Journal of the Indian Institute of Science, Bangalore 560 012, India
[ Section C: Biological Sciences ]
Volume 62
August 1980
Number 8
CONTENTS
Page
ORIGINAL PAPER
MolecuLv Biology
STUDIES
ON THE EXTRACHROMOSOMAL DNA
ensis
OF
Bacillus thuringiensis var. thuringiS. Rajalakshrni-
SHORT COMMUNICATION
Biochemistry
COMBINED
ACTION OF POLYMYXIN B AND GRAMICIDIN DUBOS ON L~POSOMES
DERIVED FROM PHOSPHOLIPIDS OF E. C O I ~
G. Radhakrishna and L. K. Ramachandran
REVIEW PAPERS
Biochemistry
DESENSITIZATION
OF ALLOSTERIC SITES : CONSEQUENCESON THE CONFORMATION
AND CATALYTIC PROPERTIES OF REGULATORY ENZYMES
A. R. Venugopala Reddy, J. Sobhanaditya and N. Appaji Rao
CARBOHYDRATE
METABOLISM
IN RIPENING BANANA AND ITS ALTERATION ON GAMMA
IRRADIATION IN RELATION TO DELAY IN RIPENING
K. K. Surendranathan and P. M . Nair
BOOK REVIEW
21
tn"hq
hit. Sci. 62 (C). Aug. 1980, Pp. 21-31
,.
Institute of S ~ i e n c e ,PIinted in India.
gdes on the extrachromosomal DNA of BuciNus thnringknsis var.
ikm'ngieensis
s, RMALAKSHMI
?d;&iology
and Cell Biology Laboratory. Indian Institute of Science, Bangalore 560 012.
c enlaL-hiomosomal DNA was isolated from B. Ihuiin&iiis var. thurin$orsis and characterized.
k. :p.olsolar weight was h u n d lo he approx. 10 % 10". The covalently closed narure of the moktias confirmed hy CcCbethdium bromide centrifugation, agarose gel electlophoreiis and electron
wro,copy. The clrrach~-omo?ornalD N A wa rsoiated at various vages ool gxoirrh, undw ditfemni
gsmiwndirions (Svitli varying concentrations of cys). Two mode\ of existence foi tl~cexttachron~oemel DNA were ~ugsested: (0)an autonomous cylopiasrnic fmm and (h) a cryplic form in which ~t
:~emm.bly was integrued into the smomic DNA. It is suggested that some oi the c r y s d protein
and major spore cont protcin could have been coded for by llilc cxtrarluomosonial DNA of B. 2l~rrrint k m var. fhuriiigimsis whde it ia in an extrachromosomal state. I n t k integrated stale the extrahomoron~dDNA is non-functionai.
mtesaI;CV ~ w d s: B. tharing~ensis, extrachromosomal DNA, spole coat, crystal protein, inhib~l~on,
r,m. cytoplasmic state.
foe presence of extrachrornosomal DNA has been reported in several badli,
Like 6. subfilis and B. rneg~tsriurnj--7.l~.~~.'8.Stahly er ul'" have reported the
~~tmc
ofemultiple copies of such extrachromosomal DNA in B. flzuringiensis. The
Ektional role of these DNAs has not been clearly established. It was noticed that
?$en B. thuringiensis strain 612 was transferred from a sporulating medium to a non-
sgorulating medium, no extrachronlosomal DNA was round, and the crystal rormation
was also negligible%. Based on these results, it wes suggested that there could be a
:rlationship between the extrcchromosorral DNA and the formation of the toxic crystalhe inclusion in B. fhuring;ensis8J5.
En the present study attempts were made to isolate and characterize the extrachromo.
DNA from B. tlzuringiensis var. fliuringiensis at various stages of the growth of
!he organism, under different growth conditions. so as to examine its existence and
disappearance observed with respect to the nutritional status of the medium*. cyst&
was used as a probe to study the relationship between the extrachromosomal DNA and
the formation of the crystal toxin in B. thuringiensis var. thringiensis and the results are
discussed here.
2. Materials and methods
2.1. Organism and culture conditions
Bacillus thuringiensis var. fhuringiensis serotype I was obtained from Prof. H. de Barb,
Institut Pasteur, Paris. Inoculum and the minimal medium containing mineral satti
and 1% glucose were prepared as mentioned earlierL3. For further studies the m i n d
medium was supplemented with low (0.05%), medium (0.15%) and high (0,Wk
concentration of cys.
