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The Primitive Thylakoid-Less Cyanobacterium
Gloeobacter
Is a Common Rock-Dwelling Organism
Jan Mares
ˇ
1,2
*, Pavel Hrouzek
3
, Radek Kan
ˇ
a
3
, Stefano Ventura
4
, Otakar Strunecky
´
1,5
,Jir
ˇ
ı
´
Koma
´
rek
1,2
1 Institute of Botany ASCR, Centre for Phycology, Tr
ˇ
ebon
ˇ
, Czech Republic, 2 Department of Botany, Faculty of Science, University of South Bohemia, C
ˇ
eske
´
Bude
ˇ
jovice,
Czech Republic, 3 Institute of Microbiology ASCR, Department of Autotrophic Microorganisms - ALGATECH, Tr
ˇ
ebon
ˇ
, Czech Republic, 4 CNR-ISE Istituto per lo Studio degli
Ecosistemi, Sesto Fiorentino, Italy, 5 Centre for Polar Ecology, Faculty of Science, University of South Bohemia, C
ˇ
eske
´
Bude
ˇ
jovice, Czech Republic
Abstract
Cyanobacteria are an ancient group of photosynthetic prokaryotes, which are significant in biogeochemical cycles. The
most primitive among living cyanobacteria, Gloeobacter violaceus, shows a unique ancestral cell organization with a
complete absence of inner membranes (thylakoids) and an uncommon structure of the photosynthetic apparatus.
Numerous phylogenetic papers proved its basal position among all of the organisms and organelles capable of plant-like
photosynthesis (i.e., cyanobacteria, chloroplasts of algae and plants). Hence, G. violaceus has become one of the key species
in evolutionary study of photosynthetic life. It also numbers among the most widely used organisms in experimental
photosynthesis research. Except for a few related culture isolates, there has been little data on the actual biology of
Gloeobacter, being relegated to an ‘‘evolutionary curiosity’’ with an enigmatic identity. Here we show that members of the
genus Gloeobacter probably are common rock-dwelling cyanobacteria. On the basis of morphological, ultrastructural,
pigment, and phylogenetic comparisons of available Gloeobacter strains, as well as on the basis of three new independent
isolates and historical type specimen, we have produced strong evidence as to the close relationship of Gloeobacter to a
long known rock-dwelling cyanobacterial morphospecies Aphanothece caldariorum. Our results bring new clues to solving
the 40 year old puzzle of the true biological identity of Gloeobacter violaceus, a model organism with a high value in several
biological disciplines. A probable broader distribution of Gloeobacter in common wet-rock habitats worldwide is suggested
by our data, and its ecological meaning is discussed taking into consideration the background of cyanobacterial evolution.
We provide observations of previously unknown genetic variability and phenotypic plasticity, which we expect to be
utilized by experimental and evolutionary researchers worldwide.
Citation: Mares
ˇ
J, Hrouzek P, Kan
ˇ
a R, Ventura S, Strunecky
´
O, et al. (2013) The Primitive Thylakoid-Less Cyanobacterium Gloeobacter Is a Common Rock-Dwelling
Organism. PLoS ONE 8(6): e66323. doi:10.1371/journal.pone.0066323
Editor: John R. Battista, Louisiana State University and A & M College, United States of America
Received January 12, 2013; Accepted May 3, 2013; Published June 18, 2013
Copyright: ß 2013 Mares
ˇ
et al. This is an open-access artic le distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported as a long-term research development project no. RVO 67985939, by the grant no. GA JU 135/2010/P, GAC
ˇ
R P506/12/1818
and by the Center for Algal Biotechnology Tr
ˇ
ebon
ˇ
- ALGATECH (CZ. 1.05/21.00/03.0110). The authors acknowledge MetaCentrum v.o. for providing
supercomputing facilities under the research agreement MSM6383917201. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: mares@butbn.cas.cz
Introduction
Cyanobacteria are the most significant group of photosynthetic
prokaryotes, generating global impact effecting biogeochemical
cycles since ancient Earth history [1,2]. As one of the most
important sources of atmospheric oxygen and crucial carbon
fixers, they have been intensively studied by experimental and
evolutionary science [3,4,5]. Gloeobacter violaceus Rippka et al. 1974,
the most primitive among living cyanobacteria, has been subjected
to both of these approaches. In its original description [6], the
authors studied a single cyanobacterial strain (PCC 7421) isolated
from the surface of a limestone rock in Kernwald (Switzerland). A
peculiar simple cell organization with complete absence of
thylakoids and an unusual structure of the photosynthetic
apparatus supported the description of a new, separate monotypic
genus Gloeobacter [6,7,8]. Following studies of the phylogenetic
comparison of SSU rRNA gene and other loci [9,10,11]
demonstrated that G. violaceus diverged very early during the
cyanobacterial radiation, in an ancient lineage preceding the
cyanobacterial chloroplast ancestors [12]. These findings were in
accordance with its primitive morphology and cell ultrastructure.
Since then, G. violaceus PCC 7421 has become one of the key
species in evolutionary studies of (cyano)bacteria [13,14,15] and
plant life in general [16,17,18]. Gloeobacter was also among the first
cyanobacterial strains having its complete genome sequenced [19].
