RESEARCH ARTICLE
The genome sequence of Synechocystis sp. PCC 6803
substrain GT-T and its implications for the evolution of
PCC 6803 substrains
Satu Koskinen1 , Juha Kurkela1 , Mark
eta Linhartov
a2 and Taina Tyystj€arvi1
1 Department of Life Sciences/Molecular Plant Biology, University of Turku, Finland
2 Institute of Microbiology of the Czech Academy of Sciences, Trebon, Czech Republic
Keywords
chromosome sequence; cyanobacteria;
Synechocystis sp. PCC 6803
Correspondence
T. Tyystj€arvi, Department of Life Sciences/
Molecular Plant Biology, Turku FI-20014,
Finland
Tel: +358 400 546 259
E-mail: taityy@utu.fi
(Received 1 December 2022, revised 2
February 2023, accepted 7 February 2023)
doi:10.1002/2211-5463.13576
Edited by Alberto Alape-Gir
on
Synechocystis sp. PCC 6803 is a model cyanobacterium, glucose-tolerant
substrains of which are commonly used as laboratory strains. In recent years,
it has become evident that ‘wild-type’ strains used in different laboratories
show some differences in their phenotypes. We report here the chromosome
sequence of our Synechocystis sp. PCC 6803 substrain, named substrain GT-
T. The chromosome sequence of GT-T was compared to those of two other
commonly used laboratory substrains, GT-S and PCC-M. We identified 11
specific mutations in the GT-T substrain, whose physiological consequences
are discussed. We also provide an update on evolutionary relationships
between different Synechocystis sp. PCC 6803 substrains.
The cyanobacterium Synechocystis sp. PCC 6803
(hereafter Synechocystis) is a unicellular, non-nitrogen-
fixing, freshwater cyanobacterium that is widely
used as a model organism. It was isolated in California
in 1968 and deposited to the Pasteur Culture Collec-
tion of Cyanobacteria (Synechocystis sp. PCC 6803)
and to the American Type Culture Collection (ATCC
27184) [1].
The popularity of Synechocystis as a model organism
is based on a few advantages. The complete chromo-
some sequence of Synechocystis was available already in
1996 [2]. Construction of mutant strains is easy in this
naturally competent cyanobacterium [3]. The original
PCC 6803 or ATCC 27184 strains do not tolerate glu-
cose in the light, but later glucose-tolerant substrains
allowing photoheterotrophic, mixotrophic, and hetero-
trophic growth have been isolated [4–7]. A large variety
of biophysical, biochemical, and molecular biology tech-
niques have been optimized to Synechocystis.
The 1996 sequenced strain was a substrain Kazusa
(GT-Kazusa). GT-Kazusa is a derivative of the
glucose-tolerant Williams strain [4]. Phenotypic varia-
tions and some known sequence differences between
Synechocystis substrains led to the sequencing of the
substrain GT-S (substrain from Sato’s laboratory in
Tokyo) in 2011 [8]. GT-S originated from the same
laboratory strain as GT-Kazusa. Due to their close
history, it was assumed the GT-S and GT-Kazusa
would have identical sequences. However, a compar-
ison of the GT-S sequence and re-sequenced frozen
Abbreviations
ATCC, American Type Culture Collection; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; PPFD, photosynthetic photon flux density; SNP,
single nucleotide polymorphism.
1FEBS Open Bio (2023) 
 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
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DNA samples of GT-Kazusa revealed that these
strains contain a few differences [8].
Thereafter, more Synechocystis substrains have been
sequenced: GT-I, PCC-P and PCC-N [9], PCC-M [10],
GT-O1 and GT-O2 [11], GT-G [7], and GT-P and
GT-W [12]. Based on sequence data, Synechocystis
substrains can be divided into two main clades, the
GT clade comprise nonmotile, glucose-tolerant descen-
dants of the ATCC 27184 strain, whereas PCC strains
are descendants of Synechocystis sp. PCC 6803 strain
in the Pastor Culture Collection of Cyanobacteria.
Some sequence differences between GT and PCC
strains are common to all sequenced substrains, but on
the top of these common features, all sequenced
strains contain numerous substrain-specific mutations.
To allow full comparison of the results obtained
with different substrains and mutants constructed
using them as host strains, we have now sequenced
our substrain, named as GT-T.
