RESEARCH ARTICLE
Molecular genetic and chemotaxonomic characterization of the
terrestrial cyanobacterium Nostoc commune and its
neighboring species
Hiromi Arima1, Noriomi Horiguchi1, Shinichi Takaichi2, Rumiko Kofuji1, Ken-Ichiro Ishida1,
Keishiro Wada1 & Toshio Sakamoto1
1
Division of Biological Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan; and
of Biology, Nippon Medical School, Kawasaki, Japan.
Correspondence: Toshio Sakamoto, School
of Natural System, College of Science and
Engineering, Kanazawa University, Kakuma,
Kanazawa 920-1192, Japan. Tel.: +81
76 264 6227; fax: +81 76 264 6215;
e-mail: [email protected]
MICROBIOLOGY ECOLOGY
Present addresses: Ken-Ichiro Ishida,
Graduate School of Life and Environmental
Sciences, University of Tsukuba, Tsukuba,
Ibaraki, 305-8572, Japan;
Keishiro Wada, Ishikawa Study Center,
University of the Air, Ohgigaoka 7-1,
Nonoichi-Machi, 921-8812, Japan.
Received 21 April 2011; revised 12 July
2011; accepted 24 August 2011.
Final version published online 27 September
2011.
DOI: 10.1111/j.1574-6941.2011.01195.x
Editor: Riks Laanbroek
2
Department
Abstract
The phylogeny of the terrestrial cyanobacterium Nostoc commune and its neighboring Nostoc species was studied using molecular genetic and chemotaxonomic approaches. At least eight genotypes of N. commune were characterized
by the differences among 16S rRNA gene sequences and the petH gene encoding ferredoxin-NADP+ oxidoreductase and by random amplified polymorphic
DNA analysis. The genotypes of N. commune were distributed in Japan without
regional specificity. The nrtP gene encoding NrtP-type nitrate/nitrite permease
was widely distributed in the genus Nostoc, suggesting that the occurrence of
the nrtP gene can be one of the characteristic features that separate cyanobacteria into two groups. The wspA gene encoding a 36-kDa water stress protein
was only found in N. commune and Nostoc verrucosum, suggesting that these
Nostoc species that form massive colonies with extracellular polysaccharides can
be exclusively characterized by the occurrence of the wspA gene. Fifteen species
of Nostoc and Anabaena were investigated by comparing their carotenoid composition. Three groups with distinct patterns of carotenoids were related to the
phylogenic tree constructed on the basis of 16S rRNA sequences. Nostoc commune and Nostoc punctiforme were clustered in one monophyletic group and
characterized by the occurrence of nostoxanthin, canthaxanthin, and myxol
glycosides.
Keywords
Anhydrobiosis; carotenoid; ferredoxin-NADP+
oxidoreductase; nitrate/nitrite transporter;
random amplified polymorphic DNA; water
stress protein.
Introduction
Cyanobacteria are photoautotrophic prokaryotes that perform oxygen-evolving photosynthesis with water as the
primary electron donor. Requiring only light, water, carbon dioxide, and inorganic salts, cyanobacteria have very
simple nutrient requirements that allow these organisms
to occupy highly diverse ecological niches. Cyanobacteria
are found in almost all terrestrial, freshwater, and marine
habitats. Some cyanobacteria form symbiotic associations
with hosts, such as fungi (lichens), bryophytes, cycads,
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mosses, fern (Azolla), and one angiosperm, Gunnera (Rai
et al., 2000). Interestingly, most of the cyanobacterial
photobionts in lichens belong to the genus Nostoc (Rikkinen et al., 2002).
The terrestrial cyanobacterium Nostoc commune is a
well-known species of the genus Nostoc, and N. commune
may be a cosmopolitan species that is adapted to terrestrial environments (Potts, 2000). In natural habitats, N.
commune forms nonspherical, irregularly spreading, macroscopic colonies that consist of trichomes of cells and
extracellular matrix (Potts, 2000). Nostoc commune is very
FEMS Microbiol Ecol 79 (2012) 34–45
35
Phylogeny of Nostoc
unique because of its extreme desiccation tolerance; desiccated colonies have no metabolic activity and sustain the
capacity for cell growth for over 100 years (Lipman,
1941; Cameron, 1962). This phenomenon is termed
‘anhydrobiosis’ (Crowe et al., 1998; Clegg, 2001; Potts,
2001; Crowe, 2002). Nostoc commune is considered an anhydrobiotic microorganism that has oxygenic photosynthetic capabilities and does not differentiate into akinetes
(spores) (Potts, 2000, 2001). The mechanism of extreme
desiccation tolerance by this species is thought to involve
multiple processes that include extracellular polysaccharide (EPS) production, compatible solute accumulation,
regulation of photosynthesis, and protection from UV
radiation. Removal of EPS in N. commune causes a significant reduction in the stress tolerance of photosynthesis
during desiccation (Tamaru et al., 2005). The cessation of
photosynthesis and trehalose accumulation occurs as an
acclimatizing response to matric water stress (desiccation)
and osmotic water stress (high salt concentration) (Sakamoto et al., 2009; Yoshida & Sakamoto, 2009). With
UV-absorbing pigments of mycosporine-like amino acids
and scytonemin (Wright et al., 2005; Sakamoto et al.,
2011), N. commune may be able to adapt to terrestrial
environments with high levels of solar radiation.
To investigate a unique mechanism responsible for
the acquisition of the extreme desiccation tolerance of
N. commune, a taxonomic study of this organism is necessary. In this study, we investigated the phylogeny of N. commune within the genus Nostoc using a molecular phylogenic
approach that uses genetic techniques and a chemotaxonomic approach that uses carotenoid patterns to characterize N. commune and its neighboring Nostoc species.
