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MICROBIOLOGY
LETTERS
EJSWIER
FEMS Microbiology
Lettera 133 (1995) 187-193
A comparison of gene organization in the zwf region of the
genomes of the cyanobacteria Synechococcus sp. PCC 7942 and
Anabaena sp. PCC 7 120
Julie Newman, Haydar Karakaya, David J. Scanlan, Nicholas H. Mann
Department
oj’Biologica1
qf
Sciences, Uniwrsip
Received 21 August 1995; revised 7 September
Warwick.
Cm,entp
1995; accepted
*
CV4 7AL, UK
I4 September
I995
Abstract
The region of the genome encoding the glucose-6-phosphate
dehydrogenase gene zwf was analysed in a unicellular
S~~~choco~u.s
sp. PCC 7942, and a filamentous, heterocystous cyanobacterium,
Anabaena sp. PCC 7 120.
Comparison of cyanobacterial
zwf se q uences revealed the presence of two absolutely conserved cysteine residues which
may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene flp, encoding
fructose- 1,6_bisphosphatase,
upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these
organisms may function to completely oxidize glucose 6-phosphate to COz. The amino acid sequence of fructose- I ,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide
exchange mechanism. In the case of
Anahaena sp. PCC 7120, the ral gene, encoding transaldolase, lies between ;M?f and &J.
cyanobacterium,
Kepvordx
Glucose-6-phosphate
dehydrogenase;
;v$: Fructo se- I ,6-bisphosphatase;
1. Introduction
Swechococcus;
Anabaena
* Corresponding
author. Tel.: +44 (1203) 523 526; Fax: f44
(1203) 523 701; E-mail: [email protected].
chococcus sp. PCC 7942 exhibited similar dark respiratory activity, as measured by oxygen uptake, to
that of the wild-type [21]. Thus cyanobacteria may
employ an alternative respiratory pathway when the
OPP is non-functional.
The OPP is also thought to be
largely responsible for the supply of reductant, in the
light, to nitrogenase in the heterocyst [2,24]. Because
of the central physiological importance of this transition from phototrophic metabolism to heterotrophic
metabolism in the dark, the mechanisms involved in
regulating the activity of key reductive and oxidative
pentose phosphate cycle enzymes have been the
focus of much attention. As is the case with higher
plants, cyanobacteria exhibit light/dark
activation/
inactivation of Calvin cycle enzymes. In Nostoc sp.
MAC experiments
with permeabilized
cells have
037%1097/95/$09.50
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The dominant nutritional mode of cyanobacteria
is photoautotrophy involving the assimilation of CO?
through the reductive pentose phosphate pathway
(RPP). However, these organisms are also capable of
generating
maintenance
energy during periods of
darkness via the dissimilation of fixed carbon stored
as glycogen. Dark respiration is thought to proceed
exclusively through the oxidative pentose phosphate
pathway (OPP) (for review see [22]). However, it has
been shown recently that a zwf mutant of Sync-
0 1995 Federation
SSDI 037%1097(95)00369-X
of European
Microbiological
suggested the light activation of fructose- I ,6-bisphosphatase, sedoheptulose- 1,7-bisphosphatase, ribulose-5phosphate
kinase and NADP-linked
glyceraldehyde-3-phosphate
dehydrogenase, possibly via a
thioredoxin-based
mechanism [3]. However, there is
as yet little detailed information as regards the mechanism(s) of light/dark
enzyme activation/inactivation. In this study, gene organization
in the ?Mif
regions of the genomes of two cyanobacteria
was
analysed to establish whether other genes encoding
enzymes of dark metabolism are located close to ZVV~
and to verify whether the predicted amino acid sequences of the proteins are consistent with thioredoxin control of activity.
DNA sequence analysis was performed using both
random and directed cloning of fragments in Ml3
mp18 and M 13 mp19. Reactions were primed using
universal - 40 primers or synthetic oligonucleotides,
using Sequenase II polymerase (Amersham plc). The
nucleotide
sequence
was determined
using the
dideoxy chain termination method. Analysis of DNA
and protein sequence information
was carried out
using the Wisconsin Package version 8 [ 171.