Materials and methods
Synechocystis sp. PCC 6803 substrains and
growth conditions
The GT-T substrain of Synechocystis sp. PCC 6803 was
brought to Turku from Christer Jansson’s laboratory in the
early 90s [13,14]. To Stockholm, Christer Jansson brought
it from McIntosh’s laboratory in the late 80s [15]. The AR
mutant strain, derived from the glucose-tolerant McIn-
tosh’s strain, contains interrupted psbA1 and psbA3 genes
and the antibiotic resistance cassette after the psbA2 gene
[14]. Substrain Nishiyama is used as a control strain in
Nishiyama’s laboratory [16] and the substrain GT-P is from
Nixon’s laboratory [12].
Cells were grown in our standard growth conditions. The
BG-11 medium was supplemented with Hepes-NaOH pH 7.5
and cells were grown at 32 °C in ambient air. Thirty-mL cell
cultures were grown in 100-mL Erlenmeyer flasks under con-
stant illumination with the photosynthetic photon flux density
(PPFD) of 40 lmol m
2 s
1. The light source was a mixture
of fluorescence tubes, light colors 840 and 865 [Osram
(Munich, Germany)/Philips (Amsterdam, The Netherlands)].
For photoheterotrophic conditions, cultures were supple-
mented with 5 mM glucose and 10 lM 3-(3,4-dichlorophenyl)-
1,1-dimethylurea (DCMU). Growth was followed by measur-
ing OD730 once a day. Dense cultures were diluted (OD730 did
not exceed 0.4 in measurements) and dilutions were taken into
account when the results were calculated.
DNA sequencing
Genomic DNA was isolated using the phenol extraction
method [4]. Sequencing was done by Eurofins Genomics
Europe Sequencing Gmbh (Konstanz, Germany) with
NovaSeq 6000 platform and 150 bp paired-end configura-
tion. For library preparation, TruSeq Adapter sequences
were utilized using a self-validated and established protocol
of Eurofins Genomics, based on NEBNext Ultra II Direc-
tional DNA Library Prep Kit for Illumina. Default param-
eters were used in the following analysis tools. The quality
control for the 3,651,578 Illumina raw reads was done with
FastQC v.0.11.3 [17].
Mapping of DNA reads to the chromosomes of
Synechocystis sp. PCC 6803 substrains GT-S and
PCC-M
Reads (30 9 sequence coverage) were mapped to the refer-
ence genome sequence of Synechocystis sp. PCC 6803 sub-
strain GT-S (https://www.ncbi.nlm.nih.gov/nuccore/NC_
017277) using BWA-MEM aligner v.0.7.12-r1039 [18] using
the following parameter setting: Minimum seed length 19,
Maximum gap length 100, Match score 1, Mismatch penalty
4, Gap opening penalty 6, Gap extension penalty 1, Penalty
for end clipping 5. GT-S reference genome was modified by
removing a transposase gene sequence (nucleotide position
2047218 to 2048400), as only GT-Kazusa and GT-S contain
this transposase [19]. Variant calling was done with SAM-
tools v.1.2 + htslib-1.2.1 [20] using the following setting: Min-
imum mapping quality for an alignment to be used 0,
Minimum base quality for a base to be considered 20, Output
per sample read depth yes, Output per sample number of
high-quality nonreference bases yes. All data analysis steps
were performed with Chipster v4 platform [21]. The chromo-
some was annotated using NCBI Prokaryotic Genome Anno-
tation Pipeline (PGAP) with the best-place reference protein
set and GeneMarkS-2+ v6.1 [22–24]. In addition, the reads of
GT-T were mapped to the genome sequence of the substrain
PCC-M (https://www.ncbi.nlm.nih.gov/nuccore/CP003265).
Negative staining of pilus structures
Cells (1 mL, OD730 = 0.4) were pelleted at 3000 g for 5 min
and fixed in 1 mL of 1% glutaraldehyde, pelleted again, and
resuspended in fresh BG-11 medium. Cells were allowed to
adhere to formvar-coated copper grids (300 mesh, Agar Scien-
tific, Stansted, UK) for 5 min. Then, the grid was drained and
negatively stained for 1 min with 3% aqueous uranyl acetate
and examined in a JEOL JEM-1010 transmission electron
microscope equipped with a CCD Sis MegaView III (Olym-
pus, Tokyo, Japan) at Laboratory of electron microscopy
(Biology centre CAS, Cesk
e Budejovice, Czech Republic).
