FEMS MicrobiologyLetters 50 (1988) 11-16
Published by Elsevier
11
FEM 03130
Distribution of sym-homospermidinein eubacteria, cyanobacteria,
algae and ferns
Koei H a m a n a
a
Shigeru M a t s u z a k i b a n d M a s a k i S a k a k i b a r a c
a College of Medical Care and Technology and b Institute of Endocrinology, Gunma University, Maebashi, Gunma, Japan,
and c BiochemicalLaboratory, DIC Ltd., Ichihara, Japan
Received and accepted 2 December1987
Key words: Polyamine; Homospermidine; Nitrogen-fixing eubacterium; Cyanobacterium; Alga; Fern
1. SUMMARY
Polyamines were analyzed in 12 species of N 2fixing aerobic eubacteria and other eubacteria,
cyanobacteria, algae and ferns, s y m - H o m o spermidine (homospermidine) was found to be
widely distributed as a major polyamine in various
Nz-fixing eubacteria which belong to Azospirillum,
Agromonas,
Beijerinckia,
Bradyrhizobium,
Rhizobium and Xanthobacter. 3 species of Azotobacter contained spermidine but not homospermidine, though they are Nz-fixing eubacteria.
Homospermidine is also distributed in some
eubacteria, i.e., the photosynthetic Rhodopseudomonas rutila and the sulfur-oxidizing Thiobacillus denitrificans, a cyanobacterium, Synechococcus
sp., and in the cyanobacterium-symbiotic ferns,
Azolla imbricata and Azolla japonica.
2. INTRODUCTION
Homospermidine [NH2(CH2)4NH(CH2) 4
NH2] has been found as a major polyamine in
many N2-fixing species of cyanobacteria [1,2] and
Rhizobiurn, a N2-fixing eubacterium [3]. These
Correspondence to: Koei Hamana, Collegeof Medical Care and
Technology, Gunma University,Maebashi, Gunma 371 Japan.
findings suggested a possible correlation between
N 2 fixation and the presence of homospermidine
[1]. This triamine is rarely present in heterotrophic
eubacteria [4,5], but present as a predominant
polyamine in some photosynthetic eubacteria
[6-8], a sulfur-oxidizing eubacterium [9], and some
methanogenic archaebacteria [10]. Previously we
reported that some autotrophic bacteria which are
able to fix N 2 contained homospermidine as their
major polyamine [1,6]. Homospermidine is
sporadically detected in eukaryotic algae and ferns
as a minor polyamine [11-13]. In the present
study we have analyzed polyamines in various
N2-fixing, photosynthetic or sulfur-oxidizing
eubacteria, thermophilic or halophilic cyanobacteria and green algae, and cyanobacterium-symbiotic ferns. The aim of this study was to examine
the relationship between the distribution of homospermidine and the ability to fix nitrogen or autotrophic growth activity in these organisms. Polya m i n e s in an a c i d o p h i l i c e u b a c t e r i u m ,
Acidiphilium, were also analyzed.
3. MATERIALS AND METHODS
3.1. Cultures and growth conditions
Azospirillum brasilense (JCM1224), Azospirillurn lipoferum (JCM1247), Xanthobacter autotrophicus (JCM1202), Xanthobacter flavus (JCM
0378-1097/88/$03.50 © 1988 Federationof European MicrobiologicalSocieties
12
1204), Klebsiella oxytoca (JCM1655) and
Klebsiella pneumoniae (JCM1662) were heterotrophically grown in a nutrient medium containing meat extract and polypeptone (NM) [14].
