chihuahua
2489 9 J. Moreno, University p2489
of Granada
a The sizes of strains were determined by comparison with E. coli plasmids
of known molecular weight. MDa, Megadaltons.1984
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Maia, Mauricio; Sanchez, Juan M. & Vela, G. Roland, 1927-. Plasmids of Azotobacter vinelandii, article, April 1988; [Washington, D.C.]. (https://digital.library.unt.edu/ark:/67531/metadc287021/m1/1/: accessed April 25, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT College of Arts and Sciences.