Determination of chlorophyll
Cells from 1 mL of culture (OD730 = 0.6) were collected by
centrifugation at 9400 g for 1 min. The pellet was
2 FEBS Open Bio (2023) 
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Federation of European Biochemical Societies.
Sequence of GT-T substrain of Synechocystis 6803 S. Koskinen et al.
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resuspended into 1 mL of 100% methanol and incubated
for 30 min. After centrifugation at 13,500 g for 5 min,
OD665 was measured and chlorophyll content was calcu-
lated using the extinction coefficient as in [25].
Immunological detection of the x subunit of RNA
polymerase
Cells were grown in the standard growth conditions (30 mL,
OD730 = 0.6) and collected by centrifugation at 4000 g for
5 min at 4 °C, and proteins were isolated as in [26]. Protein
concentration was determined with the DC protein assay kit
(Bio-Rad, Herculaes, CA, USA). Samples containing 25 lg
of total proteins were solubilized with Laemmli’s solubiliza-
tion buffer at 75 °C for 10 min and separated by Next Gel
SDS/PAGE according to the manufacturer’s instructions
(Amresco, Radnor, PA, USA). Thereafter, proteins were
transferred to Immobilon-P membrane (Millipore, Burling-
ton, MA, USA) and the a and x subunits of the RNA poly-
merase (Agrisera, V€ann€as, Sweden; custom antibodies
described in [19]), and the large subunit of Rubisco (Agris-
era, AS03037) was immunodetected with the specific anti-
bodies as in [19]. The goat anti-rabbit IgG (H + L) alkaline
phosphatase conjugate (Zymed, ThermoFisher, Waltham,
MA, USA) and CDP-star chemiluminescence Reagent (Per-
kinElmer, Waltham, MA, USA) were used for the signal
detection. Immunoblots were quantified using Epson perfec-
tion V600 photo scanner (Epson, Suwa, Nagano, Japan) and
IMAGE J program [27].
Results
The DNA from the GT-T substrain was isolated using
the phenol extraction method [4] and sent for sequenc-
ing to Eurofins Genomics Europe Sequencing Gmbh.
The genome of the GT-S substrain [9] was selected as
a reference genome and reads from the GT-T genome
were mapped to the GT-S genome. The length of the
GT-T chromosome was 3,569,929 bp with an overall
GC content of 47.74%. Mapping reads of GT-T gen-
ome to the GT-S reference genome revealed 12 differ-
ences between the chromosomes of GT-T and GT-S
(Table 1). The GT-T chromosome contains one large
1183 bp long deletion, one 6 nt-long insertion, 3 one
nucleotide insertions, and seven single nucleotide sub-
stitution mutations (Table 1), altogether making the
chromosome of GT-T 1174 bp shorter than that of
GT-S.
The long deletion of the GT-T chromosome com-
prises transposase slr1635 (Table 1) that is only found
in the substrains GT-S and Kazusa [9–11,19]. The
6 bp long insertion of the GT-T chromosome locates
in the pilT2 gene causing Ile Asn duplication after
amino acid 224 in the PilT2 protein (Table 1). PilT2 is
homologous to other conserved PilT motor proteins
functioning in the depolymerization process of type IV
pili [28]. The pilM gene contained the A to T substitu-
tion, leading to a Glu89Val mutation in the PilM pro-
tein. The PilM protein is suggested to be part of the
plasma membrane type IV pili core complex [29]. To
figure out if mutations in pil genes cause defects in
pilus structures, pilus structures were studied by trans-
mission electron microscopy of negatively stained GT-
T, GT-W, and PCC-M cells. Representative electron
micrographs are shown in Fig. 1. The pili structures of
the GT-T (Fig. 1A–C) closely resemble those of other
nonmotile, glucose-tolerant substrains GT-W (Fig. 1D,
E) or GT-P [30] but differ from the motile PCC-M
substrain (Fig. 1F,H; for motile strains see also
[29,31]). The GT-T, GT-W, and GT-P substrains dis-
play thin pili and thin pili bundles but lack typical
type IV thick pili in contrast to the motile substrains.
Three one bp insertions were detected in the GT-T
substrain. The insertion of an extra A nucleotide in
position 3276804 located in an intergenic region
Table 1. Location and effects of single nucleotide changes and indels found in the comparison of the nucleotide sequence of the GT-T sub-
strain to that of the GT-S substrain in the database.
