Báo cáo khoa học: Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteria potx

Comparative biochemical and functional studies of
family I soluble inorganic pyrophosphatases from
photosynthetic bacteria
Marı
´
aR.Go
´
mez-Garcı
´
a*, Manuel Losada and Aurelio Serrano
Instituto de Bioquı
´
mica Vegetal y Fotosı
´
ntesis, Centro de Investigaciones Cientı
´
ficas Isla Cartuja, CSIC-Universidad de Sevilla, Spain
Soluble inorganic pyrophosphatase (sPPase) (inorganic
diphosphatase, EC 3.6.1.1) is an essential and ubiquit-
ous metal-dependent enzyme that cleaves inorganic
pyrophosphate (PP
i
), producing inorganic orthophos-
phate (P
i
). Its role in metabolism is thought to be the
removal of PP
i
, a byproduct of many vital anabolic
reactions, especially those involved in the synthesis of
polymers, making them thermodynamically irreversible

[1]. sPPases belong to two nonhomologous families:
family I, widespread in all types of organism [2], and
family II, so far confined to a limited number of
bacteria and archaea [3,4]. The families differ in many
functional properties; for example, Mg
2+
is the pre-
ferred cofactor for family I sPPases studied, whereas
Mn
2+
confers maximal activity to family II sPPases
[5,6]. Although no sequence or overall structural
similarity is observed between these two protein
classes, there is a striking conservation of key active
Keywords
anoxygenic photosynthetic bacteria;
cyanobacteria; functional complementation;
ppa; soluble pyrophosphatases
Correspondence
M. R. Go
´
mez-Garcı
´
a, Department of
Biochemistry, Stanford University School of
Medicine, Beckman Center B413, 300
Pasteur Dr., Stanford, CA, 94305-5307, USA
Fax: +1 650 725 6044
Tel: +1 650 723 5348
E-mail:

A. Serrano, Instituto de Bioquı
´
mica Vegetal
y Fotosı
´
ntesis, CSIC-Univ. de Sevilla, Avda.
Ame
´
rico Vespucio 49, 41092 - Sevilla, Spain
Fax: +34 954460065
Tel: +34 954489524
E-mail:
*Present address
Department of Biochemistry, Stanford Uni-
versity School of Medicine, Beckman Center
B413, Stanford, CA, USA
(Received 17 April 2007, revised 23 May
2007, accepted 8 June 2007)
doi:10.1111/j.1742-4658.2007.05927.x
Soluble inorganic pyrophosphatases (inorganic diphosphatases, EC 3.6.1.1)
were isolated and characterized from three phylogenetically diverse cyano-
bacteria ) Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and
Pseudanabaena sp. PCC 6903 – and one anoxygenic photosynthetic bacter-
ium, Rhodopseudomonas viridis (purple nonsulfur). These enzymes were
found to be family I soluble inorganic pyrophosphatases with c. 20 kDa
subunits with diverse oligomeric structures. The corresponding ppa genes
were cloned and functionally validated by heterologous expression. Cyano-
bacterial family I soluble inorganic pyrophosphatases were strictly Mg
2+
-

dependent enzymes. However, diverse cation cofactor dependence was
observed for enzymes from other groups of photosynthetic bacteria.
Immunochemical studies with antibodies to cyanobacterial soluble inor-
ganic pyrophosphatases showed crossreaction with orthologs of other
main groups of phototrophic prokaryotes and suggested a close relation-
ship with the enzyme of heliobacteria, the nearest photosynthetic relatives
of cyanobacteria. A slow-growing Escherichia coli JP5 mutant strain,
containing a very low level of soluble inorganic pyrophosphatase activity,
was functionally complemented up to wild-type growth rates with ppa
genes from diverse photosynthetic prokaryotes expressed under their own
promoters. Overall, these results suggest that the bacterial family I soluble
inorganic pyrophosphatases described here have retained functional similar-
ities despite their genealogies and their adaptations to diverse metabolic
scenarios.
Abbreviations
A. 7120 sPPase, Anabaena sp. PCC 7120 soluble inorganic pyrophosphatase; Ec-sPPase, Escherichia coli soluble inorganic pyrophosphatase;
P. 6903 sPPase, Pseudanabaena sp. PCC 6903 soluble inorganic pyrophosphatase; PCC, Pasteur culture collection; S. 6803 sPPase,
Synechocystis sp. PCC 6803 soluble inorganic pyrophosphatase; sPPase, soluble inorganic pyrophosphatase.
3948 FEBS Journal 274 (2007) 3948–3959 Journal compilation ª 2007 FEBS. No claim to original US government works
site residues, a remarkable example of convergent
enzyme evolution [5–10]. The two best-studied exam-
ples of family I enzymes are the hexameric sPPase of
Escherichia coli (Ec-sPPase) and the dimeric enzyme of
Saccharomyces cerevisiae, prototypes of prokaryotic
and eukaryotic family I sPPases, respectively [11]. Bac-
terial and archaeal family I sPPases are usually homo-
hexamers, whereas eukaryotic sPPases are homodimers
or monomers [12]. The subunit size is generally 19–
22 kDa in prokaryotic sPPases and 30–34 kDa in
their dimeric or monomeric eukaryotic counterparts

[2,11,12]. In a previous study, it was shown that
sPPases of photosynthetic plastids from microalgae
and plants are eukaryotic family I enzymes, and it was
suggested that during the evolutionary history that
gave rise to these organelles, the prokaryotic sPPase of
the ancestral cyanobacterial-like endosymbiont was
functionally substituted by its host cell homolog [12].
In this context, the sPPases of photosynthetic bacteria,
a polyphyletic and very diverse assembly of prokaryo-
tes, are worth characterizing.
An increasing body of biochemical and genetic evi-
dence suggests that PP
i
plays an important role in the
bioenergetics of many archaea, bacteria, and protists
[13,14]. In these organisms, two types of inorganic
pyrophosphatases, sPPases and proton-translocating
PPases, H
+
-PPases, with different subcellular localiza-
tions, hydrolyze PP
i
generated by cell anabolism, and
replenish the P
i
pool needed for phosphorylation reac-
tions. The widespread presence of these key enzymes
of PP
i
metabolism in photosynthetic organisms, except

cyanobacteria, strongly supports the ancestral nature
of bioenergetics based on this simple energy-rich com-
pound that may play an important role in survival
under different biotic and abiotic stress conditions
[13,15].
This work shows that cyanobacterial strains as well
as diverse anoxygenic photosynthetic bacteria possess
family I sPPases with different catalytic and physico-
chemical properties (i.e. divalent cation dependence,
oligomeric structure), and extends prior work on
cyanobacterial counterparts [16], as no detailed com-
parative studies of these enzymes from prokaryotic
photosynthetic organisms have been performed so far.
The only previous study on cyanobacterial sPPases
reported that the enzyme from the unicellular cyano-
bacterium Microcystis aeruginosa NIES-44 was a
trimeric protein with a 28 kDa subunit [16], in contrast
to the well-characterized hexameric structure of
Ec-sPPase [2]. The characterization of photobacterial
sPPases has also allowed us to establish phylogenetic
and evolutionary relationships between prokaryotic
enzymes and homologs from photosynthetic plastids.
Results and Discussion
Detection of sPPase activity in photosynthetic
prokaryotes ) enzymatic features of isolated
sPPases
Family I is the most widespread and probably the
most ancestral sPPase group [2,11]. Molecular phylo-
genetic analyses indicate the existence of two divergent
evolutionary lineages in this protein assembly: the