2 . 2 . Isohtion and characterization of the extrachromosormrl DNA of B. thuringid
vur. tburingiensis
The extraohromosomal DNA was isolated from B. thuringiensis var. thuringiensis by
acid-phenol extractionls and also by the lysozyme-EDTA technique of Gerry et d
The covalently closed nature of the extrachromosomal DNA was confirmed by QC%
ethidium bromide centrifugation"^ well as by agarose gel electrophoresisu. Rum@
graphy of the agarose gels was carried out according to Bonner and StedmanY.The
molecular weight of ihe extrachromosomal DNA was calculated by subjecting the saqk
to electrophoresis on 4% polyacrylamide gel, using I-DNA, colitis bacteriophage, and
colitis bacteriophage DNA as marked4. The cells were labelled with [aH]-thymi&
for 2 hr prior to the isolation of the extrachromosomal DNA. The bacteriophq$ i
was propagated in B. coli 621 as the host and the 1 phage was puriiied on a lim
sucrose density gradient (15-60% w/v, sucrose in 0.1 M tris-HCl b a r , pR 7.2). Thc
colitis bacteriophage was propagated and purified as per the standard procedure". DNA
from the bacteriophage I and the colitis bacteriophage DNA were isolated by the treat
ment of the phages with SDS (1%) and repeated phenol extraction (thrice) followed by
extensive dialysis against 0.1 M NaCI. The &DNA was labelled with [SH]-thymidine
ie., the addition of radioactive material was made 10 min after the phage infedion.
The radioactive thymidine was used at a concentration of 0.1 mCi per 100 ml cultatctc
2.3. lrolation of the ertrachromoJomal DNA under different growth condition^
The extrachromosomal DNA was isolated under varying growth conditions (i.e.,
gown with 0.05%, 0.15% and 0.25% cy s in a glucose-salts medium), at different W
of growth s t a r h g from the mid-lag phase, with a regular interval of U) min. A W ' J
was paid to observe the appearance or the disappearance (ifany) of the extrachromosom*i
DNA depending on the nutritional status of the medium as reported by Errnakova et &.
Besides, excess cys was added at, and after the stationary phase to arrest the F
coat and tbe crystal formation, or the spore-crystal formation completely ( u n P u W
PLASMID DNA O F
B. thuringiensis
23
md the existence of the extrachromosomal DNA was studied. Reversal of
ipOm~ation-inhibiti~n
was carried out by the addition of glutamate or divalent metal
the intermediates of Krebs cycle (unpublished data) and the isolation of the extradvomosomalDNA was carried out under such conditions also.
~ h r o r n o s o m a lDNA was analysed by electron microscopy as described by Bak
dl. The extrachromosomal DNA (2yg per ml) in TES buffer (0.05 M NaCl +
&35M tris t 0.005 M EDTA, p H 8 4) was mixed with an equal volume of a 0.04%
&tion of cytochrome-c. and twice the volume of 4 M ammonium acetate. This solu(ion was spread on the surface of distilled water. The monolayer was transferred to
?mcopper grids (300 mesh) covered with formvar. The preparation was fixed in
&lute alcohol for 30 seconds, stained with 3% uranyl acetate (in absolute alcoho~)
J examined under Philips 301 electron microscope with 60 kV accelerating voltage
u j ~ ag magnification of approx. 7,500 X . Contour lengths of the circular DNA were
rn9ured with a Hewlett-Packard digitizer (model 9107 A), after magnification (10 X )
d &e negatives on to a white screenG.
d
fHEThymidine was obtained from the Bhabha Atomic Research Centre, Bombay, India.
&.rose, SDS, tris-(hydroxymethyl) aminomethane, lysozyme, CsCI, ethidium bromide,
aerylamide, methylene bis-acrylamide, cytochrome-c and EDTA were obtained from
Sigma Chemical CO., St. Louis, MO., USA. L-Cystine was obtained from E. Merck,
Darmstadt. Formvar and uranyl a.cetate were obtained from Polaron Equ~pmentLtd.,
London, N3, UK. All the other chemicals were of analar or reagent grade.
aowth of the culture was monitored by the absorbance at 600 nm in Bausch and
Lomb Spectronic-20 colorimeter. Density gradient centrifugations were carried out
ha Beckman L2-50 ultracentrifuge. Philips 301 transmission electron microscope was
usedfor the electron microscopic studies of the extrachromosomal DNA. Radioactivity
was measured by Beckman LS-100 liquid scintillation spectrometer.