2. Materials
3. Results and discussion
2. I. Bacterial
tions
and methods
struins, plasmids
and culture
condi-
Synechococcus
sp. PCC7942 and Anabaena sp.
PCC7 120 were grown at 30°C under white fluorescent light (20 PE m-2 s- ‘) in liquid BGl 1 medium
[ 181. Escherichia coli MC1061 and TG 1 were used
for plasmid constructions and DNA sequencing respectively and were grown in LB medium and 2 X
YT medium [14].
2.2. DNA manipulations
Chromosomal
DNA from the cyanobacterial
strains was isolated using a method described previously [ 191. Plasmid isolation from E. cd, restriction
digestion, ligation using T4 ligase and transformations in E, cdi were performed using standard
molecular biological
techniques
[ 141. DNA fragments were isolated from agarose gels using the
Geneclean kit (Bio 101 Inc). Chromosomal
DNA
was restricted using various restriction enzymes, according to the manufacturers’ instructions. Southern
blotting of this DNA was performed using nitrocellulose (HybondC,
Amersham plc) as described by
Maniatis et al. [ 141. DNA fragments used for probes
were restricted, isolated from low melting point
agarose and labelled with [j2P]dCTP using the random priming method [9]. Filters were hybridised
under low stringency conditions (.55”C, 5 X SSPE,
5 X Denhardt’s, 0.1% SDS) [ 141 and washed at 55°C
in 2 X SSC. unless otherwise stated.
2.3. DNA sequence determinution
3.1. Gene organization
7942
and analysis
in Syzechococcus
sp. PCC
We had previously cloned and sequenced the zyf
gene encoding glucose-6-phosphate
dehydrogenase
from Synechococcus
sp. PCC 7942 [20] and were
interested in establishing
whether genes encoding
other enzymes associated with respiratory metabolism
were clustered in this region of the genome. Complete sequencing
of the 2.8-kb Hind111 fragment
containing the Synechococcus zwf gene revealed an
incomplete ORF upstream (5’) of Z~I$ A Sal1 site
occurs in the middle of the ZW~gene and so the two
corresponding
Sal1 fragments (approx. 6 kb and 5
kb) were cloned in plasmid pUC19 to yield plasmids
pDA and pDB. The sequence of the upstream 1033
bp ORF was completed using oligonucleotide primers
and a 3-kb FsfI fragment from pDA sub-cloned into
Ml3 mp18 and 19. This ORF was identified as
coding for fructose- 1,6-bisphosphatase
on the basis
of the similarity of its translation product to known
sequences including E. coli and several plant sources
and was hence designated as jbp. Subsequently, the
jbp gene from the cyanobacterium
Nostoc sp. strain
ATCC 29 133 was cloned and sequenced 1231 and the
two cyanobacterial proteins exhibited 8 I % similarity
(67% identity). Approximately
4.5 kb of sequence
upstream from jbp has been analysed and surprisingly no other ORFs were detected. Sequence information downstream of wf was obtained by sub-
.‘_vnechoroccuv sp. PCC 7942
petD petB
fapl
Anahaena
sp P(‘(‘ 7 I20
tal
fbP
W-
1 kb
Fig.
I. Gene organization
in the :n:f’ region of the genomes 01
Slnrchoc,oc,~u.\ sp. PCC 7942 and A~7ahtrrno
sp. PCC
7 120. The
nucleotide sequence information on which this diagram is based
has the Genbank accession numbers U33282
7 120) and U33285
(S~ilec,ho~oc,c,rrs
sp. PCC
( Adxrrmr
hp. PCC
7942).
cloning a contiguous 1.7-kb Hind111 fragment from
pDB into Ml 3 mpl8 and 19 and extended using a
2.8-kb Hind111 fragment from pDB. Three further
ORFs were identified. This sequence information is
summarized in Fig. 1. The two ORFs furthest downstream from :“:f and on the complementary
strand
are identified as encoding cytochrome b, ( petB) and
subunit IV (perD) of the cytochrome b6/‘f‘ complex
by virtue of the similarities with Swechococcw
sp.
