ISSN 1063-0740, Russian Journal of Marine Biology, 2016, Vol. 42, No. 3, pp. 252–257. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © O.A. Shokur, T.Yu. Magarlamov, D.I. Melnikova, E.A. Gorobets, I.A. Beleneva, 2016, published in Biologiya Morya.
Life Cycle of Tetrodotoxin-Producing Bacillus sp. on Solid and Liquid
Medium: Light and Electron Microscopy Studies
O. A. Shokur
, T. Yu. Magarlamov
, D. I. Melnikova
, E. A. Gorobets
, and I. A. Beleneva
Zhirmunsky Institute of Marine Biology, Far East Branch, Russian Academy of Sciences,
ul. Pal’chevskogo 17, Vladivostok, 690041 Russia
School of Biomedicine, Far Eastern Federal University, ul. Oktyabrskaya 29, Vladivostok, 690950 Russia
Received November 25, 2015
Abstract—The lifecycle of the Bacillus sp. 1839 cultivated during a long period on solid and liquid Youschi-
mizu-Kimura medium was investigated, and then bacteria and spores were studied by light and transmission
electron microscopy. Sporulation in this strain is distinguished by engulfment of forespore by mother cell. In
the liquid medium, bacteria have the decondensed nucleoid and the loose granular component of cytoplasm;
bacteria and spores are generally smaller; the outer coat of spores includes 2 concentric rings. On the solid
substratum, the nucleoid is condensed, and the cytoplasmic region is extensive and dense; a longer cultivation
stimulates transition of vegetative cells into the spore form; spores have a thicker outer coat with 3–5 rings.
On the solid substratum, sporulation in Bacillus sp. 1839 is spontaneous, without additional stimulation;
spores have a larger diameter and thicker layers than those in the liquid medium. This research contributes to
the current understanding of biotechnological tetrodotoxin production from a bacterial raw material.
Keywords: tetrodotoxin, Bacillus, forespore, tetrodotoxin-producing bacteria, transmission electron micros-
The methods of tetrodotoxin production attract
growing attention, as this substance is considered a
promising anesthetic and antiarrythmic agent for
medical practice . A biotechnological production
based on tetrodotoxin-producing bacteria is one of the
most attractive methods for the toxin recovery. How-
ever, due to the lack of a culture that can retain its pro-
ductive properties for sufficient time, it has not been
possible to use the tetrodotoxin-producing strains for
biotechnological production for a long period .
The tetrodotoxin-producing strain Bacillus sp.
1839, genetically the closest to Bacillus asahii [2, 8],
was isolated from marine worms (Nemertea). The
strain can produce the toxin during a long period at
multiple culture passages. Tetrodotoxin is accumu-
lated mostly in endospores and free spores, i.e. its
amount directly depends on the bacterial biomass .
By now, the lifecycle of bacteria of the genus Bacil-
lus has been studied mainly for soil-dwelling species
such as Bacillus subtilis [4, 12]. Some works consider
sporulation in Bacillus сereus (, Lysinibacillus
sphaericus , and Bacillus anthracis  at the level of
No ultramicroscopic investigations into the lifecy-
cle of bacilli associated with marine organisms have
been conducted previously. Thus, the goal of this work
is to study the lifecycle of the halophilic, aerobic tetro-
dotoxin-producing bacterium Bacillus sp. 1839.
Understanding the lifecycle and sporulation in this
strain is necessary to obtain the maximum amount of
highly toxic spores. Moreover, this knowledge can
help to develop biotechnological production of anes-
thetic agent, based on a culture of spore-forming
MATERIALS AND METHODS
The sequenced bacterial strain Bacillus sp. 1839
(KF444411-KF444416) was taken from the Collec-
tion of Marine Heterotrophic Bacteria, Zhirmunsky
Institute of Marine Biology, Far Eastern Branch, Rus-
sian Academy of Sciences. To cultivate it, the solid and
liquid Youschimizu–Kimura (Y–K) media  were
used. For the study of sporulation dynamics under
normal conditions, bacteria were cultivated during a
long period, 6 days; the material was collected at the
following time periods: 12 and 24 h later, and then on
the 3rd and 6th days after the beginning of cultivation.
The cell morphology was studied on Gram-stained
The text was submitted by the authors in English.