2.7. Other parameters
%re and crystal formation were monitored by observing a wet smear of the cdture
phase microscope. Heat-stable spores were estimated by plating the sample on
Wient agar plates after exposing the spore suspension at 80" C for 15 min. Heat
labile spores were estimated by plating the sample on nutrient agar plaks after an approP%te dilution, without any heat treatment. Heat lability was confirmed by the lysoEyme sensitivity of the spores16. Toxicity was checked by feeding the crude cell lysate
2nd or 3rd instar larvae of B. mori. One mg protein was sprayed on mulberry leayes
fed to 20 worms of B. mori. After 4-5 hr the mortality was scored.
0.25-w.
0.5
1.5
Log Mol. W t . ;: lo-"
I-LC.L .
MolecuLar uXeightdctermmation of the ex~rachuornosornalD N A hy agarox gel clcctropharrur.
1-colitis bacreriophage DNA (md. wt. = 10 ;: loX) ; 2--coli(is bactcriopl~age(mol. wt. = 20 . lki.
3-;\-DNA ; A - s x i r a c h ~ ~ o m o ~ o mDNA
; ~ ~ l or B. rlr~rrinfiiensisvac. t h a r i n ~ i ~ n s i s .
3.1. Existence of the extraehroinosomal D N A zcrrrlpr different growth conditions
When the cells were provided with low concentration of cys (O.OYX), s p o r e - ~ d
formation was observed; under such conditions the exirachromosornal DNA was found
in an ' axtrachronlosomal state ', until 2 h r after the stationary phase. Whenthecel!~
were provided with 0.15% cys, no spore coat and crystal Formation was noticed @li'
heat labile spores were produced); whereas, when the cells were providcd wih h$
concentration of cys (0.25:;) n o spore and crystal Cormation was obcervcd (unpublisM
data). Under thz conditions of 111ediun-i alld high concsntrations of cya, the emchroinosomal DNA could not I?e isolated from 60 min prior to the cessation of tfit
vegetative growth (Table I ) ; when the sporulation inhibition was reveraed by
addition of reversal agents like glutamate, dival:nt metal ions or intermediates of Kreb,
cycb (unpubhshzd data), the extrachromosomal DNA reappeared and the organisif
retained toxicity. The extrachrornoso~nalDNA, present in the isolatabk form until
the addition of excess cys, disappeared as soon as the excess cys was added after
stationary phase. When the reversal agents werz added 10 t o 15 min prior to tk
addition of excess cys, the extrachromoson~alDNA was in an isolatable form 2nd there
was spore and crystal formation (Table I).
3.2.
ChaI'aCtePistics o j the extrachromosomal DNA
The molecular weight of the extrachromosolnal DNA was found to be approx. 10 * IN
(Fig. 1). a coval*tly closed nature of the lnolecule was confirmed by akWose
Growth
Minerel
salts
-1%
zlucose
niedium
0.05
0.25
p15
0.05
Cys;;
+
.
-
.-
.7
-
-k
+
Reversal a ~ e n t s
Re-!ersai
agents
O.~%C>S
0.15 or
0.15 or
0 . 2 % ~s)
-
.-
-
-$
-
-
+
-
-I
0
-
-. .
--
-
-60
..
Reversal agents
L,
--90
-
I-
-8-
4-
midlog
Extrachromosomal DNA
3
-
-
--
SC:
XLS or
-SC
-sC
SC
SC
SC
HLS
112
-
'cys = CYstine; Reversal agents = glutamate, divalent metal ions or intermediates of TCA cycle; SC =spore and crystal; HLS =
h ~ labile
t spore; -SC = n o spore and crystal: -90 = 90 min prior to stationary phatc (SP): -60
60 min prior to SP; 0 =At
SP; -!-I, ~ 2 etc.
,
1, 2, 3 ... hr after SP.
.rharingiefwis
Bacilhrs
lirurir~giertsisvar,
Organism
Exi~tonceof the cxtrnchromosomal DNA under different groa'th conditions
2
*.
2.
5.