Agromonas oligotrophica (JCM1494) was cultured
in a diluted nutrient medium (DNM) [15]. Yeast
extract-mannitol medium (YM) [16] a n d / o r
nitrogen-free mannitol medium (M) (No. 207
medium on the List of Cultures of Institute for
Fermentation, Osaka, 1984) were used for the
culture of Azotobacter beijerinckii (IAM12683),
Azotobacter chroococcum (IFO12994), Azotobacter
t,inelandii (IAM1078), Bradyrhizobium japonicum
(IAM12034), Rhizobium rneliloti (IAM12035) and
Rhizobium trifolii (IFO13337). The yeast extractglucose medium (YG) (No. 15B medium on the
Catalogue of Institute of Applied Microbiology,
University of Tokyo, 1986) and synthetic NH4C1containing mineral medium (SM) supplemented
with 0.2% sodium succinate (SM-1) [17] were used
for Beijerinckia indica (IAMl195). The microorganisms were cultured aerobically at 30 °C and
then harvested at 3 days of culture. For heterotrophic growth of the 2 species of Xanthobacter,
SM was supplemented with 0.2% sodium succinate
[17]. For chemolithotrophic growth, 0.05%
NaHCO 3 was added to SM and incubated under
an atmosphere of 2% O2/10% CO2/28% N2/60%
H 2 (SM-2) [17]. For growth on nitrogen-free
medium, NH4C1 was omitted, 0.2% sodium succinate was added to SM and the cultures were
incubated under 2% 02/98% N 2 (SM-3) [14].
Rhodopseudomonas rutila (JCM2524) was grown
in yeast e x t r a c t / m a l a t e / g l u t a m a t e medium
(YMG) or malate/glutamate containing 199
medium (MG-199) at 30°C in the light under
anaerobic conditions [18].
T. denitrificans (JCM3869) and Thiobacillus
neapolitanus (JCM3861) were cultured chemolithotrophically in inorganic medium containing
0.1% Na2S203, S-8 medium or S-6 medium, respectively, or in organic medium containing yeast
extract and 0.1% Na2S203 (YT) [9] at 30°C.
An acidophilic eubacterium, Acidiphilium
cryptum (IFO14242) was heterotrophically cultivated in a medium containing (NH4)2SO4, KC1,
K2HPO4, MgSO4, polypeptone and glucose, pH
3.0 by aerobically static culture at 30 °C [19].
A thermophilic cyanobacterium, Synechococcus
sp., isolated from the Beppu Hot Springs, Kyushu,
Japan by Yamaoka et al. [20], was grown photoautotrophically in an inorganic medium with illumination at 55 ° C [20]. The culture was continuously bubbled with air containing 5% CO 2. A
halophilic cyanobacterium, Aphanothece halophytica (7418), purified and numbered by R,Y. Stainer
[21], was cultured photoautotrophically in a high
salt-containing inorganic medium with illumination at 30°C [21]. Halophilic green algae,
Dunaliella bardawil (ATCC30861) and Dunaliella
sp, isolated from the Salton Sea (California,
U.S.A.) by DIC Ltd., were cultured photoautotrophically in inorganic medium containing 2 M
NaC1 at 25 °C in the light [22].
The cyanobacterium-symbiotic ferns, A. imbricata and A. japonica, isolated from the field in
Japan by Kito and Shiorni [23], were cultured in
inorganic medium in the light at 25 °C [23].
3.2. Polyamines
Homospermidine and thermospermine were
synthesized in our laboratories [12]. Aminopropylhomospermidine and canavalmine were kindly
supplied by Dr. K. Samejima of Josai University.
Aminopropylcadaverine was kindly supplied by
D.W. Worth of Parke-Davis (Detroit, MI, U.S.A.).
Norspermidine and norspermine were purchased
from Eastman Organic Chemicals (Rochester, NY,
U.S,A.). Diamines, spermidine and spermine were
purchased from Wako Pure Chemicals (Osaka,
Japan).
3.3. Analysis of polyamines
The pellets of organisms were repeatedly washed
with distilled water and then homogenized in equal
volumes of cold 1.0 M HC104. Polyamines in
HC104 extracts were analyzed by high-performance liquid chromatography (HPLC) on an ionexchange column (Kyowa Seimitsu, 62210F) [24].