Position GT-S GT-T AA change Locus Gene Function
157961 C T Val219Met sll0223 ndhB NAD(P)H-quinone oxidoreductase subunit 2
619782 – T frameshift S56 slr1916 Esterase
938475 T C Gln589Arg sll1732 ndhF3 NAD(P)H-quinone oxidoreductase subunit F
1290831 G A Silent slr1813 Hypothetical protein
1840410 G A Silent sll1374 melB
1864740 A T Glu89Val slr1274 pilM Type IV pilus assembly protein
2047228 Del 1183 bp slr1635 Transposase
2044508 – ATCAAC Insertion Ile225Asn226 sll1533 pilT2 Pilus retraction protein
2398167 – C frameshift G28 sll0771 glcP Glucose transporter
2465926 G T Gln194Lys sll0020 clcP Clp protease ATP binding subunit
2786264 G A Ala61Thr slr0914 Hypothetical protein
3276804 – A Intergenic between ORFs sll1342-slr1423
3FEBS Open Bio (2023) 
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(Table 1) is unlikely to cause any phenotype, but two
other insertions cause frameshift mutations. The first
one is located in the slr1916 gene that encodes a puta-
tive esterase (Table 1). This esterase has been shown
to have chlorophyll dephytylase activity in vitro and
Dslr1916 was found to have higher chlorophyll content
than the control strain [32]. We compared the chloro-
phyll content of GT-T, GT-P, and Nishiyma strains,
and detected no difference between the strains
(Fig. 2A). Recently, a protein family consisting of
DphA1-3 proteins with dephytylase activity, was char-
acterized in Synechoccus elongatus PCC 7942 [33].
Orthologous dphA genes were found in numerous
cyanobacteria including Synechocystis sp. PCC 6803
[33]. Thus DphA enzymes might compensate the miss-
ing Slr1961 in the GT-T substrain.
The second frameshift mutation is located in the
sll0771 gene that encodes a glucose transporter [34].
To test whether our GT-T strain is able to use exter-
nally added glucose, we grew the GT-T substrain in
photoheterotrophic conditions by blocking PSII activ-
ity with 10 lM DCMU and simultaneously supple-
menting the growth medium with 5 mM glucose. The
GT-T cells were not able to grow photoheterotrophi-
cally (Fig. 2B) indicating that, indeed, the detected fra-
meshift mutation prevents the function of the glucose
transporter. We also tested a cell culture started from
the oldest GT-T glycerol stock cells we have (prepared
in 1999), and also those cells contained a nonfunc-
tional glucose transporter. The mutation in the glucose
transporter gene has appeared in Turku in 1990s, as
the glucose-tolerant strain in McIntosh laboratory [5],
Fig. 1. Pili on the surface of Synechocystis sp. PCC 6803 nonmotile and motile substrains. Transmission electron microscopy micrographs
of nonmotile GT-T substrain (A–C), nonmotile GT-W substrain (D, E), or motile PCC-M substrain (F–H) cells that were negatively stained with
3% aqueous uranyl acetate. Thick pili are indicated with white arrows. Bundles of thin pili are indicated with black arrows.
4 FEBS Open Bio (2023) 
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Federation of European Biochemical Societies.
Sequence of GT-T substrain of Synechocystis 6803 S. Koskinen et al.
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and A2 and AR mutants constructed on that strain
were able to grow photoheterotrophically [14,15,35].
We also confirmed that AR cells still grow photo-
heterotrophically (Fig. 2B).
Two of the seven-point mutations in the GT-T
strain are located in ndh genes (Table 1). The NDH-1
core protein NdhB [36,37] contains the Val219Met
mutation in GT-T and the NdhF3 protein Gln589Arg
mutation; the NdhF3 is a specific subunit for an
NDH-1 complex functioning in carbon acquisition
[36,37]. Next point mutation cause Gln194Lys change
in the ATP binding subunit of the Clp protease. The
Clp protease is involved in phycobilisome degradation
[38], and in addition, some mutations in the ClpC pro-
tein have been shown to increase the thermotolerance
of Synechocystis cells [39]. However, the degradation
of phycobilisomes during nitrogen deficiency occurred
normally in GT-T cells [40], and we have not observed
increased thermotolerance either [41,42] suggesting the
normal function of the mutated Clp protease. Two
open reading frames slr1813 and sll1374 contain one
silent mutation each and the hypothetic protein
Slr0914 contains Ala61Thr mutation.
