CHARACTERIZATION OF FACTORS CONTROLLING THE FORMATION
OF AKINETES IN THE CYANOBACTERIUM,
CYLINDROSPERMUM LICHENIFOBME KfiTZ.

BY

Takayasu Hirosawa

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology
1978

ABSTRACT

CHARACTERIZATION OF FACTORS CONTROLLING THE FORMATION
OF AKINETES IN THE CYANOBACTERIUM,
CYLINDROSPERMUM LICHENIFORME KfiTZ.

BY
Takayasu Hirosawa

Substances which stimulate the formation of akinetes
(spores) in Cylindrospermum Zicheniforme Kfitz. are
secreted into a phosphate-free sporulation medium by
filaments of that cyanobacterium. One such substance,
which is able to initiate sporulation in a phosphate-
containing culture medium, was purified from the
centrifugal supernatant fluid of sporulating cultures.
High resolution mass spectrometry showed that the

chemical formula of the substance is C7HSOSN. Other

peaks of high intensity found at m/e 123 and m/e = 96
in the mass spectrum were produced by loss of CO from

the molecular ion, and by additional loss of HCN. Proton
nuclear magnetic resonance spectroscopy of the

substance showed a complex of peaks in the region of

6 = 7.19 to 6 = 7.29 ppm. Peaks in the infrared absorp—

tion spectrumwere attributable to methylene C—H bonds and

O H Takayasu Hirosawa

III
to C=S and cyclic -C-N-groups. The most probable

structure consistent with the above findings embodies two
fused, five-membered rings, one of which is a lactam and
the other of which has a thioketone group.

The addition of 12.5% H2 stimulated sporulation in
:0

air or under CO :Ar (O.1:l9.9:90, v/v) by up to

2 2
2.5-fold. The same concentration of hydrogen did not
significantly affect the reduction of acetylene by

intact filaments. These data indicate that the
stimulation of sporulation by hydrogen was not mediated
by an effect upon the fixation of nitrogen. Hydrogen
uptake in a cell-free suspension derived from whole
filaments was detected manometrically with phenazine
methosulfate as electron acceptor,at a rate of 5.8 umoles

-1 -1

H (mg chlorophyll a) h . The uptake hydrogenase

2
activity derived from isolated heterocysts accounted
for 84 i 2% of the uptake hydrogenase activity from
whole filaments, on a per heterocyst basis, whereas no
activity was detected in a fraction derived from the
vegetative cells.

The formation of the pattern consisting of spores
contiguous with heterocysts may be controlled by
either, or a combination of, (i) a sporulation-
stimulatory substance, if that substance is synthesized

solely in heterocysts, or (ii) some substance which is

reduced by the uptake hydrogenase in heterocysts.

ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to
his major professor, Dr. C. Peter Wolk, for his patience
and guidance throughout these investigations. The author
also wishes to extend his gratitude to Dr. Philip Filner,
Dr. Michael Jost, Dr. Kenneth Poff, and Dr. Harold L.
Sadoff for their participation in guidance committee.

The assistance of Mr. Bernd Soltmann with the mass
spectrometer and of Dr. Frank J. Bennis with the NMR
spectrometer is warmly acknowledged.

The author wishes to thank all of the members of the
PRL community, whom I cannot name individually, for
friendship and assistance; Dr. Thomas Currier for his
correction of the English of this thesis; and Dr. Richard
B. Peterson for his assistance during assays of hydro-
genase.

The continuous encouragement by Dr. Masayuki Takeuchi
and Dr. Hisashi Matsushima is gratefully appreciated.

Finally the author wishes to thank his wife Reiko for
her continuous moral support throughout these years.

This work was supported by the 0.8. Department of

Energy under Contract EY—76-C-02—1338.

ii

TABLE OF CONTENTS

Page
LIST OF FIGURES . . . . . . . . . . . . . . . . . . v

LIST OF TABLES O O O O O O O O O I O O O O O O O O O Vij-

ABBREVIATIONS . . . . . . . . . . . . . . . . . . Xi
INTRODUCTION. . . . . . . . . . . . . . . . . . . . 1
Structure and Chemical Composition of Akinetes . 2
Metabolic Activities of Akinetes . . . . . . . . 5
Factors Controlling the Formation of Akinetes
in Cyanobacteria . . . . . . . . . . . . . . 5
Nutritional factors. . . . . . . . . . . . . 5
Genetic factors. . . . . . . . . . . . . . . 7
Extracellular products . . . . . . . . . . . 7
Heterocysts. . . . . . . . . . . . . . . . 9
MATERIALS AND METHODS . . . . . . . . . . . . . . . 15
Culture Conditions . . . . . . . . . . . . . . 15
Bioassay of Substances Inducing the Formation
of Akinetes. . . . . . . . . . . . . . . . . 16
Chemical Fractionation . . . . . . . . . . . . . 17
a) Gel filtration. . . . . . . . . . . . . . 17
b) Charcoal-celite column fractionation. . . 18
c) Paper chromatography. . . . . . . . . . . 18
d) Silica gel thin layer chromatography. . . 18
e) Cellulose thin layer chromatography . . . 19
f) Microdistillation . . . . . . . . . . . . 19
g) Mass spectroscopy . . . . . . . . . . . 19
h) Nuclear magnetic resonance (NMR)
spectroscopy. . . . . . . . . . . . . . . 20

1) Infrared (IR) and ultraviolet spectroscopy 20

Assay for Hydrogenase. . . . . . . . . . . . . . 20
Isolation of Heterocysts . . . . . . . . . . . . 21
Chemicals. 0 O O O C O O O O O O O O O O O O O O 22
RESULTS
Part I. Isolation and Characterization of a
Substance which Stimulates the Formation of
Akinetes in C. licheniforme. . . . . . . . . 23

iii

Page
Production of factors stimulating the

formation of akinetes. . . . . . . . . . . 23
Gel filtration . . . . . . . . . . . . 28
Extraction with methanol . . . . . . . . . . 28
Stability. . . . . . . . . . . . . . . . . 31
Charcoal adsorption chromatography . . . . . 33
Paper chromatography . . . . . . . 37

Chromatography on thin layers of silica gel. 39
Chromatography on thin layers of cellulose . 43

Determination of dry weight. . . . . . . . . 47
Isolation, from cells, of material

stimulating the formation of akinetes. . . 53
Mass spectral analysis . . . . . . . . . . . 53
A further step of purification . . . . . . . 61
NMR spectroscopy . . . . . . . . . . . . . 61
IR spectroscopy. . . . . . . . . . . . . . 65
UV absorption spectroscopy . . . . . . . . . 65

Part II. Effects of Various Environmental
Factors upon the Formation of Akinetes in
C. licheniforme. . . . . . . . . . . . . . . 70

Effect of hydrogen upon the formation of
akinetes . . . . . . . . . . . . . . . . . 7O
Uptake hydrogenase and its localization. . . 70
The effects, upon the formation of
akinetes, of various substances of low
molecular weight . . . . . . . . . . . . . 73
Cytochemistry with a redox dye . . . . . . . 77

DISCUSSION. . . . . . . . . . . . . . . . . . . . . 84
The Substance Inducing the Formation of

Akinetes O O O O O O C O O O O O I O O O O O 84

Other Factors which Control the Formation
Of AkiflEteS. o o o o o o o o o o o O o O Q 0 89
REFERENCES 0 O O O O O O O O O O O O O O O O O O O O 96

iv

Figure

10

11

12

LIST OF FIGURES
Page

Growth of Cylindrospermum Zioheniforme
and formation of akinetes, in SSM. . . . . . . 24

Time course of production of material

stimulating the formation of akinetes, as
measured by bioassay of its stimulatory

activity . . . . . . . . . . . . . . . . . . . 26

Gel filtration, on a column of Sephadex G-25,
of 20 ml of a culture filtrate concentrated
25-f01do o o o I o o o c o o o o o o o o o o o 29

UV absorption spectra between 260 and 300 nm,
of charcoal-treated culture filtrates. . . . . 35

Paper chromatography of the combined

ethanolic eluates from a charcoal-celite

column developed with a solvent system of
isopropanol:58% NH4OH:water (9:1:1, v/v) . . . 40

Silica gel thin layer chromatogram of the
active extract from a previous paper
chromatogram . . . . . . . . . . . . . . . . . 44

Chromatogram, on a thin layer of cellulose,
of the active extract from a silica gel
thin layer chromatogram. . . . . . . . . . . . 48

Purification procedure for the sporulation—
stimulatory substance. . . . . . . . . . . . . 51

Mass spectral fragmentogram of (a) the active
extract from a cellulosic thin layer

chromatogram and (b) an inactive extract of
material with slightly lower Rf. . . . . . . . 55

Mass spectrum of the material evaporating
into the electron beam at 150 C. . . . . . . . 58

Proton magnetic resonance spectrum of the
vacuum-distilled sporulation-stimulatory
substance 0 O O O O O O O O O O O O O O O O O O 6 3

Infrared spectra of (a) the vacuum-distilled
sporulation-stimulatory substance in a KBr
pellet and (b) a KBr pellet without additional
material . . . . . . . . . . . . . . . . . . . 66

Figure
13

14

15

Page

UV absorption spectrum of the sporulation—
stimulatory substance dissolved in 0.5 m1
of acetonitrile. . . . . . . . . . . . . . 68

Reduction of nitro blue tetrazolium chloride
by (a) intact, (b) bent, and (c) cut
filaments. . . . . . . . . . . . . . . . . 82

Approximate structure of the substance which
stimulates the formation of akinetes in
C. Zieheniforme. . . . . . . . . . . . . . 90

vi.

Table

II

III

IV

VI

VII

VIII

IX

XI

XII

LIST OF TABLES

Page
Biological activities of the fractions
derived by extraction, with methanol,
of a dried culture supernatant fluid. . . . . 32

Sporulation-stimulatory activities of a
supernatant fluid (methanol-soluble fraction)
following treatment of that fluid with

different amounts of activated charcoal . . . 34

Activities of the fractions from the column
of charcoal and celite. . . . . . . . . . . . 38

Bioassay of the substances extracted from a
paper chromatogram. . . . . . . . . . . . . . 42

Activities in extracts from sections of
a silica gel thin layer chromatogram. . . . . 46

Activities of the extracts from the sections
of a cellulosic thin layer chromatogram . . . 50

Dry weights and biological activities 0f
samples from different stages of
purification. O O O O O I O O O O O O O O O O 52

Biological activity of the methanolic
extract from cells. . . . . . . . . . . . . . 54

Molecular weights, measured with high

resolution, and possible chemical formulae

of the major ions in the mass spectrum of

the active material . . . . . . . . . . . . . 60

Biological activity, in SSM and AA/8, of the
vacuum—distillate of the sporulation-

stimulatory material purified by the entire
procedure of Figure 8 . . . . . . . . . . . . 62

Effect of hydrogen gas upon the formation of
akinetes O O O O O O O O I O O O O O I O O O O 71

Effect of hydrogen gas upon acetylene
reduction in air. . . . . . . . . . . . . . . 72

vii

Table
XIII

XIV
XV

XVI

XVII

XVIII

Uptake of hydrogen by a cell-free
suspension, measured manometrically, with
various electron acceptors at a

concentration of 10 mM.

Localization of hydrogenase .

Effects of amino acids upon the formation

of akinetes .

Effect of ammonium upon, added to SSM or
to sporulation-stimulatory supernatant

O

fluid, the formation of akinetes.

Effects of cyclic nucleotides upon the

formation of akinetes

Effects of Csz and C2H4 upon the formation

of akinetes .

Viii

Page
. . 74
. 75
. 76
. . 78
. . 79
. . 80

AA/8

c-AMP
Chl
DCPIP

Dibut-c-AMP

Dibut-c-GMP

PIPES
PMS
SSM

TES

ABBREVIATIONS

Medium of Allen and Arnon (1955), diluted
eight-fold

Adenosine 3':5'-cyclic monophosphoric acid
Chlorophyll a
2,6-Dichlorophenolindophenol

I
N6,O%-Dibutyryl adenosine 3':5'-cyclic

monophosphoric acid

I
N2,02-Dibutyry1 guanosine 3‘:5'-cyclic
monophosphoric acid

(Ethylenedinitrilo) tetraacetic acid,
disodium salt

N-2-Hydroxyethylpiperazine—NN-Z-ethane-
sulfonic acid

Mass to charge ratio

Nitro blue tetrazolium chloride, or 2,2‘—
diphospho-nitrophenyl—S,5'-dipheny1-3,3'—
(3,3‘-dimethoxy-4,4'-dipheny1ene)
ditetrazolium chloride

Piperazine-N,N'-bis(2-ethanesulfonic acid)
Phenazine methosulfate
Standard sporulation medium

N-Tris(hydroxymethyl)methyl—Z-aminoethane-
sulfonic acid '

Chemical shift relative to tetramethylsilane

;iX_

INTRODUCTION

In many multicellular organisms, patterns are formed
by temporally and spatially controlled differentiation of
certain cells. Intercellular communication can play an
important role in the control of differentiation. In
most of these organisms, there is a large and heterogen-
eous population of cells, interactions among which are
organized in three dimensions.

In certain filamentous cyanobacteria (blue-green
algae) there are three distinct types of cells: the
vegetative cell, the heterocyst, and the akinete (spore).
The latter two types of cells arise by differentiation
of vegetative cells. The three types of cells are
arrayed in one-dimensional patterns along the
filaments: the heterocysts are spaced apart, whereas
the akinetes form either next to the heterocysts, as in
Cylindrospermum Zieheniforme Kfitz., or remote from the

heterocysts.