The 16S rRNA gene is the most highly studied gene in
phylogenic analysis and is useful in determining the identification of cyanobacterial species (Rasmussen & Svenning,
2001; Svenning et al., 2005; Rehakova et al., 2007). Ferredoxin-NADP+ oxidoreductase (FNR) catalyzes electron
transfer from the reduced ferredoxin to NADP+ to produce NADPH in the final step of photosynthetic electron
transfer. The cyanobacterial FNR contains a unique N-terminal domain similar to the 9-kDa phycocyanin-associated
linker polypeptide CpcD (Nakajima et al., 2002; Morsy
et al., 2008a). Thus, the petH gene that encodes cyanobacterial FNR was used for the phylogenic study, serving as a
specific and characteristic gene of cyanobacteria. Two distinct types of nitrate/nitrite transporters (NRT) have been
identified in cyanobacteria thus far. An ATP-binding cassette (ABC)-type NRT, in which NrtA functions as a highaffinity nitrate/nitrite-binding protein, occurs in freshwater
strains (Omata, 1995), whereas marine cyanobacterial
strains have an NrtP-type permease (Sakamoto et al., 1999;
Wang et al., 2000). Because the freshwater cyanobacterium
Nostoc punctiforme has the NrtP-type permease (Meeks
FEMS Microbiol Ecol 79 (2012) 34–45
et al., 2001; Aichi et al., 2006), the attribution of the types
of NRT to the habitats of cyanobacteria is questioned.
Hence, the distribution of the types of NRT was studied.
The groEL gene encoding chaperonin 60 is transcribed at
similar levels in both hydrated and desiccated cells of N.
punctiforme (Yoshida & Sakamoto, 2009). Thus, the groEL
gene was studied as a constitutively expressing gene. The
wspA gene encodes a 36-kDa water stress protein in the
extracellular matrix of N. commune (Wright et al., 2005;
Morsy et al., 2008b; Sakamoto et al., 2011). There is no
homologous gene to the wspA gene in the genome of
N. punctiforme ATCC 29133 (Meeks et al., 2001), although
N. punctiforme is believed to be closely related to N. commune. In our recent study, the wspA gene was found in
the aquatic cyanobacterium Nostoc verrucosum (Sakamoto
et al., 2011). Thus, the occurrence of this gene was studied
in N. commune and its neighboring species. Carotenoids in
cyanobacteria have two main functions: as accessory pigments for photosynthesis and in protection against photooxidative damage (Hirschberg & Chamovitz, 1994).
Cyanobacteria synthesize unique ketocarotenoids and glycosyl carotenoids that are not found in higher plants (Takaichi et al., 2005). Thus, carotenoid patterns were studied
in the genus Nostoc as a chemotaxonomic marker.
Materials and methods
Organisms and culture conditions
Nostoc commune colonies growing naturally in the field
were collected in Japan, and samples from China and
France were kindly provided for this study. Nostoc commune strain IAM M-13 (NIES-24), Nostoc muscorum
strain IAM M-14, N. punctiforme strain IAM M-15
(NIES-2108), Nostoc linckia strain IAM M-30 (NIES-28),
and Nostoc carneum strain IAM M-35 (NIES-2107) were
retrieved as cultivated samples from the Institute of
Molecular Biosciences at the University of Tokyo. Antarctic strains of Nostoc cf. commune SO-42, SO-36, KG-54,
and KG-102 were provided by Dr S. Ohtani at Shimane
University. Nostoc sp. strain PCC 7120 (also known as
Anabaena sp. strain PCC 7120) was provided by Dr
A. Higo at Saitama University. The domestic strains of
Nostoc sp. KU001 and N. commune KU002 were isolated
from the Kakuma Campus at Kanazawa University, and
they were purified by streaking on agar plates. These
cyanobacterial strains originally isolated by the authors
will be available upon request. Cells were grown at 25
–30 °C under constant illumination from fluorescent
lamps (5–10 lmol m 2 s 1) on modified BG110 liquid
medium (without a nitrogen source) with shaking for
aeration or BG110 solid medium with 1.5% agar supplemented with a vitamin mix to give final concentrations of
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36
1 lg L 1 of biotin, 2 mg L 1 of thiamin, and 1 lg L 1
of cyanocobalamin (Castenholz, 1988). These culture
media were buffered with 20 mM HEPES-NaOH (pH
7.5). For culturing N. carneum strain IAM M-35,
1.5 g L 1 of NaNO3 was added as a nitrogen source.
These cyanobacterial cultures were unialgal but not completely bacteria-free.