The HPLC patterns show no change after hydrolysis in the presence of 6 M HC1 at l l 0 ° C for 24 h
or with 2 M Ba(OH)2 at 120°C for 20 h. After
fractionation by column chromatography [25], the
identity of isolated polyamines was confirmed by
thin-layer chromatography (TLC) on cellulose
(Avicel SF, Funakoshi) with 2-propanol-ammonia
13
(7:3) [11] or silica gel (Merck) with n-butanol/
acetic acid/ pyridine/ formalin (3 : 3 : 2 : 1) [26]. To
confirm the identity of the polyamines, the HPLC
analysis was also performed before and after the
enzymatic cleavage using putrescine oxidase from
Micrococcus (Tokuyama Soda Co.) [27] and polyamine oxidase from Aspergillus (Amano Pharmaceutical Co.) [28].
4. RESULTS AND DISCUSSION
Two types of N2-fixing eubacteria were found
on the basis of their polyamine distribution patterns. One type, e.g. Beijerinckia indica (formerly
Azotobacter indicus) contained homospermidine as
major triamine, while another type, e.g.A, chroococcum, contained spermidine (Fig. 1).
H~,pa
1-A
Put
2-A
Spd
S~n
Put
$Pw,
U I-BPZ"
~
Pl
2-BA
PT
Sore
2-C
SPm
~
1-C
put
C~
Cad
__1
Fig. 1. Column chromatograms of polyamines in Beo'erinckia
indica (l-A) and Azotobacter chroococcum (2-A), and the products (P1 or P2) formed from the triamine (spermidine or
homospermidine) of B. indica (l-B) and A. chroococcum (2-B)
after putrescine oxidase treatment, and the products from the
triamines and tetraamine (spermine) of B. indica (l-C) and A.
chroococcum (2-C) after polyamine oxidase treatment. Elution
patterns were followed by o-phthalaldehyde. Abbreviations:
Put, putrescine; Cad, cadaverine; Spd, spermidine; Hspd, homospermidine; and Spm, sperrnine.
After the putrescine oxidase treatment of the
polyarnines, the P1 peak was formed as a product
of homospermidine and the P2 peak from
spermidine (Fig. 1; l-B, 2-B). Diamines were degraded, while spermine was not completely cleaved
by this enzyme. Putrescine was formed from homospermidine, spermidine and spermine, while diamines such as putrescine and cadaverine were
not cleaved by polyamine oxidase (Fig. 1, 1-C,
2-C). The data show that the triamines are
spermidine and homospermidine and that the diamines are putrescine and cadaverine. The identity of the triamines was further confirmed by two
different TLC methods (data not shown).
Some other aerobic N2-fixing eubacteria were
found to contain homospermidine as major polyamine (Table 1). These include symbiotic N2-fixing R. meliloti, R. trifolii, and Bradyrhizobium
japonicurn (formerly Rhizobium japonicum).
Rhizobium phaseoli and Rhizobiurn leguminosarum
have already been reported to contain homo°
spermidine as a major polyamine [3]. On the other
hand, spermidine was the major polyamine in the
3 species of Azotobacter (Table 1), though they are
typical aerobic Nz-fixing eubacteria. Homospermidine was not detected under either N2-nonfixing (YM medium) or N2-fixing (M medium)
growth conditions. Putrescine and spermidine were
found to be major polyamines in the N2-non-fixing strains of K. pneurnoniae and K. oxytoca (Table 2).
Xanthobacter, Azospirillum, and Bradyrhizobium
are not only N2-fixing eubacteria but also chemolithotrophic H2-oxidizing organisms [29]. In
Xanthobacter, homospermidine was constantly observed under heterotrophic (N2-non-fixing, SM-1
medium), chemolithotrophic (H2-oxidizing, N 2non-fixing, SM-2 medium), and N2-fixing (SM-3
medium) growth conditions. These findings suggest that N2-fixation and homospermidine synthesis are not directly related, though homospermidine was detected mostly in N2-fixing species of
both autotrophs and heterotrophs. Recently,
Kneifel et al. also found homospermidine in X.
autotrophicus [30].
