Plant Cell Physiol. 46(3): 539–545 (2005)
doi:10.1093/pcp/pci045, available online at www.pcp.oupjournals.org
JSPP © 2005
Short Communication
Some Cyanobacteria Synthesize Semi-amylopectin Type α-Polyglucans
Instead of Glycogen
Yasunori Nakamura 1, 2, 8, Jun-ichiro Takahashi 2, Aya Sakurai 2, Yumiko Inaba 1, Eiji Suzuki 1, 2, Satoko
Nihei 3, Shoko Fujiwara 2, 3, Mikio Tsuzuki 2, 3, Hideaki Miyashita 4, Hisato Ikemoto 5, Masanobu Kawachi 6,
Hiroshi Sekiguchi 7 and Norihide Kurano 7
1
Faculty of Bioresource Sciences, Akita Prefectural University, Akita-City, 010-0195 Japan
CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012 Japan
3
Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, 192-0392 Japan
4
Department of Technology and Ecology, Kyoto University, Sakyo-ku, Kyoto, 606-8501 Japan
5
Beverage Development Department, Suntory Limited, Shimamoto-cho, Mishima-gun, Osaka, 618-8503 Japan
6
Environmental Biology Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, 305-8506 Japan
7
Marine Biotechnology Institute, Kamaishi, Iwate, 026-0001 Japan
2
;
glucosidic linkages are localized within the cluster so that α1,4-glucosidic side chains are regularly arranged to form a double helix (Kainuma and French 1972) when the degree of glucose polymerization (DP) of neighboring chains reaches more
than 10 (Gidley and Bulpin 1987). The tandem-cluster structure of amylopectin is considered to be synthesized by concerted
reactions catalyzed by three classes of enzymes, i.e. starch synthase, starch branching enzyme and starch debranching enzyme,
each of which is composed of multiple isozymes making a different contribution to the cluster structure (Nakamura 2002,
Ball and Morell 2003). In contrast, it is generally accepted that
glycogen can be synthesized by a single form of glycogen synthase and glycogen branching enzyme in animals and bacteria.
It has been reported that cyanobacteria synthesize glycogen while red algae produce floridean starch with a structure
that is intermediate between that of starch and glycogen, and
that green algae accumulate amylopectin-like polysaccharides
(Manners and Sturgeon 1982, Manners 1991, Ball and Morell
2003). These observations tempted us to postulate that the fine
structure of α-polyglucan evolved from the random-branched
structure of glycogen in cyanobacteria to the highly regulated
tandem-cluster structure of amylopectin in green plants, possibly because the acquisition of cluster structure in α-polyglucan
would be advantageous for organisms to survive. Therefore,
detailed analysis of changes in α-polyglucan structure in various algae could be of great help to elucidate the essential factors for the amylopectin structure.
The present study was conducted to examine the variation
of the fine structure of polyglucans in cyanobacteria by measuring chain length profiles for polyglucans by a fluorescencelabeled capillary electrophoresis method (O’Shea et al. 1998).
Table 1 summarizes the structural features of α-polyglucans in
cyanobacteria (26 species) and rice plants. Fig. 1A–D and
Table 1 show that most cyanobacterial polysaccharides had
It is widely accepted that green plants evolved the
capacity to synthesize the highly organized branched αpolyglucan amylopectin with tandem-cluster structure,
whereas animals and bacteria continued to produce random branched glycogen. Although most previous studies
documented that cyanobacteria accumulate glycogen, the
present study shows explicitly that some cyanobacteria such
as Cyanobacterium sp. MBIC10216, Myxosarcina burmensis and Synechococcus sp. BG043511 had distinct α-polyglucans, which were designated as semi-amylopectin. The
semi-amylopectin was intermediate between rice amylopectin and typical cyanobacterial glycogen in terms of chain
length distribution, molecular size and length of the most
abundant α-1,4-chain. It was also found that Cyanobacterium sp. MBIC10216 had no amylose-type component in its
α-polyglucans. The evolutionary aspect of the structure of
α-polyglucan is discussed in relation to the phylogenetic
evolutionary tree of 16S rRNA sequences of cyanobacteria.