When the chromosome sequence of GT-T was com-
pared to that of PCC-M, 29 variations were detected
between the chromosomes (Table 2). Eleven of those
variations were the same as detected in the comparison
of GT-T and GT-S suggesting that those are specific
to GT-T. Eight of the variations are typical for all
PCC substrains, and 9 are specific for PCC-M
(Table 2). Both GT-T and PCC-M are missing trans-
poson slr1635, but only PCC-M is missing adjacent
18 bp, just eight nucleotides upstream of the rpoZ
gene (Fig. 3A). These 18 nucleotides are also missing
in PCC-P, PCC-N, and GT-I but not in other GT
strains (Fig. 3A). A putative promoter region of the
rpoZ gene that encodes the nonessential x subunit of
RNA polymerase [19] locates within this area, and
therefore we measured the amount of the x protein in
PCC-M. PCC-M cells are able to produce the x sub-
unit, although the amount has decreased circa 60%
compared to that in GT-T (Fig. 3B). The amounts of
the a subunit of RNA polymerase and the large sub-
unit of Rubisco (RbcL) were measured as control pro-
teins. Similar amounts of the a subunit of RNA
polymerase and RbcL were detected in GT-T and
PCC-M substrains (Fig. 3B).
Discussion
The laboratory substrains of Synechocystis can be
divided into two main clades, the GT clade originates
from the ATCC 27184 strain comprising nonmotile
glucose-tolerant strains, the majority of which are
descendants of the glucose-tolerant substrain selected
by Williams [4] but include also independently isolated
GT-G strain by Wei’s laboratory [7]. The PCC clade
containing motile strains originated from the PCC
6803 strain, some PCC substrains being glucose-
sensitive and some glucose-tolerant [10]. The chromo-
some sequence of our Synechocystis sp. PCC 6803 sub-
strain places it among the GT strains (Fig. 4).
Two frameshift mutations separate all GT and PCC
strains. A frameshift mutation in the spkA gene
Fig. 2. The properties of Synechocystis sp. PCC 6803 substrain
GT-T. (A) Chlorophyll a content of substrains GT-T, GT-P, and a
glucose-tolerant substrain from Nishiyama’s laboratory. (B) Auto-
trophic and photoheterotrophic growth of the GT-T substrain and
the AR mutant strains (interrupted psbA1 and psbA3 genes and
the antibiotic resistance cassette after the psbA2 gene). For auto-
trophic conditions, 30-mL cell cultures were grown in 100-mL
Erlenmeyer flasks in the BG-11 medium supplemented with
Hepes-NaOH pH 7.5 at 3 °C in ambient air with gentle agitation of
90 rpm. Cells were constantly illuminated, PPFD 40 lmol m
2 s
1.
For photoheterotrophic conditions, cell cultures were supple-
mented with 5 mM glucose and 10 lM DCMU. The results are the
average of three independent biological replicates and the error
bars denote SEM.
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destroys the function of the SpkA protein in all GT
substrains (Fig. 4). The Spk kinase has been shown to
function as a regulator of cell motility [43,44], and the
lack of the SpkA kinase can be considered to be a rea-
son for nonmotile phenotype of GT strains. The fra-
meshift mutation in the hlyA gene causes a lack of the
full-length HlyA surface layer protein in PCC strains
and might increase the sensitivity of the PCC strains
to external stress conditions, although Dsll1951 cells
grow well in standard conditions [45].
Three-point mutations separate all GT and PCC
strains (Fig. 4). To figure out which lineage has a
mutated version, we compared nucleotide sequences of
these three genes in Synechocystis substrains to those
of close homolog genes in other cyanobacteria. In the
slr1865 gene, the mutation has most probably occurred
in the GT lineage as homologous genes in Synechocys-
tis sp. CACIAM 05, Synechocystis sp. PCC7339, Syne-
chocystis sp. PCC7338, Synechocystis sp. PCC6714,
Nostocales HT-58-2, and Halothece sp. PCC 7818 all
contain A nucleotide in position 340 just like the PCC
substrains. Whereas in the hik25 gene, the mutation
has occurred in PCC lineage, as Synechocystis sp.
CACIAM 05, Synechocystis sp. PCC7339, Synechocys-
tis sp. PCC7338 and Synechocystis sp. PCC6714
strains contain T in position 2692, just like the GT
substrains. Position 674 in the putative phosphatase
gene, slr1983, shows variation between the strains
being C in GT lineage and in Synechocystis sp.
CACIAM 05 and Synechocystis sp. PCC6714, T in
PCC lineage, and A in Synechocystis sp. PCC7339 and
Synechocystis sp. PCC7338.