Structure and Chemical Composition of Akinetes

 

Mature akinetes are spherical or cylindrical cells
surrounded by a thick envelope. In C. licheniforme, they
average 20 um in diameter and 40 um in length, i.e.,
their diameter is twice, and their length is three to four
times, as great as the corresponding dimensions of
vegetative cells.

Akinetes retain many of the cytological and chemical
features of vegetative cells. Like vegetative cells, the
akinetes may contain photosynthetic thylakoids. Fay
(1969b) reported that there is lower content of photo-
synthetic pigments, per dry weight, in akinetes than in
either heterocysts or vegetative cells, in Fogg's strain
of Anabaena cylindrica Lemm. He also reported that
chlorophyll is largely replaced by pheophytin and that
the carotenoid composition is different from that of
vegetative cells. However, because the akinete has a
thick and dense envelope, it is unclear whether the
content of photosynthetic pigments differs from the
corresponding values for the other two types of cells
per dry weight of protoplasm. Also, no: controls were
presented to test whether the degradation of chlorophyll
to pheophytin and the difference in carotenoids were
artifacts of the extraction procedure. Wolk and Simon

(1969), working with a different strain of A. cylindrica,

found that the in vivo absorption spectra of isolated
akinetes and of vegetative cells were virtually super-
imposable. On the other hand, a complete breakdown of
photosynthetic pigments is a common feature of the
differentiation of akinetes in other cyanobacteria
(Geitler, 1932). Whether, in the latter cyanobacteria,
there is also a disappearance of the thylakoids is
unknown. It has also been reported that the akinetes of
Fogg's strain of A. cylindrica have a lower content of
lipids and fatty acids than have vegetative cells, but
that their lipids are more saturated than those of
vegetative cells (Yamamoto, 1972). These lipids are
presumably present principally in the thylakoids,
although lipid granules have been reported to be
present in the cytoplasm of the akinetes of other species
(Miller and Lang, 1968; Clark and Jensen, 1969).

The only known qualitative difference between the
structures of akinetes and of vegetative cells is the
occurrence of a thick enve10pe surrounding the cell wall
in the akinete and, seemingly of lesser significance,
the apparent absence of deposits of polyphosphate in the
akinete (Talpasayi, 1963; Wildon and Mercer, 1963;

Leak and Wilson, 1965; Miller and Lang, 1968; Jensen and
Clark, 1969). The chemical composition of a wall—plus-

envelope fraction from akinetes of A. cylindrica was

shown to be 41% carbohydrate, 24% amino compounds, 11%
lipid, 2% ash and the balance unaccounted for. The
sugar composition of the carbohydrate moiety is 70%
glucose, 17% mannose, 4% xylose and 3% galactose (Dunn
and Wolk, 1970). Cardemil and Wolk (1976) reported that
the backbone polysaccharides from both akinetes and
heterocysts consist of repetitions of the same structural
unit, B(l+3)-linked glucosyl-glucosyl-glucosyl-mannose.
The xylose and galactose, as well as part of the

mannose and part of the glucose, are present in side
branches.

Cyanophycin granules, consisting of copolymers of
aspartic acid and arginine, are present in akinetes, as
they are in vegetative cells, but are larger and more
numerous in the akinetes. Unique to the cyanobacteria,
these granules probably function as a nitrogenous reserve
(Simon, 1971). The relative amount of DNA per cell in
akinetes, compared with the vegetative cells, is either
variable or controversial. On the basis of staining
with the fluorescent dye coriphosphin, Ueda (1971) and
Ueda and Sawada (1971, 1972) estimated that akinetes
of Cylindrospermum sp. contain 30-fold more DNA than do
vegetative cells. Simon (1977a), however, found no
great difference between direct measurements of

the amount of DNA per cell in isolated akinetes and in

intact filaments (consisting mostly of vegetative cells)
of Anabaena cylindrica. Akinetes, like vegetative

cells, appear to contain glycogen (Zastrow, 1953).

Metabolic Activities of Akinetes

 

Akinetes are considered to be resting cells. They
have been shown to be capable of germination even after
five years of storage in the dark in a desiccated
condition (Yamamoto, 1975). Fay (1969a) reported that
isolated akinetes from A. cylindrica fix carbon dioxide
in the light at a lower rate and evolve carbon dioxide
in the dark at a higher rate than do intact filaments,
per mg dry weight. Nitrogenase activity was not
detected in the isolated akinetes. Whether the measured
activities had been affected by the isolation procedures
employed - i.e., whether these might have been the
activities of germinating or immature akinetes -— was

not determined.

Factors Controlling the Formation of Akinetes in
Cyanobacteria

 

Nutritional factors

 

Although the effects of various environmental
conditions on the formation of akinetes differ from
species to species, one of the most important factors

appears to be the concentration of phosphate in the

medium. Generally, the formation of akinetes is promoted
by the absence of phosphate (Glade, 1914; Wolk, 1964,
1965; Gentile and Maloney, 1969). Glade (1914) reported

that a medium consisting of 0.05-0.1% Ca(NO 0.02%

3’2'
MgSO4, 0.02% KZHPO4, and trace of iron was best for
formation of akinetes in Cylindrospermum, after growth.
He suggested that the formation of akinetes was initiated
by the depletion of the components of the medium. Sucrose
and calcium nitrate inhibited the formation of akinetes
in Nostoc punctiforme (Harder, 1917). Supraoptimal
concentrations of nitrate and sulfate promoted the
formation of akinetes in several species of Anabaena
(Canabaeus, 1929). The formation of akinetes by

A. cylindrica was Optimal under the following conditions:
concentration of phosphate less than 50 uM, presence of

a buffer (ca. 5.7 mM DL-alanyl-DL-alanine or DL-alanyl-
glycine), a high concentration of acetate (25 mM), a

high concentration of calcium ions (2.5 mM), and a large
amount of inoculum; light intensity of 80 fc; and a
temperature of 25-30 C (Wolk, 1965).

In A. doZioZum, however, the supply of nitrogen may
be more important, because nitrate, nitrite and ammonium
nitrogen inhibit the formation of akinetes (Singh and
Srivastava, 1968; Tyagi, 1974). Glucose (25 mM) promotes

the formation of akinetes in A. doZioZum (Tyagi, 1974).

Genetic factors

 

Singh and Sinha (1965), working with Cylindrospermum,
have reported that genetic recombination between a
streptomycin-resistant mutant unable to form akinetes and
a penicillin-resistant mutant was able to form akinetes
resulted in a strain resistant to both antibiotics that
was, in addition, able to form akinetes at low frequency.
Singh (1967) reported that two distinct mutants of
A. doliolum which were incapable of forming akinetes
could recombine to form a strain which could form
akinetes capable of germination. The author concluded
that the formation of akinetes in that organism is under

the control of more than one genetic determinant.

Extracellular products

 

Cyanobacteria secrete biologically active substances
into culture media. Harder (1917) first described
auto-inhibition of growth in an old culture of Nostoc
punctiforme. Because addition of new salts and sugar
did not reverse the inhibition, he suggested that the
inhibition is due to a growth-inhibitory metabolite
accumulated in the medium. Certain of the secreted
substances inhibit the growth of other organisms
(Prescott, 1960; Hartman, 1960). Jakob (1961) reported
that it is a dihydroxyanthroquinone secreted by Nostoc

muscorum which is responsible for inhibition of the

growth of Cosmarium, Phormidium and EugZena. Toxins
from Aphanizomenon ons-aquae show chemical characteris-
tics similar to those of the toxin from the marine
dinoflagellate Gonyaulax cateneZZa (Jackim and Gentile,
1968).

On the other hand, some of the secreted materials
exert growth-stimulatory effects. Polypeptides
secreted by A. cylindrica complex with metal ions (Fogg,
1952; Walsby, 1974a), reducing the toxicity of - in
particular -— copper. A substance with a high affinity
for iron has been isolated from a culture filtrate of
Anabaena sp. grown in iron-containing medium (Simpson and
Neilands, 1976). In addition, the non-dialyzable
extracellular products of A. cylindrica decrease the
toxic effect of polymixin B against A. cylindrica and
Anacystis nidulans. The same material showed no effect
upon the formation of akinetes by the Anabaena or upon
the uptake of phosphate by either of the organisms
(Whitton, 1965, 1967).

Fisher and Wolk (1976) reported that the culture
filtrate from an akinete—forming culture of
Cylindrospermum Zieheniforme in phosphate-free standard
sporulation medium (SSM) stimulates the formation of
akinetes in a fresh inoculum of the cynobacterium,
without affecting the growth of the organism.

Supplementation of the culture filtrate with all of the

constituents of SSM did not affect the stimulation by

the filtrate. They therefore concluded that the
stimulation of sporulation by the culture filtrate was
due not to depletion of the medium, but to a substance or

substances released into the medium.

Heterocysts

 

The participation of heterocysts in the formation of
akinetes has long been suggested for those instances in
which there is a close spatial relationship between the
two types of cells. Carter (1856), having recognized
the relationship, first suggested that heterocysts were
supplying substances to neighboring, developing akinetes.
Other researchers have subsequently made similar
suggestions (Fritsch, 1904, 1951; Bharadwaja, 1933; Wolk,
1965). Wolk (1966) provided the first experimental
evidence in support of the idea that heterocysts play a
role in the sporulation of contiguous vegetative cells.
Having separated heterocysts from neighboring vegetative
cells by gentle agitation, he observed that the
vegetative cells detached from heterocysts failed to
differentiate into akinetes. Although the detached
vegetative cells appeared undamaged, it could not be
excluded that the detachment procedure had -— in fact -—
damaged the vegetative cells, and had thereby prevented

sporulation.

10

Knowledge of the metabolic activities of hetero-
cysts has led to elucidation of certain interactions
between heterocysts and vegetative cells. Fay et al.
(1968) proposed that heterocysts are sites of nitrogen
fixation. Use of 15N had provided no evidence in favor
of nitrogen fixation by isolated heterocysts (Fay and
Walsby, 1966), but subsequent work (Fay et aZ., 1968)
showed that nitrogenase activity had been lost completely
during the initial stages of the isolation procedure
employed. Using the more sensitive acetylene reduction
technique, Stewart et al. (1969) demonstrated, nitrogenase
activity in heterocysts in the presence of ATP and sodium
dithionite. Wolk and Wojciuch (1971) interpreted
certain kinetic experiments as implying the occurrence
of light-dependent nitrogenase activity in heterocysts.
In recent reports, .10 to 40% of the nitrogenase
activity of intact filaments of Anabaena 7120 and
A. cylindrica, and 60% of the nitrogenase activity of
intact Anabaena variabilis, could be recovered in
isolated heterocysts (Peterson and Burris, 1976; Thomas
et aZ., 1977; Peterson and Wolk, 1978b). Moreover, an
average of 91% of the MoFe-protein and 70% of the more
oxygen-labile Fe-protein of nitrogenase of the intact
filaments could be recovered in the heterocysts isolated

from A. variabilis (Peterson and Wolk, 1978b). Thus,

11

although it appears clear that nitrogenase can be present
in vegetative cells under anaerobic cn: microaerobic
conditions (Stewart and Lex, 1970; Rippka et aZ., 1971;
Rippka and Stanier, 1978), heterocysts are the major, and
very possibly the sole, sites of nitrogen fixation by
heterocyst-forming species under aerobic conditions. The
initial pathways of assimilation of fixed nitrogen by
nitrogen-fixing cyanobacteria have recently been eluci-
dated (Wolk et aZ., 1976; Meeks et aZ., 1977, 1978).
Fixed nitrogen is assimilated by glutamine synthetase
into the amide group of glutamine; the amide group is
then transferred to a-ketoglutarate by glutamate synthase,
to form the a-amino group of glutamate. Glutamine
synthetase is present in both heterocysts and vegetative
cells, although at slightly higher specific activity

in heterocysts (Dharmawardene et aZ., 1973; Thomas et aZ.,
1977) . However, the kinetics of solubilization of
glutamate synthase indicate that little, if any, of that
enzyme is located in heterocysts (Thomas et aZ., 1977).
These results suggest that nitrogen fixed in heterocysts
leaves the heterocysts (Wolk et aZ., 1974) as glutamine,
and that some of the glutamate formed by glutamate

synthase is transported back to the heterocysts.

12

Heterocysts lack two major photosynthetic functions.
Fay (1969b) and Wolk and Simon (1969) have shown that
heterocysts of A. cylindrica contain very little or no
phycocyanin, a pigment characteristic of photosystem II.
Microspectrophotometric analysis of individual
vegetative cells and heterocysts confirmed this result
for heterocysts in situ in Anabaena sp. (Thomas, 1970).
A relatively high ratio of P700 to chlorophyll, low
fluorescence by chlorophylch a low intensity of
emission of delayed light, and lack of a Hill reaction
(Donze et aZ., 1972), further suggested that heterocysts
lack photosystem II. The absence of photosystem II from
heterocysts was further confirmed by the lack of 02
evolution by heterocysts isolated from A. cylindrica
(Bradley and Carr, 1971; Tel-Or and Stewart, 1977). The
suggestion by Fay and Walsby (1966) that there is a
deficiency of COz-fixing activity in heterocysts was
confirmed by autoradiography (Wolk, 1968) and by the
demonstration (Winkenbach and Wolk, 1973; Stewart and
Codd, 1975) that ribulose-l,5-bisphosphate carboxylase
is absent from heterocysts. Because of these deficien-
cies, heterocysts have to depend on vegetative cells for
the electron donors and carbon skeletons necessary for
nitrogen fixation. In fact, Wolk (1968) showed by
autoradiography that part of the carbon fixed by

photosynthesis in vegetative cells moves into heterocysts.