Keywords: Amylopectin — Cyanobacteria — Glycogen —αPolyglucan — Starch.
Abbreviations: DP, degree of polymerization; MP, maximum parsimony; NJ, neighbor joining.
The structure of starch in higher plants differs from that of
glycogen in animals and bacteria in that amylopectin, a major
component of starch, has a highly organized structure designated as a tandem-cluster structure, in which the structural unit
clusters are tandem linked to form an amylopectin molecule
(Manners 1991, Gallant et al. 1997, Thompson 2000). The
length of the amylopectin cluster is almost constant among
plant species (Jenkins et al. 1993), and the positions of α-1,68
Correponding author: E-mail, [email protected]; Fax, +81-18-872-1681.
539
540
Some cyanobacteria synthesize semi-amylopectin
Table 1
Characterization of chain-length distribution of α-polyglucans from various cyanobacteria and rice
Species
Polyglucan structure
Chain
distribution
(area%)
Most abundant
Type of
chain (DP)
polyglucan
(ΣDP ≤8)
(ΣDP ≥37)
I. Cyanobacteria
1 a Anabaena (No stoc) sp. PCC7120
2 a Cyanobacterium sp. MBIC10216
3 a Dermocarpa sp.
4 a Dermocarpa sp.
5 a Dermocarpa sp.
6
Fischerella major
7 a LPP-group
8 a LPP-group
9 a LPP-group
10
Myxosarcina burmensis
11
Oscillatoria limnetica
12 a Phormidium sp.
13 a Phormidium sp.
14 a Phormidium sp.
15 a Phormidium sp. MBIC10025 (7-OP14)
16
Spirulina platensis
17 a Synechococcus sp.
18 a Synechococcus sp.
19 a Synechococcus sp.
20 a Synechococcus sp.
21 a Synechococcus sp.
22 a Synechococcus sp.
23 a Synechococcus sp.
24 a Synechococcus sp. BG043511
25 a Synechococcus sp. PCC7942
26 a Synechocystis sp. PCC6803
6
11
8
8
8
8
6
4
6
10
6
6
6
8
7
6
6
8
6
6
6
8
8
11
6
6
49.5
14.8
39.3
34.6
36.3
33.8
69.5
74.6
40.1
22.9
46.9
50.6
40.4
40.4
55.9
42.3
36.2
36.9
50.9
41.9
41.4
38.0
32.0
15.4
61.7
56.5
0.31
4.62
0.38
0.52
0.00
0.99
0.00
0.00
0.51
3.82
0.64
0.25
0.19
0.22
0.49
0.30
0.00
0.25
0.20
0.00
0.14
0.21
0.29
3.26
0.00
0.00
Glycogen
Semi-amylopectin
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Semi-amylopectin
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Glycogen
Semi-amylopectin
Glycogen
Glycogen
II. Rice (Oryza sativa, L)
Kinmaze
EM914 (sugary-1 mutant)
11
9
7.42
26.6
6.71
0.80
Amylopectin
Phytoglycogen
Strain number
PCC7120
MBIC10216
MBIC10001
MBIC10766
MBIC10768
NIES-N-592
MBIC10086
MBIC10087
MBIC10597
IAM-M-246
NIES-N-36
MBIC10003
MBIC10070
MBIC10076
MBIC10025
IAM-M-135
MBIC10073
MBIC10079
MBIC10083
MBIC10089
MBIC10456
MBIC10459
MBIC10770
b
PCC7942
PCC6803
The parameters for polyglucan structure were obtained by the same methods as described in Fig. 1.
Strain Collections: IAM, Institute of Applied Microbiology of the University of Tokyo (Japan); MBIC, Marine Biotechnology Institute Culture
Collection (Japan); NIES, National Institute of Environmental Studies (Japan); PCC, Pasteur Culture Collection (France).
a
Strains used for the 16S rRNA gene sequence analysis.
b
See Ikemoto and Mitsui (1994).
the chain length distribution pattern typical for glycogen, differing from typical amylopectin found in rice cv. Kinmaze, but
rather similar to phytoglycogen in the isoamylase1-deficient
sugary-1 mutant line EM914 of rice (Nakamura et al. 1997).
