JOURNAL OF BACTERIOLOGY, Jan. 1983, p. 436-442
Copyright C 1983, American Society for Microbiology
Vol. 153, No. 1
Dielectric Characterization of Forespores Isolated from
Bacillus megaterium ATCC 19213
R. E. MARQUIS,'* G. R. BENDER,1 E. L. CARSTENSEN,2 AND S. Z. CHILD2
Departments ofMicrobiology1 and Electrical Engineering,2 The University ofRochester, Rochester, New
Received 21 June 1982/Accepted 12 October 1982
Isolated stage III forespores of Bacillus megaterium ATCC 19213 in aqueous
suspensions were nearly as dehydrated as mature spores, as indicated by low
dextran-impermeable volumes of ca. 3.0 ml per g (dry weight) of cells compared
with values of ca. 2.6 for mature spores and 7.3 for vegetative cells. The
forespores lacked dipicolinate, had only minimal levels of calcium, magnesium,
manganese, potassium, and sodium, and were more heat sensitive than vegetative
cells. The effective homogeneous conductivities and dielectric constants mea-
sured over a frequency range of 1 to 200 MHz indicated that the inherent
conductivities of the forespores were unusually low, in keeping with their low
mineral contents, but that the forespores could be invaded by environmental ions
which could penetrate dielectrically effective membranes. Overall, our findings
support the view that the dehydration of a forespore during stage III of
sporogenesis may be the result of ion movements out of the forespore into the
Bacterial endospores are extremely dormant
and extremely resistant to destructive influences
such as heat and radiation. They also have an
unusual degree of electrostasis (3, 4); for exam-
ple, spores of Bacillus cereus appear to be
almost nonconducting for an electrical current of
50 MHz, although they contain large amounts of
potentially mobile, ionizable mineral species.
The conductivity of these spores is well below
that of vegetative cells, although the spores are
much more extensively mineralized. In essence,
it seems that the mineral ions within spores are
somehow immobilized. In B. cereus spores, this
immobilization involves mineral ions in the en-
veloping structures as well as those in the core.
In Bacillus megaterium spores, the mineral ions
in the enveloping structures are not fully immo-
bilized, but those in the core are (3).
The physicochemical bases for immobilization
of mineral ions in the core of B. megaterium
spores, or in the core and enveloping structures
ofB. cereus spores, are not known. Immobiliza-
tion does not appear to be peculiar to the calci-
fied state (8). With acid-resistant spores of B.
megaterium ATCC 19213, it is possible to bring
about complete ion exchange involving minerals
both in the core and in the enveloping structures
by means of acid soaking at pH 2 followed by
titration with appropriate base solutions. In this
way, various salt forms of the spores can be
prepared, and, amazingly, these forms all have
high levels ofviability ifthe exchange procedure
is carried out carefully. The various forms all
show immobilization of ions, even when miner-
alized with sodium or potassium.
The various salt forms of the spores all are
dehydrated to about the same extent, as indicat-
ed by low values for dextran-impermeable vol-
ume per gram (dry weight) of spores (8). It
seems that immobilization may be more related
to dehydration than to calcification. Dehydra-
tion could result in precipitation and resultant
ion immobilization, or it could lower ion mobil-
ity by increasing cytoplasmic viscosity.
In an effort to gain some appreciation for the
timing ofwater loss and development ofextreme
electrostasis during sporogenesis, we isolated
intact stage III forespores from B. megaterium
ATCC 19213 and related their dielectric proper-
ties to their mineral and water contents.
MATERIALS AND METHODS
Preparation ofspores. Large crops ofclean, dormant
spores of B. megaterium ATCC 19213 were obtained
as described previously (23). The organism was grown
in the defined medium of Slepecky and Foster (11) at
30°C with agitation and aeration. After sporulation and
autolysis of sporangia, the spores were separated from
debris by differential centrifugation and washed re-
peatedly with deionized water.
Forespores were isolated by the procedures of Ellar
and Posgate (5) with modifications. Early stationary-
phase cultures were centrifuged, and the cells were
suspended in a 1 mM CaCl2 solution. After phase-dark
forespores had formed within the cells, they were
MARQUIS ET AL.
that isolated forespores of B. megaterium KM
are osmotically sensitive and swell and shrink in
response to changes in medium osmolality.
However, the forespore osmotic behavior dif-
fered in significant ways from that ofprotoplasts
prepared from vegetative cells ofthe same orga-
nism. The forespores became maximally con-
tracted in solutions of sucrose or NaCl with
osmolarity of only about 0.35, and further in-
creases in osmolarity resulted in little addition
contraction. Vegetative cell protoplasts were
not maximally contracted even in media with
osmolarity as high as 0.60. Moreover, the slope
of the line relating volume or extinction to
osmolarity was greater for vegetative cell proto-
plasts than for forespores. These findings are
certainly indicative oflower numbers of osmoti-
cally active molecules in forespores than in
protoplasts of vegetative cells. Certainly, if the
sporangial cytoplasm was hypertonic in relation
to the forespore cytoplasm, then water would be
displaced into the sporangium to leave a rela-
tively dehydrated forespore. The major basis for
the low osmolarity and low potassium content of
forespores could be the working of the inverted
outer forespore membrane which possibly could
move potassium from the forespore to the spo-
rangium or, at least, interfere with potassium
uptake by the inner forespore membrane.
This work was supported by award DAAG2940-C-0051
from the U.S. Army Research Office, with Philipp Gerhardt as
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