In addition to common differences between the GT
and PCC substrains, all substrains originated from the
Williams strain share common mutations (Fig. 4).
Intergenic single nucleotide polymorphism (SNP)
between adkA and infA localizes to the putative 
10
promoter region of the infA gene that encodes a trans-
lation initiator factor. However, the physiological con-
sequence of these mutations is unclear, as the infA
gene belongs to a large gene cluster whose transcript
pattern suggests multiple transcription initiation sites
Table 2. Location and effects of single nucleotide changes and indels found in the comparison of the nucleotide sequence of GT-T to that
of PCC-M in the database. Clade indicates changes found between GT-T and all PCC strains (PCC), GT-T-specific changes (T), and PCC-M-
specific changes (M).
Position Clade PCC-M GT-T AA change Locus Gene Function
144507 M G A Silent slr0242 bcp Bacterioferritin comigratory protein
157961 T C T Val219Met sll0223 ndhB NAD(P)H-quinone oxidoreductase subunit 2
489109 M C T Ser628Leu slr1609 Long-chain-fatty-acid CoA ligase
619284 T – T frameshift slr1916 Esterase
730868 PCC – A frameshift sll1574 SpkA
781267 PCC Del 154 bp
831302 M T C Intergenic and slr2031
847733 M A G Asn2Ser slr1898 argB N-acetylglutamate kinase
938130 T T C Gln589Arg sll1732 ndhF3 NAD(P)H-quinone oxidoreductase subunit F
1070494 M A T Lys298Asn sll1359 Hypothetical protein
1203086 PCC A G Tyr95Cys slr1865 Hypothetical protein
1423938 PCC – A frameshift sll1951 hlyA Haemolysin
1290484 T G A Silent slr1813 Hypothetical protein
1810888 PCC T C Val233Ala slr1983 Response regulator
1840062 T G A Silent sll1374 melB
1864392 T A T Glu89Val slr1274 pilM Type IV pilus assembly protein
2044160 T – ATCAAC Ile Asn insertion sll1533 pilT2 Pilus retraction protein
2046851 PCC – GGGTAAGGGGGACAATAT intergenic
2397801 T – C frameshift sll0771 glcP Glucose transporter
2397991 M A C intergenic
2465560 T G T Gln194Lys sll0020 clpC Clp protease ATP binding subunit
2518280 PCC C T Ser928Phe slr0222 hik25 Hybrid sensory kinase
2785898 T G A Ala63Thr slr0914 Hypothetical protein
3011933 PCC C T Silent slr0302 PleD-like protein
3095975 PCC C T Arg103Cys ssr1176 Transposase
3276439 T – A Intergenic
3364288 M – A frameshift sll1496 Mannose-1-phosphate guanyltransferase
3369206 M A T Lys239Met slr1564 sigF RNA polymerase SigF sigma factor
3419464 M T C Leu118Pro slr0753 P protein
6 FEBS Open Bio (2023) 
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within the cluster that can be also transcribed as a
large operon [46]. Physiological consequences of the
154 bp deletion in and upstream of the slr2031 gene
also remain to be studied. The slr2031 gene is a homo-
log of the rsbU gene in Bacillus subtilis in which the
RsbU phosphatase regulates the function of the stress-
responsive SigB r factor via the RsbV/RsbW anti-r
factor/anti-r factor antagonist pair [47]. In Syne-
chocystis, Dslr2031 cells have been reported to show
defects in recovery from nitrogen or sulfur deficiency
[48], but in our measurements, the GT-T substrain
enters and recovers from nitrogen deficiency similarly
as the PCC-M substrain [40,49].
All descendants of the Williams strain [4] have gained
additional mutations (Fig. 4). Surprisingly, a completely
different set of mutations was discovered when the GT-
W substrain (originating from Vermaas’s laboratory)
was sequenced in Prague (laboratories of Sobotka and
Komenda) and the GT-O1 and GT-O2 substrains, also
descendants of Vermaas strain, in Otago (laboratory of
Eaton-Rye) [11,12]. GT-W contains a large, 110 kb,
tandem duplication between the identical transposases
sll0431 and sll1397 [12]. This large duplication contains
100 genes, and in addition, GT-W contains four strain-
specific SNPs and a 5326 bp deletion in the swmB gene
[12]. The phenotype of the GT-W substrain is highly
unique with high carotenoid and low chlorophyll and
phycocyanobilin contents of the cells [12]. Interestingly,
this phenotype is promoted with glucose supplementa-
tion, as the large duplication of the GT-W chromosome
disappears if cells are grown for several months in auto-
trophic conditions [12].