PCC 7002 [61. The ORF immediately downstream
from ZW~ potentially encodes a polypeptide of 445
amino acids, and the only protein in the databases
with any significant similarity (68%) is that encoded
by the ORF immediately
downstream
of :w$ in
Nostoc sp. strain ATCC 29133 [23]. Although no
clue to the role. if any, of the protein in the oxidative
pentose phosphate pathway was forthcoming from
sequence comparisons,
it is known to be always
co-transcribed with :yf in Nostoc. sp. strain ATCC
29 133 [23]. There is recent evidence [24] (Karakaya.
Scanlan. Sundaram.
Newman and Mann. unpublished results) that the protein encoded by this ORF
(designated opcA) is involved in the functional assembly of glucose-6-phosphate
dehydrogenase.
3.2. Gerlr orgcrnix~ion
in Anabuena
sp, PCC 7942
Synechowccus
sp. PCC 7942 is a unicellular
strain and is incapable of nitrogen fixation and heterocyst formation. Since the OPP is the major supplier
of reductant to nitrogenase
in the heterocyst, we
decided to examine the zu:f region of the genome of
a filamentous,
heterocyst-producing
strain, namely
Anubuetza sp. PCC 7 120. A I.%kb BamHI/HindIII
fragment carrying the downstream (3’) half of the
Synechococms
sp. PCC 7942 ;M,-fgene was used to
probe a Southern blot of HindIII-digested
DNA
from Atmbaenu sp. PCC 7 120. A 7-kb fragment
hybridized strongly (data not shown) and a clone
carrying this fragment was isolated from a size-fractionated HitId
Atzabaenu sp. PCC 7 120 library in
pBR325. A 2.4.kb HpuI/HindIII
fragment was
sub-cloned into M 13 mpl8 and 19 for sequencing.
which was completed by random sequencing in Ml3
mpl8. This yielded the complete :\\:f gene and an
incomplete ORF upstream. The translation products
of the :\~:f genes from Anubaena sp. PCC 7120 and
S!,tzpc.hoL,oc,c,ussp. PCC 7942 exhibited 83% similarity (70% identity). The sequence of the upstream
ORF was completed by random sequencing of a
1. I-kb EcoRI/HpuI
fragment in Ml3 mp18. The
ORF encoded a polypeptide of 381 amino acids
which on the basis of 50% sequence similarity (30%
identity) to the Succhcrrotnyes
cerel,isiue protein
was identified as encoding transaldolase
and was
designated rd. Subsequently,
it was shown to be
93% similar (83% identical) to the transaldolase of
Nosmc sp. strain ATCC 29133 [23]. There was a
further ORF upstream (5’) from tal, the sequence of
which was completed by analysis of a contiguous
3.5-kb EwRI fragment. This ORF was identified as
encoding fructose- 1.6-bisphosphatase on the basis of
the similarity of its translation product with that of
the Swechococws
sp. PCC 7942 ,fbp gene. All this
sequence information is summarized in Fig. I.
3.3. Cotnparison
of gene nrgani,7don
The arrangement of the .fbp. tal and zb;f genes is
the same as that reported for Nosmc sp. strain ATCC
29133 [23], which is also a filamentous, heterocystous strain. However, in the unicellular strain Swechocwcms sp. PCC 7942 there is no ml gene between .fbp and :\z:f: An internal 0.5-kb HpaI/ClaI
fragment of the rul gene from Ambuenu
sp. PCC
7 120 was used to probe a Southern blot of Syncchococcus sp. PCC 7942 DNA under conditions of
moderate stringency
(hybridization
in 5 X SSPE,
55°C; washing in 2 X SSPE, 55°C) and yielded only
190
.I.
Newmnn
etul./~EMSMicrohiolo~~Letters 133 (IYYSI
1X7-lY3
50
1
.....
Anabaena
Nostoc
Synechococcus
Chloroplast
Cytosolic
Consensus
...