13

Jfittner and Carr (1976) concluded from pulse-labeling
experiments with H14C03_ that carbon enters the
heterocysts of A. cylindrica principally as maltose,
whereas work of this laboratory (unpublished observations
of Schilling and Wolk cited by Wolk, 1979) has been
interpreted as suggesting that sucrose and glutamate are
the major carbon compounds entering heterocysts of

A. variabilis.

High "reducing activity" is also characteristic of
heterocysts. Cytochemical studies showed that hetero-
cysts reduce 2,3,5-triphenyl tetrazolium chloride more
rapidly than do vegetative cells (Drews, 1955; Drawert
and Tischer, 1956; Talpasayi and Bahal, 1967; Stewart
et aZ., 1969; Fay and Kulasooriya, 1972). Reductant
can be generated in heterocysts by dehydrogenases:
heterocysts exhibit at least seven-fold higher specific
activity of glucose-6-phosphate dehydrogenase and of
6-phosphogluconate dehydrogenase, and two-fold higher
specific activity of hexokinase, than do vegetative
cells. Little or no activity of enzymes of the glyco-
lytic pathway was detected (Winkenbach and Wolk, 1973;
Lex and Carr, 1974). Thus, reducing power necessary
for nitrogen fixation in heterocysts can be produced by
oxidative metabolism of carbon compounds synthesized
in vegetative cells (Bothe, 1970; Smith, Noy and

Evans, 1971; Apte et aZ., 1978; Lockau et aZ., 1978).

14

Peterson and Wolk (1978a) showed that uptake hydrogenase
activity is localized solelyitheterocysts in aerobically
grown Anabaena 7120. By reassimilating hydrogen released
during nitrogen fixation,heterocysts may conserve reduc-

ing equivalents.

MATERIALS AND METHODS

Culture Conditions

 

Stock cultures of Cylindrospermum Zioheniforme Kfitz.
(ATCC 29412) were grown axenically in 50 m1 of an eight-
fold dilution (AA/8) of Allen and Arnon's medium (Allen
and Arnon, 1955) in 125-ml Erlenmeyer flasks. The
cultures were grown on a reciprocating shaker (107 RPM)

4 2s‘l) from cool

in continuous light (2.0 x 10 erg cm-
white fluorescent lamps (Sylvania, Winchester, KY) at

26 i 1 C, and were subcultured 1% (v/v) weekly in

order to prevent the formation of akinetes. Phosphate-
free standard sporulation medium (SSM) was prepared as
described previously (Wolk, 1965) except that N-tris(hy-
droxymethyl)-methy1-2-aminoethanesu1fonic acid (TES)

was used as buffer in place of DL-alanyl-glycine, and
the pH was adjusted to 7.5. For isolation of substances
for chemical analysis, stock cultures were inoculated
into five-gallon glass carboys containing 16 liters of
SSM, and were aerated with compressed air and exposed

2

to continuous light (8.8 x 104 erg cm- 5‘1) from cool

white fluorescent lamps

15

16

at 25 i 2 C. For studies of hydrogenase, the carboys

contained AA/8.

Bioassay of Substances Inducing the Formation of Akinetes

 

Bioassays were performed under the same conditions
of light, temperature and agitation as described for the
growth of stock cultures. An assay mixture consisted of
4 ml of the material being tested, dissolved in SSM, plus
1 ml of a suspension of filaments of C. Zieheniforme in
SSM, in a 50-ml Erlenmeyer flask. The pH of the test
solution was adjusted to 7.5, and the solution was
sterilized by filtration through a 0.22-um pore—size
membrane filter (Millipore Corp., Bedford, MA). The
cells to be resuspended in SSM for bioassay were
collected from one week old stock cultures by
centrifugation. The initial concentration of chlorophyll
a (Chl) in an assay mixture containing cyanobacteria was
0.11 ug Chl/ml. After three and one half to four days,
the numbers of akinetes attached to a sample of 200
heterocysts were counted. A cell was considered to be
a heterocyst if it was present at a terminal position on
a filament, had a thick envelope, and contained a polar
granule (Clark and Jensen, 1969). Cells were considered
to be akinetes only if they were at least twice as long

as a normal vegetative cell and were wider than

17

heterocysts (Simon, 1977b).

When bioassays were conducted under a variety of gas
phases, 20 ml of cell suspension in SSM (0.11 ug Chl/ml)
were placed in 250-ml filter flasks (effective volume,
280 m1) modified as follows. The side arm of a flask
was sealed with a rubber serum stopper. The mouth of
the flask was fitted with a rubber stopper penetrated by
glass tubing (1.5 mm i.d.) which reached the bottom of
the flask. The glass tubing was connected via silicon
rubber tubing to a 0.22-um pore-size Millipore filter.
The flask was then flushed with one gas or a mixture of
gases via the Millipore filter and tubing, with gas
efflux through a hypodermic needle inserted into the
serum stopper in the side arm, needle then removed and
the tubing clamped shut. Measured portions of other
gases, sterilized with a Millipore filter, were then
injected through the side arm with a gas—tight syringe.

Excess pressure was released with the injecting syringe.

Chemical Fractionation

 

a) Gel filtration. Gel filtration was performed

 

on a 2.5 cm i.d. x 40 cm long column of Sephadex G-25,
fine (Pharmacia Fine Chemicals, Piscataway, NJ). The
column was eluted with water at a flow rate of 0.5m1/min.
The void volume was 87 ml. Twenty-four 10-ml fractions

were collected.

18

b) Charcoal-celite column fractionation. Activated

 

charcoal (Darco G-60) was cleaned as follows. It was
boiled in 20% acetic acid for ten minutes, and then
washed extensively with (i) hot (95 C) double distilled
water, (ii) 50% glass-distilled ethanol containing 2%
NH4OH, and (iii) distilled water. The charcoal was then
dried at 120 C, and kept at 120 C until used. Celite 545
was cleaned in the same way except that it was boiled
with 6.1:HC1. rather than with 20% acetic acid.
Charcoal-celite column fractionation was performed
on a 0.9-cm long glass column packed with .a 1:2 (w/w)
mixture of charcoal and celite. The charcoal and celite
were mixed in an Erlenmeyer flask by vigorous shaking.
Samples were passed through the column at a flow rate
of 100 ml/hr. The adsorbed materials were eluted first
with 50% glass-distilled ethanol and then with 50%

ethanol containing 2% NH4OH.

c) Paper chromatography. Descending paper

 

chromatography was performed on Whatman No. 1 filter
paper (20 cm x 20 cm) in a solvent system of isopropanol:

58% NH4OH:water (9:1:1, v/v).

d) Silica gel thin layer chromatography. Silica
gel thin layer chromatography was performed on plates

(E. Merck, Darmstadt, W. Germany) pre—Coated with Silica

19

gel 60F-254 (which contains a fluorescent indicator) to
a thickness of 0.25 mm, in a solvent system consisting
of n-butanol:acetic acid:ether:water (9:6:3:l, v/v;

Piskornik and Bandurski, 1972).

e) Cellulose thin layer chromatography. Cellulose

 

thin layer chromatography was performed on plates pre-
coated with a layer of cellulose, 0.25 mm thick
(Analtech, Inc., Willmington, DE), in a solvent system

consisting of isopropanol:58% NH4OH:water (9:1:1, v/v).

f) Microdistillation. Microscale vacuum distil-

 

lation was performed according to R. Roper and T. S. Ma
(1957) with modifications. A methanolic solution of the
sample was introduced into the lower chamber of an L-
shaped glass tube. After evaporation of the solvent,
the end of the tube containing the sample was immersed
in mineral oil which had been placed in a well of a
heating block. The distillate was collected in a U-
shaped tube immersed in an ethanol-dry ice bath
connected to a vacuum pump. A vacuum of 30 to 40 pm of

Hg was maintained during the distillation.

9) Mass spectroscopy. Mass spectroscopic studies

 

were performed with a CHS-direct probe mass spectrometer
(Varian MAT, Bremen, W. Germany) with electron impact

ion source. This instrument was interfaced to a Digital

20

Equipment Company PDP-ll/40 computer. Exact mass
measurements were made by the peak matching technique
(Quisenberg et aZ., 1956), with perfluoroalkanes as

a reference.

h) Nuclear magnetic resonance (NMR) spectroscopy.

 

NMR spectra were taken with a Bruker Model WH-lBOS
Fourier-transform spectrometer (Bruker Spectrospin,
Wissenbourg, France). The samples were dissolved in

[2Hlacetonitrile.

i) Infrared (IR) and ultraviolet~Spectrosc0py. IR

 

spectra were taken with a Perkin-Elmer model 621 grating
infrared spectrophotometer (Perkin-Elmer Corp., Norwalk,
CT). A sample—containing solution in glass-distilled
acetone was mixed with KBr in a mortar by drop-wise
addition of the solution to the salt. The mixture was
then evacuated overnight in a desiccator. Discs were
made in an evacuable KBr die (Perkin-Elmer Corp.) pressed
with a Carver hydraulic press (F. S. Carver, Inc., Summit
NJ). Ultraviolet absorption spectra were taken with a DB
-G spectrophotometer (Beckman Instruments, Inc., Irvine,

CA).

Assay for Hydrogenase

Hydrogen uptake was measured manometrically at 30 C
using a Gilson differential respirometer (Gilson Medical
Electronics, Middleton, WI). One and eight tenths ml of

test sample, 0.2 m1 of 100 mM electron acceptor, and

21

0.1 ml of 5 N KOH were placed, respectively, in the

side arm, main chamber, and center well of a Warburg

flask filled with hydrogen.

Isolation of Heterocysts

 

Heterocysts were isolated by the method of Peterson
and Wolk (1978b). Cells harvested from five gallon
carboys of AA/8 by centrifugation at l7,300)<g (RC-2B
centrifuge equipped with an 88-34 rotor and a continuous
flow attachment: Dupont—Sorvall Inc., Norwalk, CT) were
resuspended in approximately 30 ml of medium. The
suspension, placed in a polyethylene centrifuge tube
fitted with a rubber serum stopper, was twice evacuated

to 50 pm of Hg and refilled to 1 atm with H All

2.
subsequent operations were performed under H2, except
that the buffers were thoroughly sparged with N2. Cells
were centrifuged (500 x g, 10 min), resuspended in HP
buffer containing 10 mM HEPES, 10 mM PIPES, 1 mM MgClzr
and 1 mM cysteine, centrifuged again as before, and then
resuspended in 40 m1 of the same buffer to which had been
added 10 mM NaZEDTA and 1.0 mg lysozyme/ml. The
suspension was shaken at 200 rpm in a reciprocating

water bath at 30 C for 30 minutes. The suspension was

then centrifuged at 500 x g for five minutes, and the

pellet resuspended in 30 ml of HP buffer. The

22

suspension was transferred to a stoppered 125-m1
Erlenmeyer flask and was cavitated in a sonic cleaning
bath at 12 to 13 C for 20 to 30 minutes until the
ratio of vegetative cells to heterocysts decreased to
0.02. Heterocysts were sedimented at 500 x g for five
minutes, and the supernatant fluid was collected as a
vegetative cell fraction. The heterocyst-containing
pellet was washed twice with HP buffer. The isolated
heterocysts and intact filaments were resuspended in
HP buffer and were totally disrupted with a Ribi press

(Dupont Sorvall Inc.) at a pressure of 20,000 psi.

Chemicals

 

Celite 545 was obtained from Fisher Scientific Co.
(Pittsburgh,PA); Darco G-60 activated charcoal from
Sargent-Welch (Skokie, IL); PMS from Calbiochem (Los
Angeles, CA); calcium glucuronate from Mann Research
Laboratories, Inc. (New York, NY); isopropanol, diethyl
ether, formic acid, and 58% NH4OH from Mallinkrodt
(St. Louis, MO); n-butanol from Aldrich Chemicals Co.
(Milwaukee, WI); and glass-distilled methanol from
Burdick and Jackson Lab., Inc.(Muskegon, MI). All gases
were obtained from Matheson Gas Products (East Rutherford,
NJ). All other chemicals were obtained from Sigma
Chemical Co. (St. Louis, MO). Solvents were always

redistilled before use.

RESULTS

PART I. Isolation and Characterization of a Substance
which Stimulates the Formation of Akinetes in

C. licheniforme

Production of factors stimulating the formation of

 

akinetes

As is illustrated in Fig. 1, C. Zicheniforme,
inoculated from a stock culture into SSM, grows actively
for 12 days. Akinetes are produced during the period
of from four to 12 days. After 12 days, more than 80% of
the heterocysts are accompanied by akinetes. After the
14th day, the filaments fragment and the heterocysts
become detached from the filaments. Portions of
supernatant fluid taken every other day from the above
culture were lyophilized and extracted with methanol,
and the methanolic extracts dried, redissolved in SSM,
and bioassayed. Figure 2 shows the percentage of
heterocysts with contiguous akinetes after four days of
bioassay. A stimulation of the formation of akinetes is
first demonstrable using the methanolic extract of the
supernatant from a two day old culture, and then

increases monotonically for ten days.

23

Figure 1.

24

Growth of Cylindrospermum Ziaheniforme

and formation of akinetes, in SSM. Growth
is expressed as ug Chl/ml. Chl content

(

 

) was determined by measurement of
the optical density at 665 nm of
methanolic extracts of C. Zicheniforme
(Mackinney, 1941). The formation of
akinetes ( ----- ) is expressed as the
percent of heterocysts with a contiguous
akinete. One hundred m1 of suspension
were sampled from a one-liter culture.

every other day.