‘eukaryotic’ (fungi, plants, metazoa, and most pro-
tists), and the ‘prokaryotic’ (bacteria, archaea, and
photosynthetic eukaryotes) [12]. Family I is therefore
an ancient conserved group of orthologs from evolu-
tionarily very distant organisms. Cell-free protein
extracts from all photosynthetic prokaryotes studied
(Table 1) contain substantial levels of an alkaline
Table 1. Strains used in this work.
Section
a
Strain
Cyanobacteria I Synechocystis sp. PCC 6803
b
Synechococcus sp. PCC 7942
Microcystis aeruginosa NIES-44
c
Microcystis aeruginosa HUB5-2-4
d
II Dermocarpa sp. PCC 7437
III Pseudanabaena sp. PCC 6903
Phormidium laminosum
(argardh)Gom.H-1pC11
e
Spirulina sp. PCC 6313
IV Anabaena sp. ATCC 29413
f
Anabaena sp. PCC 7120
Nostoc sp. PCC 7107
Calotrix sp. PCC 7601
V Fischerella sp. UTEX 1829

g
Anoxygenic
photosynthetic
bacteria
Purple nonsulfur
bacteria
Rhodopseudomonas palustris
h
Rhodopseudomonas viridis
h
Rhodospirillum rubrum S1
Rhodobacter sphaeroides
DSM 158S
i
Rhodobacter capsulatus E1F1
Purple sulfur
bacteria
Amoebobacter roseus
j
Chromatium vinosum
j
Green sulfur
bacteria
Chlorobium limicola
j
Chlorobium tepidum ATCC 49652
Chlorobium phaeobacteroides
j
Heliobacteria Heliobacterium chlorum
DSM 1132

a
Sections in the classification of Rippka et al. [17].
b
PCC, Pasteur
Culture Collection.
c
NIES, National Institute of Environmental Stud-
ies, Japan.
d
Humbodt University Berlin, Professor T. Bo
¨
rner.
e
Univ. Pais Vasco (Dr J. L. Serra).
f
ATCC, American Type Culture
Collection.
g
UTEX, Culture Collection University of Texas.
h
Dr A.
Verme
´
glio, CEA-Caradache, France.
i
DSM, Deutsche Sammlung
von Microorganismen, Germany.
j
University of Girona, Spain, Pro-
fessor Jordi Mas.

M. R. Go
´
mez-Garcı
´
a et al. Pyrophosphatases from photosynthetic bacteria
FEBS Journal 274 (2007) 3948–3959 Journal compilation ª 2007 FEBS. No claim to original US government works 3949
sPPase activity (0.2–2.5 UÆmg
)1
protein) that abso-
lutely requires a divalent metal cation. Cyanobacterial
sPPases from different taxonomic groups [17] (Table 1)
are all strictly Mg
2+
-dependent enzymes with
c. 22 kDa subunits (Table 2, and data below). They
exhibit fairly constant specific activity levels (0.2–
0.4 UÆmg
)1
protein). On the contrary, a marked vari-
ability of cation dependence was found among anoxy-
genic bacteria sPPases; other cations, such as Zn
2+
,
Mn
2+
or Co
2+
, replace Mg
2+
efficiently in extracts of

the purple, nonsulfur and sulfur (not shown) anoxy-
genic bacteria studied (Table 2). Thus, Rhodospirillum
rubrum and Rhodopseudomonas viridis enzymes are
Zn
2+
-dependent, whereas Rhodobacter capsulatus
sPPase is Mn
2+
-dependent. Interestingly, the green
(sulfur and nonsulfur) photosynthetic bacteria and the
Heliobacterium strain tested exhibit sPPase activity
with a marked preference for Mg
2+
, being similar in
this respect to their cyanobacterial counterparts
(Table 2 and data not shown). On the whole, specific
activity levels in extracts of anoxygenic bacteria (1.0–
2.5 UÆmg
)1
protein) were higher than in cyanobacterial
extracts.
A purification procedure, similar to the one des-
cribed for the isolation of sPPase isoforms from the
unicellular alga Chlamydomonas reinhardtii [12], was
used to isolate the sPPases from the cyanobacteria
Synechocystis sp. PCC 6803 (S. 6803 sPPase), Anabae-
na sp. PCC 7120 (A. 7120 sPPase), and Pseudanabaena
sp. PCC 6903 (P. 6903 sPPase), and the purple bacter-
ium Rhodop. viridis. The method yielded electrophoret-
ically pure sPPases with specific activities in the range

120–300 UÆmg
)1
protein and recovery levels of 20–
30%. In all cases, the analysis by SDS ⁄ PAGE and
native PAGE of purified preparations showed only
one protein band of 20–22 kDa (Figs 1 and 2; supple-
mentary Fig. S1). Analytical gel filtration FPLC of
S. 6803 sPPase revealed one active hexameric sPPase
(native molecular mass of 110 ± 5 kDa), in accordance
with the oligomeric state of the archetypal Ec-sPPase
[2,18]. A subunit molecular mass of 19 187 Da ±
0.1% was determined by MALDI-TOF MS for
S. 6803 sPPase, somewhat lower than but in fair agree-
ment with the apparent molecular mass values estima-
ted by indirect measurements (Fig. 1). Native PAGE
showed small differences in the migration of purified
S. 6803 sPPase and A. 7120 sPPase (Fig. 2), in accord-
ance with the strong acidic character of the native
S. 6803 sPPase (pI 4.70) determined by column chro-
matofocusing (data not shown). The same oligomeric
states were found for A. 7120 sPPase (not shown)
and P. 6903 sPPase (supplementary Fig. S1) (native
molecular masses of 114 ± 5 kDa and 120 ± 5 kDa,
respectively). In all cases, both the subunit molecular
masses and oligomeric states are similar to those
described for Ec-sPPase [2,18]. Interestingly, the
sPPase from the purple nonsulfur photobacterium
Rhodop. viridis exhibits a clearly larger native molecu-
lar mass (240 ± 15 kDa), suggesting a higher oligo-
meric state (dodecameric) (Fig. 1B).