5
Pj
4:
i-
L
ti
3
2
26
S. RAJALAKSHMI
electrophoresis followed by fluorography (Fig. 3, and by CsC1-ethidium bromide
fugation (Fig. 2a). Since the genoinic D N A could n o t penetrate the gel, it rema&
at the top of the gel (0.70/, agarose); once the covalently closed molecoles xineariic,
they are susceptible t o endonuclease digestion, which would result in no sharp band
formation on agarose gel. Such digested molecules and the broken pieces of D N ~
during the isolation procedure moved along the dye band. Hence the sharp band
~ b t a i ~ con
d agarose gel electrophoresis was due to covalently closed molecub of
B. thuringierzsis var. thzrringiensis (Fig. 3 ) . As the intercalating dye ethidium b r o d e
would relax the supercailed nature o r the extrachromosomal DNA, the density of the
molecules would decrease on CsCl density gradient. Hence shift in the band density
was observed when the centrifugation was carried out with ethidiurn bromide, as
pared to the band obtained without ethidium bromide (Fig. Zu, b). For agaros
electrophoresis and CsCI-ethidium bromide centrifugation, &DNA labelled with [Q
thymidine was used as marker (Figs. 2 b and 3). Colitis bacteriophage and W&
bacteriophage DNA were used without radioactive labelling.
3.3. Electron microscopy
Electron mmoscopy also proved the covalently closed nature of the molecule (Fig 4)
The length of the extrachromosomal DNA was found to be 4.4,um. The cai&,M
molecular weight bssed on the length was found to be 8 x lo6 (Table 11) Some of
Molecular weight determination of the extrachromosomal DNA based on the elmicroscopic observation
-
16.5 prn
16-5 x lO6A
Distance between haws = 3.5 A
No. of base pairs = (16.5 X 10L)/3.5
= 4 . 7 x to4
Length of A-DNA
Mol. wt. of A
T
G
C
=
135
126
151
I11
Take il for granred that AT : GC = 1 : I
M d wt. of >.-DNA by electron microscopy = 26 x I@
Actual mol. wt. of I-DNA = 30 r 10'
-
-
-
-
Length of extrachromosomal D N A of B thunrrgre~urr var thrrnnyrmsrs - 4 4 prn = 4 4
N o of base pacrs = (4 4 \ 10L)/3 5 = J 6 x 10'
M d wt obtamed by ekctron mrcroscopy = 8 36 x 1@
hk4. wt ob-
bY amme gel electrophaesrs = 10 x 10d
I@*
PLASMID D N A OF
I
I
0
2 .S
10
,
I
0
I
10
B. ti~uringiensis
I
30
FRACTION N U M B E R
I
I
20
30
FRACTION NUMBER
FIG.2. CsC1-ethidium bromide centrifugation of (2a) extrachromosomal DNA of B. thuii~~gienris
on a 5-20%.sucrose
and (2b) &DNA. The extrachromosomal DNA and the ;\-DNA were ~ruriiid
iintah gradient containing 0.1 M NaCI, O.W5 M EDTA and 0.05 M phosphate buffer pH 7.5. To
Study the ethidium bromide binding, 1.654 g per ml CsC1, 15 pg of ethidium bromide per ml of the
bufferand 2 pg per ml of DNA were used. The centrifugation was carried out at 1 9 C in a Beckman
SW 50 rotor for 24 hr (44,000 rpm). The tubes were punctured, 0.1 ml fractions were collected and the
ndioactivity was -wed.
0-0 without cthidium bromide ; 0with ethidium bromide,
~ ~ U i o n1sto 40 correspond to the fractions from bottom to top.