Besides its significance in evolutionary research, G. violaceus PCC
7421 has been frequently used as a model organism for
experimental studies of oxygenic photosynthesis [20]. A unique
molecular structure of photosystems I and II [21,22,23] and an
unusual morphology of its phycobilisomes (PBS) [24] enable
Gloeobacter to harvest light and transfer energy in a manner, which
is different from other photosynthetic organisms. Unlike in other
cyanobacteria, its PBSs are composed of six peripheral phycocy-
anin/phycoerythrin rods bound as a bundle to five horizontal rods
of an allophycocyanin core, allowing atypical energy transfer
pathways [25]. Apart from the field of photosynthesis research, a
pentameric ligand-gated ion channel (GLIC) was cloned from G.
violaceus and has become an important molecular model of
membrane receptors in general biological and clinical studies
[26,27,28].
PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e66323
In contrast to detailed knowledge of cell structure, physiology
and genetics of the experimental model strain PCC 7421, data on
the ecology, distribution, life strategy and overall variability in the
genus Gloeobacter have, until now, remained extremely limited.
Apart from PCC 7421, a few more populations were collected and
isolated from nearby localities (PCC 9601, PCC 8105). The only
cyanobacterium morphologically and phylogenetically corre-
sponding to G. violaceus found, aside from the original locality,
was reported to be from a surface of a fountain in Florence, Italy
[29]. The unexplained biological identity of Gloeobacter and its
relationship to other cyanobacteria has been under discussion ever
since the establishment of the genus. Already in its original
description, Rippka et al. [6] suggested a close relationship to a
little known botanical species Gloeothece coerulea Geitler 1927. This
latter cyanobacterium has a similar, simple cell morphology and
life cycle, and contains polar granules clearly resembling those of
G. violaceus [30]. On the basis of this assumption and the
observation of fossil cyanobacterial remnants, Golubic & Camp-
bell [31] hypothesized that the Gloeobacter/Gloeothece coerulea-like
morphotype has colonized epilithic habitats since the Precambri-
an. Another rock-inhabiting species, Aphanothece caldariorum Richter
1880, was studied in detail by Hansgirg [32] and it has quite often
been found in samples of (sub-)aerophytic microalgal biofilms since
then. As noted by Koma´rek & Anagnostidis [33], this cyanobac-
terium is almost identical to G. coerulea in both morphology and life
strategy and thus potentially related to G. violaceus.
Under the current cyanobacterial taxonomy [33], the genus
Gloeobacter can be relatively easily distinguished from the morpho-
logically similar genera Aphanothece, Anathece, Cyanobium and
Gloeothece by its phylogenetic position and by the absence of
thylakoids. However, until now, Gloeobacter was studied exclusively
by observing its cultures, while similar morphospecies from other
genera (e.g., Gloeothece coerulea, Aphanothece caldariorum) have never
been isolated into cultures, making it impossible to study them
using electron microscopy and molecular analysis to provide the
necessary evidence concerning their phylogeny and cell ultra-
structure.
In this study, we provide extensive evidence for three original
isolates of Gloeobacter violaceus, a botanical type specimen of A.
caldariorum, and two reference strains G. violaceus PCC 7421 and
PCC 9601 to uncover their taxonomic identity, phylogeny, and
biology in natural populations.
Methods
Strains and Samples
Two strains of Gloeobacter violaceus (PCC 7421 and PCC 6901)
were obtained from the Pasteur Culture Collection of Cyanobac-
teria, Paris, France. Another strain (VP3-01) was purified from a
biofilm growing on a surface of a fountain in Florence, Italy, as
previously described [29]. Two new strains were isolated from
samples dominated by cyanobacteria corresponding to the
morphospecies Aphanothece caldariorum from wet rocks in artificial
waterfalls in tropical greenhouses of botanical gardens in Liberec
and Teplice, the Czech Republic, in the years 2009–2010. The
collection of samples of cyanobacteria in the greenhouses of the
public Botanical Gardens in Liberec and Teplice, the Czech
Republic was approved by their directors Miloslav Studnic
ˇ
ka
(Liberec) and Jir
ˇ
ı
´
R. Haager (Teplice). No additional permissions
were required by the legal regulations of the Czech Republic. No
protected species were sampled. For isolation, a portion of an
environmental sample was spread on the surface of an agar plate
enriched with BG 11 medium [34]. Colonies that emerged from
individual Aphanothece caldariorum-like cells (checked by optical
microscopy) were then sequentially transferred to fresh plates until
unicyanobacterial strains were obtained. The holotype of A.
caldariorum var. cavernarum Hansgirg 1889 was provided by the WU
(Institute of Botany, University of Vienna) herbarium. The type
material of the nominate variety of this species was not found in
the respective herbaria, as it was probably lost.
The strains were cultivated in both liquid and agar BG11
medium for morphological, ultrastructural and molecular analysis.
For the purpose of pigment analysis and statistical analysis of cell
dimensions, the strains were grown in 50 mL liquid batch cultures
(inoculated with 1 mL of a previous batch at growth maximum),
under constant temperature (23uC) and irradiance (2065
mmol
m
22
s
21
). Our original cultures were deposited in Culture
Collection of Autotrophic Organisms (CCALA) of the Institute
of Botany ASCR, which is accessible to the public, under accession
codes CCALA 979 ( = G. violaceus VP3-01 from Florence), CCALA
980 ( = G. violaceus [A. caldariorum morphotype] from Teplice) and
CCALA 981 ( = G. violaceus [A. caldariorum morphotype] from
Liberec).