Hexameric Ec-sPPase has been described as a dimer
of trimers, and the formation of these structures
involves residues such as H136, H140 and D143, which
participate in strong ionic interactions mediated by
Mg
2+
[2,19]. Changes in the residues involved in the
interactions between subunits could explain the unusual
oligomeric states found for some photosynthetic bac-
teria and photosynthetic eukaryote sPPases [12], so the
trimeric structure reported by Kang & Ho for Mi. aeru-
ginosa NIES-44 sPPase [16] is probably due to the pres-
ence of trimers in solution. All determinations of native
molecular masses reported here were performed with
excess of Mg
2+
in solution to allow the interactions
involved in hexamer formation. The Rhodop. viridis
sPPase (Fig. 1B) shows differences in the oligomeric
state (a dodecameric structure), probably due also to
changes in the residues involved in the formation of the
dimer of trimers. Although these proposals require
Table 2. Cation dependence of sPPase enzymes. The level 100 is assigned to the activity determined with Mg
2+
in each case. Assays were
performed in the presence of 4 m
M divalent cation using purified enzyme (partially purified, in the cases of Rhodob. capsulatus, Rhodop.
palustris and Chlorob. tepidum) in the range 20–30 UÆmL
)1
.

Cation
Synechocystis sp.
PCC 6803
Anabaena sp.
PCC 7120
Pseudanabaena sp.
PCC 6903
Rhodos.
rubrum
Rhodop.
viridis
Rhodop.
palustris
Rhodob.
capsulatus
Chlorob.
tepidum E. coli
Mg
2+
100 100 100 100 100 100 100 100 100
Mn
2+
3 3 1 115 97 97 180 13 13
Cu
2+
4 2 2 42 94 105 50 20 3
Fe
2+
3 1 5 150 134 131 21 9 4
Zn

2+
13 1 2 165 140 40 90 15 10
Co
2+
2 3 2 154 125 41 84 17 10
No cation 0 0 0 0 0 0 0 0 0
Pyrophosphatases from photosynthetic bacteria M. R. Go
´
mez-Garcı
´
a et al.
3950 FEBS Journal 274 (2007) 3948–3959 Journal compilation ª 2007 FEBS. No claim to original US government works
further study, it should be noted that all sPPase
sequences from photosynthetic bacteria found in data-
bases (see below) show nonconservative substitutions
in most residues corresponding to those six involved
in Ec-sPPase hexamer stabilization [2,19] (data not
shown).
The catalytic properties of photosynthetic bacterial
sPPases studied in this work are shown in Table 3.
Similar to Ec-sPPase [20], the cyanobacterial enzymes
exhibit a high affinity for the substrate; however, the
anoxygenic bacteria sPPases show K
m
values one order
of magnitude higher than the cyanobacterial counter-
parts. The catalytic efficiencies (estimated as k
cat
⁄ K
m

ratios) of the sPPases from purple photosynthetic bac-
teria are in the same range as that found for Ec-sPPase
and their homologs from photosynthetic eukaryotes
[12], but the cyanobacterial enzymes show values one
order of magnitude higher, due to their lower K
m
.
sPPases among diverse cyanobacteria and
anoxygenic photosynthetic bacteria
Western blot analyses performed using a monospecific
polyclonal antibody against S. 6803 sPPase with sol-
uble protein extracts from cyanobacteria belonging to
all different taxonomic sections [17] revealed that the
product of the ppa gene was present in all of the strains
tested (Fig. 3A). All of them exhibit Mg
2+
-dependent
sPPase activity (Table 2 and data not shown) and
possess 20–22 kDa polypeptides, which strongly cross-
reacted with the antibody to S. 6803 sPPase, suggesting
that cyanobacteria have tightly related prokaryotic fam-
ily I sPPases. P. 6903 sPPase, in accordance with the
greater length of its ppa gene, and orthologs in other
strains of section III (see Table 1), showed an immuno-
detected band with a higher molecular mass (Fig. 3A,
middle). Two different strains of Mi. aeruginosa
Elution volume (ml)
ALD
BSA
OVA

CYT
2
1
CAT
9. 10.
8.
3
Log molecular mass
0
0.01
0.02
0
0246810
5
10
15
20
25
30
sPPase activity (U/ml) ( )
35
0
5
10
15
20
25
30
sPPase activity (U/ml) ( )
35

A
B
Elution volume (ml)
02
4
6 8 10 12
Elution volume (ml)
*
45
29
20
66
kDa
Absorbance at 280 nm
0
0.01
0.02
Absorbance at 280 nm
Mass/Charge
Relative intesity
3000010000 20000
40000
M
M
2+
M
3+
2M
+
100

50
+
45
29
20
14
kDa
Synechocystis
sPPase
5. 6. 7.
4
Elution volume (ml)
Log molecular masss
5
FER
CAT
ALD
LPD
BSA
OVA
CYT
6
Rps. viridis sPPase
*
Fig. 1. Gel filtration FPLC of purified native
Synechocystis sp. PCC 6803 (A) and Rho-
dop. viridis (B) sPPases. Aliquots (0.5 mL) of
S. 6803 sPPase purified preparations were
applied to a Superose 12HR 10 ⁄ 30 column.
Isocratic elution was performed at a flow

rate of 1 mLÆmin
)1
, and 0.2 mL fractions
were collected. The Coomassie Blue-stained
SDS ⁄ PAGE gels of the indicated fractions
around the activity peaks (highest activity
fraction marked with an asterisk) show a
single 22 kDa protein that coeluted with
sPPase activity in both cases. The positions
and molecular masses of protein standards
are indicated on the left side of the gels. (A)
The upper right inset shows column calibra-
tion protein standards (CAT, catalase; ALD,
aldolase; BSA, bovine seroalbumin; OVA,
ovoalbumin; CYT, cytochrome c) and the
positions of the cyanobacterial sPPase peak
(black circle), which corresponds to a native
molecular mass of c. 110 kDa. The MALDI-
TOF MS profile of S. 6803 sPPase is shown
on the left. (B). The upper inset shows col-
umn calibration with protein standards
(FERR, ferritine; CAT, catalase; ALD, aldo-
lase; LPD, lipoamide dehydrogenase; BSA,
bovine seroalbumin; OVA, ovoalbumine;
CYT, cytochrome c). A native molecular
mass of 240 kDa was estimated for the
Rhodop. viridis sPPase.
M. R. Go
´
mez-Garcı