PCR direct sequencing and phylogenic analysis
The DNA fragments containing the 16S rRNA gene, the
petH gene encoding FNR, the nrtP gene encoding nitrate/
nitrite permease, and the groESL gene encoding chaperonin 60 were amplified by PCR, and their nucleotide
sequences were determined. The field samples of N. commune and cultivated cyanobacterial cells were lyophilized
until dry and ground to a powder. A genomic DNA template was extracted from approximately 15 mg of the
powder using the DNA Plant Mini Kit (QIAGEN)
according to the manufacturer’s instructions. The genespecific primers for the 1.4-kb 16S rRNA gene, the 1.9-kb
petH gene, the 1.3-kb nrtP gene, the 2-kb groESL gene,
and the 1.7-kb groEL gene (Table S1) were designed and
synthesized based on the nucleotide sequence of the
N. punctiforme strain ATCC 29133 genome (Meeks et al.,
2001). The reaction mixture contained 5 lL of 109 PCR
buffer (100 mM Tris–HCl pH 8.3, 500 mM KCl, and
15 mM MgCl), 4 lL of 2.5 mM dNTP mixture, 50 pmol
of a forward primer, 50 pmol of a reverse primer, 1.25 U
of Taq polymerase (R001A; TaKaRa, Kyoto, Japan), and 5
–20 ng of the genomic DNA template in a total volume
of 50 lL. PCR was performed under the following conditions: initial denaturation at 94 °C for 5 min, followed by
30 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for
30 s with a final extension step at 72 °C for 5 min and
final soak step at 4 °C. The PCR product was purified
using a QIAquick PCR Purification Kit (QIAGEN) to
remove excess PCR primers. Cycle sequencing reactions
were performed to determine the nucleotide sequence by
3′ dye-labeled dideoxynucleotide triphosphates (BIG DYE
terminators; Applied Biosystems company) using the
internal specific primers and the template DNA amplified
by PCR and run on an ABI PRISM377 DNA Sequencer
(Perkin-Elmer ABI, Foster City, CA). The nucleotide
sequences determined in this study have been submitted
to GenBank/EMBL/DDBJ with the accession numbers
listed in Table S2. The accession numbers of the 16S
rRNA gene sequences obtained from the GenBank/EMBL/
DDBJ database are listed in Table S3. The phylogenic
analysis was performed using PHYLIP (Phylogeny Interference Package) version 3.6 (Felsenstein, 2004). The
sequence data were aligned using CLUSTAL X version 1.83
and corrected manually using MACCLADE version 4.0. The
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H. Arima et al.
distance matrix was computed using RESTDIST program on
the Kimura-2-parameter model, and a phylogenic tree
was constructed using NEIGHBOR program. Bootstrap test
was performed using SEQBOOT program.
PCR–restriction fragment length
polymorphisms (RFLP) analysis
To characterize the genotypes of the field-isolated natural
colonies of N. commune, PCR–RFLP analysis was performed using 16S rRNA and petH genes (Table S4). The
1.4-kb DNA fragment of the 16S rRNA gene was amplified from the genomic DNA template by PCR using the
specific primers (Table S1) and digested by the restriction
enzymes Ase I, Bsm I, and Hpa I, respectively. The 1.8-kb
DNA fragment of the petH gene was amplified by PCR
and digested by Sau3A I. These digestion products were
separated by electrophoresis on a 1.5% agarose gel and
stained with ethidium bromide. The DNA fragment patterns were compared with those from the known genotypes. Four distinct types designated A, B, C, and D
could be recognized by this analysis (Table S4).
Random amplified polymorphic DNA (RAPD)
analysis
Three oligonucleotide primers, 5′-CCA(A/G)TCCCCA(A/
G)TCCCC-3′ containing short tandem repeat repetitive
sequences (STRR; Mazel et al., 1990), 5′-GCGATCGCACC-3′ (Bhaya et al., 2000), and 5′-TAAACCTTTAGGC
GATCGCG-3′ containing highly iterated palindrome
sequences (HIP1; Gupta et al., 1993), were used as a single primer for the random amplification of the genomic
DNA template. The reaction mixture contained 5 lL of
109 PCR buffer (100 mM Tris–HCl pH 8.3, 500 mM
KCl, and 15 mM MgCl), 4 lL of 2.5 mM dNTP mixture,
100 pmol of a RAPD primer, 1.25 U of Taq polymerase
(R001A; TaKaRa), and 5–20 ng of the genomic DNA
template in a total volume of 50 lL. The PCRs were performed using the following PCR profile: an initial denaturation at 95 °C for 6 min, followed by 30 cycles with
denaturation at 94 °C for 1 min, annealing at 56 °C for
1 min, and extension at 65 °C for 5 min with a final
extension step at 65 °C for 16 min and final soak step at
4 °C (Mazel et al., 1990). The amplified DNA fragments
were separated by electrophoresis on a 2% agarose gel
and stained with ethidium bromide.
PCR amplification of the wspA gene
A 0.8-kb DNA fragment containing the wspA gene was
amplified by PCR from the genomic DNA templates as
previously described (Sakamoto et al., 2011). The degenFEMS Microbiol Ecol 79 (2012) 34–45
37
Phylogeny of Nostoc
erate primers used a forward primer (23-mer with 96-fold
degeneracy) 5′-TA(T/C) GGI TA(T/C) ACI AT(T/C/A)
GGI GA(A/G) GA-3′ derived from the amino acid
sequence YGYTIGED and a reverse primer (23-mer with
96-fold degeneracy) 5′-TC (T/C)TG (A/G)TA ICC IGT
(A/G/T)AT (T/C)TC (A/G)TA-3′ derived from the amino
acid sequence YEITGYQD. PCR was performed under the
following conditions: initial denaturation at 94 °C for
1 min, followed by 40 cycles of 94 °C for 1 min, 50 °C
for 2 min, and 72 °C for 3 min with a final extension
step at 72 °C for 5 min. The amplified DNA fragments
were separated by electrophoresis on a 1.2% agarose gel
and stained with ethidium bromide. The nucleotide
sequences of the wspA genes from N. commune and
N. verrucosum have been submitted to GenBank/EMBL/
DDBJ with the accession numbers listed in Table S2.
Southern hybridization analysis
Genomic DNA (2 lg) was digested by Hind III and EcoR
I, separated by electrophoresis on a 0.8% agarose gel, and
transferred onto a nylon membrane (Hynbond N+; GE
Healthcare UK Ltd.). For the digestion of the genomic
DNA from Nostoc sp. KU001, Hind III and BamH I were
also used. The 1.4-kb nrtA probe was amplified by PCR
using the specific primers (Table S1) and the genomic
DNA template of Nostoc sp. strain PCC 7120. The 1.3-kb
nrtP probe was amplified by PCR using the specific primers (Table S1) and the genomic DNA template of N. commune strain KU002. The 0.7-kb wspA probe was
amplified by PCR using the specific primers (Table S1)
and the genomic DNA template of N. commune strain
KU002. The DNA fragments were labeled using AlkPhos
Direct Labelling Module (GE Healthcare), and hybridizations with these probes were preformed in AlkPhos Direct
Hybridization Buffer (GE Healthcare). After excess probes
were washed, the hybridization signals were detected by a
chemiluminescence detection system (CDP-Star Detection
Reagent, GE Healthcare) and visualized with a luminoimage analyzer (LAS-3000; FUJIFILM).