25

80:26 30:92.8 0
5.3 3.3322»... Co 285a

 

m m

 

 

IOI2|4

6

 

Culture age (days)

Figure 1

Figure 2.

26

Time course of production of material
stimulating the formation of akinetes,

as measured by bioassay of its stimulatory
activity. Supernatant liquids from the
culture of Fig. 1 were lyOphilized and
extracted with methanol, and the
methanolic extracts dried and redissolved
in SSM. The activities are expressed as
the percent of heterocysts with
contiguous akinetes after four days of
incubation. The concentrations of solids
in the suspension used for bioassay of
the culture filtrate are shown,

relative to their concentration in the
original filtrate: x2, x1, x8. The
control value (3.5% heterocysts with a
contiguous akinete) obtained with 0 x

filtrate, i.e., with SSM, have been

subtracted from the data as presented.

27

 

 

 

I004

C)
I)

agaumo snonbuuoa 0
(mm sgsfloomaq 5o waned

Figure 2

Culiure age (days)

28

Gel filtration

 

After Sephadex G-25 gel filtration of the
supernatant fluid from a 12 day old culture in SSM,
most of the activity (capacity to stimulate the formation
of akinetes) was eluted from the column between 140 and
200 ml (Fig. 3), i.e., within the internal volume of
the gel. This suggests that the molecular weight of

the stimulatory factor is less than 5000 D.

Extract with methanol

 

Cell-free supernatant fluids from 12 day old 16-
liter batch cultures in SSM were obtained by centrifuga-
tion at 17,300 x g with a Sorvall SS-34 rotor equipped
with a continuous flow attachment. Because an oily
substance was present in the supernatant, the
supernatant could not be evaporated completely to a
powder. The supernatant dired in a Bfichi rotary
evaporator (Flawil, Switzerland) at 55 C, was extracted
three times with 800 ml of absolute methanol. The
insoluble residue was removed by filtration through
Whatman No. 1 filter paper and was reextracted with an
additional 500 to 600 ml of absolute methanol. The
residue was denoted the methanol—insoluble fraction.
The methanol-soluble material was concentrated approx-
imately five-fold in a rotary evaporator at 35 C.

During this condensation a large amount of white

Figure 3.

29

Gel filtration, on a column (2.5 cm i.d.
x 40 cm long) of Sephadex G-25, of 20 ml
of a culture filtrate concentrated 25-fold.
Each 20 m1 fraction in the eluate was
diluted 25—fold, and its sporulation-

stimulatory activity assayed.

30

240

 

200

 

PM; womwadns emuna
mugbuo ,0 human ’0 0,.

Figure 3

80 I20 ISO

40

 

Elution volume (ml)

31

material precipated. The precipitate was removed by
centrifugation at 20,000 x g for 20 min, and the pellet
was washed once with a small volume of absolute methanol.
The combined supernatants were stored in the freezer

(-20 C) overnight. A precipitate appeared during this
cold treatment, and was removed by centrifugation at
20,000 x g for 20 min. The pellet, combined with that
from the previous step and dried under a current of

air, was designated the methanol-precipitate fraction.
The final centrifugal supernatant fluid was evaporated to
dryness at 35 C with a rotary evaporator. The dried
methanol-soluble materials were redissolved in 160 ml

of double distilled water and stored as necessary. This
fraction was designated the methanolic extract fraction.
Table I shows the results of bioassay of the above
fractions. The methanol-soluble fraction shows 75 to
82% of the original sporulation—stimulatory activity of
the culture supernatant. Only three to 12% of the
original activity is seen either in the methanol-
insoluble fraction or in the methanol-precipitate

fraction (Table I).

Stability

 

The methanolic extract can be stored in a

refrigerator at 4 C for months without loss of activity.

32

Table I. Biological activities of the fractions derived
by extraction, with methanol, of a dried
culture supernatant fluid. The methanol
precipitate appeared during condensation and
cold treatment of the methanolic extract.

Each value presented is the percent of
heterocysts with contiguous akinetes, out of
200 heterocysts surveyed.

 

 

 

 

0x kxa _ 1x 2x 3x
Control 3.5
Original supernatant fluid 38 . 0 72 . 3 b
Methanol-insoluble 7.5 5.5 3.5 3.0
Methanol-precipitate 6.0 9.0 5.5 2.0
Methanol-soluble 29.5 60.0

 

 

 

aConcentration of the active substance, relative to its
concentration in the original supernatant fluid, based
on the assumption that all of the substance is present
in the fraction being assayed.

bExtensive fragmentation of filaments occurred in these
flasks.

33

Eighty percent or more (see Fisher and Wolk, 1976) of
the original activity survives autoclaving at 121 C for
20 min. Ninety-four percent of the original activity

was lost upon treatment with 6 N HCl at 110 C for 24 hr.

Charcoal adsorption chromatography

 

A major disadvantage of charcoal chromatography is
that solutes may be bound irreversibly to the charcoal.
In order to minimize the loss by irreversible binding,
the minimum amount of charcoal required to remove the
sporulation-stimulatory substances from the culture
supernatant was determined. Culture supernatant fluid
concentrated 40-fold was incubated with different
amounts of charcoal for 15 min, and the stimulatory
activity remaining in the filtrates was assayed
(Table II). With a concentration of 13 ug of charcoal
per ml of the original supernatant, 82 to 85% of the
original activity was removed from the supernatant.
Figure 4 shows the UV absorption spectra of the same
samples. A decrease in the absorption between 270 nm
and 290 nm coincides with the loss of activity. The
concentrated methanolic extract was diluted with
distilled water to from 0.04 to 0.1 of the volume oftflma
original culture supernatant and passed through the

column at a flow rate of 100 ml/hr. It was necessary

Table II.

34

Sporulation-stimulatory activities of a
supernatant fluid (methanol-soluble fraction)
following treatment of that fluid with
different amounts of activated charcoal. The
culture filtrate was treated with different
amounts of charcoal for 15 min each,the
charcoal removed by filtration, and the fluid
then assayed. The activities are expressed
as the percent of heterocysts with contiguous
akinetes, out of 200 heterocysts surveyed.

 

0x

1x

2x

3x

 

Control 1.0
Original filtrate

Amount of charcoal used
(us/m1)

2
6
9
13
39

30.5

40.0

25.0
10.5
5.5
8.5
4.0

 

35

Figure 4. UV absorption spectra between 260 and
300 nm, of charcoal-treated culture
filtrates. In each of the scans,

260 nm is at the left and 300 nm at

the right.

Absorbance

36

 

3959.620

Amount of charcoal used (pg/ml)

Figure 4

37

to dilute the methanolic extract in order to obtain
complete adsorption of the sample.

Table III shows the result of adsorption of the
active material to a column of charcoal and celite, and
its subsequent elution from the column with 50% ethanol

and then with 50% ethanol plus 2% NH OH. The first and

4
second 500 m1 of unadsorbed effluents from the column
exhibitedJJLtol4% of the activity of the original
methanolic extract. However, the activities associated
with the effluents did not increase with concentration.
Elution of the column with 50% ethanol removed 50 to 60%
of the original activity. An additional 24 to 28% of
the original activity was eluted with a solution of 50%
ethanol containing 2% NH4OH. The combination of the
four fractions resulted in full recovery of the original
activity of the methanolic extract applied to the column.
The activities in the ethanolic eluate and ethanol-

ammonia eluate increased with concentration, as did the

activity associated with the methanolic extract.

Paper chromatography

 

Chromatography papers (20 cm x 20 cm, Whatman No. 1)
were washed by descending chromatography with both of two
solvent systems. Solvent I consisted of isopropanol:

formic acid:water (20:1.~1“:5,v/v) , and solvent II of

38

Table III. Activities of the fractions from the column
of charcoal and celite. One liter of a
27.5-fold concentrate of an aqueous solution
of the dried methanolic extract was passed
through a 0.9 cm i.d. x 4.5 cm long column
of a 1:2 (w/w) mixture of charcoal and
celite. The following samples of eluate
were collected: two samples of unadsorbed
effluent, 500 ml each; a 50% ethanolic
eluate, 1 liter; and an eluate consisting of
50% ethanol with 2% NH4OH, 1 liter.
Activities are expressed as the percent of
heterocysts with contiguous akinetes,out of
200 heterocysts surveyed.

 

 

0x 8x 1x 2x
Control 3.5
Methanolic extract 23.0 37.0 50.0
First 500 m1 unadsorbed 2.5 4.0 4.0
Second 500 ml unadsorbed 6.5 5.0 6.5
Ethanol eluate 12.5 23.0 26.5
Ethanol-ammonia eluate 5.0 14.0 14.0

Combination of all four eluate fractions 37.5

 

39

isopropanol:58% NH4Oszater (9:1:1, v/v). The paper
was dried between and after the two washings. A toxic
effect upon the growth of Cylindrospermum exhibited by
the extracts from unwashed papers was completely
prevented by washing, and neither did the extracts
show growth-stimulatory effects.

Figure 5 shows a drawing of a paper chromatogram
of the combined ethanolic eluates from the charcoal-
celite column, in solvent system II. The bands shown
were observed under UV light or after spraying with a
solution of ninhydrin. The brown-colored material
observed at the origin in visible light could not be
extracted from the chromatogram. The chromatogram was
divided into three sections (Fig. 5), and each section
was cut into small pieces and extracted with solvent
system II. The results of bioassay of the extracts is
shown in Table IV. The eluate from section #1 was in-
active, that from section #2 exhibited 71 to 90% of the
original activity of the ethanolic eluates, and that
from the third section exhibited one to five percent of

the applied activity.

Chromatography on thin layers of silica gel

 

All silica gel 60F-254 plates were prewashed by
chromatography with solvent system III, n-butanolzacetic

acid:ether:water (9:6:3:l, v/v).

40

Figure 5.. Paper chromatography of the combined
ethanolic eluates from a charcoal-celite
column,developed with a solvent system of
isopropanol:58% NH4Oszater (9:1:1, v/v).
The bands were located under UV light or
by subsequent spraying with a 2% solution
of ninhydrin in acetone acidified with 2 N
formic acid. The chromatogram was cut
into three sections and extracted for
bioassay. The results of the bioassay

are shown in Table IV.

41

 

W 0-00

 

— g O.2|

 

W 0-40

 

 

 

 

 

C’ ’i) (”49
0.78
OBI

 

 

 

H L00

Figure 5

Visible brown colour T
UV fluorescent (light blue)

 

UV fluorescent (blue)

Ninhydrin positive
UV fluorescent (blue)

UV fluorescent (dark blue)_¥_
2

UV fluorescent (sky blue)
UV absorbing

l
UV'fluarescent (pde) _L

 

42

Table IV. Bioassay of the substances extracted from a
paper chromatogram. Three sections of the
paper chromatogram (see Fig. 5) were extracted
with the solvent system used for chromatography.
Activities are expressed as the percent of
heterocysts with contiguous akinetes, out of
200 heterocysts surveyed.

 

 

0x 1x 2x 4x 8x
Control 7.0
Original eluate 26.0 43.0 -——- -——-
Section #1 7.0 7.0 6.0 -——-
Section #2 24.3 32.5 18.3 8.0

Section #3 8.0 11.5 13.0 -———

 

43

The extract from section #2 of the paper chromat-
ogram was chromatographed on silica gel in solvent
system III. Figure 6 shows bands visualized against
the fluorescent background by means of UV light. The
chromatogram was divided into six sections. Each
section was scraped from the plate, and extracted three
times with 50% ethanol. The results of bioassay of the
extracts from the six sections are shown in Table V.
The extract from section #1 exhibited 80 to 98% of the
activity of the extract applied to the plate. The
extract from section #4 exhibited 11 to 12% of the
original activity. Section #1 included a band showing a
mixture of UV absorbance and fluorescence. When it was
dried, the extract from this section contained a large
amount of crystals. These crystals were shown by a
separate experiment to have come from the plate itself
rather than from the extract which had been applied to
the plate, and to be without effect on the formation of

akinetes or on the growth of an assay culture.

Chromatography on thin layers of cellulose

 

All cellulosic thin layer plates were prewashed
with solvent systems I and II. The active extract from
section #1 of a silica gel chromatogram was further

chromatographed on a prewashed cellulosic plate in

Figure 6.

44

Silica gel thin layer chromatogram of
the active extract from a previous
paper chromatogram. The chromatog-
raphy was performed in a solvent
system of n-butanol:acetic acid:
ether:water (9:6:3:1, v/v). The
chromatogram was divided into six
sections for bioassay. The results

of the bioassay are shown in Table V.

 

 

 

 

 

 

 

 

 

 

 

 

C233 . :12?)

 

 

 

45

l.00

0.79
0.72

0.63

0.43

0.l8

0.00

Figure 6

UV fluorescent (blue),

UV flsorbing

UV fluorescent (blue)

T
1

2

u——.|+_u_+

lo—a)

46

 

 

Table V. Activities in extracts from sections of a
silica gel thin layer chromatogram. Six
sections were scraped from the plate (see
Fig. 6) and extracted with 50% ethanol.
Activities are expressed as the percent of
heterocysts with contiguous akinetes out of
200 heterocysts surveyed.