´
a et al. Pyrophosphatases from photosynthetic bacteria
FEBS Journal 274 (2007) 3948–3959 Journal compilation ª 2007 FEBS. No claim to original US government works 3951
(NIES-44 and HUB5-2-4) also showed an immunode-
tectable band of c. 22 kDa and exhibited Mg
2+
-
dependent activity (data not shown), suggesting that
their sPPase subunits might be similar to those of
typical cyanobacterial homologs (Fig. 3A, right). This
is in disagreement with previously reported data on
Mi. aeruginosa NIES-44 sPPase, where a larger 28 kDa
subunit was estimated by SDS ⁄ PAGE and a trimeric
structure was found by gel filtration [16].
The antibody against S. 6803 sPPase also crossreact-
ed with soluble extracts from nearly all anoxygenic
photosynthetic bacteria tested, which belong to differ-
ent taxonomic groups (Table 1). They display, how-
ever, greater heterogeneity in the molecular mass of
the detected protein band, as is also the case for metal
cation dependence, as the sPPase activity of many of
these bacteria can efficiently use other divalent cations
as cofactors, e.g. Zn
2+
,Co
2+
and Fe
2+
(Table 2). The
enzymes of rhodospirillacean species Rhodos. rubrum,

Rhodop. palustris and Rhodop. viridis had 22 kDa
subunits, suggesting that they should be members of
family I (Figs 1B and 3B), in agreement with the
genome database sequences available (see below).
However, the sPPase of the closely related species
Rhodob. capsulatus shows Mn
2+
-dependent activity
and is not recognized by the antibody raised against
S. 6803 sPPase, as expected, because Rhodob. capsula-
tus and Rhodop. sphaeroides sPPases have already been
described as family II enzymes [21]. It can be specula-
ted that this could be a singular case of horizontal
gene transfer in photosynthetic prokaryotes, as it has
been shown that marine unicellular cyanobacteria pos-
sess two paralogous ppa genes of different phylogeny;
one of them, similar to the proteobacterial homologs,
was probably obtained by horizontal gene transfer [22]
(see below). Highly degenerate family I sPPase-enco-
ding pseudogenes are also present in the genomes of a
number of prokaryotes from diverse taxonomic groups
with functional family II sPPase genes, thus illustrating
functional substitution by nonhomologous sPPases in
a context of gene degradation and displacement, which
is proposed to be of major importance in microbial
genome evolution [22,23]. The clearly larger size of the
Chromatium vinosum immunodetected protein band
(60 kDa) (Fig. 3B) is an exception among photosyn-
thetic bacteria; nevertheless, there are no data in the
literature regarding sPPases from purple sulfur photo-

synthetic bacteria, or genome sequence projects of any
organisms of this phylogenetic group, that could sug-
gest that its sPPases form a subfamily with distinctive
features.
The high variability in cation dependence and oligo-
meric states found for the anoxygenic bacteria may
reflect adaptations to specific metabolic scenarios. This
proposal is supported by the striking differential
inhibition of enzymatic activity by phosphorylated
metabolites shown by S. 6803 sPPase and the Rho-
dop. viridis sPPase: the purple bacterial enzyme was
strongly inhibited by fructose 1,6-bisphosphate or
2-phosphoglycerate in the assays (70% and 40%,
respectively; 1 mm in the assay); however, its cyano-
bacterial counterpart was not affected at all; ATP was
also inhibitory (c. 50%; 1 mm in the assay) to both
photobacterial enzymes, probably by virtue of its
321
AB
3'2'1'
Anabaena
Synechocystis
Fig. 2. Native PAGE analysis of S. 6803 sPPase and A. 7120
sPPase. (A) Coomassie Blue-stained nondenaturing PAGE of puri-
fied S. 6803 sPPase (lanes 1 and 2) and A. 7120 sPPase (lane 3)
resolved in 7% polyacrylamide gel. (B) In situ sPPase activity assay
of the same preparations in a native gel run in parallel (1¢,2¢ and
3¢). Six micrograms of protein was loaded per lane.
Table 3. Kinetic parameters of sPPase enzymes. Data are an average of at least three independent determinations.
Synechocystis sp.

PCC 6803
Synechocystis sp.
PCC 6803
recombinant
Anabaena sp.
PCC 7120
Pseudanabaena sp.
PCC 6903
Rhodop.
viridis
Rhodos.
rubrum
a
Rhodop.
palustris
a
E. coli
b
K
m
(lM) 2.8 3.1 3 2.9 27.4 25.2 30.1 4.5
K
cat
(s
)1
) 916 800 850 850 750 750 800 200
K
cat
⁄ K
m

327 258 283 293 27 30 27 44
a
Data obtained from partially purified preparation.
b
Salminen et al. [20].
Pyrophosphatases from photosynthetic bacteria M. R. Go
´
mez-Garcı
´
a et al.
3952 FEBS Journal 274 (2007) 3948–3959 Journal compilation ª 2007 FEBS. No claim to original US government works
chelating properties. It is interesting to note in this
respect that the pioneering work of Klemme and Guest
in the early 1970s already identified two classes of
sPPase in rhodospirillaceae with different biochemical
and metabolite-dependent regulatory characteristics,
which may correspond to the currently identified fam-
ily I and II enzymes [24].
It is noteworthy that antibody to S. 6803 sPPase
showed a strong crossreaction with a 20 kDa protein
band in the extract of Heliobacterium chlorum, a mem-
ber of the only group of photosynthetic Gram-positive
bacteria known so far (heliobacteria) (Fig. 3B, right).
This is in agreement with the close relationship
between cyanobacteria and heliobacteria determined
by phylogenetic analysis of photosynthetic genes [25].
No protein bands were immunodetected in cell extracts
from E. coli K12 and DH5a or nonphotosynthetic pro-
tists (data not shown). Genomic DNA from strains
representative of all cyanobacterial taxonomic groups