We previously reported the occurrence of homospermidine in N2-fixing species of cyanobacteria and of photosynthetic eubacteria [1,61 and a
14
Table 1
Cellular concentrations of polyamines in N2-fixing eubacteria
Abbreviations: Put, putrescine; Cad, cadaverine; Spd, spermidine; Hspd, homospermidine; Spm, spermine; ND, not detectable;
IAM, Institute of Applied Microbiology, University of Tokyo; JCM, Japan Collection of Microorganisms, RIKEN (Wako, Saitama);
IFO, Institute of Fermentation, Osaka; ATCC, American Type Culture Collection; and DSM, Deutsche Sammlung yon
Mikroorganismen.
Organism
Polyarnines (/~mol/g wet weight)
Medium
Put
Cad
Spd
Hspd
Spm
A zotobacter beijerinckii
(IAM12683, ATCC19360)
YM
M
0.111
0.006
0.006
0.003
0.035
0.020
ND
ND
ND
ND
A zotobacter chroococcum
(IFO12994, ATCC9043)
YM
M
1.444
0.067
0.218
0.001
0.500
0.052
ND
ND
0.001
ND
A zotobacter t,inelandii
(IAM1078, ATCC9046)
YM
M
0.944
0.277
0.272
0.065
0.280
0.096
ND
ND
0.025
ND
Azospirillum brasilense
(JCM1224, ATCC29145)
NM
0.067
ND
0.230
0.310
0.010
Azospirillum lipoferum
(JCM1247, ATCC29708)
NM
0.305
ND
0.080
0.600
0.042
A grornonas oligotrophica
(JCM1494, ATCC43045)
DNM
0.250
ND
0.020
1.950
0.005
Be~erinckia indica
(IAM1195, ATCC9540)
YG
SM-1
0.029
0.050
0.003
ND
0.032
0.001
0.800
0.200
0.042
ND
Bradyrhizobium japonicum
(IAM12034)
YM
0.556
0.227
0.250
2.200
0.125
Rhizobium rneliloti
(IAM12035)
YM
0.444
0.109
0.520
2.600
0.200
Rhizobium trifolii
(IFO13337)
YM
0.178
0.027
0.200
2.250
0.030
Xanthobacter autotrophicus
(JCM1202, DSM432)
NM
SM-1
SM-1
SM-3
0.083
0.067
0.040
0.003
0.010
ND
ND
ND
0.046
0.010
0.004
0.010
0.260
0.860
0.184
0.720
0.020
0.010
ND
0.015
Xanthobacter fiat)us
(JCM1204, DSM338)
NM
SM-1
SM-2
SM-3
1.244
0.061
0.044
0.005
ND
ND
ND
ND
0.240
0.001
0.003
0.001
3.120
0.770
0.180
0.100
0.020
0.005
ND
ND
sulfur-oxidizing Thiobacillus novellus [9]. H o m o spermidine occurs as a predominant polyamine in
a N2-fixing photosynthetic Rhodopseudomonas
rutila, a sulfur-oxidizing T. denitrificans, and a
thermophilic cyanobacterium, Synecococcus sp.
(Table 2). Some strains belonging to Thiobacillus
and Synechococcus are also able to fix N 2 [2,31],
but the N2-fixing activity of the Thiobacillus and
Synechococeus used in this study have not been
tested. It is likely that the distribution of homospermidine is rather correlated to taxonomic differences in these photosynthetic, sulfur-oxidizing
or N2-fixing microorganisms than to their ability
to fix N 2 [2].
Homospermidine has been sporadically detected in eukaryotic algae [11,12] and ferns [13].