GT-T-specific mutations have consequences on the
phenotype of cells. A frameshift mutation has
destroyed the glucose transporter, and GT-T cells do
not grow heterotrophically (Fig. 2). We have not real-
ized that earlier, because we have not grown GT-T
cells in photoheterotropic or heterotrophic conditions.
When testing psbA mutants in 1990s, we often used
photoheterotrophic conditions, but in those experi-
ments, we always used the AR strain as a reference
strain [50]. Our RNA polymerase mutants have been
constructed using GT-T as a host strain [19,26,51,52],
but those mutants have not been tested in photo-
heterotrophic conditions. Obviously, the GT-T sub-
strain or mutants constructed using GT-T as a host
strain should not be used in studies utilizing externally
added glucose. However, the lack of the functional
glucose transporter does not have overall effects on
sugar metabolism. An overdose of the SigE r factor
that regulates sugar catabolic genes causes similar
transcriptomic responses whether GT-T [52] or the
glucose-tolerant strain of Tanaka’s laboratory is used
as a host strain [53].
Fig. 3. Sequence of the upstream region of the rpoZ gene and the content of the a and x subunits of the RNA polymerase and the large
subunit of Rubisco (RbcL) in Synechocystis sp. PCC 6803 substrains. (A) Comparison of the upstream region of the rpoZ gene in Synechocystis
substrains. Transposase in violet, target site duplication in red, putative 
10 region of the rpoZ gene in bold blue, and the initiation codon of the
rpoZ gene in blue. (B) GT-T and PCC-M substrains were grown in standard conditions, total proteins were isolated, solubilized and 25 lg of pro-
teins were separated by Next gel SDS/PAGE. The x and a subunits of RNA polymerase and the large subunit of Rubisco (RbcL) were immun-
odetected by Western blotting using specific antibodies. Three independent biological replicates are shown.
7FEBS Open Bio (2023) 
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In the GT-T substrain, PilM and PilT2 proteins con-
tain mutations on the top of pili regulating kinase
SpkA (Table 2). The insertion of Ile225 and Asn226 in
the PilT2 protein is located in a conserved domain in
the proximity to the crucial Lys212 residue in a
Walker A ATP/GT-P-binding motif [28] and a
manganese-binding pocket [54], thus potentially affect-
ing the function of the PilT2 protein. The pilT2 dele-
tion strain, constructed in the motile PCC host strain,
remains motile and competent but shows an opposite
response to unidirectional light than the host strain
[28]. Cells without functional PilM lost both motility
and competence [29]. Mutations in pilT and pilM
genes can be assumed to play only minor roles, as pili
of GT-T resemble those of the other nonmotile sub-
strains (Fig. 1, [30]) and GT-T is competent, just like
all other GT substrains, excluding GT-Kazusa, which
has lost competence due to the frameshift mutation in
the pilC gene [8].
Two ndh genes of the GT-T substrain contain point
mutations (Table 1). The NdhB protein is supposed to
function both in cyclic electron flow and in CO2 acqui-
sition, whereas NdhF3 functions only in carbon
acquisition [36,37]. The GT-T substrain shows typical
responses to high and low CO2, indicating that the
GT-T substrain contains functional NDH complexes
[55].
It is noteworthy that the movement of transposons
is one driving force for substrain-specific mutations.
Furthermore, single nucleotide point mutations (in-
cluding only few silent mutations) or single nucleotide
deletion or insertions seem to concentrate on the cod-
ing sequences of genes with known function and show
high variation between substrains. It should also be
noted that we sequenced DNA isolated from storage
cells that are currently in use, whereas the majority of
other sequencing projects have been started from old
storage cells from the early 1990s. In 1990s, we pre-
pared new storage cells after the old ones had run out,
and therefore our current storage cells have been
grown for numerous generations after the GT-T sub-
strain was brought from Stockholm to Turku. After
understanding how easily new mutations appear in the
genome, we have been more careful in producing stor-
age cells only after a minimal number of cell genera-
tions.