....
.....
.
.
.
.I........
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
,.........
.
.
.
.
.
.
.
.
.
.
maasaattts shlllsssrh vasssqpsil sprslfsnng kraptgvrnh
.. .... .. .. ..... ..
.
.
. ... ..
__________ _____..... _________. __________ __________
Anabaena
NOStOC
Synechococcus
Chloroplast
Cytosolic
consensus
51
..... ...MA KAsESLDlSV
NEsTDkALDR
DCTTLSRHVL
QQLQSFSaDA
100
. . . . . . ..MA
KtpESLEsSI
NEiTDPALDR
DCTTLSRHVL
QQLQSFSpDA
. . . . . . ..MA
qsttS.....
.EthtRdLDR
DCTTLSRHVL
eQLQSFSpEA
qyasgvrcMA
VAaDasETkt
aarkksgyE1
q..TLtgwlL
r.qemkgeid
. . . . . . ..Md
hAgDsNrT..
. . . . . . ..Dl
m..TitRyVL
neqskrpesr
--------MA
KA--SLETS-
NE-TDRALDR
DCTTLSRHVL
QQLQSFS-DA
QDLsALNnRI
ALAgKIVARR
LSRAGLMEGv
LGFTGEVNVQ
150
GEsVKKMDVY
QDLSAIMnRI
ALAgKIVARR
MSRAGLMEGV LGFTGHVNVQ
GEsVKKMDVY
QDLsALMqRI
gLAaKLIARR
LShAGLvDda
LGFTGEINVQ
GEaVKrMDVY
aELtivMss1
sLAcKqIAs1
vqRAGi.snl
tGvqGaINIQ
GEdqKKLDVi
gDFtiLLsh1
vLgcKFVcsa
vnkAGL.akl
iGLaGEtNIQ
GEeqKKLDVl
QDL-ALM-RI
ALA-KLVARR
LSRAGLMEG-
LGFTGE-NVQ GE-VKKMDVY
BEMDEPYYIP
ENCPIGRYTL
101
Anabaena
Nostoc
Synechococcus
Chloroplast
Cytosolic
consensus
t
151
ANDVFISVFk QSGLVCRLAS
l
Anabaena
Nostoc
Synechococcus
Chloroplast
Cytosolic
consensus
ANDVFISVFk
QSGLVCRLAS
sNEVFsncLr sSGrtgiiAS
sN!JVFVkaLt SSGrtCiLvS
AN-VFISVF- QSGLVCRLAS
t
dnabaena
Nostoc
Synechococcus
Chloroplast
Cytosolic
consensu*
alignment,
Arabidopsis
in upper
case.
redox-sensitive
produced
sp. PCC
thaliancl
The
7942
using
(this
study),
cysteines
residues
250
.._..
.GdDsDGqAK
DLLtnGRkQI
.GtDsDGkAt
DLLanGRkQl
.fyDEsheAK
1GsEEqrciv
...fEtatle
-G-DEL%-AK
7
DLLQPGdrQI
nvcQPGnnl1
DvLQPGknmV
DLLQPGR-QI
t
300
RIPdHGaVYS
RIPNIiGsVYS
qlPNsGqIYS
eIPkaGrIYS
kIPNkGkIYS
RIPNHG-IYS
tDnNLSlGS1
FaIRQQE...
.
VDvdLnvGSI
IDaavStGSI
IDcgvSiGtI
ID-NLS-GSI
FaVRrQE...
FgIyspnDec
FgIymvkD..
F-IRQQED--
. .... ...
ivddsddisa
. ..___..