Identification and composition of carotenoids
Colonies of N. verrucosum growing naturally in the
stream were collected from Shishiku Park, Hakusan-shi,
Ishikawa, Japan (36°26′8″N, 136°38′37″E) (Sakamoto
et al., 2011). Colonies of N. commune growing naturally
in the field were collected, washed with tap water to
remove soil, air-dried, and stored at room temperature.
Cells of N. muscorum strain IAM M-14, N. linckia strain
IAM M-30 (NIES-28), and Nostoc sp. strain KU001 were
grown diazotrophically for 4 weeks at 25 °C under constant illumination from fluorescent lamps (5–10
FEMS Microbiol Ecol 79 (2012) 34–45
lmol m 2 s 1) on modified BG110 liquid medium (without a nitrogen source) buffered with 20 mM HEPESNaOH (pH 7.5) with shaking for aeration. Cells of
N. carneum strain IAM M-35 were grown for 2 weeks on
BG11 liquid medium containing 1.5 g L 1 of NaNO3 as a
nitrogen source otherwise under identical conditions. The
light intensity was measured using a photometer (model
LI-185B; LI-COR, Lincoln, NE).
Pigments were extracted from wet cells with acetone/
methanol (7 : 2, v/v) and analyzed by the HPLC system
equipped with a lBondapak C18 column (8 9 100 mm,
RCM-type; Waters, Milford, MA) and an MCPD-3600
photodiode array detector (Otsuka Electronics; Osaka,
Japan). After elution with methanol/water (9 : 1, v/v) for
20 min at a flow rate of 2.0 mL min 1, the mobile phase
was changed to 100% methanol. For quantitative analysis,
the molar extinction coefficient in the HPLC eluent at the
maximum wavelengths of each carotenoid was assumed
to be the same (Takaichi et al., 2001; Iwai et al., 2008).
For the identification of the carotenoids, their absorption spectra and their specific retention times on HPLC
were compared with N. commune strain IAM M-13
(NIES-24) (Takaichi et al., 2009). For the identification
of the glycoside moieties of myxol glycosides, they were
purified using silica gel TLC and the HPLC system as
described earlier. Their relative molecular masses were
measured by FD-MS using an M-2500 double-focusing
gas chromatograph-mass spectrometer (Hitachi, Japan)
equipped with a field-desorption apparatus. The proton
nuclear magnetic resonance (1H-NMR) (500-MHz) spectra of carotenoids in CDCl3 at 24 °C were measured
using the UNITY INOVA-500 system (Varian).
Results and discussion
Genetic diversity of Nostoc commune and
phylogenic analysis
The 1.4-kb DNA fragment of the 16S rRNA gene was
amplified and sequenced using 34 samples of fieldisolated, naturally grown colonies of N. commune collected in Japan. In total, eight distinct sequences of the
16S rRNA gene were found. These nucleotide sequences
obtained from the field samples of N. commune were
> 96% identical to each other. These sequences were also
highly similar to the 16S rRNA gene sequences obtained
from N. commune strain IAM M-13 and the Antarctic
strains of N. cf. commune SO-42, SO-36, KG-54, and
KG-102 with a sequence identity of > 96%. These data
confirmed a high level of genetic similarity among all
field samples of N. commune. The 1.8-kb DNA fragment
of the petH gene encoding FNR was amplified and
sequenced, and seven distinct sequences of the petH gene
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
38
were found in the 26 field samples of N. commune
examined. The deduced amino acid sequences of PetH
were highly similar to each other with a sequence identity
of > 91%. These PetH sequences in N. commune exhibited < 84% sequence identity to other cyanobacterial
PetH sequences obtained from the GenBank database,
suggesting that N. commune can be recognized as a discriminative species by the PetH sequence. When the identical 16S rRNA gene sequence was obtained, an identical
or very similar sequence of the petH gene was found,
indicating that these genotypes are consistently recognizable by either the 16S rRNA gene sequence or the petH
gene. Thus, these genotypes were designated A1, A2, B1,
B2, C1, C2, D1, and D2 and characterized further.
By comparing the nucleotide sequences of 16S rRNA
and the petH gene obtained from the genotypes of N.
commune, RFLP in these genes were consequently found
(Table S4). These RFLPs allowed us to characterize the
four distinct genotypes, designated A, B, C, and D, of the
field-isolated natural colonies of N. commune by PCR–
RFLP analysis (Table S4). In total, more than 45 colonies
were examined by the 16S rRNA gene sequence and PCR
–RFLP analysis, and all of the colonies were recognizable
as N. commune with the characteristics of this organism
as previously described (Rehakova et al., 2007). Colonies
of N. commune were found throughout Japan, and there
was no specific regional distribution of these genotypes
(Supporting Information, Fig. S1). Moreover, three genotypes were found in a small area at the Kakuma Campus
of Kanazawa University, and occasionally, two distinct
genotypes were mixed together in a small fragment of N.
commune colonies (data not shown). It is noteworthy that
these genotypes of N. commune were hardly distinguishable morphologically, and their genetic differences were
not great enough to recognize these genotypes as distinct
species.