Morphology and Ultrastructure
Fresh cyanobacteria were observed under 4006 and 10006
magnifications using Olympus BX 51 microscope equipped with
differential interference contrast, an Olympus DP71 camera, and
the QuickPhoto Micro v. 2.3 image analysis software. Statistical
analysis of cell dimensions in cultures was based on photographs
taken at 10006magnification. Length and width of 100 randomly
selected mature cells (excluding the stage of fission or just after it)
from each sample were measured with an accuracy of 60.1
mm.
Statistical differences in cell length and length:width ratio between
the batch cultures of individual strains was assessed, using
Statistica v. 9.1 [35], by a Kruskal-Wallis test; pair-wise differences
among the batches were evaluated by standard 2-tailed t-tests.
For ultrastructural studies, biological material of cyanobacteria
was fixed with 6% glutaraldehyde and kept at room temperature.
Samples were washed with 0.05 M phosphate buffer (pH 7.2) and
postfixed with 2% osmium tetroxide in the same buffer at room
temperature for 2 hours, then repeatedly washed with 0.05 M
phosphate buffer. Finally, cells were dehydrated with a graded
isopropanol series and embedded in Spurr’s resin [36] using
propylene oxide as an intermediate stage. Thin sections were
stained with uranyl acetate and lead citrate and observed in a Jeol
JEN 1010 transmission electron microscope at 80 kV.
Table 1. List of PCR and sequencing primers.
Primer Name Sequence (59to 39)
359F
1
[39] GGG GAA TYT TCC GCA ATG GG
23S30R
1
[38] CTT CGC CTC TGT GTG CCT AGG T
Cyano6r
2*
GAC GGG CCG GTG TGT ACA
T7
2
TAA TAC GAC TCA CTA TAG GG
SP6r
2
TAT TTA GGT GAC ACT ATA G
rpc/MF
1
[9] GGT GAR GTN ACN AAR CCA GAR AC
rpc/CR-1
1
[9] CCA GAR TAG TCN ACC CGT TTA CC
1
PCR primers,
2
sequencing primers,
*reverse complement to primer 14 [38].
doi:10.1371/journal.pone.0066323.t001
Gloeobacter violaceus Is a Common Organism
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Molecular Analysis
The biomass was dried for 48 hours over silica gel and crushed
to powder in a Retsch MM200 laboratory mill with wolfram
carbide beads (3 minutes, 30? s
21
). Total genomic DNA was
isolated following the modified xanthogenate-SDS buffer extrac-
tion protocol with addition of 3% PVPP and PEG-MgCl
2
precipitation [37]. Alternatively, for the historical type specimen
of A. caldariorum var. cavernarum, a small piece (1 mm
2
) of the
herbarium material was pulverized as previously, rehydrated in
50
mL of TE buffer, and 2 mL of the suspension were directly
added to the PCR mix. A section of the rRNA operon containing
the partial SSU rRNA gene and the ITS region was amplified with
primers 359F and 23S30R [38,39] (Table 1). Ten ng of template
DNA was mixed with 6 pmol of each primer in a commercial
PCR mix with Taq polymerase (Plain PP Master Mix, Top Bio,
the Czech Republic), and amplified with an initial denaturation
Figure 1. Morphology of
Gloeobacter violaceus
and
Aphanothece caldariorum
-like samples. (A) and (B) A. caldariorum-like samples from the
botanical gardens in Liberec and Teplice, respectively, showing typical rod-shaped cells with polar granules and layered mucilaginous envelopes; (C)
cell morhology in the perfectly preserved herbarium type specimen of A. caldariorum var. cavernarum; (D) ‘‘nanocytes‘‘ in the environmental sample
from Liberec dominated by A. caldariorum-like morphotype; (E) and (F) batch cultures ordered by increasing age (from the left) of A. caldariorum-like
strain CCALA 981 and G. violaceus PCC 7421, respectively, showing a gradual color shift from grey-violet to yellow-orange; (G–J) and (K–N) change in
cell morphology from subspherical nanocyte-like cells to rod-shaped cells with occasional mucilaginous envelopes in the batch cultures of CCALA
981 and PCC 7421, respectively (the batches are the same as in panels E and F). Scale bars, 10
mm.
doi:10.1371/journal.pone.0066323.g001
Gloeobacter violaceus Is a Common Organism
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step (5 minutes at 95uC), 35 cycles of denaturation (1 min at
94uC), primer annealing (45 s at 55uC) and elongation (2 min at
72uC), and final elongation for 10 min at 72uC. The PCR product
was cloned using the standard pGEM-T Easy (Promega Corp.,
WI, USA) vector system according to supplier instructions. The
plasmid containing the required insert was purified from the
bacterial culture using Zyppy Plasmid Miniprep kit (Zymo
Research Corp., CA, USA). The rpoC1 gene fragment was
amplified using primers rpc/MF and rpc/CR-1 (Table 1)
following the published protocol [9] (initial denaturation for
5 min at 94uC, 35 cycles of 1 min denaturation at 94uC, primer
annealing for 1 min at 52uC, elongation for 2 min at 72uC, and
final elongation step for 10 min at 72uC), and cloned as above.