:ovalently closed molecules had a length of 8.8 flm (Fig. 5 ) 1 and some soch dimershad
' 8-shaped ' structure (Figs. 6 , 7 and 8). These ' %shaped ' molecules could
e&r
replicating molecules of the extrachromosal DNA. They could also have formed
due to recombination between the molecules of the extrachromosomal DN.A.3.13. ~ i , ~ ,
the existence of the ' 8-shaped ' molecules proves the possibility of the presence of more
than one copy oC the extrachro~nosoinalDNA per cell of B. fltwifgiensis var. rhtriingipnsi.,
4. Discussion
A number of bacilli have been reported to contain thc extrachromosomal DNA".~.~.IO.I~.~X
Stahly e t nilj have coniirnled the presence of multiple copicb of extrachrolnosomal DNA
in 8. t/~urb~,uicnsis.Ermakova e f uln have isolated three plasmids of n~olecularweight
of 5.9 x lo':, 10 x 10"nd
10.9 x 108 frorn B. fhuringiensis var. gulleriue strain 612,
The functional role of these plasmid DNAs has not been clearly established. It vra,
notizzd that when B. thtirirt,yiensis strain 612 was grown in a liquid or solid medium,
there was spore and crystal formation ; the existence of the plasmid DNA in those cell,
was also establisheds: when those cells were transferred to Spizizen salts medium (SN
medium) or basal salts medium (BM medium) supplemented with 0.2% w/v sodiun
oitrars. no extrachromosomal DNA was found and the crystal formation was also negligible" In the presenr study, disagpearance o r the extrachromosomal DNA was observed
depending on cys concentration. The presence of the cxtrachromosomal DNA war
rehted to the apore coat and the crystal formation (Table I). The spore coat fornuticjn
and the crystal protein synthesis were observed only when the extrachromosomal DNA
was present in an ' extrachromosomal state ' (Table I). When the sporulation inhibition was reversed by various means (unpublished data), the extrachro~nosomal DNA
was found to reappear. These results suggest that therc could bc a correlation between
the presence of the extrachron~osornalDNA and the Cormation of the spore coat and the
crystal protein. It could be possible that under certain physiological conditions, like
high concentration of cys or transferring of the culture to a non-sporulating medium as
performed by Ermakova ct u15, the extrachromoso~nalD N A gets integrated into tht
genomic DNA, so that it is not isolatable (Table I). Further, it is conceivable that tb:
senes of the extrachromosomal DNA could be expressed only when it is in the extrachromosomal state, and not in an integrated state. Thesc results show that there is s
clear relationship between the presence of the extrachromosomal DNA and the produo
tion of the crystal and the spore coat in B. thuringicnsis var. tl~uringiensis(Table I).
Probably, the crystal and some of the major spore coat protein are synthesized from
similar species of mRNA coded for by the extrachromosomal DNA of B. thuringiemis
wr. thlaingimsis.
T h e calculated molecular weight of the extrachromosomal DNA based on the stwk
on electron microscopy was found to be less than that obtained by agarose gel eledrophoresis. Such an altered molecular weight was obtained for I-DNA also (Tabb U!J!.
Multiple copies of extrachrolnosomal DNA'have been isblated from B. th~ringiensir
by Stably ct alls, The results obtained suggest that only one copy of extrachromosod
FIG 4. Elec~ron miciagraph of rhe exrrachi-ornosomal DNA. Rar
I-enl-rrmt;
1
.,
FIG. 5 The dmer n:olecule. Bar represents Igm.
FIG.6. The '8-sllaped' molecule. Bar represents 1jim.
FluorOgraphic partern of t h e exrracllro~nonomaiDNA. Btt-B. riirrviigitnsi~var. f h u r i ~ i e n s i s;
DNA. The top spot corresponds to the contarninaxion from the I~osrDNAs. The bortom spot
the bioken aieces of rhe DNA which move along with the dye band, i.e., bromophe~lolblue The
niddie spot corresponds t o tho estracluomoiomal DNA of B. thevi~~giensis
var. fhtirin&'ieiisis and rhe
-DNA.
'IG.?.
-
3
IGS. 7
and 8. The ' 8-shaped ' molecule. Onc of (he circles of ' 8-sl~uped' slructurc is broken,
robably during isolation. Bar represents 1 urn.
)NA was isolatcd in B. thuringie~zsisv a . t i ~ u i i n g i c t ~ s i s .This could be due to the fat
1at the culture was inaiutained routinely on nutrient agar and extellsivcly subcultured.
luring such process the other copies of the extrachromosomal DNAs, which would bay?
Ieen present earlier, could have lost, leaving the molecules of molecular weight 10 x 108,
'he presence of the dimer molecules and the ' 8-slmped ' rnoleculcs suggest the presence
,f more than one copy of the extra.chromoson1al DNA per bacillus, since the presence
)f the ' 8-shaped ' molecules has been recognised as intermediate lnolecules durlng
enetic recombination3J2.
Acknowledgement
'he author thanks Drs. Y. I. Shethna, M. S . Shaila, J . D. Padayatty, G. N. Subhama,
S. Panchapagesan and Mr. Mohan L. Gope for their excellent cooperation in this
tudy.
'.
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~
~
i