The triamine was detected also in a halophilic
green alga, Dunaliella, as a minor polyamine com-
15
Table 2
Cellular concentrations of polyamines in eubacteria, cyanobacteria, eukaryotic algae and ferns
Organism
N2-non-fixing eubacteria
Klebsiella oxytoca
(JCM1655, ATCC13182)
Klebsiella pneumoniae
(JCM1662, ATCC13883)
Polyamines (g m o l / g wet weight)
Medium
Put
Cad
Spd
Hspd
Spm
NM
0.444
0.800
0.260
ND
0.007
NM
0.455
0.743
0.150
ND
0.007
0.326
0.07l
ND
ND
0.209
0.055
0.690
0.400
0.020
ND
N2-fixing photosynthetic eubacterium
Rhodopseudornonas rutila
YMG
(JCM2524, ATCC33872)
MG-199
Sulfur-oxidizing eubacteria
Thiobacillus denitrificans
(JCM3869, ATCC23642)
YT
S-8
0.123
0.010
0.031
ND
0.050
ND
0.227
0.666
0.080
ND
Thiobacillus neapolitanus
(JCM3861, ATCC23640)
YT
S-8
0.876
0.886
0.115
0.009
0.045
0.003
ND
ND
0.035
ND
0.010
ND
0.629
ND
0.008
0.048
ND
0.024
0.058
ND
0.003
ND
0.296
ND
0.002
Dunaliella bardawil
(ATCC30861)
0.064
0.015
0.369
ND
0,002
Dunaliella sp. c
(DIC Ltd.)
0.108
0.001
0.584
0.016
0.006
0.062
0.369
ND
0.011
0.151
0.036
0.030
0.068
0.240
0.005
0.080
0.132
ND
ND
0.168
0.054
0.056
0.052
ND
ND
Acidophilic eubacterium
A eidiphilium crypturn
(IFO14242, ATCC33463)
Thermophilic cyanobacterium
Synechoeoccus sp. a
(Tokyo Univ.)
Halophilic cyanobacterium
Aphanothece halophytica b
(7418)
Halophilic eukaryotic algae
Cyanobacterium-symbiotic ferns
Azolla imbricata d (leaf)
(root)
Azolla japoniea a (leaf)
(root)
Isolated from the Beppu Hot Springs, Kyushu, Japan by Yamaoka et al. [20].
b Purified and numbered by Steiner [21] and maintained at Nagoya University.
c Isolated from Salton Sea (California, U.S.A.) by DIC Ltd.
d Isolated from the field in Japan by Kito and Shiomi [23].
p o n e n t a n d i n c y a n o b a c t e r i a ( A n a b a e n a azollae)s y m b i o t i c f e r n s , A z o l l a , as a m a j o r p o l y a m i n e , as
s h o w n i n T a b l e 2. H o w e v e r , A n a b a e n a is a n N 2-
fixing cyanobacterium and contains a high conc e n t r a t i o n o f h o m o s p e r m i d i n e [1].
M a j o r p o l y a m i n e o f t h e a c i d o p h i l e , Acidiphili-
16
um cryptum was spermidine. Homospermidine and
tetraamines were not detected in the eubacterium.
Homospermidine had not been found in
acidophilic archaebacteria belonging to Sulfolobus
and Thermoplasma [32,33], an acidophilic eubacterium, Bacillus acidocaldarius [34] and an
acidophilic eukaryotic alga, Cyanidium caldarium
[12], suggesting no correlation between the occurrence of homospermidine and the acidophily of
these microorganisms.
ACKNOWLEDGEMENTS
We are indebted to Institute of Applied Microbiology, University of Tokyo (IAM), Institute of
Fermentation, Osaka (IFO), Japan Collection of
Microorganisms (JCM), Dr. T. Akazawa of School
of Agriculture, Nagoya University, and Dr. K.
Satoh of College of Arts and Sciences, University
of Tokyo for kindly supplying bacteria. We thanks
also Dr. N. Shiomi of Radiation Center of Osaka
Prefecture for kindly supplying Azolla. Thanks
are also due to Drs. M. Okada and K. Isobe for
supplying putrescine oxidase and polyamine
oxidase, respectively.
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