However, three cyanobacteria species, namely Synechococcus
sp. BG043511 (Ikemoto and Mitsui 1994), Cyanobacterium sp.
MBIC10216 (formerly Synechocystis aquatilis SI-2) and Myxosarcina burmensis, had clearly different chain distributions for
these polyglucans. Since glycogen lacked the unit structure-like
cluster in the amylopectin molecule, it contained only a small
fraction of long chains with a DP ≥37 (<1% of the total α-1,4chains), while short chains with DP ≤8 were enriched (32–
75%) (Table 1). On the contrary, the polysaccharides in the
three cyanobacteria contained significant amounts of the long
chains of DP ≥37, and the proportion ranging from 3.3 to 4.6%
of the total chains was intermediate between those in amylopectin of rice endosperm (6.7%) and glycogen in other cyanobacteria and phytoglycogen, as shown in Table 1. Based on
these observations, the fine structure of these three cyanobacteria polyglucans was distinct from that of typical cyanobacterial
glycogen (Fig. 1E). The most abundant chains in these three
cyanobacteria were markedly longer (DP10–11) than those
(DP4-8) in glycogen of most cyanobacteria (Table 1). The results
indicate that the structure of these three cyanobacterial polyglu-
Some cyanobacteria synthesize semi-amylopectin
541
Fig. 1 Comparison of chain length distribution of α-polyglucans in cyanobacteria and rice. Polyglucans were debranched by Pseudomonas amyloderamosa isoamylase and their isoamylolysates were labeled with 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) at their reducing ends. The
APTS-labeled α-1,4-glucans were separated by capillary electrophoresis by the method of O’Shea et al. (1998) and quantified on a molar basis, as
described previously (Wong et al. 2003). The peak area of a fraction of α-1,4-chain with a specific chain length was calculated as a percentage of
total peak area up to a degree of polymerization (DP) of 70. The chain length distributions in α-polyglucans from rice (wild-type japonica cultivar
Kinmaze) endosperm (A), Synechococcus sp. PCC7942 (B) and Cyanobacterium sp. MBIC10216 (C) are shown. (D) Differences in the proportions of α-polyglucan chain lengths between Synechococcus sp. PCC7942 or Cyanobacterium sp. MBIC10216 and rice (cv. Kinmaze). Numbers
on top of bars in (A–D) indicate DP values. (E) The relationship between the proportion of long α-1,4-glucan chains with DP ≥37 and that of
short α-1,4-glucan chains with DP ≤8 in cyanobacteria polyglucans and rice amylopectin and phytoglycogen. The organisms examined were the
same as in Table 1. #2, Cyanobacterium sp. MBIC10216; #10, Myxosarcina burmensis; #24, Synechococcus sp. BG043511; #25, Synechococcus
sp. PCC7942.
cans was intermediate between those of amylopectin and typical
glycogen or phytoglycogen, and we designated the polyglucan
as ‘semi-amylopectin’. Cyanobacterium sp. MBIC10216 was
selected as a representative cyanobacterium with the semiamylopectin, and we used its polyglucan for further analysis.