(Chroococcales, Synechocystis sp. PCC 6803 and Syn-
echococcus sp. PCC 7942; Oscillatoriales, Pseudanabae-
na sp. PCC 6903; Nostocales, Nostoc sp. PCC 7107
and Calothrix sp. PCC 7601; Stigonematales, Fischerel-
la sp. UTEX182) and the green anoxygenic photobac-
teria Chlorobium tepidum and Chlorob. limicola were
found to possess homologous ppa genes by Southern
blot analysis using the Synechocystis sp. PCC 6803 ppa
gene as a probe (data not shown). Hence, ppa genes
and their products (family I sPPases) are widely distri-
buted among diverse photosynthetic prokaryotes.
Functional complementation studies
sPPase appears to be essential for cell anabolism, and
it has not been possible to generate a mutant totally
lacking this activity in E. coli [26] or Synechocystis sp.
PCC 6803 [27]. However, some reconstitution studies
have been performed with a thermosensitive E. coli
mutant [28]. Here, we used an E. coli JP5 strain [29]
obtained by chemical mutagenesis, lacking c. 90% of
its native sPPase activity, as a host for in vivo comple-
mentation experiments to test the functionality of ppa
genes cloned from Synechocystis sp. PCC 6803, Anab-
aena sp. PCC 7120, Pseudanabaena sp. PCC 6903 and
Chlorob. tepidum, using their native promoters. As can
be observed from the growth phenotype (Fig. 4), the
photobacterial sPPases produced from pRGS, pRGA,
pRGP and pRGCT plasmids restored normal E. coli
PCC
6903
PCC 7437

PCC 7601
PCC 7120
PCC 6803
PCC 7942
UTEX 1829
PCC 6803
PCC
6903
PCC 6313
Phormidium
laminosum
PCC
6803
NIES-44
HUB5-2-4
C.7601
Rsp
.
rubrum
Rsp
.
palustris
Rsp
.
viridis
Rb
.
capsulatus
Chr
.

vinosum
Am.
r
oseus
H
b
.
chlorum
Chl
.
limicola
Chl
.
tepidum
Chl
.
phaeobacter
24 kDa
A
B
22 kDa
24 kDa
22 kDa
22 kDa
22 kDa
Fig. 3. Western blot analysis of sPPases in cell-free extracts from diverse cyanobacteria and anoxygenic photosynthetic bacteria. (A) Western
blots probed with a monospecific polyclonal antibody to S. 6803 sPPase showing crossreaction with sPPase orthologs of phylogenetically
diverse cyanobacteria. A single 22 kDa protein band was immunodetected in all unicellular and filamentous strains of sections I, II, IV and V
[5] tested, including the unicellular strain Mi. aeruginosa NIES-44. Note that the three section III strains tested, namely Pseudanabaena sp.
PCC 6903, Spirulina sp. PCC 6313 and Phormidium laminosum, showed one band of slightly higher apparent molecular mass (c. 24 kDa). The

Synechocystis sp. PCC 6803 (section I) sPPase was used as an internal standard in all blots (left-hand lanes). Strains are identified by their
bacterial collection numbers. About 40 lg was loaded per lane. (B) Western blots probed with the monospecific antibody to S. 6803 sPPase
showing crossreaction with sPPases in soluble protein extracts from diverse anoxygenic photosynthetic bacteria. A single 22–24 kDa band
was immunodetected in purple nonsulfur (Rhodospirillaceae) and sulfur (Chromatiaceae) and in green sulfur (Chlorobiaceae) strains, as well as
in Helio. chlorum (Heliobacteriaceae). Remarkably, Chr. vinosum (purple sulfur) showed a protein band of c . 60 kDa, and no band was
detected in Rhodob. capsulatus, which has a family II sPPase. About 80 lg of protein was loaded per lane, except for Helio. chlorum,
Rhodos. rubrum, Rhodop. palustris and Rhodop. viridis extracts, when 40 lg was loaded.
M. R. Go
´
mez-Garcı
´
a et al. Pyrophosphatases from photosynthetic bacteria
FEBS Journal 274 (2007) 3948–3959 Journal compilation ª 2007 FEBS. No claim to original US government works 3953
growth rates and sPPase activity levels (Table 4). The
antibody against S. 6803 sPPase also recognized the
sPPases expressed in the mutant (Fig. 4). This expres-
sion is similar to that found by Lahti et al. [29] with a
thermosensitive E. coli mutant [30]. The complementa-
tion studies demonstrate that photobacterial sPPases
are functionally equivalent to that of the host organ-
ism, and that the promoters seem to be regulated by
the same factors. Analysis of the promoter regions of
these bacterial ppa genes may be helpful for under-
standing their regulation in future studies.
Sequence and phylogenetic analysis
Knowledge of the N-terminal sequences of the three
cyanobacterial sPPases purified and characterized in
this work allowed us to identify the correct first codon
of transcription of the Synechocystis sp. PCC 6803 ppa
gene, as in the genome sequence of this cyanobacterium

[31], it was assigned an ATG situated upstream of
the real GTG used, actually encoding a formyl-Met,
which was determined by Edman degradation sequen-
cing of the N-terminal region of the native pro-
tein (MDLSRIPAQP KAGLINVLIE IPAGSKNKYE
FDKDMNNFAL DRV). A few ppa genes from Syn-
echocystis sp. PCC 6803 and also Anabaena sp.
PCC 7120 share this feature [32]. The encoded 170
amino acid polypeptide has a predicted molecular mass
of 19 216 Da and a pI of 4.69, in good agreement with
MALDI-TOF MS (Fig. 1A) and chromatofocusing
data, respectively. The other three N-terminal
sequences determined in this work, of Anabaena sp.
PCC 7120 (MDLSRIPAQP KPGVINILIE IAG) (with
an initial formyl-Met also encoded by a GTG
codon), Pseudanabaena sp. PCC 6903 (MDLSRIPPQP
KAGILNVLIE IPAG), and Rhodop. viridis (MRIDA
IDXA), and that of Mi. aeruginosa NIES-44 (MDL
SRKPAQP IPGLKNVLVE TAGSINIT) [16], show a
high degree of similarity with other cytosolic sPPases
and conservation of residues localized in the active site
(shown in bold) and involved in catalysis in Ec-sPPase
[2,11]. In all cases, the molecular mass determined by
SDS ⁄ PAGE and ⁄ or MS is in good agreement with
values estimated from the DNA sequence.
The heterogeneity of the sPPases from photosyn-
thetic prokaryotes is clearly in accordance with the
phylogenetic analysis presented in Fig. 5. Two well-
defined groups of family I sPPases cluster on the
phylogenetic tree shown: on one side, cytosolic and

organellar eukaryotic sPPases, and on the other side,
0
1.0
2.0
0
1.0
2.0
0
1.0
2.0
0
0 200 400 600 800 1000 1200
1.0
2.0
ppa Chl.tep.
JP5
DH
AB
5
!
Time (min)
Absorbance at 600 nm
C
ppa S.6903
ppa A.7120
ppa S.6803
P. 6903
22 kDa
22 kDa
22 kDa