PCR gel pictures are given in Figure S4. The clones were
sequenced using primers T7 and SP6r (Table 1) in Laboratory of
Genomics, Biology Centre of the Academy of Sciences of the
Czech Republic, C
ˇ
eske´ Bude
ˇ
jovice (with an ABI PRISM
3130 XL, Applied Biosystems, Life Technologies Corp., CA,
USA). Sequences were deposited in GenBank under accession
numbers KC004017–KC004023 and KC866356.
Phylogenetic Analysis
Sequences of the SSU rRNA and rpoC1 gene from thirty-eight
cyanobacteria were collected from published studies and mined
from the whole genome database available in GenBank. The
sequence matrices were assembled to include equal number of
strains from all cyanobacterial orders. Three bacterial strains, E.
coli K12, Salmonella enterica str. UK-1, and Cronobacter turicensis
z3032, were used as out-group taxa. The sequences were aligned
via MAFFT v. 6 [40] using the FFT-NS-I strategy, and manually
corrected. For the final analysis of the SSU rRNA gene data, a
region of alignment that is common to all collected sequences was
used. It spanned 1041 positions from 377 to 1432 (Escherichia coli
numbering) after ambiguous gap columns were removed. Phylo-
genetic analysis was conducted employing Bayesian inference in
MrBayes 3.1.2 [41], maximum likelihood analysis in RAxML
7.3.2 [42], and maximum parsimony analysis in PAUP* 4.0b10
Figure 2. Morphometric analysis of cells in
Aphanothece caldariorum
-like and
Gloeobacter violaceus
cultures. (A) and (B) increasing cell
length corresponding to batch cultures of increasing age of A. caldariorum-like strain CCALA 981 and G. violaceus PCC 7421 (Kruskal-Wallis test,
p,0.001), difference between individual pairs of batches was also significant (two-tailed t-test, p,0.02 except for a pair of batches 41 and 50 days
old in PCC 7421, for which the difference was at the edge of statistical significance, p = 0.045); (C) and (D) increasing cell length:width ratio in the
same batch cultures as previously (Kruskal-Wallis test, p,0.001), difference between individual pairs of batches was also significant (two-tailed t-tests,
p,0.001) except for a pair of batches 41 and 50 days old in PCC 7421. One hundred cells were measured in each sample.
doi:10.1371/journal.pone.0066323.g002
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[43]. For the Bayesian analysis, two runs of four Markov chains
were executed for 1 000 000 generations with default parameters,
sampling every 100 generations (the final average standard
deviation of split frequencies was lower than 0.01). The maximum
likelihood calculation was executed upon the generalized time-
reversible (GTR) substitution model with discrete gamma distri-
bution in six categories. The gamma shape parameter a as well as
the proportion of invariable sites were estimated from the data set
(GTR+G+ G model), and 1000 bootstrap replicates were
calculated to evaluate the relative support of branches. A
maximum parsimony analysis involved one hundred replicate
searches with starting trees obtained by random stepwise addition,
using the tree bisection-reconnection (TBR) branch swapping
algorithm; one thousand nonparametric bootstrap replications
were run with the same settings to evaluate the relative branch
support. All bases and base changes were equally weighted, and
gaps were coded as missing data. MetaCentrum (www.
metacentrum.cz) and CIPRES (www.phylo.org) supercomputing
facilities were used for fast calculation of Bayesian and likelihood
trees.
For the combined analysis of both loci, a 778 bp long region of
partial rpoC1 data was merged with an aligned SSU rRNA gene
from corresponding strains into a final concatenated alignment of
1988 bp. Phylogenetic analysis of the concatenated alignment was
conducted using Bayesian inference, maximum likelihood and
maximum parsimony methods as mentioned previously. Phyloge-
netic trees were visualized using FigTree v. 1.3. (http://tree.bio.
ed.ac.uk/software/figtree/). The alignments were uploaded to the
TreeBase web (http://purl.org/phylo/treebase/phylows/study/
TB2:S14106 ).
Pigment Analysis
The relative concentrations of the two phycobiliproteins,
phycocyanin (PC) and allophycocyanin (APC) were estimated
from the mean absorption of light between 616–624 nm and 650–
658 nm respectively according to Krogmann et al. [24]. The
relative concentration of phycoerythrin (PE) was calculated at
662 nm, by utilizing an equation, which was used in the same
study, as [PE] = A
662
/e
662
with e
662
(extinction coefficient) at
456 mM cm
21
. Absorption spectra were recorded in vivo with a
Unicam UV 550 (Thermospectronic, UK) following the method
described by [44].