The 16S rRNA gene phylogenic tree of N. commune
and its neighboring species of Nostoc was constructed by
the neighbor-joining method (Fig. 1). Anabaena cylindrica
strain PCC 7122 was included as an outgroup to root the
tree. All of the genotypes of field-isolated, naturally grown
N. commune were clustered in a monophyletic group with
other strains of N. commune, Nostoc flagelliforme, and
N. punctiforme. This clade including N. commune and
N. punctiforme was significantly exclusive from other
Nostoc species. Symbiotic Nostoc strains that are thought
to belong to N. punctiforme were also included in this
clade when included in the phylogenic analyses (data not
shown). This analysis is in agreement with previous studies of the phylogeny of symbiotic Nostoc strains (Svenning
et al., 2005). The phylogenic trees constructed using the
petH, nrtP, and groEL genes were essentially the same as
the trees constructed using the 16S rRNA gene (data not
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Published by Blackwell Publishing Ltd. All rights reserved
H. Arima et al.
shown), confirming the result of the 16S rRNA gene analysis (Fig. 1). These results suggest that N. commune can
be considered a synonym of N. punctiforme, and N. commune and N. punctiforme, including symbiotic Nostoc
strains, are closely related, although their habitats are
superficially different. Nostoc commune forms free-living
macroscopic colonies in terrestrial environments, and
N. punctiforme is found as a phycobiont in the root of
cycads (Rehakova et al., 2007).
RAPD analysis using the primers derived from cyanobacterial repetitive sequences of STRR (Mazel et al., 1990)
and HIP1 (Gupta et al., 1993) is a powerful tool to study
the genetic diversity of cyanobacterial strains. Because the
DNA profile generated by random amplification using
cyanobacterial-specific repetitive sequences resolves the
small differences in the entire genome in an enhanced
manner, this analysis is one of the most sensitive methods
for distinguishing among closely related strains (Prabina
et al., 2005). The eight genotypes of N. commune recognized by the 16S rRNA gene were analyzed by RAPD
(Fig. 2). Each primer produced a distinct set of DNA
fragments for each of the eight genotypes of N. commune
(Fig. 2a,c,d). When seven samples of the type A1 (characterized by the laboratory culture strain of N. commune
KU002) from distant geographic locations were examined,
all of the DNA profiles obtained were identical to each
other (Fig. 2b). The band patterns shown in Fig. 2a were
converted into binary data to construct a dendrogram
(Fig. S2). The dendrogram graphically represents the differences among the genotypes according to their band
patterns. These results indicate that the genotypes of
N. commune can be distinguished by the DNA profiles
specific to genotype using this technique.
In this study, the genetic diversity of the cosmopolitan
terrestrial cyanobacterium N. commune was analyzed, and
we designated eight genotypes of N. commune in Japan
based on the nucleotide sequences of the 16S rRNA gene.
The eight genotypes of N. commune are genetically similar, and the morphological features of the macroscopic
colonies and microscopic trichomes are almost indistinguishable. These genotypes are easily recognized by PCR
direct sequencing of the 16S rRNA gene and PCR–RFLP
or RAPD analysis as described earlier. Our results support
the results of other studies that found that N. commune,
N. punctiforme, and symbiotic Nostoc strains are clustered
in a monophyletic group and originate from a common
ancestor (Rikkinen et al., 2002; Svenning et al., 2005;
Rehakova et al., 2007). The aquatic cyanobacterium
N. verrucosum forms visible colonies and retains a large
amount of EPS. The appearance of N. verrucosum colonies is superficially similar to that of N. commune, but
N. verrucosum is sensitive to desiccation (Sakamoto et al.,
2011). The 16S rRNA gene sequence of N. verrucosum
FEMS Microbiol Ecol 79 (2012) 34–45
39
Phylogeny of Nostoc
Anabaena cylindrica PCC7122
Aphanizomenon sp. PCC7905
Anabaena sp. 86
Anabaena sp. 14
65.1
Nodularia sphaerocarpa Fae19
Nostoc sp. KU001
Nodularia harveyana CDAC1983/300
Anabaena bergii 283A
Nostoc carneum IAM M-35
Nostoc sp. YK-01
Nostoc sp. HK-01
Nostoc sp. KK-01
Anabaena variabillis IAM M-3
Nostoc sp. strain PCC7120
Nostoc muscorum I
77.5
Nostoc muscorum II
Nostoc muscorum IAM M-14
Nostoc entophytum IAM M-267
Nostoc linckia IAM M-251
Nostoc linckia IAM M-30
67.2
Nostoc commune C2 type Saga
Nostoc commune C1 type Akashi
Nostoc commune A3 type Hokkaido
Nostoc commune A1 type KU002
Nostoc commune A2 type Rokko
Nostoc flagelliforme IMGA0408
100.0
Nostoc commune China
Nostoc commune France
Nostoc punctiforme PCC73102
Nostoc commune B1 type Osaka
Nostoc commune B2 type Okinawa
Nostoc commune D1 type Yamaguchi
Nostoc commune D2 type Kanazawa
Nostoc commune IAM M-13
Nostoc commune SO-42
Nostoc commune KG-54
0.01
NrtP
NrtA
Fig. 1. Phylogenic position of Nostoc commune in relation to its neighboring Nostoc species. The phylogenic tree was constructed on the basis
of 16S rRNA sequences using the neighbor-joining (NJ) algorithm. The root of the tree was determined using the 16S rRNA sequence of
Anabaena cylindrica strain PCC 7122. Bootstrap percentages (> 65%) are indicated for the nodes. The occurrence of the characteristic
carotenoids and the types of nitrate/nitrite transporters are indicated.
strain KU005 showed 95% identity to the sequence of
N. commune strain KU002, suggesting that these strains
are genetically similar but different species of cyanobacteria. Nostoc verrucosum may be the origin of the terrestrial
cyanobacterium because of the morphological resemblance. However, we could not precisely resolve the phylogeny of N. verrucosum within the genus Nostoc by
phylogenic analysis using the 16S rRNA gene sequence
(data not shown).