24 kDa
Fig. 4. Functional complementation of E. coli JP5 mutant with
pRGS, pRGA, pRGP and pRGCT plasmids. (A) Growth curves,
checked by absorbance at 600 nm, of E. coli DH5a (control, C),
E. coli JP5 mutant and E. coli JP5 expressing the Chlorob. tepidum
ppa gene, and the cyanobacterial Synechocystis sp. PCC 6803,
Anabaena sp. PCC 7120 and Pseudanabaena sp. PCC 6903 ppa
genes. Growth of the complemented E. coli JP5 mutant recovered
rates up to those of the wild type. (B) Western blot analysis of
E. coli JP5 transformed with an empty plasmid (C, control) and
plasmids expressing the Chlorob. tepidum, Synechocystis sp.
PCC 6803, Anabaena sp. PCC 7120 and Pseudanabaena sp.
PCC 6903 ppa genes. The recombinant photobacterial sPPases
were immunodetected in cell-free extracts from the transformed
clones. Forty micrograms of protein was loaded per lane, except in
the case of the Chlorob. tepidum sPPase clone, when 70 lg was
loaded.
Table 4. sPPase specific activities of E. coli JP5 strains. Data are
means ± standard errors of three independent determinations.
Strain (plasmid) Specific activity (UÆmg
)1
)
DH5a 4.05 ± 0.15
JP5 0.32 ± 0.10
JP5 (pRGS) 4.25 ± 0.10
JP5 (pRGA) 3.85 ± 0.15
JP5 (pRGP) 4.30 ± 0.10
JP5 (pRGCT) 5.30 ± 0.15
JP5 (pBS SK
+

) 0.35 ± 0.05
Pyrophosphatases from photosynthetic bacteria M. R. Go
´
mez-Garcı
´
a et al.
3954 FEBS Journal 274 (2007) 3948–3959 Journal compilation ª 2007 FEBS. No claim to original US government works
the prokaryotic (bacterial and archaeal) sPPases and
prokaryotic-type homologs of photosynthetic eukaryo-
tes [12] (Fig. 5). Typical studied cyanobacteria, such
as Synechocystis sp. PCC 6803, Anabaena sp.
PCC 7120, or Pseudanabaena sp. PCC 6903, form a
compact group that is different from other clusters of
photosynthetic bacterial sPPases. As we previously
reported, Prochlorococcus marinus MED4 and Syn-
echococcus WH8102 have two ppa genes in their
genomes: PPA1 codes for an inactive sPPase that
0.1
Mycoplasma
genitalium
Mycoplasma
pneumoniae
Gloeobacter violaceus
Pseudanabaena PCC 6903
Synechocystis PCC 6803
Thermosynechococcus elongatus. BP-1
Trichodesmium erythraeum IMS101
Nostoc punctiforme
Anabaena PCC 7120
Microcystis

aeruginosa NIES44
B
a
c
i
l
l
u
s
s
t
e
a
r
ot
h
e
r
m
o
p
h
i
l
u
s
B
a
c
i

l
l
u
s
h
a
l
o
d
u
r
a
n
s
Helicobacter pylori
Sulfolobus
acidocaldarius.
Aquifex aeolicus
Rickettsia prowazekii
Escherichia coli
Vibrio cholerae
Caulobacter crescentus
Rhodopseudomonas
palustris
Rhodospirillum rubrum
Thermus thermophilus
Dehalococcoides
ethenogenes
Streptomyces
coelicolor

Mycobacterium
leprae
Mycobacterium
tuberculosis
Chloroflexus
aurantiacus
Halobacterium
NCR1
Thermoplasma
acidophilum
Methanobacterium thermophilus.
Thermococcus litoralis
Pyrococcus horikoshii
Pyrococcus furiosus
Chlorobium
tepidum
C
h
l
a
m
y
d
o
mo
n
a
s
r
e

i
n
h
a
r
d
t
i
i
(
s
P
P
a
s
e
I
I
)
Arabidopsis thaliana
Oryza
sativa
Zea
mays
Solanum
tuberosum
Chlamydia
pneumoniae
Chlamydia
trachomatis

Chlamydomonas reinhardtii
CHLOR.(sPPase I)
Arabidopsis thaliana
CHLOR.
Oryza sativa
CHLOR.
S. cerevisiae (PPA1)
Mus musculus
MIT.
Bos taurus
Mus musculus
Synechococcus
WH8102 (PPA2)
Prochlorococcus marinus
MED4 (PPA2)
Haemophilus influenzae
Neisseria meningitidis
Eukaryotic
Eukaryotic
Family I
Family I
sPPases
sPPases
Synechococcus
WH8102 (PPA1)
Prochlorococcus marinus
MED4 (PPA1)
C
y
a

n
o
b
a
c
t
e
r
i
a
C
y
a
n
o
b
a
c
t
e
r
i
a
*
*
Ba
c
t
e
r

ia
l
Ba
c
t
e
r
ia
l
-
-
l
i
k
e
l
i
k
e
p
l
a
n
t
p
l
a
n
t
s

P
P
a
s
e
s
s
P
P
a
s
e
s
A
r
c
h
a
e
a
A
r
c
h
a
e
a
S. cerevisiae
(PPA2) MIT
1000

*
*
989
1000
1000
760
823
651
518
925
923
Purple
Purple
non
non
-
-
sulfur
sulfur
phot
phot
.
.
bact
bact
.
.
Green
Green
non

non
-
-
sulfur
sulfur
phot
phot
.
.
bact
bact
.
.
Green sulfur
Green sulfur
phot
phot
.
.
bact
bact
.
.
Prokaryotic
Prokaryotic
Family I
Family I
sPPases
sPPases
Fig. 5. Molecular phylogenetic analysis of family I sPPases of photosynthetic prokaryotes. Amino acid sequences deduced from prokaryotic

