FEMSMicrobiologyLetters 3 (1978) 139-141
139
© Copyright Federation of European MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
MULTIPLE EXTRACHROMOSOMAL DEOXYRIBONUCLEIC ACID MOLECULES IN
THURINGIENSIS
BACILLUS
D.P. STAHLY, D.W. DINGMAN, R.L. IRGENS *, C.C. FIELD *, M.G. FEISS and G.L. SMITH
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 and * Department of Life Seienees, Southwest Missouri
State University, Springfield, Missouri 65802, U.S.A.
Received8 December1977
1. Introduction
Bacillus thuringiensis is a commercially important
microorganism due to its production of a parasporal
body, an intraceUular proteinaceous crystal, that has
insecticidal properties [1 ]. Nothing is known conceming the gene(s) coding for the crystal. Several
investigators have reported that loss of the ability to
form crystals occurs at a much higher frequency than
that ordinarily due to spontaneous mutation [2-4].
Such instability of a phenotypic property is suggestive of plasmic inheritance [5], although other
viable hypotheses certainly are possible. As indicated
below, B. thuringiensis does contain extrachromosomal covalenfly closed circular deoxyribonucleic
acid (DNA) molecules. It remains to be determined
whether these closed covalent circular DNA molecules
are plasmids and whether one or more of the size
classes of these circular molecules is missing from
crystal-minus dissociants.
2. Materials and Methods
K2HPO4 (2A g), and KI-I2P04 (0.5 g). Radioactive
labeling of the DNA was accomplished by addition of
5 mCi of [methyl.SH]thymidine and 0.25 g of 2deoxyadenosine per liter of growth medium. Cells
were grown at 37°C on a rotary shaker and were harvested by centrifugation when the turbidity was
400 Klett units (No. 54 filter).
Covalenfly closed circular DNA was isolated by
the procedure of Currier and Nester [6] and the dyebuoyant density procedure of Radloff et al. [7]. Density fractions containing extrachromosomal DNA
were examined by electron microscopy using preparative procedures described by Davis et al. [8]. A
JEOL model 100B electron microscope was employed. Contour lengths of the molecules were measured directly on 8 X 11 inch prints using ~X174
duplex DNA as an internal length standard (3.4.109
daltons [8]). Examination of covalently closed circular molecules from B. thuringiensis in the absence of
~bX174 RF DNA showed no molecules of the size of
~X174 RF DNA. Measurements of contour length
were made using a Hewlett-Packard 9107A digitizer
and 9100B calculator generously provided by Dr.
Charles Wunder.
The microorganism used in this study is a strain of
B. thuringiensis var. alesti anduze doubly resistant to
penicillin and streptomycin. This strain was kindly
provided by H.J. Somerville. It sporulates and forms
parasporal crystals normally. The growth medium
adjusted to pH 7.15, contained the following in one
liter: glucose (5.0 g), Casamino acids (Difco; 5.0 g),
ammonium sulfate (1.88 g), ferric citrate (0.05 g),
MgC12 • 6 H20 (0.1 g), MnSO4. H20 (0.05 g), CuSO4
(0.01 mg), ZnS04 (0.01 mg), CaC12 (0.1 mg),
3. Results
Cesium chloride-ethidium bromide equilibrium centrifugation of [14C]DNA from B. thuringiensis
revealed two bands, the most dense containing covalently closed circular DNA and the least dense containing linear strands of chromosomal DNA (Fig. 1).
Examination of the extrachromosomal DNA by
140
electron microscopy revealed approx. 12 size classes
of molecules (Fig. 2. Table 1). Some of the size classes
may represent multimers of smaller molecules. The
relative n u m b e r of molecules in each size class is a
very rough approximation. The contour lengths of
molecules with many twists were not determined.
Since twists obviously occur more frequently as the
length of the molecules increases, there may be some
bias in the recorded data against molecules of high
molecular weight.
9l~0
8,0O0
r.000
6,O00
®
5,0O0
0
~E 4.o00
o
3,O00
2,O00
1,000
4
8
12
2o 2', 2~ 3~ 3;
16
Fraction Number
,'0 ,'.
4. Discussion
Fig. 1. Ethidium bromide-cesium chloride equilibrium centri-.
fugation of DNA from B. thuringiensiz The solution to be
centrifuged contained 3.5 rrd of DNA (previously dialyzed
against pH 8.0 buffer containing 0.05 M Tris-hydroxymethylaminomethane,5.0 mM ethylenediamine tetraeetic acid,
and 0.05 M NaC1), ethidium bromide (1.5 ml of an aqueous
solution containing4 mg/ml), and cesium chloride (3.75 g).