----------
251
AAGYILYGpS
AAGYILYGpc
AAGYVLYGaS
AAGYcMYssS
AAGYcMYGsS
AAGYILYG-S
c
TMLVYTMGtG
TMLVYTiGKG
TLLVYsMGqG
viFVlTLGKG
CtLVlstGsG
TMLVYTMGKG
VHSFTLDPSL GEFILseENI
VIiSFvLDPSL GEFILTeENI
VHvFvLDPSL GBFVLaqsdI
VfSFTLDPmy GEFVLTqENI
VngFTLDPSL GEYILThpdI
VHSFTLDPSL GBFILT-EN1
l
EgyRqYIRem
HRrEa....Y
SgRYSGALVa
DfHRILMQGG
351
VFLYPGTIqN PeGKLRLLYE sAPLAFLIqQ AGGrAtTGLV
VFLYPGTIqN PeGKLRLLYE tAPIdFLIEQ AGGrAtTGLV
VFLYPeTVFN PtGKLRLLYE aAPMAFLaEQ AGGkAsdGqk
IYgYPrdaKs
knGKLRLLYE
cAPMsFiVEQ AGGkgsdGhs
IFLYPGdkKs PnGKLRvLYE VfPMsFLmEQ AGGqAfTGkq
VFLYPGTIKN P-GKLRLLYE -APMAFLIEQ AGG-A-TGLV
400
dILDWPkKL
nILDWPkKL
pILl+qPqaL
RVLDIqPtei
RaLDlIPtKi
RILDVVP-KL
401
429
HQRTPLIIGS KEDVaKVESF iqNGH....
the
HQRTPLIIGS
KEDVaKVEsF
iqNGH
HeRcPLIIGS
aaDVDfVEac
1Aesvp
tEEVEKlEkY
lA.......
HeRsPvflGS
yDDVHdIkaL
yAsqekta
HQRTPLIIGS
KEDVHKVE-F
-ANGH----
PILEUP
enzyme
._.
HQRvPLyIGS
Ancrhaenrr
implicated
in the cytosolic
l
._....._..
[ 171, of the amino
programme
sp. PCC
7120
[I I] and the cytosolic
form from Spinoceu
cysteine
LYDPiDGSSN
LYDPLDGSaN
vFDPLDGSSN
vFDPLDGSSN
LYDPLDGSSN
fNEGNYqmWD DkLkkYIddl kdpgptgkPY SARYiGsLVg DfHRtLLyGG
VNEGNaknWD gpttkYVekc kfptdgsspk SlRYiGsMVa DVHRtLLyGG
VNEGNFWQW- ES-R-YIR-- HR-EG---PY SARYSGAMV- DIHRILLQGG
Anabaena
Nostoc
Synechococcus
Chloroplast
Cytosolic
consensus
An
ENCPIGRYTL
ENCPIGRYTL
EesysGnYw
EpslrGkYcv
ENCPIGRYTL
201
tDtNLSlGS1 FsIRQQE...
VNEGNFNQNp
Armbaena
Nostoc
Synechococcus
Chloroplast
Cytosolic
consensus
2.
BEMEnPWIP
EEHEkPYYIP
BEeDvPvaV.
EEdEEatFI.
EEMEEPYYIP
301
350
VNEGNFWQWE ESMReYIRyv HRtEG....Y tARYSGAMVs DIHRILvQGG
VNEGNFWQWE ESiReYIRyv HRtEG....Y SARYSGAMVs DIHRILVQGG
Anabaena
Nostoc
Synechococcus
Chloroplast
Cytosolic
consensus
S\n&rococcuu
QSGLVCRLAS
ANqVFISVFr
Anabaena
Nostoc
Synechococcus
Chloroplast
Cytosolic
consensus
Fig.
200
LYDPiDGSSN
in activation
are
marked
of
(this
olrrc~ru
study),
[12].
the chloroplast
(* ).