Since the size of amylopectin is known to be larger than
that of glycogen or phytoglycogen (Wong et al. 2003), the
MBIC10216 polyglucan was subjected to Sephacryl S-1000SF
gel filtration chromatography (Fig. 2A). The MBIC10216 polyglucan eluted faster than Synechococcus sp. PCC7942 and
542
Some cyanobacteria synthesize semi-amylopectin
Fig. 2 (A) Sephacryl S-1000 SF gel filtration chromatography of αpolyglucans in cyanobacteria, amylopectin in rice (cv. Kinmaze) and
phytoglycogen in rice sugary-1 mutant EM914. The polyglucans were
applied onto a Sephacryl S-1000 (Pharmacia Biotech) column (2 cm
diameter; 60 cm length), and eluted with the 0.1 M NaOH, 0.2% NaCl
solution. Fractions were taken at about 6.6 ml intervals and the total
sugar content in each fraction (shown by the vertical axis) was measured by an enzymatic method (Nakamura and Miyachi 1982). X shows
the λmax value of the absorption spectrum of iodine–polyglucan complex in each fraction, as described by Nakamura et al. (1997). (B) Toyopearl HW55S gel filtration chromatography of α-1,4-glucans after
debranching treatment of α-polyglucans with Pseudomonas amyloderamosa isoamylase. The polyglucans were debranched by treatment
with P. amyloderamosa isoamylase and the resulting linear polyglucans were applied onto a Toyopearl HW55S (Tosoh Corporation) column (2 cm diameter; 60 cm length), and eluted with the 0.1 M NaOH,
0.2% NaCl solution. Fractions were taken at 1.0 ml intervals and the
total sugar content in each fraction as shown by the vertical axis was
measured by an enzymatic method (Nakamura and Miyachi 1982).
Synechocystis PCC6803 glycogen-type polyglucans, as fast as
rice amylopectin. It is interesting to note that the phytoglycogen of the sugary-1 mutant endosperm of rice was slightly
larger in size than cyanobacterial glycogen.
Amylose, the additional component of starch, is basically
composed of linear α-1,4-glucosidic linkages, and is characterized by its distinct absorption of visible light in an iodine solution with a λmax value about 600–630 nm, as shown in rice
starch preparations (Fig. 2A). In contrast, the Cyanobacterium
sp. MBIC10216 polyglucan did not show such a λmax value,
suggesting the absence of an amylose-type constituent in the
cyanobacterium. The presence of amylose-type linear polyglucans was examined by Toyopearl HW55S (Tosoh Corporation,
Tokyo, Japan) gel filtration chromatography of debranched α1,4-glucan chains after treatment of total polyglucans with isoamylase. The amylose in rice starch eluted first as a peak
because it is basically a non-branched linear polyglucan,
whereas no such peak was found in total debranched polyglucans of Cyanobacterium sp. MBIC10216 (Fig. 2B), indicating
that the cyanobacterium has no amylose-type polyglucan. In
addition, the amylose–iodine complex specific high λmax value
at about 600–630 nm was not found in fractions 25–27 of
Cyanobacterium sp. MBIC10216 polyglucans (Fig. 2B).
Fredrick (1951) reported that the absorption spectrum of
the iodine–polyglucan complex in Oscillatoria princes exhibited a single peak with λmax of 550 nm, the maximum absorption peak value being distinctly higher than that in animal
glycogen (420 nm) and comparable with that of the Cyanobacterium sp. MBIC10216 polyglucan (Fig. 2A). Although they
did not measure the chain length profile for the O. princes polyglucan, the results suggest that O. princes may also produce
semi-amylopectin.
In summary, the present results established that some
cyanobacteria such as Synechococcus sp. BG043511, Cyanobacterium sp. MBIC10216 and M. burmensis have semi-amylopectin that is clearly different from typical glycogen found in
most cyanobacteria in terms of the chain length profile and
molecular size, but is an intermediate-type polyglucan between
glycogen and amylopectin.
In an attempt to infer the phylogenetic positions of the
cyanobacteria that produce semi-amylopectin, we determined
the 16S rRNA gene sequences of 22 species of cyanobacteria
marked in Table 1 and constructed phylogenetic trees using
these sequences and those obtained from the DNA data bank of
Japan (Fig. 3). Sequences from 85 cyanobacterial strains, and
Agrobacterium tumefaciens and Rhodospirillum rubrum as the
Fig. 3 NJ tree of 16S rRNA sequences from cyanobacteria. Numbers shown at the branches are bootstrap percentages using NJ (left) and MP
(right). Cyanobacteria subjected to the 16S rRNA gene sequence analysis in this study are shown in blue, and those with semi-amylopectin are
boxed. Percentages are shown only for the branches having credibility values >80%. The scale bar is for 0.01 substitutions per site. Taxa marked
with # are listed in Table 1. Algal cells harvested by centrifugation were homogenized using FastPrep (Qbiogene, U.S.A.). Genomic DNA was
extracted using FastDNA kit (Qbiogene, U.S.A.). The almost complete 16S rDNA gene was amplified by PCR protocols with the primer set of
Miyashita et al. (2003). PCR products, purified by QIAquick gel extraction kit (QIAGEN Sciences, U.S.A.), were directly sequenced using with a
DNA Analyzer 3730 (Applied Biosystems, U.S.A.).