Nitrate/nitrite transporters in Nostoc
Two distinct types of NRT, an ABC-type NRT in freshwater strains (Omata, 1995) and an NrtP-type permease in
marine strains (Sakamoto et al., 1999; Wang et al., 2000),
have been identified in cyanobacteria. The nrtA gene
encodes a high-affinity nitrate/nitrite-binding protein that
is a component of the ABC-type NRT (Omata, 1995).
The distribution of these types of NRT in the genus
FEMS Microbiol Ecol 79 (2012) 34–45
Nostoc was characterized. Figure 3 shows the results of
Southern hybridization using nrtA (Fig. 3a) and nrtP
(Fig. 3b) gene probes. The signals hybridizing with the
nrtA gene probe were detected in Nostoc sp. strain
KU001, in N. carneum strain IAM M-35, and in the positive control Nostoc sp. strain PCC 7120 (Fig. 3a, lanes 1
–4). The signals hybridizing with the nrtP gene probe
were detected in N. carneum strain IAM M-35, N. linckia
strain IAM M-30, N. commune strain KU002, and
N. commune strain IAM M-13 (Fig. 3b, lanes 4–7).
According to these results, the nrtP gene was isolated by
PCR using the gene-specific primers designed from the
nucleotide sequence of the N. punctiforme strain ATCC
29133 genome (Meeks et al., 2001). As summarized in
Table S2, the nrtP genes from 15 strains of the genus
Nostoc were PCR-amplified and sequenced, indicating
that the nrtP gene is widely distributed in the genus
Nostoc. Because the signals hybridizing with the nrtA
probe were also detected in N. carneum strain IAM M-35,
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Published by Blackwell Publishing Ltd. All rights reserved
40
H. Arima et al.
(a)
(c)
(b)
(d)
Fig. 2. Diagnostic PCR distinguishing the genotypes in Nostoc commune by RAPD analysis. Using the genomic DNA template extracted from
N. commune samples of the 8 genotypes recognized by their 16S rRNA sequences, the DNA fragments were amplified by the following primers:
5′-CCA(A/G)TCCCCA(A/G)TCCCC-3′ (a), 5′-GCGATCGCACC-3′ (c), and 5′-TAAACCTTTAGGCGATCGCG-3′ (d). To assess the consistency of the
band pattern of the amplified DNA, PCR was performed using the genomic DNA of the A1 type with 7 different sampling localities in Japan as
the template DNA and the primer 5′-CCARTCCCCARTCCCC-3′ (b). Amplified PCR products were separated by agarose gel electrophoresis and
stained with ethidium bromide (a, c, and d). M, kHind III maker; lane 1, A1 type; lane 2, A2 type; lane 3, B1 type; lane 4, B2 type; lane 5, C1
type; lane 6, C2 type; lane 7, D1 type; lane 8, D2 type. Cluster analysis using the band patterns in a is shown in Fig. S2.
we tried to isolate the nrtA gene by PCR using the primers designed from the nucleotide sequence of the nrtA
gene of Nostoc sp. strain PCC 7120. However, the nrtA
gene could not be isolated from N. carneum strain IAM
M-35 by this approach (data not shown). Thus, future
studies need to determine whether N. carneum is an
intermediate species having two distinct nitrate/nitrite
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
transport systems; N. carneum clearly has the nrtP gene
(Table S2).
The distribution of the nrtP gene in the genus Nostoc
was related to the phylogenic tree (Fig. 1), but the habitats of cyanobacteria were unrelated to the type of NRT.
The nrtP genes were found in all of the marine, freshwater, and terrestrial cyanobacteria. These results suggest
FEMS Microbiol Ecol 79 (2012) 34–45
41
Phylogeny of Nostoc
(a)
kb
1 2 3 4 5 6 7
(b)
kb
9.4
9.4
6.6
6.6
4.4
4.4
2.3
2.0
2.3
2.0
0.56
0.56
1 2 3 4 5 6 7
Fig. 3. Detection of the nrtA gene (a) and the nrtP gene (b) by
Southern hybridization. The nrtA probe from Nostoc sp. strain PCC
7120 and the nrtP probe from Nostoc commune strain KU002 were
prepared as described in Materials and methods. Lane 1, Nostoc sp.
strain PCC 7120; lane 2, Nostoc sp. strain KU001 (digested by Hind III
and EcoR I); lane 3, Nostoc sp, strain KU001 (digested by Hind III and
BamH I); lane 4, Nostoc carneum strain IAM M-35; lane 5, Nostoc
linckia strain IAM M-30; lane 6, N. commune strain KU002; lane 7,
N. commune strain IAM M-13. The predicted DNA fragment
containing the nrtA gene in Nostoc sp. strain PCC 7120 is Hind IIIHind III 8.3 kb but multiple signals cross-hybridized with the nrtA
probe were detected (a, lane 1). In Nostoc sp. strain KU001, a single
hybridized signal of approximately 5 kb and two signals of 0.7 and
0.5 kb were detected (a, lanes 2 and 3). In N. carneum strain IAM M35, two signals of 2.1 and 0.8 kb were detected because of the
internal Hind III site in the nrtP gene of this strain (b, lane 4). In N.
linckia strain IAM M-30, the predicted Hind III 0.3-kb signal was
detected (b, lane 5).
that the occurrence of the nrtP gene can be one of the
characteristic features that separate cyanobacteria into
two groups. Future studies need to examine how these
two systems of NRT were originated and selected in cyanobacteria.
acid polypeptide with 73% sequence identity to the
WspA of N. commune KU002 (Sakamoto et al., 2011).