Fluorescence emission spectra of living cells at room temper-
Figure 3. Light spectroscopy analysis of photosynthetic
pigments in
Aphanothece caldariorum
-like strain and
Gloeobacter
violaceus
. (A) fluorescence emission spectra of A. caldariorum-like strain
CCALA 981 in comparison with G. violaceus PCC 7421 for excitation to
phycobilisomes. Higher content of phycoerythrin in CCALA 981 is
documented by a relative increase in its fluorescence emission at
574nm. (B) and (C) whole cell absorption spectra of CCALA 981 and PCC
7421 normalized to chlorophyll a content. Clear accumulation of
carotenoids (wide absorbance peak between 450–500 nm) at the
stationary phase of growth (third and fourth batch) is obvious in both
strains. The absorbance of 41-day old strain PCC 7421 is raised due to
the beginning of accumulation of carotenoids in this batch, but
phycobilin peaks are still recognizable. Individual absorbance peaks are
as noted; CAR, carotenoids; PE, phycoerythrin; PC+APC, phycocyanin
and allophycocyanin; Chl a, chlorophyll a.
doi:10.1371/journal.pone.0066323.g003
Table 2. Relative concentration of particular
phycoerythrobilins in phycobilisomes of Aphanothece
caldariorum-like and Gloeobacter violaceus strains calculated
from absorption spectra.
PE/APC PC/APC PE/PC
A. caldariorum
-like CCALA 981 1.7160.16 1.3460.21 1.2860.08
G. violaceus
PCC 7421 1.2660.16 1.0660.22 1.260.11
PE, phycoerythrin; APC, allophycocyanin; PC, phycocyanin. See Metho ds for
precise description of calculation of the values.
doi:10.1371/journal.pone.0066323.t002
Table 3. Number of phycoerythrin and phycocyanin trimers
in a single phycobilisome rod of Aphanothece caldariorum-like
and Gloeobacter violaceus strains.
n(PE) n(PC)
A. caldariorum
-like CCALA 981 4.560.4 3.660.6
G. violaceus
PCC 7421 3.460.4 2.860.6
PE, phycoerythrin; PC, phycocyanin. Values were calculated on the basis
absorbance changes taking into account a model of the phycobilisome for G.
violaceus PCC 7421 consisting of the phycobilisome core (16 allophycocyanins
trimers) with 6 attached rods with different amount of PE and PC trimers [24].
doi:10.1371/journal.pone.0066323.t003
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ature were measured using an Aminco-Bowman Series 2
spectrofluorometer, in the standard instrument geometry with
excitation at 492 nm. Fluorescence emission was scanned between
550–750 nm with a 4 nm bandwidth.
To assess carotenoid composition, the biomasses of CCALA 981
and PCC 7421 were extracted with an acetone/methanol mixture
(7/3 v/v) in the dark into Eppendorf tubes and centrifuged. The
samples were subjected to HPLC analysis on the Agilent
Technologies 1200 series chromatographic system with a diode-
array detector. Separation was performed on a Luna C8 column
(3
mm, 100A, 10064.6 mm –00D-4248-E0, Phenomenex, USA)
using methanol (A) and 28 mM amonium acetate in 80%
methanol (B) as solvents (from 30% to 100% of solvent A in
30 min.). The pigments were detected on the basis of their
retention times (internal database) and absorption spectra.
Results and Discussion
Natural Morphology and Habitat
Material collected from artificial waterfalls in greenhouses of
two botanical gardens revealed the presence of a morphotype
exactly matching the species Aphanothece caldariorum as described in
botanical literature [32,33]. In both cases, it was a component of
an epilithic gelatinous cyanobacterial mass. In the native state, the
collected material (from both greenshouses) showed all of the
morphological characteristics typically described for A. caldariorum
[33], such as dimensions and shape of cells, concentrically
lamellated mucilaginous envelopes, and polar granules
(Figure 1A, B). Compared to the typical properties of Gloeobacter
violaceus strains [6,33], the A. caldariorum-like cells were significantly
longer (up to more than 10
mm versus 2–3 mminG. violaceus), often
somewhat bent or arcuate (straightly rod-shaped in G. violaceus),
with broader and more conspicuously lamellated sheaths. We also
recorded frequent occurrence of small subspherical cells,
(Figure 1D) clearly corresponding to ‘‘nanocytes‘‘ [33], which
were not reported for G. violaceus. According to our observations,
Figure 4. Comparison of cell ultrastructure in
Aphanothece caldariorum
-like strains and
Gloeobacter violaceus
. Typical cell ultrastructure
of G. violaceus PCC 9601(A), G. violaceus CCALA 979 (B), A. caldariorum-like strain CCALA 981(C) and A. caldariorum-like strain CCALA 980 (D),
respectively. Cells did not contain any thylakoid, the photosynthetic pigments accumulated in an electron-dense layer near the multi-layered cell wall.
Cells typically contained two large polyphosphate granules in polar positions. Observed ultrastructure was identical to the reference strain G.
violaceus PCC 7421 [6]. Scale bars, 500 nm.
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these cells do not seem to represent any specialized reproductive
stage. Apparently, they were produced by fast serial binary fission
under favorable conditions. A. caldariorum-like morhotype was
found on wet rocks in waterfalls, which corresponded well to the
original description of this species from botanical gardens in
Prague and wet rocks in Bohemia [32], and also to other reports of
this species from numerous similar localities worldwide
[45,46,47,48,49,50,51,52,53]. The similarity of the morphotype
to A. caldariorum was further confirmed through microscopic
examination of the well-preserved botanical type specimen of A.
caldariorum var. cavernarum (Figure 1C). Unfortunately, the type
specimen of the nominate variety caldariorum was lost or destroyed
during the Second World War, and is not available for study.
Thus, a definite proof of identity (by molecular methods) of our
material to A. caldariorum cannot be given.