Nostoc
Amino
enzyme
acid
sequences
sp. ATCC
acid
residues
are indicated
of
29133
fructose-1,6-bisphosphatase
from
[23],
from
agreeing
by
the chloroplast
with
vertical
enzyme
the consensus
arrows
and
those
are shown
potential
191
J. Newman et al./ FEMS Microhiolog~ Letter.~ 133 ClYY5) 1X7-193
CO!lSeL¶US
1
.mvsLLENPL
.mvsLLENPL
mtpkLLENPL
----LLENPL
RVGLqQqgmP
RVGLqQqgmP
RIGLrQdkvP
RVGL-Q---P
50
EPQIiVIFGA sGDLTwRKLV PAlYkLrrER
EPQIiVIFGA sGDLTwRKLV PAlYkLrrER
EPQIlVIFGA tGDLTqRKLV PAiYeMhlER
EPQI-VIFGA -GDLT-RKLV PA-Y-L--ER
Anabaena
Nostoc
Synechococcus
consensus
51
RiPPEtTIVG
RiPPEtTIVG
RlPPElTIVG
R-PPE-TIVG
VARREWShEY
VARREWShEY
VARRDWSdDY
VARREWS-EY
100
FREqMqkGmE eahssVelgE 1WqdFsQGLF
FREqMqkGmE eahpdVdlgE 1WqdFsQGLF
FREhLrqGvE qfgggIqaeE vWntFaQGLF
FRE-M--G-E -----V---E -W--F-QGLF
Anabaena
Nostoc
Synechococcus
consensus
150
101
YcPGdIDnPe sYQkLknlLs eLDEkRGTRG NRmFYLSVAP nFFpEAiKQL
YsPGdIDnPe sYQkLktlLs eLDEkRGTRG NRmFYLSVAP sFFpEAiKQL
FaPGnIDdPq fYQtL+drLa nLDElRGTRG NRtFYLSVAP rFFgEAaKQL
Y-PG-ID-P- -YQ-L---L- -LDE-RGTRG Ni-FYLSVAP -PF-EA-KQL
Anabaena
Nostoc
S'ynechococcus
COIX3HX3U.5
200
151
GgaGMLdDPy KhRLVIEKPF GRDLaSAQsL NaVvQkyCkE hQVYRIDHYL
GsgGMLeDPy KhRLVIEKPF GRDLaSAQsL NqVvQkyCkE hQVYRIDHYL
GaaGMLaDPa KtRLVVEKPF GRDLsSAQvL NaIlQnvCrE sQIYRIDHYL
G--GML-DP- K-RLVIEKPF GP.DL-SAQ-L N-V-Q--C-E -QVYRIDWn
Anabaena
Nostoc
Symchococcus
consensus
201
GKETVQNLLV FRFANAIFEP LWWRQFVDHV QITVAET'VGv
GKETVQNLLV FRFANAIFEP LWNRQFVDHV QITVABTVGv
GKETVQNLLV FRFANAIFEP LWNRQYIDHV QITVAETVGl
GKETVQNLLV FRFANAIFEP LWNRQFVDHV QITVAETVG-
Anabaena
Nostoc
Synechococcus
consensus
251
GALRDMlQNH
GALRDMlQNH
GALRDMvQNH
GALRDM-QNH
LMQLYcLTAM
LMQLYcLTAM
LMQLFsLTAM
LMQLY-LTAM
Anabaena
Nostoc
Synechococcus
consensus
301
SrSAIRGQYs
SrSAVRGQYs
SlSAVRGQYk
S-SAVRGQY-
AGWMkGqqVP gYRtEpGvDP nSsTPTWgM
AGWMkGqaVP gYRtEpGvDP nStTPTYVaM
AGWMnGrsVP aYRdEeGaDP qSfTPTYVaM
AGWM-G--VP -YR-E-G-DP -S-TPTYV-M
Anabaena
Nostoc
Synechococcus
CCJllSellsUs
351
GVPFYLRTGK
GVPFYLRTGK
GVPFYLRTGK
GVPFYLRTGK
RMPKKVsEIs
RMPKKVsEIa
RMPKKVtEIa
RMPKKV-EI-
IhFrdVPsrM
IhFreVPsrM
IqFktVPhlM
I-F--VP--M
Anabaena
Nostoc
Synechococcus
consensus
401
NEGISLRFDV
NEGISLRFDV
NEGVSLRFEV
NEGISLRFDV
KmPGaefRsR
KmPGaefRtR
KtPGssqRtR
K-PG---R-R
SVDMDFsYgs fgieaTsDAY
SVDKDFsYgs fgiqaTsDAY
SVDMDFrYdt afgspTqEAY
SVDMDF-Y-- -----T-DAY
Anabaena
Nostoc
Synechococcus
consensus
451
DQTLFTRADE
DQTLFTRADE
DQTLFTRADE
DQTLFTRADE
Anabaena
Nostoc
Synechococcus
consensus
501
INqDG..rrw rRl.......