No DNA fragment was obtained in the other 12 strains
of Nostoc tested, including strains closely related to
N. commune (Fig. 4, lanes 4–15). Using the 0.7-kb probe
of the wspA gene from N. commune strain KU002,
Southern hybridization analysis was performed to confirm the result of the PCR analysis using the degenerated oligo DNA primers, and hybridization signals were
detected in N. commune strain KU002 and N. verrucosum but not in the other cyanobacteria tested (data not
shown). These results suggest that the wspA gene exclusively occurs in two Nostoc species, N. commune and
N. verrucosum, and these species form massive colonies
with a large amount of extracellular matrix in natural
habitats. WspA is a major extracellular matrix protein
with b-galactosidase activity (Morsy et al., 2008b) and is
assumed to maintain the structure and/or functions of
the extracellular matrix (Wright et al., 2005). The primary structure of WspA shows no similarity to the
known proteins in the database and is thought to be a
unique protein in N. commune. It is noteworthy that
several N. commune strains lack the wspA gene (Fig. 4).
This distribution of the wspA gene raises new questions
surrounding the origin of this gene and genetic relationships between N. commune and N. verrucosum.
kb
M 1 2 3 4 5 6 7 8 9 1 0 11 12 13 1 4 1 5 1 6
23
9.4
6.6
4.4
2.3
2.0
0.56
Occurrence of the wspA gene in Nostoc
To examine the distribution of the wspA gene in the
genus Nostoc, the wspA gene was amplified by PCR
using the degenerated oligo DNA primers that were
designed according to the deduced amino acid sequence
of WspA (Fig. 4). The 0.8-kb PCR product of the wspA
gene was obtained from the genomic DNA template of
N. commune strain KU002 (Fig. 4, lane 1), N. verrucosum strain KU005 (Fig. 4, lane 2), and the field-isolated,
naturally grown N. verrucosum (Fig. 4, lane 3). The
wspA gene of N. commune strain KU002 encoded a 333amino-acid polypeptide with 77% sequence identity to
WspA of N. commune strain DRH1, and the wspA gene
of N. verrucosum strain KU005 encoded a 330-aminoFEMS Microbiol Ecol 79 (2012) 34–45
Fig. 4. Detection of the wspA gene by PCR using degenerated oligo
DNA primers. The PCR primers were designed according to the
deduced amino acid sequence of WspA of Nostoc commune strain
DRH1 (Wright et al., 2005) as described in Materials and methods.
The genomic DNA templates of 15 Nostoc strains were used. M,
kHind III maker; lane 1, N. commune strain KU002; lane 2, Nostoc
verrucosum strain KU005; lane 3, N. verrucosum (field-isolated
sample from Takaoka, Japan); lane 4, N. commune strain IAM M-13;
lane 5, Nostoc punctiforme strain IAM M-15; lane 6, N. cf. commune
strain SO-36; lane 7, N. cf. commune strain SO-42; lane 8, N. cf.
commune strain KG-54; lane 9, N. cf. commune strain KG-102; lane
10, Nostoc muscorum strain IAM M-14; lane 11, Nostoc linckia strain
IAM M-30; lane 12, Nostoc carneum strain IAM M-35; lane 13,
Nostoc sp. strain KU001; lane 14, Nostoc sp. strain HK-01; lane 15,
Nostoc sp. strain PCC 7120; lane 16, no template DNA control.
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Published by Blackwell Publishing Ltd. All rights reserved
42
H. Arima et al.
Table 1. Carotenoid composition (mol%) of cyanobacteria
Species
Group 1
Anabaena variabilis
ATCC 29413‡
Nostoc sp. KU001
N.cameum IAM M-35
Nostoc sp. YK-01†
Nostoc sp. HK-01†
Nostoc sp. KK-01†
Anabaena variabilis
I AM M-3†
Nostoc cf. verrucosum
Group 2
N. muscorum IAM M-14
N. linckia var. arvense
IAM M-30
Group 3
Nostoc sp. PCC 7120†
N. punctiforme PCC
73102†
N. commune
(field, Nonoichi)§
N. commune IAM M-13§
N. punctiforme IAM
M-15
Hydroxylated carotenoids
Ketocarotenoids
b-cryp
Echine
Cantha
51
20
22
5¶
58
43
64
55
39
38
27
27
20
18
40
33
14
2
5
2
4
15**
15**
14**
22††
8**
11††
10**
13††
40
12
6
32††
9††
2
10
1
1
25
17
1
13
8††
11††
4††
13††
b-caro
69
69
62
45
7
5
<1
<1
18
50
50
Zea
<1
Calo
Nosto
12
9
9
7
<1
<1
Myxol glycosides
3′-OH-E
1
<1
M-G
KM-G
2OH-M-G
4OH-M-G
2¶
< 1‡‡
< 1‡‡
< 1‡‡
< 1‡‡
1
3
4
43
12
9**
11**
2
5
5
8
11
11
20
20
2
4††
3**
5††
2**
Data from a single experiment are shown but the occurrence of the carotenoids was confirmed at least twice.
b-caro, b-carotene; b-cryp, b-cryptoxanthin; Zea, Zeaxanthin; Calo, Caloxanthin; Nosto, Nostoxanthin; Echine, Echinenone; Cantha, Canthaxanthin; 3′-OH-E, 3′-OH-Echinenone; M-G, Myxol glycoside; KM-G, 4-Ketomyxol glycoside; 2OH-M-G, 2-hydroxymyxol glycoside; 4OH-M-G, 4-hydroxymyxol glycoside.
†
Takaichi et al. (2005),
‡
Takaichi et al. (2006),
§
Takaichi et al. (2009),
¶
free (not glycoside),
**methyl pentoside,
††
fucoside,
‡‡
these might be 4OH-M-G due to lack of nostoxanthin.