Strain Morphology and Pigment Analysis
Very high similarity in basic morphometric characteristics was
found between typical Gloeobacter PCC 7421 strain and all the
studied Apahnothece caldariorum morphotype samples. The observed
cyanobacterial isolates also exhibited almost identical changes in
morphology and pigmentation during their life cycle, as described
below.
In cultures grown in nutrient-rich media (BG 11), cells
resembling Aphanothece caldariorum, immediately after inoculation,
started accelerated proliferation (Figure S1), resulting in compact
clusters of small subspherical ‘‘nanocyte-like‘‘ cells, mostly with
two distinct granules inside (Figure 1G). As long as it was cultured
in fresh media, this morphotype was maintained. Upon consulting
available literature [6,34], this strain morphology obviously
matched that of the reference strain Gloeobacter violaceus PCC
7421. Our optical microscopy observations verified identical
appearance of all gathered strains (PCC 7421, PCC 9601,
CCALA 979, CCALA 980, CCALA 981– Figure 1G–N, Figure
S2).
During the growth of batch cultures on both solid and liquid
media, a dramatic color shift was observed in all strains (Figure 1E,
F). The color changed from various shades of grey, greyish-blue-
green and greyish-violet in young cultures, through bright violet or
pinkish-violet at growth maxima, to green or yellow-green in older
cultures, ending with yellow and orange in their stage of
senescence. These observations were in accordance with reports
of an extreme color variability of A. caldariorum in natural habitats
[32,33].
In order to determine the principles and symptoms of these
changes, we compared series of batch cultures grown in
standardized conditions for one representative of typical G.
violaceus (PCC 7421) and one representative of a strain derived
from the A. caldariorum-like morphotype (CCALA 981). Four
batches of different ages spanning the color range from young and
thriving (grey, violet) to senescent cultures (yellow, orange) were
chosen for morphometric and pigment analysis for each of the two
strains (Figure 1E, F). As shown by the statistical analysis of cell
dimensions (Figure 2, p,0.001), the cells of both strains changed
their shape from subspherical to short cylindrical of ‘‘nanocytes‘‘
in young cultures, to rod-shaped cells in old cultures (Figure 1G–
N). Cultivated cells never reached a length of over 5
mm, which is
a dimension frequently exceeded in natural populations of
A.caldariorum (Figure 1A–C, [33]). A probable reason for such a
difference lies in the unnatural chemical composition of the culture
medium, and, possibly, also in the different physical properties of
the artificial medium as compared to natural substrates. However,
in the oldest batches the cells were often somewhat arcuate and
enclosed in gelatinous envelopes (Figure 1J, N) clearly resembling
the typical A. caldariorum morphotype.
To elucidate the process of color shift, we performed a light
spectroscopy analysis of the main photosynthetic pigments
(phycobilins and chlorophyll a) and HPLC analysis of carotenoids
in the same batch cultures that were used for morphometry.
In general, the pigment composition of CCALA 981 and PCC
7421 was similar. Nevertheless, the A. caldariorum-like strain had
slightly different molar ratios of particular phycobiliproteins within
the PBS in comparison to G. violaceus PCC 7421 (Table 2). The
molar ratio (measured in young cultures) in PCC 7421 was about
1APC: 1.3PC: 1.2 PE, while in CCALA 981 we found relatively
higher contents of phycoerythrins and phycocyanins in compar-
ison to allophycocyanins (1APC: 1.3 PC: 1.7 PE). The higher
content of phycoerythrin in the latter strain was also confirmed by
a relative increase in its fluorescence emission at 574nm
(Figure 3A). The observed discrepancy in the phycobilin ratios
corresponded well to a slightly different color of the cultures
(violet-grey vs. bright violet). This finding suggests a relative
increase in the length of PBS rods in CCALA 981 (Table 3). The
PBS in G. violaceus PCC 7421 was previously shown to consist of
16 APCs in the core, with 6 attached rods of different length and
PE/PC ratio [24,54] depending on growth conditions. Assuming
an identical core structure, the PBS rods of the A. caldariorum-like
strain CCALA 981 were almost 40% longer in comparison to
PCC 7421 (In Table 3, almost 8 PC+PC trimers per single rod of
CCALA 981 and only five PC+PC trimers in PCC 7421).
Alternatively, some PBSs without the APC core could occur
similarly to the so called Cpc-G2 phycobilisomes recently
described for Synechocystis sp. [55].
Our data clearly suggests that the color variability in young
cultures (grey/blue-green/violet hues) can be explained by
different ratios of individual phycobilins depending on actual
growth conditions and physiological state of the cultures
(Figure 3A, Figure S3). The versatile phycobilin ratio can also
explain the greyish color of young PCC 7421 (Figure 1F), which
differs from bright violet color typically described for this strain
[6]. As documented by absorption spectra (Figure 3B, C), in older
batch cultures the PBSs degraded while carotenoids accumulated.
The decrease, and finally the total absence of phycoerytrin
(565 nm) and phycocyanin (620 nm) peak, could be seen in the
whole cell spectra of older cultures in both CCALA 981 and PCC
7421, where the phycobilins were replaced by an intensively
absorbing band in the wavelength range of 400–550 nm,
corresponding mainly to carotenoid absorption. This process was
reflected by a gradual shift in color from greenish (chlorophyll) to
yellowish and orange (carotenoids).