Some cyanobacteria synthesize semi-amylopectin
543
544
Some cyanobacteria synthesize semi-amylopectin
outgroup, were first aligned by Clustal X (1.83) and manually
refined. Ambiguous sequences were removed, and finally 1218
sites were used for the phylogenetic analysis. Two independent
types of data analysis were performed using PAUP* (4.0b10):
neighbor joining (NJ) and maximum parsimony (MP). The tree
demonstrates seven major evolutionary lineages in cyanobacteria as detected by Honda et al. (1999), and suggests that the
cyanobacteria with the semi-amylopectin are all included in
group 5 on the assumption that the 16S rRNA gene sequence of
M. burmensis is the same or very similar to that of Myxosarcina sp. PCC7312 or Myxosarcina sp. PCC7325 (Fig. 3).
The bootstrap analysis of the phytogenetic tree was done
with 1,000 bootstrap replicates each by NJ and MP methods.
Relatively high bootstrap values were obtained in group 5. This
group corresponds to the SY/PLEU/MY (Synechocystis/Pleurocapsa/Microcystis) subtree of Turner’s grouping (Turner
1997), which is the largest group and contains both unicellular
(e.g. Synechocystis sp. PCC6803) and non-heterocyst-forming
filamentous strains (e.g. Oscillatoria and Spirulina). The relatively weak support for the monophyly of this group was
reported by Turner (1997). Nevertheless, comprehensive phylogenetic studies done by Honda et al. (1999) and Miyashita et al.
(2003), together with the present results (Fig. 3) evidently
show the topological similarity. Honda et al. (1999) referred to
this group as a single evolutionary lineage.
Waterbury and Rippka (1989) suggested that Synechococcus sp. BG043511 should be classified under the genus Cyanothece. The phylogenetic tree generated in the present study
strongly supports their suggestion.
Cyanobacterium sp. MBIC10216 is characterized by an
extracelullar polysaccharide layer visible under the microscope, while the type strain of this genus, Cyanobacterium
stanieri, has no such obvious feature (Rippka et al. 2001).
Reddy et al. (1993) isolated two marine unicellular cyanobacteria, Cyanothece sp. strain BH63 and strain BH68, from intertidal sands of the Texas Gulf coast. Strain BH63 produces no
extracellular material, whereas strain BH68 secretes slime.
Philippis et al. (1998) collected 15 exopolysaccharide-producing Cyanoteche strains. It is possible that some cyanobacteria
could extracellularly release the α-polyglucans analyzed in this
study. Thus, the polyglucans might play an ecological role.
However, it is unlikely that all the semi-amylopectin is
excreted, because numerous polyglucan granules were detected
in cells of Synechococcus sp. BG043511 (Ikemoto and Mitsui
1994).
The acquisition of the ability to synthesize semi-amylopectin might be a unique property in group 5 lineage. Otherwise, the ability, once acquired, is retained due to some
physiological advantages in some species in group 5. Thus, it
would be very interesting to investigate how semi-amylopectin-producing cyanobacteria acquired and maintained the ability to synthesize semi-amylopectin, as well as to clarify how
green algae and plants acquired the ability to synthesize amylopectin and amylose.
Acknowledgments
We thank Professor Shigetoh Miyachi for his encouragement in
this project. We are also grateful to Dr. Perigio P. Francisco, Jr for correcting the English version.
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