Identification and composition of carotenoids
in Nostoc
b-Carotene, its hydroxyl derivatives of zeaxanthin, caloxanthin, and nostoxanthin, and its keto derivatives of
echinenone and canthaxanthin were identified based on
the compatible absorption spectra and the specific
retention times on HPLC of N. commune strain IAM
M-13 (NIES-24) (Takaichi et al., 2009). Two carotenoid
glycosides were purified from N. verrucosum and were
identified as myxol 2′-fucoside and 4-ketomyxol 2′-fucoside based on their relative molecular masses of 730
and 744, respectively, and 1H-NMR spectra (Takaichi
et al., 2005). Nostoc cameum IAM M-35 and Nostoc sp.
strain KU001 contained myxol methylpentoside that
was identified based on a relative molecular mass of
730, which was thought to be fucoside, rhamnoside, or
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
quinovoside. Nostoc punctiforme strain IAM M-15 contained myxol methylpentoside and 2-hydroxymyxol
methylpentoside that were identified based on the
presence of nostoxanthin. Minor components in Nostoc
species lacking nostoxanthin were thought to be 4-hydroxymyxol methylpentoside. Of note, 2,2′-b-hydroxylase, CrtG, catalyzes the reactions of zeaxanthin to
nostoxanthin and myxol to 2-hydroxymyxol (Iwai et al.,
2008).
Carotenoid compositions in N. commune and its neighboring Nostoc and Anabaena species are summarized in
Table 1. The most common carotenoids in cyanobacteria
are b-carotene, zeaxanthin, the ketocarotenoid echinenone, and the myxol glycosides (Takaichi & Mochimaru,
2007). In all of the cyanobacterial species examined,
b-carotene was found to be a major component, and the
ketocarotenoids of echinenone and canthaxanthin were
FEMS Microbiol Ecol 79 (2012) 34–45
43
Phylogeny of Nostoc
also present. Three groups of distinct carotenoid patterns
were found. Group 1 was characterized by the absence of
hydroxylated molecular species, such as b-cryptoxanthin,
zeaxanthin, caloxanthin, and nostoxanthin. Group 2 was
characterized by the absence of myxol glycosides. Nostoc
commune was found to belong to Group 3 and was characterized by the presence of hydroxylated carotenoids and
myxol glycosides (Table 1). This carotenoid composition
is in agreement with a previous report of the pigment
analysis in N. commune (Potts et al., 1987), in which bcarotene and echinenone predominate. These three groups
designated by carotenoid composition were strongly
related to the phylogenic tree constructed on the basis of
16S rRNA gene sequences. Each group was discriminated
from the others and recognized as a separated clade
(Fig. 1), indicating that this chemotaxonomic approach
using the carotenoid patterns is a valid approach and is
useful for distinguishing Nostoc strains.
As a chemotaxonomic tool to characterize Nostoc species, fatty acid profiling has been reported (Liu et al.,
2003; Temina et al., 2007). Because a quantitative and
statistical analysis is required for fatty acid profiling, this
approach may be complex and it is difficult to avoid
the fluctuation in fatty acid composition that depends
on the growth conditions. Compared with the previously
reported fatty acid profiling, the differences in the carotenoid patterns found by this study can be supported by
the presence and/or absence of the specific genes
involved in the carotenogenesis pathways. These comparative studies of the genes involved in the carotenogenesis
pathways in the genus Nostoc are important to confirm
the validation of this chemotaxonomic tool. This confirmation remains to be directly demonstrated in future
studies.
Concluding remarks
The taxonomic characters for the genus Nostoc are limited, and these characters are hardly distinguishable
morphologically. Thus, molecular genetic and chemotaxonomic tools are useful and convenient for the characterization of Nostoc species. The terrestrial cyanobacterium
N. commune can be discriminated from the other Nostoc
species by a combination of the presence of genes related
to the structure and functions of the extracellular matrix,
such as the wspA gene, and the presence of the specific
carotenogenesis pathways for both hydroxylated carotenoids and myxol glycosides. To elucidate the mechanisms
of terrestrial cyanobacteria adaptation to the extreme
environments and expansion of their cosmopolitan distribution, the freshwater Nostoc species that is genetically
closest to N. commune must be determined. Further studies are required to identify the characteristic genes attribFEMS Microbiol Ecol 79 (2012) 34–45
utive to the unique habit of anhydrobiosis of the
terrestrial cyanobacterium N. commune.
Acknowledgements
The authors thank Dr T. Maoka (Research Institute for
Production Development) for the NMR analysis of certain myxol glycosides and Y. Tamaru (Kanazawa University), F. M. Morsy (Kanazawa University), Y. Andoh
(Kanazawa University), E. Hiratani (Kanazawa University), and S. Matsumura (Kanazawa University) for their
technical assistance. This work was supported by Japan
Society for the Promotion of Science (KAKENHI
22510227), Suntory Institute for Bioorganic Research
(SUNBOR Grant), Micro Algae Corporation, and HABA
Laboratories, Inc.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
FEMS Microbiol Ecol 79 (2012) 34–45
45
Fig. S1. Sampling locality of field-isolated, naturally
grown colonies of Nostoc commune in Japan.
Fig. S2. Cluster analysis using RAPD patters in the 8
genotypes of N. commune.
Table S1. PCR primers.
Table S2. Accession numbers for the nucleotide sequences
of 16S rRNA gene, petH, groESL, nrtP, and wspA.
Table S3. Accession numbers of 16S rRNA gene
sequences retrieved from the EMBL/GenBank/DDBJ database.
Table S4. PCR–RFLP analysis.
Please note: Wiley-Blackwell are not responsible for the
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material) should be directed to the corresponding author
for the article.
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