0x lx 2x 4x 8x

Control 3.5

EXtraCt 0f Paper 48 o 55 o 50 o 26.0

chromatogram ’ ' ’

Section #1 39.0 49.5 49.0 26.0

Section #2 5.0 4.5 5.5 5.5

Section #3 4.0 3.5 4.5 3.5

Section #4 8.5 9.5 8.5 8.5

Section #5 4.5 3.5 5.5 4.5

Section #6 3.5 4.5 4.5 3.5

Combined

40.5 47.5 40.5 30.5

 

47

solvent system II. Three bands, all of which were
present in the first paper chromatogram (Fig. 5), can
be seen, better separated, on the plate (Fig. 7). The
chromatogram was divided into four sections; each was
scraped from the plate and extracted three times with
solvent system II. The UV-absorbing band with an Rf of
0.82 exhibited 76 to 91% of the activity of the extract
applied to the plate (Table VI).

As will be discussed in a later section, further
purification was achieved by means of vacuum distillation
at 150 C.

Figure 8 illustrates the entire purification

procedure.

Determination of dry weight

 

Samples were lyophilized in tared culture tubes
(1 cm i.d. x 6 cm long), and the tubes with dried
samples weighed with a SS microbalance (Mettler, Zfirich,
W. Germany). Table VII shows the dry weights and
biological activities of samples from successive
purification steps starting with one particular culture
supernatant. The loss of dry weight during paper
chromatography is due to irreversible binding of
brown-colored material to the paper. The increase
in dry weight during chromatography on thin layers of

silica gel is due to the extraction of crystalline

48

Figure 7. Chromatogram, on a thin layer of cellulose,
of the active extract from a silica gel
thin layer chromatogram. The solvent
system was isopropanol:58% NH4OH:water
(9:1:1, v/v). The chromatogram was
divided into four sections for bioassay.
The results of the bioassay are shown in

Table VI.

 

 

 

 

 

 

 

49

LCD

0.82
0.78

0.65

0.00

Figure 7

UV absorbing
UV fluorescent (sky blue)

UV fluorescent (bl ue)

+l+—-|

 

l1

50

 

 

Table VI. Activities of the extracts from the sections
of a cellulosic thin layer chromatogram.
Four sections scraped from the plate (see
Fig. 7) were extracted with the same solvent
system used for the chromatography.
Activities are expressed as the percent of
heterocysts with contiguous akinetes.
0x 1x 2x 4x 8x
Control 4.0
Silica gel extract 33.5 44.5 48.0 40.5
Section #1 4.5 5.5 7.5 4.5
Section #2 27.0 35.5 44.0 40.0
Section #3 6.0 6.5 6.6 5.5
Section #4 5.5 4.5 4.0 4.5

 

51

Akinete-forming culture in SSM
Centrifugation
Culture supernatant fluid

Evaporation

 

Dried supernatant fluid

Extraction with 100% methanol

 

 

. Methanol-insoluble
Condensation
substances
Methanolic extract Methanolic precipitate

Chromatography on a column of charcoal and celite

 

Elution with 50% EtOH and 50% EtOH + 2% NH4OH Solution of
unadsorbed
Eluate materials

Paper chromatography with a solvent system of
isopropanol:58% NH4OH:water (9:1:1, v/v)

 

Active section, extracted Inactive sections
of chromatogram

TLC on silica gel 60F-254 in n-BuOH:HAc:ether:water
(9:6:3:1, v/V)

 

Active section, extracted Inactive sections
of chromatogram

TLC on cellulose in isopropanol:58% NH OH:water

 

 

 

 

4
(9:1:1, v/v)
Active section, extracted Inactive sections
of chromatogram
Vacuum distillation at 150 C
|
Distillate Residue

Figure 8. Purification procedure for the sporulation-stimulatory
substance.

52

Amufl>wuom Houusoo I cumuucmocou xa mo >uw>fiuoay

.0

uco>H0m a once pupfl>wonsm mm3 b a o .mwm no at cofiuocm .Q
ImcHomdm ousuaso Hmcflmfluo on» no wEsHo> on» no cowuuom van» 0» sawuomum can no conusawv so comma munda> .6

tasbnmfimz he 9:

.cofluuam on» «0 “Emmy Huccwmsuu can can Ahmv econ acouu
.mhamma HaUHmOH0wn Mom owns 90: owsam usuu

 

 

 

 

 

 

o.oa o.mH o.vH m.HH omm.o v:

o.HH m.ma c.4H 0.4H mao.o ms

owHH m.o~ m.Hm m.h~ o.v~ HHo.o mt

o.>H o.mH o.mH o.va moo.o QEBHH*
o . m." o . «a o . .2 m . m moo . o ppm .3 33m 303:8 £03 30336

o.oa o.mH o.ma o.w Hom.o mt

m.m m.ma o.mH o.~H 5mm.o mt

m.oa m.mm o.mm o.om o.Hm Hom.o afifimfit
o . ma m . S o . 3 o . 3 3m . o mm 2. 32m Hum 828 scum 3833B

o.m o.oa m.m m.m onw.m m:
n.HH m.hm o.av o.o¢ o.H~ mam.o m:
o . ma m . «H o . NH o . va omN . o 3 5639320 modem Baum muomuuxm
I m . 3 o . «om . m nmsofipummma 30330
mmmd mév 05o m.mv o.mv o.mm Hooch—dab 50.5 33am oflocanum
III II I m . ma 0 . new cumuafiooum Hosanna:
I m . ma 0 . Ham cowuounm 03302735562
«So . o I II II o . me o . omm . m uomuuxm oflocmfimz
Shoo . o l o . em o . omm . m 33.... ufiumcnoasm canvas
m.HH_ Houucco
owwwwwwmm xw xv xm oxa xo UHE\unowc3 asp m:

.HOU 00% NO SOflUMHHCOOGOU

 

.coflumowwwusm mo mummum ucaucmmwo sown moamficm mo mufiufi>fiucc;awofimoHoHn use musmwm3 hue

.HH> manna

53

material from the plates. The dry weight-based specific
activity of the most purified sample is 1.5 x 105 times

higher than that of the original culture supernatant.

Isolation, from cells, of material stimulating the

 

formation of akinetes

 

Cells from the same 100 liters of sporulating
cultures used for the experiment whose data are
tabulated in Table VII were collected by centrifugation,
washed with fresh SSM, and lyophilized. The dry weight
of the cells was 15.45 g. The lyophilized cells were
extracted three times with 250 m1 of absolute methanol.
The combined extracts were evaporated to dryness and
suspended in 20 m1 of distilled water. Low concentra-
tions of the extract exhibited significant sporulation

-stimu1atory activity (Table VIII).

Mass spectral analysis

 

The active, UV-absorbing band extracted from the
cellulosic thin layer plate and used for mass spectral
analysis was contaminated with a slight amount of
material from a band of lower Rf. Figure 9a shows a
mass spectral fragmentogram of seven fragments pre-
dominant (m/e = 41, 44, 56, 69, 123, 151 and 265) in
the sample. Among them, only the fragment with m/e

ratio of 96, 123, and 151 are both dominant in the

54

Table VIII. Biological activity of the methanolic extract
from cells. 15.45 g of lyophilized cells
from 100 liters of culture (i.e., 0.154 mg
dry weight of cells per m1 of original
suspension), were extracted with absolute
methanol, and the methanolic extract dried
and redissolved.

 

 

 

 

Mg dry weight of cells extracted, Percent of heterocysts with a
per m1 of assay-suspension contiguous akinete
618 T
309
154 -cells lysed
77.2
38.6
19.3 - 35.0
3.86 44.0
1.93 33.0
0.386 29.0
0,193 16.5

0.000 7.0

 

55

Figure 9. Mass spectral fragmentograms of (a) the
active extract from a cellulosic thin
layer chromatogram and (b) an inactive

extract of material with slightly lower

Rf.

56

 

o 0.9.39.2.th

 

 

 

 

 

 

 

 

 

075r00_

mMWHHHHHU ”HM“ HHHUO:EquOO_ _

up...

 

 

 

ConquOO_

222. .68
m8: $83.3

oo .22anth

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a can
322 E Wazyom .D

Figure 9

57

sample containing the sporulation-stimulatory substance
and not characteristic of the fragmentogram (Fig. 9b)

of the neighboring UV-fluorescent band extracted
separately from the cellulosic plate. The other
fragments represent ca. six percent of the material
present in the active sample. The ions with m/e =

96, 123, and 151 show peaks at 150 C. The mass spectrum
of material evaporating into the electron beam at 150 C
is shown in Figure 10. Besides the parent peak at m/e =
151, high intensity peaks are found at m/e = 123 and

m/e = 96. The high ratios of the heights of the peaks
at m/e +2 to the heights of the parent (m/e) peaks
indicate that an atom of sulfur is present in the
molecule. Masses were measured with high resolution by
peak matching, using perfluoroalkanes as internal
standard. The masses of the three ions, measured with
high resolution, and the possible chemical composition
of those ions are shown in Table IX. The chemical
formula for the only molecular ion peak which can account
for the fragmentation pattern is C7HSOSN. The peak at
m/e = 123 is produced by the loss of CO from the
molecular ion, and the peak at m/e = 96 is produced by
the additional loss of HCN. The paucity of hydrogen
atoms relative to carbon atoms indicates that the

molecule contains unsaturated bonds.

58

Figure 10. Mass spectrum of the material evaporating

into the electron beam at 150 C.

l00

59

8

MPH-l mad wales

 

IZO I40 |60 "‘xe

I00

60

4.0

Table IX.

60

the active material.

Molecular weights, measured with high
resolution, and possible chemical formulae

of the major ions in the mass spectrum of

 

Molecular weight

 

151.00931

123.01418

96.00314

Possible chemical
formulae a

 

C1H3°5N4

C2H752N4

C4H7°451

C5H351N4

C7H5°151N1

H3°4N4

C2H505N1

C3H7O3S1

C6HSSlNl

C1H4°5

C5H451

bass loss
difference

 

-0.00104
-0.00191
0.00281
0.00145
0.00012
-27.99513(CO)
-0.00125
-0.00258
0.00260
0.00009
-26.98278(HCN)
-0.00273

-0.00023

 

a Permissable tolerance of mass, relative to the measured molecular
weight: : 0.0030 amu.

61

A further step of purification

 

Mass spectroscopy indicated that the active material
eluted from the cellulosic thin layer chromatogram
contained predominately a substance vaporizing at
approximately 150 C under high vacuum. The active
extract prepared from the cellulosic thin layer plate
was therefore further purified by distillation under
vacuum. The distillate, which formed a single UV-
absorbing band at an Rf of 0.81 on a cellulosic thin
layer chromatogram developed with solvent system II,
exhibited 52% of the activity of the original
methanolic extract, in SSM (Table X). The distillate
also was active in stimulating akinete formation in

phosphate-containing AA/8.

NMR spectroscopy

 

A proton magnetic resonance spectrum of ca. 0.5 mg
of the distillate is shown in Figure 11. Peaks at
6 = 1.940, 2.165, 7.845, 7.840, 7.157, 5.84, and 5.73
ppm originate from the solvent. Peaks from the sample
are confined to the region 7.19 to 7.29 ppm, and form
a complex pattern. The absence of resonance peaks at a
low ppm range together with the complex pattern at
ca. 7.2 ppm suggest that the sample has a conjugated

ring structure (Paudler, 1971).

62

.np3oum u>fiumuamw> How penance“ mucufiuam can no ads mcflmucoo aw awn» uomu ecu
muflmmco adsows was» cw coflumusHME anaemoHoanOE muonEoo a» mans uuoz m\¢¢ cw coshom £0w£3 muuucwxmo

 

 

o.h o.h

m.mH m.hN

M\<< efimw
xw

 

m.m o.m

m.sm o.ve

m\<< 2mm
x«

 

 

m.m m.m

m.sa m.m~

m.nm m.mv

m>2 2mm
xm

o.m o.m

m.sa o.s~

o.mm m.mm

«\«4 2mm
xa

cowueaafiumfip assoc> scum oaofimum
cucaawumavlausom>

macaw

unsuccuumdm cusuaso Aooflupv

Hogwmfluo mo uomuuxu aflaocmnuoz

o . o o . h Houusou

 

 

.mmuocflxm msosmwucoo

cuw3 mummooumums mo unmoumm may no owmmmumxm ma >uw>fluo¢ .m wusmflm

mo musooooum chance on» ma commando Hafiuoumfi muoumaseflumlcoflucasnomm
an» we mnmaafiumflcusssom> can no wm\m¢ can 2mm a“ .sufl>fluom Hmofimoaofim .x manna

63

Figure 11. Proton magnetic resonance spectrum
of the vacuum-distilled sporulation-

stimulatory substance.

64

E

 

.HH musmflm

 

 

.xp

 

 

 

 

x ou—

 

 

 

 

 

 

 

 

65

IR spectroscopy

 

Infrared spectra were taken of ca. 0.5 mg of the
vacuum-distilled sporulation-stimulatory material in a
disc of KBr (Fig. 12). A peak at 3429 cm.1 is referred
to an -NH or -0H stretch. Although in some cases KBr
itself shows a peak at this frequency, perhaps due to
water absorbed by the salt, the peak in the spectrum
of the sporulation-stimulatory material is attributable
to the sample because a control spectrum of a disc con-
taining only the same amount of KBr as in the sample disc
shows no peak at that frequency. Peaks were observed
consistently at 800, 1010, 1085, 1257, 1627, 1725, 2855,
2930, 2988, and 3429 cm-1 (Fig. 12a).

UV absorption spectroscopy

 

An ultraviolet absorption spectrum was taken of the
sporulation stimulatory material in acetonitrile (Fig.
13). Peaks were observed at 242, 247, 253, 263, and

268 nm.