The carotenoid composition of the A. caldariorum-like strain
CCALA 981 was identical to that of G. violaceus PCC7421 as
assessed by HPLC analysis. The dominant carotenoids were b-
Carotene and (2S,29S)-oscillol 2,29-di(a-l-fucoside), while echine-
none was found as a minor component. Our results matched those
in the report for G. violaceus PCC7421 by Tsuchiya and co-workers
Figure 5. Phylogenetic position of
Gloeobacter violaceus
and
Aphanothece caldariorum
. (A) Phylogenetic tree based on a SSU rRNA gene
alignment. (B) Phylogenetic tree based on a concatenated SSU rRNA gene+rpoC1 alignment. Sequences generated in this study are printed in bold
font. Branch support values (%) are given at nodes in this format: Bayesian inference/maximum likelihood/maximum parsimony. A well supported
basal clade of cyanobacteria consisting of G. violaceus and A. caldariorum is highlighted by blue colour.
doi:10.1371/journal.pone.0066323.g005
Gloeobacter violaceus Is a Common Organism
PLOS ONE | www.plosone.org 8 June 2013 | Volume 8 | Issue 6 | e66323
[16]. Presence of the rarely occurring carotenoid (2S,29S)-oscillol
2,29-di(a-l-fucoside) in the studied strains further supported their
isolated position in phylogeny of cyanobacteria. Carotenoids
belonging to oscillol 2,2-diglycosides were found only in limited
number of cyanobacteria and bacteria [56,57].
Ultrastructure
Perhaps the most remarkable feature in Gloeobacter, that
separates it from other cyanobacteria, is the complete absence of
thylakoid membranes [6,58]. Hence, the study of cell ultrastruc-
ture by TEM was one of the crucial components of our analysis.
Our results unambiguously showed an identical cellular structure
in all of the studied strains (Figure 4A–D), exactly matching that of
the reference strain PCC 7421 [6]. The multi-layered cell wall was
fringed by a band of electron-dense material (photosynthetic
pigments). No thylakoid membranes were registered but usually
two large polyphosphate granules were present, one at each pole of
the cell.
Phylogeny and Taxonomy
The phylogenetic reconstruction based on both, the SSU rRNA
gene and a combination of SSU rRNA gene with a protein-coding
housekeeping gene like rpoC1, provided congruent results regard-
ing the position of Gloeobacter violaceus and Aphanothece caldariorum-
like strains. All studied samples clustered in a single, distinct, fully
supported basal clade, which clearly corresponded to the genus
Gloeobacter (Figure 5). All the cultured strains of G. violaceus and A.
caldariorum-like isolates, including the reference strain PCC 7421,
formed an extremely tight cluster, which has to be regarded as the
single species - G. violaceus (SSU rRNA gene similarity over 99%).
This conspicuous taxon is characterized by a specific combination
of characters, i.e. phylogenetic position, absence of thylakoids,
pigment composition, life cycle, and ecology (life strategy). Given
the fact that direct comparison with the type material of A.
caldariorum var. caldariorum by molecular methods is impossible, the
decision whether this species is truly identical with G. violaceus
depends on interpretation of indirect evidence. In our opinion, at
least the assignment of A. caldariorum to the genus Gloeobacter is
clear, and the identity of the two species is quite possible.
Unfortunately, the name Gloeobacter violaceus was never validly
published under the rules of Bacteriological Code, and if identity
with A. caldariorum was assumed, the epithet ‘‘caldariorum‘‘ (and
probably also ‘‘coerulea‘‘ from Gloeothece coerulea) would have priority
over ‘‘violaceus‘‘ under the Botanical Code. Thus, the nomencla-
toric status of the species Gloeobacter violaceus is rather unclear and
has to be amended by a dedicated study. Interestingly, the SSU
rRNA gene sequence of the A. caldariorum var. cavernarum type
specimen was slightly different from the rest of Gloeobacter
sequences. Considering the SSU rRNA gene similarity (, 96%),
it could be regarded as a separate species of Gloeobacter. However,
our observations did not reveal any obvious difference (other than
DNA sequence) when compared to the material of A. caldariorum
from the greenhouses in Liberec and Teplice. The original
description [32] distinguished between the varieties on the basis of
slight differences in cell dimensions and color, characteristics,
which were proven in this study to show great variability. In our
opinion, a decisive taxonomic conclusion would require analysis of
fresh samples, isolated strains of the var. cavernarum, and collection
of more data. Nevertheless, based on the relatively common
occurrence of the A. caldariorum morphotype (as mentioned in
scientific reports worldwide), it is quite probable that members of
the genus Gloeobacter are much more common than previously
thought. This hypothesis is yet to be tested by careful study of
epilithic cyanobacterial communities in future.
While the picture of Gloeobacter as an isolated ancient lineage
agrees with most published results [4,12,13,59,60], a recent study
[61] proposed a possible relationship to other simple coccoid
cyanobacteria. In their report, Courdeau et al. [61] described a
phylogenetic cluster with moderate branch support, consisting of
Synechococcus-like morphotypes related to Gloeobacter, which they
called ’Gloeobacterales’. It also included the peculiar candidatus
Gloeomargarita lithophora that forms intracellular calcite precipitates.