sPPases were aligned using
CLUSTALX. Most sequences have all the amino acids reported to be functionally important for sPPase activity and
the PROSITE motif of family I sPPases. Numbers in nodes are bootstrap values (1000 replicates) supporting representative groups. Asterisks
indicate the pairs of sPPase paralogs present in marine unicellular cyanobacteria [32]. The circled P indicates a partial N-terminal sequence.
The 0.1 bar represents amino acid substitutions per site. Accession numbers for the sequences are (reading clockwise): Chlamydo. rein-
hardtii CHLOR sPPase I, AJ298231; Arabidopsis thaliana CHLOR sPPase I, Atg09650; Oryza sativa CHLOR, BAD 16934; Sa. cerevisiae PPA1
(cytosolic), YBR011C; Sa. cerevisiae PPA2 MIT (mitochondrial), YMR267W; Mus musculus MIT (mitochondrial), Q91VM9; Bos taurus,
P37980; Mus musculus, BAB25754; Synechococcus WH8102 PPA2, CAE08303; Pr. marinus MED4 PPA2, CAE18953; Hae. influenzae,
P44529; Neisseria meningitidis, F81175; Mycoplasma genitalicum, P47593; Mycop. pneumoniae, P75250; Gloeobacter violaceus, grl4227;
Pseudanabaena PCC 6903, P80898; Synechocystis PCC 6803, P80507; Thermosynechococcus elongatus BP-1, BAC09435; Trichodesmium
erythraeum IMS101, ABG50803; Nostoc punctiforme, ZP_00112287; Anabaena PCC 7120, P80562; Pr. marinus MED4 PPA1, CAE18970;
Synechococcus WH8102 PPA1, CAE08284; Mi. aeruginosa NIES44, 29 amino acid partial sequence, AAB19891; Bacillus stearothermophilus,
O05724; Ba. halodurans, AP001512; Helicobacter pylori, AE001439; Sulfolobus acidocaldarius, P50308; Aquifex aeolicus, O67501; Rickettsia
prowazekii, CA15034; E. coli, P17288; Vibrio cholerae, AAF95686; Caulobacter crescentus, AE005679; Rhodop. palustris, CAE25855; Rho-
dos. rubrum, AAF21981; Chlorob. tepidum TLS, AAM72059; Chlorof. auranticus, EAO59327; Thermus thermophilus, P38576; Dehalococco-
ides ethenogenes, AAW40363; Streptomyces coelicolor, CAB42762; Mycobacterium leprae, O69540; Mycob. tuberculosis, O06379;
Halobacterium NCR, AAG18854.1; Thermoplasma acidophilum, P37981;
Methanobacterium thermoautotrophicus, O26363; Thermococcus
litoralis, P77992; Pyrococcus horikoshii, O59570; Py. furiosus, Q8U438; Chlamydo. reinhardtii (sPPase II), AJ298232; Ar. thaliana prokaryotic-
like, At2g18230; O. sativa prokaryotic-like, O22537; Zea mays prokaryotic-like, O48556; Solanum tuberosum prokaryotic-like, CAA85362;
Chlamydia pneumoniae, AAD19056; Chlamydia trachomatis, AAC68367.
M. R. Go
´
mez-Garcı
´
a et al. Pyrophosphatases from photosynthetic bacteria
FEBS Journal 274 (2007) 3948–3959 Journal compilation ª 2007 FEBS. No claim to original US government works 3955
clusters with the ‘typical’ sPPases from freshwater
cyanobacteria, whereas PPA2 codes for an isoform
that is, so far, characteristic of marine unicellular

cyanobacteria, and constitutes a second cyanobacteri-
al sPPase group that is closely related to several non-
photosynthetic proteobacteria (Haemophilus influenzae
and Neisseria spp.) and the members of which are
expressed as active sPPases [22]. It remains to be clar-
ified whether PPA2 sPPases result from horizontal
gene transfer or represent an ancestral cyanobacterial
enzyme that was lost during the evolutionary history
of the cyanobacterial lineage.
The sPPases from nonsulfur purple bacteria (Rho-
dos. rubrum, Rhodop. palustris) that show different
cation dependence and oligomeric structure [Rho-
dos. rubrum sPPase is a tetramer of c. 90 kDa (data
not shown), and Rhodop. viridis sPPase appears to be
a dodecamer; see Fig. 1B] are clustered with the main
proteobacterial assembly. It can be speculated that
these peculiar properties of the enzymes from photo-
synthetic proteobacteria may result from functional
adaptations to specific metabolic scenarios.
The sPPases from the two classes of green photosyn-
thetic bacteria associate with different branches, in
agreement with their different molecular genealogies.
The enzyme of the green nonsulfur bacterium Chlorofl-
exus auranticus clusters weakly with archaeal homologs
but it is also Mg
2+
-dependent. sPPases of the green
sulfur bacteria Chlorob. tepidum and Chlorob. limicola
are clearly Mg
2+

-dependent enzymes, and unexpect-
edly cluster with the bacterium-like Mg
2+
-dependent
sPPases of photosynthetic eukaryotes (Fig. 5), suggest-
ing an interesting evolutionary relationship between
the plant sPPase subfamily and a possible counterpart
from a bacterial ancestor.
Experimental procedures
Organisms and growth conditions
The photosynthetic bacteria used in this study are summar-
ized in Table 1. Cyanobacteria were cultured at 30 °Cin
BG11 liquid medium [17] supplemented with 1 g sodium
bicarbonateÆL
)1
and bubbled with 1.5% (v ⁄ v) CO
2
in air
under continuous white light (25 WÆm
)2
). Nonsulfur purple
bacteria were grown in modified Hutner medium [33], pur-
ple and green sulfur bacteria were grown in Tru
¨
per and
Pfenning medium [34], and Helio. chlorum was grown in
heliobacteriaceae medium [35].
The E. coli JP5 strain [36], containing only c. 10–15%
of wild-type sPPase activity levels, was cultured in LB
medium supplemented with ampicillin when necessary

(100 lgÆmL
)1
), and was grown at 37 °C with continuous
shaking at 200 r.p.m. Competent E. coli JP5 cells were
obtained using the protocol described previously [37].
Protein techniques
Assay for sPPase activity
sPPase was assayed by the colorimetric determination of P
i
produced by the enzymatic hydrolysis of PP
i
at 22 °C
[12,38] with PP
i
as a substrate. The reaction mixtures con-
tained 50 mm Tris ⁄ HCl (pH 7.5), 4 mm cation salt (MgCl
2
)
and 2 mm Na
4
PP
i
(standard assay conditions). The reaction
was started by the addition of enzyme, and the PP
i
released
after 10 min was determined. When the efficiencies of other
divalent metal cations as cofactors were tested, the corres-
ponding chloride salts were used in the assays instead of
the Mg