66

Figure 12. Infrared spectra of (a) the vacuum-
distilled sporulation-stimulatory
substance in a KBr pellet and (b) a
KBr pellet without additional

material.

67

SN

08—

Son

 

 

 

 

 

 

l
—<L —<L—f— 1,—4
0

 

 

 

 

 

8

 

 

“HE
A l

I

,

 

 

 

 

9

 

 

. _.__.. .....__._ v.1-
__...

 

 

,___k
”*4

F M o l
i v

 

 

8

 

 

 

 

 

 

 

 

 

 

 

DDDDDDD
IIIII

 

 

1
8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

,m .
a. .t.

8—

 

8 777"“
3 V

9

m.

08

89

2.

8w—

... m 0
3206.3 5023223

..5 237m>(
83A 1 _ 382

3200.2. Eozwm><3

95*

 

UNJDUBd) aoNvmwswm

(lNEDle) DNVLUWSNVM

68

Figure 13. UV absorption Spectrum of the
sporulation-stimulatory substance

dissolbed in 0.5 ml of acetonitrile.

V

05

.0
.a

Relative Abscrbance

r

OJ

 

 

D
g!

.0
I»
I

69

 

L 1 1 l I I

l
250 300
Wavelenth (nm)

Figure 13

70

PART II. Effects of Various Environmental Factors on
the Formation of Akinetes in C. Zieheniforme

Effect of hydrogen on the formation of akinetes

 

As shown in Table XI, gassing of a suspension of
filaments in SSM with a mixture of 12.5% hydrogen, 87.5%
air stimulates the formation of akinetes in SSM 2.5-fold
relative to a control gassed with air. Growth is not
affected. Table XI also shows that the addition of
hydrogen to a gas mixture consisting of 19..9%.02: 0.1% C02,
balance Ar has the same stimulatory effect on the
formation of akinetes. Hydrogen has no significant
effect upon the reduction of acetylene by C. licheniforme

(Table XII).

Uptake hydrogenase and its localization

Peterson and Wolk (1978a showed that uptake
hydrogenase activity in aerobically grown Anabaena
strain 7120 is localized solely in heterocysts.

Ferricyanide, which served as electron acceptor in a

71

Table XI. Effect of hydrogen gas upon the formation of
akinetes. The gas mixtures in the flasks used
for the assays were replaced every 12 hr.

 

Remaining gas phase

 

 

 

 

% Hydrogen -
Air 19.9% 02, 0.1% C02, 79.9% Ar
0 13.5a 13.0
5.0 26.5 16.5
10.0 26.0 18.0
12.5 34.0 18.5
15.0 25.5 27.5
20.0 25.5 28.0

 

aSporulation-stimulatory activity, expressed as the percent of
heterocysts with contiguous akinetes.

72

Table XII. Effect of hydrogen gas upon acetylene
reduction in air. Each 5-ml assay vial
contained a gas phase of air, 0.3 ml C2H2'
and hydrogen as shown, plus 2 ml of a
suspension of C. Zicheniforme in SSM.

The reactions were stopped after 30 min,
by addition of 1.0 m1 of 20% TCA.

 

 

 

% Hydrogen umoles C2H4 produced (mg Chl)-lh-l
0 12.3
5 11.2
10 13.1
12.5 12.1

15.0 11.2

 

73

cell-free preparation of Anabaena 7120, could not do so
in a cell-free preparation of C. Zieheniforme. Of
several alternative electron acceptors tried (Table
XIII), only 10 mM phenazine methosulfate (PMS) was found
to be active as an acceptor. Fujita et al. (1964),
working with the uptake hydrogenase from A. cylindrica,
reported that the most effective electron acceptor tried
was PMS, and that methylene blue, toluidine blue, and
DCPIP were less than 25% as effective. One method for
localizing uptake hydrogenase in filaments is presented
in Table XIV; on a per heterocyst basis and as measured
by in vitro assays, 84 i 2% (two experiments) of the
hydrogenase activity in whole filaments was recovered in
isolated heterocysts, whereas no activity was detected

in a fraction derived from vegetative cells.

The effects, upon the formation of akinetes, of various

 

substances of low molecular weight

 

Table XV shows the effects of the 20 usual amino
acids found in proteins on the formation of akinetes. One
mM L-tryptophan shows particularly high stimulatory
activity. Aspartic acid and pheylalanine, at a
concentration of 16 mM, also show high activity. Lower,
but significant, stimulatory activity was produced by l6mM

proline and 15 mM isoleucine. Although ammonium inhibits

74

Table XIII. Uptake of hydrogen by a cell-free

suspension, measured manometrically, with

various electron acceptors at a concentra-
tion of 10 mM.

 

Acceptor

Ferricyanide
DCPIP

PMS

Methylene blue

Methyl viologen

 

 

' Activity
E0 (mv) umole H2 (mg Chl)-1h_1
+429 0

+217 0

+80 5.8

+11 0

—550 0

 

75

Table XIV. Localization of hydrogenase. Hydrogen uptake
by cell free suspensions derived from whole
filaments, from heterocysts, and from
vegetative cells was measured manometrically.
The Warburg flasks used contained 1.8 ml of
sample suspension and 0.2 ml of 100 mM PMS.

 

 

ul H2 taken up-(lo9 heterocysts)n1min-l
Whole filaments 3.03
Heterocysts 2.48
Vegetative cells <0.01a

 

a This figure is calculated on the basis of the heterocysts in the
filaments from which the vegetative extract was derived.

76

Table XV. Effects of amino acids on the formation of
akinetes. Values shown are the percent of
heterocysts with contiguous akinetes.

 

Amino acid concentration, mM 16 10 5 1 0.5 0.1

 

Control

Glu 8.0 10.0 7.5 6.0

Gln 10.0 10.0 10.0 1.5

Pro 31.5 10.0 9.0 6.0

Arg 20.5 8.0 6.0 5.0

Asp 47.0 25.5 26.5 22.5

Asn —a — 6.0 18.5

Lys 1.52) 3.017 0.0 0.0

Thr -— — — 4.67 3.513 0.0
Met —— — —— -— -- —
TYI "“ "‘ "' "' '-

Trp -—— -—- -—- 60.5 42.0 36.0
Phe 68.5 38.0 20.5 19.0

His 0.0b 0.0 0.0 3.0

Ser —- -—- 12.5 13.5

Gly -—- -—- 0.0 4.5

Cys -—— -—— 14.0 8.5

Ala -— -—- 8.0 5.0

Val -—- ——— 0.0 0.0

Leu 2.5b 0.01) 0.0 0.0

Ile -—— -—- 35.0 17.0

 

a -—- : cells lysed

Inhibitory to growth

77

the formation of akinetes in Anabaena doliolum (Singh
and Srivastava, 1967; Tyagi, 1974), it does not do so in
C. Zicheniforme (Table XVI) except at growth-inhibitory
concentrations. The cyclic nucleotides, dibutyryl c-AMP,
dibutyryl c-GMP, and c-AMP showed little or no sporula-
tion-stimulatory activity by themselves, and did not
substantially enhance the stimulatory effect of culture
supernatant fluid (Table XVII). As shown in Table XVIII,
the formation of akinetes is completely inhibited by
1.0% CzH2 or 0.5% C2H4.
Mann calcium glucuronate, reported by Wolk (1965)
to stimulate the formation of chains of akinetes in
A. cylindrica, did not elicit the differentiation of
chains of akinetes in C. licheniforme. However, after
four days of culture in SSM supplemented with 25 mM
Mann calcium glucuronate, an akinete was present
adjacent to every heterocyst. Growth was not affected.

In control cultures in SSM, ca. 5% of the heterocysts

had contiguous akinetes.

Cytochemistry with a redox dye

Fay and Kulasooriya (1972) reported that upon
treatment of filaments of Anabaena cylindrica under air
with nitro blue tetrazolium chloride (NBT), a gradient

of the amount of formazan produced by reduction of the

78

 

 

 

 

Table XVI. Effect of ammonium upon, added to SSM
or to sporulation-stimulatory
supernatant fluid, the formation of
akinetes. Activities are expressed
as the percent of heterocysts with
contiguous akinetes.
Sporulation-stimulatory
NH4Cl (mM) SSM supernatant fluid
5.00 5.0 28.5“
2.50 1.5 40.0“
1.00 2.0 23.5a
0.50 2.0 58.0
0.10 2.5 71.5
0.02 2.0 70.5
0.00 6.0 64.0

 

a Growth was inhibited.

79

Table XVII. Effects of cyclic nucleotides upon the
formation of akinetes. Activities are
expressed as the percent of heterocysts
with contiguous akinetes.

 

Methanolic extract of dried
SSM culture supernatant fluid assayed
at 1x concentration

 

Control 1.0 30.5
Dibut-c-AMP 5 mM 5.0 49.5
Dibut-c-AMP 2.5 mM 2.5 30.5
Dibut-c-GMP 2.5 mM 1.5

Dibut-c-GMP 0.5 mM 5.0 28.5
c-AMP 2.5 mM 7.5 34.0
c-AMP 0.5 mM 2.0 31.0

Dibut-c-AMP 2.5 mM

+ Dibut-c-GMP 0.5 mM 7'0

 

80

.coflumasuommo

 

 

lam.ov Ame.ov 188.81 1N¢.oc N N
o o o o.o m o

1G5.oi 104.01 lom.ov 1G5.ov lom.oc v N
o o 0.3 o.N m.m a o

Lam.ov

eo.N Nouucoo

s N N N
. . . . . m o no m 0

sea Nm NH mm o NH O was 0 Nfloo o No unmoumm

 

.mhao HSOM on» no one on» an HE\H£U on no .mamp Hsom msfluso

musuaso on» no nu3onm asp mmmuoxw mononucmumm may upflmcfl mumnfisc use

.mmumsfixm moosmwucoo nuw3 mumwooumumn Mo unmouwm on» no Ummmmnmxm

ma CONDMHDHOQm mo ucmuxm one

musuxwe was

.un NH wnm>m omomHomu mm3 mummm mo

v N N N

.mmfimflflxm MO COHHMSHOM 03D. COQD m UUCM m U MO mHUOMMM

.HHH>X OHQMB

81

NBT was observed, with the most formazan present in
vegetative cells next to heterocysts and with the
amount of formazan present per cell gradually
diminishing with increasing distance from heterocysts.
A very similar result was obtained with C. Zicheniforme
(Fig. 14). However, as shown in Figure 14, such a
gradient can form from any point of the filament which
is bent mechanically or is nicked by a fine glass
needle. Therefore, the gradient originally shown may
be the consequence of localized penetration of NBT

at the connection between heterocysts and vegetative

cells rather than of a gradient of reducing activity.

82

Figure 14. Reduction of nitro blue tetrazolium
chloride by (a) intact, (b) bent,

and (C) out filaments.

83

(a)

(b)

 

(c) .

i
mmmrr‘r-rmrra Wt”

Figure 14

DISCUSSION

The substance inducing the formation of akinetes

 

A substance which is capable of stimulating the
formation of akinetes has been purified from the
centrifugal supernatant fluids of akinete-forming
cultures of Cylindrospermum Zioheniforme. Evidence of
the occurrence of such a substance was reported by
Fisher and Wolk (1976). Because only the most active
fraction or fractions from each step of the purification
procedure were processed in the subsequent steps, on
the order of 70% of the total activity (i.e., activity/
volume, times volume) is lost during the purification.
Significantly, the purified compound alone, even at
high concentration, does not stimulate the formation of
akinetes as strongly as does the original culture
supernatant fluid. Thus, other substances, which may be
active by themselves or may only potentiate the
sporulation-stimulatory activity of the substance which

has been isolated, contribute to the original activity.

84

85

However, the purified substance, the specific activity of
which is 1.5 x 105-fold greater than is the specific
activity of the (dried) original supernatant fluid,
appears to be the major single substance responsible for
the activity.

It is not easy to distinguish whether a compound is
released by the breakdown of cells or is released by
healthy cells (compare Fogg, 1962, 1966). Because the
sporulation-stimulatory activity in culture supernatant
fluids increases during the early part of the growth
period (Fig. 1,. 2), i.e., at a time when few cells are
moribund or dead, it appears that hte stimulatory
substance is released by healthy cells. The interpreta-
tion that the active substance is not a product of
decomposition is further supported by the observation
that significant activity may be extracted from the
cells themselves (Table VIII). Others have observed that
the formation of akinetes is inhibited by the presence
of phosphate in the culture medium, i.e., that phosphate
is inhibitory to the formation of akinetes (Glade, 1914;
Wolk, 1965; Gentile and Maloney, 1969). Because the
substance which I have isolated stimulates sporulation
in phosphate-containing growth medium (AA/8: see Table
X), this substance is not 'merely a promoter of a devel-
opmental process which occurs in response to a

deficiency of phosphate, but instead itself initiates

86

the differentiation of akinetes. Although a variety of
substances is known to be released by cyanobacteria
(Fogg, 1962, 1966; Stewart, 1963; Pattnaik, 1966; Walsby,
1974a, b; Walsby and Fogg, 1975), none of those substan—
ces is known to affect processes of differentiation (see
especially Whitton, 1965). The substance reported here
is maximally active at a concentration of 44 ug/l (4x
value of active band from final step in Table VII). It
is the first example of an organic compound, produced by
a cyanobacterium, which specifically affects the
differentiation process in the cyanobacterium.