The average density of the solution before centdfugation was
1.5482. The samples were centrifuged at 36 000 rpm for 48 h
in a Spineo Type No. 65 rotor at 15°C. Fractions (5 drops
each) were collected from the bottom of each tube. A portion (0.01 ml) of each fraction was spotted on a circle of
Whatman 3 MM tilter paper, the papers were dried, and the
radioactivity was determined by scintillation spectrometry.
The density of fractions was determined from refractive
index measurements made by use of a Zeiss model No. 79540
refraetometer. The buoyant densities of the extraehromosomal DNA and chromosomal DNA were 1.5873 and 1.5514,
respectively.
The larger number of size classes of extrachromosomal elements in B. thuringiensis is similar to the
situation ofB. megaterium [9,10]. Six major size
classes and four minor size classes o f extraehromosomal molecules were observed in B. megaterium.
Carlton [9] recently postulated that the mini-circles
in B. megaterium represent molecular hybrids of one
or more basic plasmid elements linked to chromosomal DNA sequences. While evidence has been presented supportive of this working hypothesis, Carlton
[9] states that verification will require additional
experimentation.
Questions concerning the nature and origin of the
extrachromosomal elements in B. thuringiensis are
currently being investigated in our laboratory.
TABLE 1
Frequency distribution by number and weight of the extrachromosomal DNA molecules
Size
class
Number
of
molecules
Average contour
length ± SD
(#m)
%
SD
Mol.
wt. (" 106)
% of total
molecules
by number
% of total
circular DNA
by weight
I
II
III
14
19
87
1.289 ± 0.075
1.881 ± 0.076
2.351 ± 0.071
5.8
4.0
3.0
2.60
3.80
4.75
4.6
6.3
28.6
1.4
2.7
15.5
IV
V
38
4
2.635 + 0.101
3.116 ± 0.123
3.8
3.9
5.32
6.29
12.5
1.3
7.6
0.9
VI
VII
VIII
IX
X
XI
XII
5
47
21
45
3
15
6
2.9
2.1
3.1
2.9
1.0
4.9
2.4
7.29
8.10
8.67
9.96
18.12
36.72
44.58
1.6
15.5
6.9
14.8
1.0
4.9
2.0
1.4
14.3
6.8
16.8
2.0
20.6
10.0
100.0
100.0
Totals
304
3.607
4.009
4.294
4.932
8.968
18.178
22.070
± 0.104
± 0.084
± 0.132
± 0.145
± 0.091
± 0.892
± 0.538
141
45
40
35
r,,0
30
o
25
0
20
E
15
l,r
10
I
!
i
i
s
1.0
2.0
3.0
4.0
n
5.0
6.0
7.0
8.0
9.0
15.0
20.0
25.0
Contour Length (l~m)
Fig. 2. Contour length distributions of circular DNA molecules from electron micrograph measurements. The size classes established by these distributions are indicated by Roman numerals. A group of molecules was considered as a discrete size class ff at
least two molecules were observed with length measurements deviating by less that +6.5% from the mean (the deviation observed
with ~X174 RG molecules). The size class designations for molecules in classes I, H and XI are somehwat tenuous, due to a deviation in contour length from the mean somewhat greater than ±6.5% (±12.4, 28.5, and 210.35, respectively.
Acknowledgments
This investigation was supported b y National
Science F o u n d a t i o n grants PCM76-8168 ] and
GB37310. We very much appreciate the gift o f
~bX174 R F DNA from Dr. John Donelson.
[3]
[4]
[5]
[6]
[7]
[8]
References
[9]
[1] Bulla, L.A., Rhodes, R.A. and St. Ju]lan, G. (1975)
Annu. Rev. MicrobioL 29, 163-190.
[2] Fitz-James, P.C. and Young, I.E. (1959) J. BacterioL
78,743-754.
[10]
Toumanoff, C. (1955) Ann. Inst. Pasteur 89, 644.
Vankova, J. (1957) Folla BioL 3, 175-183.
Lacey, R.W. (1975) BacterioL Rev. 39, 1-32.
Currier, T.C. and Nester, E.W. (1976) Analyt. Biochem.
76,431-441.
Radloff, R., Bauer, W. and Binograd, J. (1967) proc.
Natl. Acad. Sci. USA 57, 1514-1521.
Davis, R.W., Simon, M. and Davidsen, N. (1971) in:
Methods in Enzymology, Vol. 21D (Grossman, L. and
Moldave, K., Eds.), pp. 413-428, Academic Press,
New York.
Carlton, B.C. (1976) in: Microbiology-1976 (Sclflessinger, D., Ed.), pp. 394-405, Am. Soc. MicrohioL,
Washington, D.C.
Carlton, B.C. and Helinski, D.R. (1969) Proc. Natl.
Acad. Sci. USA 64, 592-599.