Fig. 4. Substrain-specific mutations in glucose-tolerant nonmotile Synecocystis cells. Isolated Synechocystis sp. PCC 6803 was deposited to
two culture collections. Nonmotile glucose-tolerant strains are descendants of the ATCC 27184 strain in the American Type Culture Collection,
and the motile PCC strains originate from PCC 6803 strain in the Pastor Culture Collection of Cyanobacteria. Routes of strains to the laborato-
ries, where they were sequenced, are shown according to the information found in the literature. The sequenced strains are GT-Kazusa and GT-
S (8); GT-G (7); GT-I, PCC-P, and PCC-N (9); PCC-M (10); GT-O1 and GT-O2 (11); and GT-P and GT-W (12); GT-T (this publication).
8 FEBS Open Bio (2023) 
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Federation of European Biochemical Societies.
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Just by comparing sequences, it is not easy to figure
out why glucose-tolerant strains are glucose-tolerant,
as GT and PCC-M strains do not share mutations
that could be directly connected to glucose tolerance.
When we grew PCC-M photoheterotrophically in the
presence of DCMU and glucose, cells were growing,
but similar fast growth as in the AR strain was not
observed. Furthermore, unlike AR cells, photo-
heterotrophically grown PCC-M cells formed non-
easily-breakable aggregates, and reliable measurement
of PCC-M cell density was not possible. Full-length
HlyA protein is present in all GT stains (Fig. 4) and,
unlike PCC-M cells (truncated HlyA protein), they do
not show a clumping phenotype in the presence of glu-
cose. HlyA has been connected to glucose tolerance
earlier as HlyA-less cells do not grow mixotrophically
at low light [45]. Thus, it is tempting to speculate that
the HlyA protein plays a role in glucose tolerance. The
PCC-M chromosome contains an SNP in 
56 position
upstream of the glcP glucose transporter gene, but it
remains to be studied if that mutation plays a role in
glucose tolerance. We suggest that glucose tolerances
in GT and PCC-M strains are of different origins.
Construction of the phylogenetic tree of different
substrains is complicated by some unexpected sequence
variation. First of all, strains originating from Ver-
maas’s laboratory do not share common mutations
(Fig. 4). Furthermore, the 18 bp deletion upstream of
the rpoZ gene found in all PCC strains is also present
in GT-I strain but not in any other GT strains. The
consequences of the reduced amounts of the x subunit
in PCC and GT-I strains remain to be studied. The x
less DrpoZ strain is fully viable in standard growth
conditions [19], but DrpoZ cells do not acclimate to
high CO2 [55] or high temperature [56]. And finally,
the rpl3 gene contains the same mutation in GT-W
and GT-P but not in the other GT substrains (Fig. 4).
As Synechocystis contains many copies of the genome,
one possible explanation is that all copies are not com-
pletely identical, and depending on growth conditions,
different chromosome variants become the prevailing
forms, and only those most frequent variants are con-
sidered and published as the outcomes of the genome
sequencing projects.
Obviously, all laboratory substrains contain unique
mutations causing phenotypic differences between so-
called ‘wild-type’ strains. Comparison of results from dif-
ferent laboratories and selection of an appropriate sub-
strain for each study would be easier after sequencing all
‘wild-type’ substrains. For example, our GT-T substrain
or mutants constructed using it as a host strain are not
compatible with studies involving mixotrophic or hetero-
trophic conditions using glucose as a carbon source
because the frameshift mutation in the glucose trans-
porter prevents utilization of externally added glucose.
Acknowledgements
The project was supported by Novo Nordisk Founda-
tion grant NNF19OC0057660. The authors thank Prof.
Roman Sobotka and Dr. Martin Tich
y for advice.
Conflict of interest
The authors declare no conflict of interest.
Author contributions
TT designed and supervised this work; SK performed
the data analyses; JK isolated DNA and measured
growth, chlorophyll, and RNAP and Rubisco subunit
contents of cells; and ML performed pilus staining.
SK and TT wrote the manuscript with contributions
from JK and ML All authors have read and agreed to
the published version of the manuscript.
Data accessibility section
The genome sequence has been deposited at NCBI
under GenBank accession CP094998 https://www.ncbi.
nlm.nih.gov/nuccore/CP094998 and BioProject acces-
sion number PRJNA821690 https://www.ncbi.nlm.nih.
gov/bioproject/PRJNA821690, BioSample accession
number SAMN27121261 https://www.ncbi.nlm.nih.
gov/biosample/SAMN27121261, and SRA number
SRR18670503 https://www.ncbi.nlm.nih.gov/sra/
PRJNA821690.
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