Spectral analysis of the purified substance yielded
the following information about its composition and

structure:
1) Its molecular weight (Table IX) corresponds

to the chemical formula C7HSOSN.

ii) The two major ions resulting from its
fragmentation have the chemical formulae
C6HSSN and C5H4S, respectively. The first
fragment is produced by a loss of CO from the
molecular ion. The loss of CO cannot be
attributed to the presence of an aldehyde
group in the molecule, because the absence
of aldehydic hydrogen is clearly indicated
by the lack of proton magnetic resonance in
the region of 6 = 9 ppm to 6 = 10 ppm (Paudler,
1971), and by the absence of absorption in the
IR at ca. 2720 and 2820 cm'1 (Conley, 1972).
The composition of the second fragment is

consistent with the idea that the fragment

87

is produced by the loss of HCN from the first
fragment. The high intensity of the peak at

m/e = 96 in the mass spectrum and the absence

of other peaks of comparably high intensity at
lower values of m/e indicate that the fragment
with m/e = 96 has a stable ring structure; three
such structures are consistent with the chemical
formula (C5H4S) of that ion. Of these, a six-
membered ring (a) may be excluded because it is

structurally
’ e
H \H HUGH H H
HI /H H Sit-l H H
St + 8+
(0) (b) (c) (0)

so strained that benzene rings at both sides of
the ring, as in (b), are required to stabilize
the structure, and because the formula of the
fragment would require two unligated bonds. A
second possible structure (c), a thiophene ring
with a methylene substituent,_may also be
excluded because no fragment was observed which
corresponded to the thiophene ring itself, and
because the formula of the substituted
thiophene would also require two unligated
bonds. The third possibility, a five-membered
ring with a thioketone group (d), is consistent
with (i) the presence of a strong absorption,
attributable to c=s, at 1085 cm'l, and (ii) the
absence of absorptions, attributable to C—S and
and S-H, at 590 to 700 and at 2500 to 2600 cm-£
in the IR spectrum. Peaks attributable to C=C,

C=C-H, C-C, and methylene C-H bonds

iii)

iv)

88

are observed at 1627, 2988, 800, and 2855 plus
2930 cm-1, whereas peaks attributable to
-N=C=O and CEC are not observed.

The proton NMR spectrum shows a complex of
peaks in the region of 6 = 7.19tx>6 = 7.29 ppm,
implying that the substance has a highly
conjugated ring structure.

A peak at 3429 cm.1 in the infrared absorption
spectrum may be assigned to the N—H stretch of
a secondary amine or amide. The alternative
possibility that that peak corresponds to an
O-H stretching vibration was eliminated be-
cause no absorption attributable to an O-H
bending vibration was observed in the region
of 1300 to 1500 cm”1 or at ca. 650 cm-1.
Because only one peak is detected in the

region of 3429 cm-1, the nitrogen cannot be
present as a primary amine (-NH2). Lack of
absorption in the region of 1550 cm"1 to

1530 cm-1, which would have been attributable
to N-H bending vibration, suggests that the

N-H group is a constituent of a cyclic amide.
Absorption at 1725 cm"1 supports the
possibility that the oxygen atom in the
molecule is present as a cyclic C=O, to

which that absorption may be attributed pro—
vided that the carbonyl group is both
conjugated and part of an amide group in a
five-member ring (Conley, 1972; see pp. 168,
180). A major peak at 1257 cm.1 is
attributable to the amide III band (C-N stretch)
of a secondary amide. Taken together, these

results provide strong support for the idea

89

that the nitrogen and oxygen in the molecule
are present as a secondary amide group in a
five-membered ring. This interpretation accords
closely with the mass spectrum provided that the
nitrogen atom is not adjacent to group (d)
above, so that NCH will be lost as a group
after (or together with) loss of CO. The
structure of the substance is therefore
approximately as shown in Figure 15, although
the precise positions of the C=S and of the

C=C double bonds cannot yet be assigned.

v) The ultraviolet absorption Spectrum shows a
multiplicity of peaks over the range, but these
have not helped to elucidate the structure of
the molecule.

Other Factors which Control the Formation of Akinetes

 

Hydrogen gas is active in stimulating sporulation
adjacent to heterocysts, although less active than is a
culture supernatant fluid. A mixture of 12.5% H2 in air
stimulates the formation of akinetes next to heterocysts
2.5-fold. Peterson and work (1978a), working with
Anabaena strain 7120, reported that the kinetics of
solubilization of hydrogenase, during cavitation,
closely paralleled the kinetics of destruction of
heterocysts, and that heterocysts isolated from the
cyanobacterium accounted for 86% of the cell-free
hydrogenase activity derivable from whole filaments. In

my experiments, heterocysts isolated from Cylindrospermum

accounted for 84% of the uptake hydrogenase present in

90

Figure 15. Approximate structure of the substance
which stimulates the formation of
akinetes in C. Zicheniforme. The
positions of the thioketone group
within the left-hand ring, and of
the C=C double bonds, are not

determined by the available data.

91

\.

SHC

/

 

CH
NH

/

CHO

 

 

\

 

 

 

 

H20
H C

Figure 15

92

filaments of that organism, as assayed in vitro. It has
been reported that hydrogen can serve as electron donor
to nitrogenase (Wolk and Wojciuch, 1971; Bothe et aZ.,
1977; Tel-Or et aZ., 1977). However, H2 did not
significantly stimulate the reduction of C2H2 by
Cylindrospermum under air, and H2 stimulated sporulation
under Ar/COZ/OZ. The stimulation by H2 cannot,therefore
be mediated by an effect upon the fixation of nitrogen.
What is the role of the stimulatory substance and of

H in the formation of the pattern consisting of akinetes

2
contiguous with heterocysts? If the substance is
synthesized solely in heterocysts and is transported to
nearby vegetative cells, a concentration gradient of this
compound could be formed, so that the vegetative cells
adjacent to heterocysts would be the first to differ-
entiate into akinetes. It might be argued, however, that
exogenous supply of the stimulatory substance should
provide the substance equally to all vegetative cells,
and so should change the pattern into one in which all
vegetative cells differentiate simultaneously into
akinetes. No such change of the pattern is observed. It
is possible, however, that the substance can enter the
filaments only at the connections between heterocysts and
vegetative cells. That such a localized penetration is

possible is suggested by the results of experiments with

nitro blue tetrazolium chloride (Fig. 14). In those experiments,

93

the NBT was reduced in cells contiguous with heterocysts
and in any other cell which was bent or nicked. Thus,
vegetative cells contiguous with heterocysts may contain
loci, such as — perhaps — the junCtions to heterocysts,
which are more permeable than are other sites along the
filaments. If the stimulatory substance can enter the
filaments only through the vegetative cells contiguous
with heterocysts, the site of formation of a concentration
gradient could be unchanged, as would be the pattern of
formation of akinetes. Autoradiography with radioactive-
ly labeled sporulation-stimulatory substance may be able
to establish whether it enters filaments only at the
location of the cells which sporulate.

If heterocysts are not the sole site of the synthesis
of the stimulatory substance or if the exogenously
supplied stimulatory substance permeates into all
vegetative cells, what can control the pattern? The
stimulation of the formation of akinetes by H2 suggests
that the pattern may be controlled by some substance
which is reduced by the uptake hydrogenase in
heterocysts, and then moves into neighboring vegetative
cells. As discussed in the Introduction, reducing
conditions may well characterize the interior of hetero-
cysts, which might therefore provide an excess of some
reductant to vegetative cells. The pattern, akinetes

forming contiguous with heterocysts, could be explained

94

if the onset of sporulation requires (i) a stimulatory
substance that might enter all of the cells of the
filament, and (ii) a reductant provided by heterocysts.
Both C2H2 and the product of its reduction by
nitrogenase, C2H4,inhibit the formation of akinetes.

From the rate of reduction of C2H2r 12-3 umoles (mg

Chm-1h.l (Table XII), and the concentration of

chlor0phyll (9.6 ug Chl/flack) at the end of the period
of assaying the effect of gases upon the formation of
akinetes, the amount of C2H4 produced during the final

12 hr of that period may be calculated to be 12.3

3

(reduction rate) x 9.6 x 10- (mg Chl) x 12 (hr) x

22.4 (ul/umole) x %%% (correction to 25°) = 34 ul, or

0.012% of the 280-m1 effective volume of the flask.

The inhibition of sporulation by C is therefore too

2H2

great to be attributed solely to the C produced from

234

the C2H2' but may be ascribed to a combination of that

C2H4 and to the C2H2 itself. This is the first report

of the effect of a low concentration of C2H4 on a
prokaryote.

The fact that low concentrations of tryptophan
stimulate sporulation gains interest from the fact that
among amino acid analogues tried by Mitchison and Wilcox

(1973) , only 7-azatryptophan was found to alterthe pattern of

spacing of heterocysts. Pheylalanine, another armatic amino

95

acid, also strongly stimulated sporulation although at
a l6-fold greater concentration. It is possible that
these aromatic amino acids are normally involved in the

control of the differentiation of akinetes.

REFERENCES

REFERENCES

Allen, M. B. and Arnon, D. I. (1955) Studies on
nitrogen-fixing blue-green algae. I. Growth and

nitrogen fixation by Anabaena cylindrica Lemm.
Plant Physiol. 30:366-372.

Apte, S. K., Rowell, P. and Stewart, W. D. P. (1978)
Electron donation to ferredoxin in heterocysts of

the N -fixing alga Anabaena cylindrica. Proc. Roy.
Soc. fond. B 200:1-25.

Bharadwaja, Y. (1933) Contributions to our knowledge of
the Myxophyceae of India. Ann. Bot. 47:117-143.

Bothe, H. (1970) Photosynthetische Stickstoffixierung
mit einem zellfreien Extrakt aus der Blaualge
Anabaena cylindrica. Ber. dt. bot. Ges.83:421-432.

Bothe, H., Tennigkeit, J., Eisbrenner, G. and Yates, M.G.
(1977) The hydrogenase-nitrogenase relationship
in the blue-green algae Anabaena cylindrica.
Planta 133:237—242.

Bradley, S. and Carr, N. G. (1971) The absence of a
functional photosystem II in the heterocysts of
Anabaena cylindrica. J. gen. Microbiol. 68:xiii-xiv.

Canabaeus, L. (1929) Uber die Heterocysten und
Gasvakuolen der Blaualgen und ihre Beziehung
zueinander. Pflanzenforschung 13:1—48.

Cardemil, L. and Walk, C. P. (1976) The polysaccharides
from heterocyst and spore envelopes of a blue—green
alga. Methylation analysis and structure of the
backbones. J. Biol. Chem. 251:2967-2975.

Carter, H. J. (1856) Notes on the freshwater infusoria
of the island of Bombay. No. 1. Organization.
Ann. and Magaz. Nat. Hist. Ser. II 18:115—132, 221w
249.

Clark, R. L. and Jensen, T. E. (1969) Ultrastructure of
akinete development in a blue—green alga,
Cylindrospermum sp. Cytologia 34: 439—448.

96

97

Conley, R. T. (1972) Infrared spectrosc0py. 2nd ed.
Allyn and Bacon, Inc., Boston, MA.

 

Dharmawardene, M. W. N., Haystead, A. and Stewart, W.£L P.
(1973) Glutamine synthetase of the nitrogen-fixing
alga Anabaena cylindrica. Arch. Mikrobiol. 90:281-
295.

Donze, M., Haveman, J. and Schiereck, P. (1972) Absence
of Photosystem 2 in heterocysts of the blue-green
alga Anabaena. Biochim.Biophys. Acta 256:157-161.

Drawert, H. and Tischer, I. (1956) Uber Redox-Vorgange
bei Cyanophyceen unter besonderer Berficksichtigung
der Heterocysten. Naturwissenschaften 43:132.

Drews, G. (1955) Zur Frage der TTC-Reduktion durch
Cyanophyceen. Naturwissenschaften 42:646.

 

Dunn, J. H. and Wolk, C. P. (1970) Composition of the
cellular envelopes of Anabaena cylindrica. J.
Bacteriol. 103:153-158.

Fay, P. (1969a) Metabolic activities of isolated spores
of Anabaena cylindrica. J. Exp. Bot. 20:100—109.

Fay, P. (1969b) Cell differentiation and pigment
composition in Anabaena cylindrica. Arch. Mikrobiol.
67:62-70.

Fay, P. and Kulasooriya, S. A. (1972) Tetrazolium
reduction and nitrogenase activity in heterocystous
blue-green algae. Arch.Mikrobiol. 87:341-352.

Fay, P”.Stewart, W. D. P., Walsby, A. E. and Fogg, G. E.
(1968) Is the heterocyst the site of nitrogen
fixation in blue-green algae? Nature, Lond. 220:
810-812.

Fay, P. and Walsby, A. E. (1966) Metabolic activities
of isolated heterocysts of the blue-green alga
Anabaena cylindrica. Nature, Lond. 209:94-95.

Fisher, R. W. and Wolk, C. P. (1976) Substance
stimulating the differentiation of Spores of the
blue-green alga Cylindrospermum licheniforme.
Nature, Lond. 259:394-395.

Fogg, G. E. (1952) The production of extracellular
nitrogenous substances by a blue-green alga. Proc.
Roy. Soc. Lond. B 139:372-397.

98

Fogg, G. E. (1962) Extracellular products. In:
Lewin, R. A. (Ed.) Physiology and Biochemistry of
Algae. Pp. 475-489. Academic Press, New York.

 

Fogg, G. E. (1966) The extracellular products of algae.
Oceanogr. Mar. Biol. Rev. 4:195-212.

Fritsch, F. E. (1904) Studies on Cyanophyceae.
III. Some points in the reproduction of Anabaena.
New Phytol. 3:216-228.

Fritsch, F. E. (1951) The heterocyst: a botanical
enigma. Proc. Linn. Soc. Lond. 162:194-211.