2+
salt. Mg
2
PP
i
was utilized as substrate for kinetic
parameter estimations. Reaction rates are expressed in
terms of lmol P
i
generated per minute.
SDS ⁄ PAGE, native PAGE (12% w ⁄ v) and Bradford pro-
tein estimations were performed as described previously
[39,41]. SDS ⁄ PAGE gels loaded with purified samples of
sPPase were stained for activity as described by Kang &
Ho [16].
Purification of the sPPases from Synechocystis sp.
PCC 6803, Pseudanabaena sp. PCC 6903,
Anabaena sp. PCC 7120 and Rhodop. viridis
A purification protocol similar to the one described for
the isolation of eukaryotic sPPases [12] was used for the
native proteins from photobacteria. The recombinant
S. 6803 sPPase was isolated from E. coli XL1blue trans-
formed with pRGS plasmid and cultured in LB medium
supplemented with ampicillin (100 lgÆmL
)1
) using the
same protocol; anion exchange chromatography was essen-
tial for the separation of the overexpressed sPPase and the
native Ec-sPPase. Gel filtration FPLC (Amersham Phar-
macia, Uppsala, Sweden) was used as an analytical tech-

nique for purified enzymes. Column chromatofocusing on
a Polybuffer Exchanger (PBE94) bed was performed
according to the manufacturer’s instructions (Amersham
Pharmacia).
N-terminal sequences of purified sPPases from Synecho-
cystis sp. PCC 6803, Anabaena sp. PCC 7120, Pseudanabae-
na sp. PCC 6903 and Rhodop. viridis were obtained in this
work by the Edman degradation method, using an automa-
tic sequencer, at the protein analysis facilities of the Vienna
Biocenter (University of Vienna, Austria).
Immunochemical techniques
A rabbit was injected with 500 lg of pure S. 6803 sPPase
water ⁄ Freund’s coadjuvant (1 : 1). Antibodies were
obtained as described previously [12,40]. Immunoblot
Pyrophosphatases from photosynthetic bacteria M. R. Go
´
mez-Garcı
´
a et al.
3956 FEBS Journal 274 (2007) 3948–3959 Journal compilation ª 2007 FEBS. No claim to original US government works
assays (western blots) of protein samples were carried out
after electrophoresis in SDS ⁄ PAGE (12%).
Cloning and DNA manipulation
The reaction mixture for PCR amplification of the ppa
gene from Synechocystis sp. PCC 6803 (50 lL) contained
50 pmol of each of the following two pairs of oligonucleo-
tide primers: ipyrpro,5¢-CGCTTAAGTTAAAAGCCTTT-3¢;
and ipyrend,5¢-GCGAAGCTTATTTACCGGTTCA
GTTAGCT-3¢. The PCR product contains the Synecho-
cystis sp. PCC 6803 ppa gene and 375 bp of the upstream

sequence containing the promoter region, and was cloned
in pBS SK
+
(PRGS). For amplification of ppa from Chlo-
rob. tepidum, the oligonucleotides used were: 5¢-ppa300chlor
5¢-TCGGGAAAGTGGCTCTG-3¢ and 3¢-ppa120chlo 5¢-
CTCAGTCCTTGTCCACGGC-3¢. The PCR product was
cloned in pBS SK
+
(pRGCT).
A BclI–HindIII fragment of Synechocystis sp. PCC 6803
ppa was (460 bp) used as a probe for screening a genomic
library of Anabaena sp. PCC 7120 [42]. The genomic lib-
rary was plated at a dilution of 2000 colonies per plate.
After incubation for 12 h at 37 °C, plates were replicated
on 0.45 lm sheets, and the filters were then treated as
described before [33] and hybridized overnight at 55 °C
with the
32
P-labeled probe. One clone containing a 4.6 kb
plasmid was isolated. Restriction analysis with XmnI and
ClaI (pRGA) identified the ppa gene with an extra
sequence at the 5¢-end corresponding to the promoter
region (243 bp). This plasmid was used in heterologous
expression experiments. The same methodology allowed us
to screen a genomic library of Pseudanabaena sp.
PCC 6903 [43]; one clone was obtained with a 7.2 kb
plasmid (pRGP1) after hybridization. The plasmid was
subjected to restriction analysis with HincII and EcoRI,
and subcloned in pBS SK

+
obtaining pRGP, which con-
tains Pseudanabaena sp. PCC 6903 ppa and 233 bp of the
upstream region. The plasmids used for heterologous
expression experiments (pRGS, pRGA, pRGP and
pRGCT) containing ppa ORFs and the corresponding
upstream regions were sequenced to ensure that ppa genes
were cloned in the opposite orientation to the lacZ vec-
tor’s promoter and that the expression was achieved
under their own native promoters. Chromosomal DNA
was isolated from bacterial cells as previously described
[44]. For DNAÆDNA hybridization (Southern blotting),
the method of Ausubel et al. was used [41]. Samples of
bacterial genomic DNA were completely digested with dif-
ferent restriction enzymes, run in 0.7% (w ⁄ v) agarose gels,
and blotted onto nylon membranes (Zetaprobe; Biorad,
Richmond, CA). The filter was hybridized using the pro-
tocol described by Church & Gilbert [45] at 55 °C for
heterologous hybridization. The nylon filters were then
exposed to films (Kodak X-100 310S, Racine, Chicago,
IL) at ) 80 °C and eventually developed.
Nucleotide and N-terminal protein sequence
accession numbers
The EMBL ⁄ GenBank database accession number for the
Synechocystis sp. PCC 6803 ppa gene is AJ252207.
The accession number in the SwissProt database for both
the natural and recombinant N-terminal protein sequences
is P80507. The EMBL ⁄ GenBank database accession
number for Anabaena sp. PCC 7120 ppa is AJ252206,
and the accession number in the SwissProt database is

P80562. The EMBL ⁄ GenBank database accession num-
ber for Pseudanabaena sp. PCC 6903 ppa is AJ252205,
and the accession number in the SwissProt database is
P80898.
Protein sequence comparisons and phylogenetic
analyses
A multiple amino acid sequence alignment of the sPPases
from photosynthetic prokaryotes and other selected prok-
aryotic family I sPPases was performed using the clustalx
v.1.8 program [46]. This alignment was used to construct a
phylogenetic distance tree (neighbor-joining method, BLO-
SUM matrix) with the same program. Sequence data from
public databases or unfinished microbial genome projects
were obtained by similarity searches using blast algorithms
[47] against websites of the National Center of Biotechno-
logy Information (NCBI), USA ( />PMGifs/Genomes/allorg.html), the Joint Genomic Institute
(JGI), USA ( />the Sanger Institute, UK ( />or the Institute for Genomic Research (TIGR), USA
( />Acknowledgements
The authors gratefully thank Dr N. N. Rao and Dr J.
Josse for critical review of the manuscript, and Profes-
sor W. Lo
¨
ffelhardt (University of Vienna, Austria) for
his assistance in N-terminal protein sequencing and
some MALDI-TOF MS analyses. This work was
supported by research grants from the Spanish
(BMC2001-563 and BFU2004-00843, MEC) and
Andalusian Regional (PAI group CVI-261) Adminis-
trations, funded in part by the EU FEDER program.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Gel filtration FPLC on Superose 12HR of
Pseudanabaena sp. PCC 6903 sPPase.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-
ponding author for the article.
M. R. Go
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a et al. Pyrophosphatases from photosynthetic bacteria
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