In a manner similar to
Gloeobacter, members of this group diverged
earlier in evolution than the chloroplast ancestors. In our
evolutionary trees, this cluster, represented by the Synechococcus
strains isolated from the Yellowstone National Park hot springs
(C9, Ja-3-3Ab and Ja-2-3B9a), branched separately (Figure 5B).
Considering the major differences in cell ultrastructure and
photosynthetic apparatus, we propose that the order Gloeobacter-
ales should be reserved for the genus Gloeobacter, which is primarily
defined by the absence of thylakoids [62]. This is further supported
by a considerable SSU rRNA gene sequence distance; for
comparison, there is approximately 88% similarity between G.
violaceus PCC 7421 and Synechococcus sp. Ja-3-3Ab while the
similarity between PCC 7421 and a heterocytous cyanobacterium,
Nostoc sp. PCC 7120, is 87%. On the other hand, in all known
cyanobacteria from these ancient lineages, there is a clear common
tendency to colonize wet or submerged, mostly calcite rocks [61].
This evidence reflects a possible first appearance of cyanobacteria
in rock-associated, calcifying biofilm habitats, such as stromatolites
or travertine spring mats.
Conclusions
Our results brought new clues to solving a 40 years old puzzle
about the true biological identity of Gloeobacter violaceus,an
important model organism with a great value in several biological
disciplines. In the first place, we showed that the genus Gloeobacter is
a commonly occurring terrestrial cyanobacterium. On the basis of
detailed morphological, ultrastructural, biochemical, and phylo-
genetic comparisons of two available Gloeobacter strains, three new
independent isolates, and a botanical type specimen, we generated
complementary evidence of the identity of Gloeobacter with a long
known rock-dwelling cyanobacterial morphospecies Aphanothece
caldariorum. The life strategy of Gloeobacter/A. caldariorum is
congruent with that of other primitive coccoid cyanobacteria,
suggesting a possible origin of their cyanobacterial ancestors in
alkaline rock-associated biofilms. In this paper we provided
observations of previously unknown genetic variability and
phenotypic plasticity, which we expect to be utilized by
experimental and evolutionary researchers worldwide.
Supporting Information
Figure S1
A. caldariorum-like morphology: formation of nanocyte-
like cells in culture. The cells of A. caldariorum CCALA 981
started rapid successive binary fission shortly after inoculation on
fresh media. (A) Cell division into multiple small spherical cells. (B)
Nanocyte-like daughter cells forming clusters on solid medium.
Formation of nanocyte-like cells was observed in the initial stages
of cultivation directly on the agar plate when there was still some
contamination by bacteria and fungi. Scale bars, 50
mm.
(PDF)
Figure S2
Identical morphology of Aphanothece caldariorum-like
and Gloeobacter violaceus isolates in culture. (A) G.
violaceus PCC 9601; (B) G. violaceus CCALA 979; (C) A. caldariorum-
Gloeobacter violaceus Is a Common Organism
PLOS ONE | www.plosone.org 9 June 2013 | Volume 8 | Issue 6 | e66323
like strain CCALA 980. Strains PCC 7421 and CCALA 981 are
documented in Figure 1. Scale bars, 10
mm.
(PDF)
Figure S3
Proportion of phycobiliproteins in Aphanothece caldar-
iorum and Gloeobacter violaceus during the culture
senescence. (A) A. caldariorum CCALA 981, (B) G. violaceus PCC
7421. Both PE and PC were degraded as the culture aged. Thus,
the blue-green/violet colour at the beginning of the cultivation was
replaced by yellow-orange colour (carotenoids). Interestingly, the
PE/PC ratio was increased in old cultures at the end of the
cultivation in both strains. This was due to major decrease in PC,
as seen from the PC/APC curve. The relatively high PE
proportion at the end of cultivation also agreed with the orange
colour. PE, phycoerytrin; PC, phycocyanin; APC, allophycocya-
nin.
(TIF)
Figure S4
PCR products of SSU rRNA gene region and partial
rpoC1 visualized on 1.5% agarose gels. (A) and (B) SSU
rRNA gene region PCR products; (C) and (D) Partial rpo C1 gene
PCR products; (E) SSU rRNA gene region products amplified
from A. caldariorum var. cavernarum type specimen by direct PCR.
Sample names are indicated at loading wells. A standard 100 bp
DNA ladder with fragment sizes corresponding to 100, 200, 300,
400, 500, 600, 700, 800, 900, 1000, 1200 and 1517 bp was used in
all gels. The samples were stained by GelRed Nucleic Acid Dye
(Biotium, Hayward, USA). C, negative control (blank); X,
unsuccessful PCR.
(TIF)
Acknowledgments
The skillful technical assistance of Cristina Macalchi and Dana S
ˇ
vehlova´is
gratefully acknowledged. We greatly appreciate the opportunity to study
cyanobacteria from the Botanical Gardens in Liberec and Teplice, which
was provided by Miloslav Studnic
ˇ
ka and Jir
ˇ
ı
´
R. Haager.
Author Contributions
Conceived and designed the experiments: JM PH RK JK. Performed the
experiments: JM PH RK. Analyzed the data: JM PH RK SV OS.
Contributed reagents/materials/analysis tools: SV. Wrote the paper: JM
PH SV OS JK.
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