Fujita, Y., Ohama, H. and Hattori, A. (1964) Hydrogenase
activity of cell-free preparation obtained from the
blue-green alga, Anabaena cylindrica. Plant & Cell
Physiol. 5:305-314.

Geitler, L. (1932) Cyanophyceae, Aufl. 2, Akademische
Verlagesellshaft,.. , Leipzig.

Gentile, J. H. and Maloney, T. E. (1969) Toxicity and
environmental requirements of a strain of
Aphanizomenon flos-aquae (L.) Ralfs. Can. J.
Microbiol. 15:165-173.

Glade, R. (1914) Zur Kenntnis der Gattung
Cylindrospermum. Beitr. Biol. Pfl. 12:295-346.

Gorham, P. R. (1964) Toxic algae. In: Jackson, D. F.
(Ed.) Algae and Man. Pp. 307-336. Plenum Press, NY.

 

Harder, R. (1917) Ernahrungsphysiologische Untersuchung-
en an Cyanophyceen, hauptsachlich dem endophytischen
Nostoc punctiforme. Z. Bot. 9:145-242.

Hartman, R. T. (1960) Algae and metabolites of natural
waters. In: Tyron, C. A., Jr. and Hartman, R. T.
(Eds.) The Ecology of Algae. Pp. 38-55. Spec. Publ.
No. 2, Pymatuning Laboratory of Field Biology,
University of Pittsburgh.

 

Jackim, E. and Gentile, J. (1968) Toxins of a blue-
green alga: similarity to saxitoxin. Science 162:
915-916.

Jakob, H. (1961) Compatibilités, antagonismes et
antibioses entre quelques algues du sol. Rev. gén.

99

Jensen, T. E. and Clark, R. L. (1969) Cell wall and
coat of the developing akinete of a Cylindrospermum
species. J. Bacteriol. 97:1494-1495.

Jfittner, F. and Carr, N. G. (1976) The movement of
organic molecules from vegetative cells into the
heterocysts of Anabaena cylindrica. In: Codd,

G. A. and Stewart, W. D. P. (Eds.) Proc. Second
Internat. Symp. on Photosynthetic Prokaryotes. Pp.
121-123. FEMS, Dundee.

Leak, L. V. and Wilson, G. B. (1965) Electron micro-
scopic observations on a blue-green alga, Anabaena
sp. Can. J. Genet. Cytol. 7:237-249.

Lex, M. and Carr, N. G. (1974) The metabolism of
glucose by heterocysts and vegetative cells of
Anabaena cylindrica. Arch. Microbiol. 101:161-167.

Lockau, W., Peterson, R. B., Wolk, C. P. and Burris, R.IL
(1978) Modes of reduction of nitrogenase in
heterocysts isolated from Anabaena species.

Biochim. Biophys. Acta 502:298-308.

Mackinney, G. (1944) Absorption of light by chlorophyll
solutions. J. Biol. Chem. 140:315-322.

Meeks, J. C., Walk, C. P., Thomas, J., Lockau,‘W.,Shaffen
P. W., Austin, J. M., Chien, W.-S. and Galonsky, A.
(1977) The pathways of assimilation of 13NH4+ by the
cyanobacterium, Anabaena cylindrica. J. Biol. Chem.
252:7894-7900.

Meeks, J. C., Wolk, C. P., Lockau, W., Shilling, N.,
Shaffer, P. W. and Chien, W.-S. (1978) Pathways of
assimilation of [13N]N and NH4+ by cyanobacteria
with and without heter6cysts. J. Bacteriol. 134:
125-130.

Miller, M. M. and Lang, N. J. (1968) the fine structure
of akinete formation and germination in Cylindro—
spermum. Arch. Mikrobiol. 60:303-313.

Mitchison, G. J. and Wilcox, M. (1973) Alteration in
heterocyst pattern of Anabaena produced by
7-azatryptophan. Nature New Biol. 246:229-233.

Pattnaik, H. (1966) Studies on nitrogen fixation by
Westiellopsis prolifica Janet. Ann. Bot.30:231-238.

Paudler, W. W. (1971) Nuclear Magnetic Resonance. Allyn
and Bacon, Inc., Boston, MA.

 

Peterson, R. B. and Burris, R. H. (1976) Properties of
heterocysts isolated with colloidal silica. Arch.
Microbiol. 108:35-40.

 

100

Peterson, R. B. and Wolk, C. P. (1978a) Localization
of and uptake hydrogenase in Anabaena. Plant
Physiol. 61:688-691.

Peterson, R. B. and Wolk, C. P. (1978b) Localization
and activity of nitrogenase in heterocysts isolated
from Anabaena variabilis by a new procedure. Abst.
3rd Int. Symp. N2 Fixation, Madison, WI.

Piskornik, Z. and Bandurski, R. S. (1972) Purification
and partial characterization of a glucan containing
indole-3-acetic acid. Plant Physiol. 50:176-182.

Prescott, G. W. (1960) Biological disturbances resulting
from algal populations in standing waters. In:
Tryon, C. A., Jr. and Hartman, R. T. (Eds.) The
Ecology of Algae. Pp. 22-37. Spec. Publ. No. 2,
Pymatuning Laboratory of Field Biology) University
of Pittsburgh.

 

Quisenberg, K. S., Scolma , T. T. and Nier, A. O. (1956)
Atomic mass of H1, D , C12, and S3 . Phys. Rev.
102:1071-1075.

Rippka, R., Neilson, A., Kunisawa, R. and Cohen-Bazire,
G. (1971) Nitrogen fixation by unicellular blue-
green algae. Arch. Nikrobiol. 76:341-348.

Rippka, R. and Stanier, R. Y. (1978) The effects of
anaerobiosis on nitrogenase synthesis and hetero-
cyst development in nostocacean cyanobacteria.

J. Gen. Microbiol. 105:83-94.

Roper, R. and MA, T. S. (1957) A new microdistillation
technique. Microchem. J. 1:248-251.

Simon, R. D. (1971) Cyanophycin granules from the blue-
green alga Anabaena cylindrica: a reserve material
consisting of copolymers of aspartic acid and
arginine. Proc. Nat. Acad. Sci., U.S.A. 68:265-267.

Simon, R. D. (1977a) Macromolecular composition of
spores from the filamentous cyanobacterium Anabaena
cylindrica. J. Bacteriol. 129:1154-1155.

Simon, R. D. (1977b) Sporulation in the filamentous
cyanobacterium Anabaena cylindrica. The course of
spore formation. Arch. Microbiol. 111:283-288.

Simpson, F. B. and Neilands, J. B. (1976) Siderochromes
in Cyanophyceae: Isolation and characterization of
schizokinen from Anabaena sp. J. Phycol. 12:44-48.

 

101

Singh, H. N. (1967) Genetic control of sporulation in
the blue-green alga Anabaena doZioZum Bharadwaja.
Planta 75:33-38.

Singh, H. N. and Srivastava, B. S. (1968) Studies on
morphogenesis in a blue-green alga. I. Effect of
inorganic nitrogen sources on developmental
morphology of Anabaena doZioZum. Can. J. Microbiol.
14:1341-1346.

Singh, R. N. and Sinha, R. (1965) Genetic recombination
in a blue-green alga, Cylindrospermum majus Kuetz.
Nature, Lond. 207:782-783.

Smith, R. V., Noy, R. J. and Evans, M. C. W. (1971)
Physiological electron donor systems to the
nitrogenase of the blue-green alga Anabaena cylin-
drica. Biochim. Biophys. Acta 253:104-109.

Stewart, W. D. P. and Codd, G. A. (1975) Polyhedral
bodies (carboxysomes) of nitrogen-fixing blue-green
algae. Br. phycol. J. 10:273-278.

Stewart, W. D. P., Haystead, A. and Pearson, H. W. (1969)
Nitrogenase activity in heterocysts of blue-green
algae. Nature, Lond. 224:226-228.

Stewart, W. D. P. and Lex, M. (1970) Nitrogenase activ-
ity in the blue-green alga Plectonema boryanum
strain 594. Arch. Mikrobiol. 73:250-260.

Talpasayi, E. R. S. (1963) Polyphosphate containing
particles of blue-green algae. Cytologia 28:76-80.

Talpasayi, E. R. S. and Bahal, M. R. (1967) Cellular
differentiation in Anabaena cylindrica. Z.
Pflanzenphysiol. 56:100-101.

Tel-Or, E., Luijk, L. W. and Packer, L. (1977) An
inducible hydrogenase in cyanobacteria enhances
N2 fixation. FEBS Letters 78:49-52.

Tel-Or, E. and Stewart, W. D. P. (1975) Manganese and
photosynthetic oxygen evolution by algae. Nature,
Lond. 258:715-716.

Tel-Or, E. and Stewart, W. D. P. (1977) Photosynthetic
components and activities of nitrogen-fixing isolated

heterocysts of Anabaena cylindrica. Proc. Roy. Soc.
Lond. B 198:61-86.

 

102

Thomas, J. (1970) Absence of the pigments of photo-
system II of photosynthesis in heterocysts of a
blue-green alga. Nature, Lond. 228:181-183.

Thomas, J., Meeks, J. C., Wolk, C. P., Shaffer, P. W.,
Austin, S. M. and Chien, W.-S. (1977) Formation
of glutamine from [13N1ammonia, [13N]dinitrogen, and
[14c1glutamate by heterocysts isolated from Anabaena
cylindrica. J. Bacteriol. 129:1545-1555.

Tyagi, V. V. S. (1974) Some observations on the pattern
of sporulation in a blue-green alga, Anabaena
doliolum. Ann. Bot. 38:1107-1111.

Ueda, K. (1971) Die quantitative Bestimmung des DNS-
Gehalts in den Zellen von Cyanophyceen durch
Fluorochromierung mit Coriphosphin. Biochem.
Physiol. Pflanzen 162:439-449.

Ueda, K. and Sawada, M. (1972a) A cytochemical study of
DNA in cells of Cyanophyceae. Proc. Seventh Internat.
Seaweed Symp., pp. 201-204, John Wiley & Sons, NY.

Ueda, K. and Sawada, M. (1972b) DNA-content of akinete-
cells in Cylindrospermum. Cytologia 37:519-523.

Walsby, A. E. (1974a) The extracellular products of
Anabaena cylindrica Lemm. I. Isolation of a
macromolecular pigment-peptide complex and other
components. Br. phycol. J. 9:371-381.

Walsby, A. E. (1974b) The extracellular products of
Anabaena cylindrica Lemm. II. Fluorescent sub-
stances containing serine and threonine, and their
role in extracellular pigment formation. Br.
phycol. J. 9:383-391.

Walsby, A. E. and F099, G. E. (1975) The extracellular
products of Anabaena cylindrica Lemm. III. Excre-
tion and uptake of fixed nitrogen. Br. phycol. J.
10:339-345.

Whitton, B. A. (1965) Extracellular products of blue-
green algae. J. Gen. Microbiol. 40:1-11.

Whitton, B. A. (1967) Studies on the toxicity of
polymyxin B to blue-green algae. Can. J. Microbiol.
13:987-993.

 

103

Wildon, D. C. and Mercer, F. V. (1963) The ultra-
structure of the heterocyst and akinete of the
blue-green algae. Arch. Mikrobiol. 47:19-31.

Winkenbach, F. and Wolk, C. P. (1973) Activities of
enzymes of the oxidative and the reductive pentose
phosphate pathways in heterocysts of a blue-green
alga. Plant Physiol. 52:480-483.

Wolk, C. P. (1964) Experimental studies on the
development of a blue-green alga. Ph.D. Thesis,
Rockefeller Institute, NY.

Wolk, C. P. (1965) Control of sporulation in a blue-
green alga. Develop. Biol. 12:15-35.

Wolk, C. P. (1966) Evidence of a role of heterocysts
in the sporulation of a blue-green alga. Am. J.
Bot. 53:260-262.

Wolk, C. P. (1968) Movement of carbon from vegetative
cells to heterocysts in Anabaena cylindrica.
J. Bacteriol. 96:2138-2143.

Wolk, C. P. (1979) Heterocysts, 13N, and Nz-fixing
plants. Proc. 3rd. Int. Symp. N2 Fixation, Madison,
WI. In press.

Wolk, C. P., Austin, S. M., Bortins, J. and Galonsky, A.
(1974) Autoradiographic localization of 13N after
fixation of 13N-labeled nitrogen gasbya heterocyst-
forming blue-green alga. J. Cell Biol. 61:440-453.

Wolk, C. P. and Simon, R. D. (1969) Pigments and lipids
of heterocysts. Planta 86:92-97.

Wolk, C. P., Thomas, J., Shaffer, P. W., Austin, S. M.
and Galonsky, A. (1976) Pathway of nitrogen
metabolism after fixation of 13N-labeled nitrogen
gas by the cyanobacterium, Anabaena cylindrica.
J. Biol. Chem. 251:5027-5034.

Wolk, C. P. and Wojciuch, E. (1971) Photoreduction of
acetylene by heterocysts. Planta 97:126-134.

Yamamoto, Y. (1972) The fatty acid composition of
akinetes, heterocysts and vegetative cells in
Anabaena cylindrica. Plant & Cell Physiol. 13:
913-915.

 

104

Yamamoto, Y. (1975) Effect of desiccation on the
germination of akinetes of Anabaena cylindrica.
Plant & Cell Physiol. 16:749-752.

Zastrow, E. M. von (1953) Uber die Organisation der
Cyanophyceenzelle. Arch. Mikrobiol. 19:174-205.