INqDG..rrw rR1 _._...
INrDGavgw
sRipatqlns
IN_DG_____ _R________
Anabaena
Nostoc
synechococcus
l
250
EdRAGYYEkA
EdRAGYYEsA
EgRAGWEtA
E-RAGYYE-A
300
EaPNsMdADs IRtEKVKVlQ ATRLADVhnL
EaPNaMdADs IRtEKVKVlQ ATP.LADVhnL
SpPNsLgADg IRnEKVKVvQ ATRLADIddL
E-PN-M-AD- IR-EKVKV-Q ATRLAD'J-L
350
KFLVDNWRWq
KFLVDNWRWk
KLLVDNWRWq
KFLVDNWRW-
400
FQSAaQqrN. aNILaMRIQP
FQSAaQqtN. aNILtMRIQP
FQSAtQkvNs pNVLvLRIQP
FQSA-Q--N- -NIL-MRIQP
450
dRLFlDCMMG
dRLFlDCMMG
sRLLvDCMLG
-RLF-DCMMG
l
VEAaWqWTP
VEAaWqWTP
VSAsWrVVTP
VEA-W-WTP
aLsvWDsPad
aLsvWDaPad
1LesWDdPrq
-L--WD-P--
patIpqYEAG
pttIpqYEAG
aagIsfYEAG
---I--YEAG
500
TWEPaeAEfL
TWJZPeqAElL
TWEPaeAEqL
TWEP--AS-L
525
_....
. .. . .
sgdv
_____
Fig. 3. An aligment, produced using the PILEUP programme [ 171. of the amino acid sequences of the glucose-6-phosphate
dehydrogenases
from Anabaena sp. PCC 7120 (this study), Nostoc sp. ATCC 29133 [23] and Syechococcus sp. PCC 7942 [21]. Amino acid residues
agreeing with the consensus are shown in upper case. The two conserved cysteine residues are indicated (* ).
a faint signal with a 6-kb Hind111 fragment. Thus it
is not clear whether Synechococcus
sp. PCC 7942
contains a ful gene. Although it is risky to infer
physiological properties from DNA sequence information, the close proximity of the ,fbl, gene to :nf
in all the cyanobacterial
strains examined and their
co-transcription
in Nosfoc sp. strain ATCC 29133
[23] do suggest something about the way the OPP
may be operating. The action of transaldolase and.
presumably
transketolase,
regenerates
fructose 6phosphate, which can re-enter the cycle, and glyceraldehyde 3-phosphate.
Fructose- I ,6_bisphosphatase
ensures that glyceraldehyde 3-phosphate via aldolase
will also re-enter the cycle rather than be metabolized to pyruvate. Thus glucose 6-phosphate can be
completely oxidized to CO, with the concomitant
production of maximal amounts of NADPH. This is
in keeping with the observations of BBhme [5], in
relation to reductant supply to nitrogenase, that glycolytic degradation of hexose appears to be of minor
importance and that aldolase and fructose- I ,6-bisphosphatase function to provide the oxidative pentose phosphate pathway with additional hexose phosphates.
3.4. Implications for regulation of enzyme actir!ity
Cyanobacteria,
like plants. regulate certain enzyme activities in response to light-dark transitions.
The light activation of several enzymes including
fructose- I ,6-bisphosphatase,
sedoheptulose- I ,7-bisphosphatase,
ribulose-5-phosphate
kinase
and
NADP-linked
glyceraldehyde-3-phosphate
dehydrogenase, possibly via a thioredoxin based mechanism,
has been reported for permeabilized cells of Nostoc
sp. [3]. In plants there are two distinct fructose-l,6bisphosphatases,
chloroplast and cytosolic, with different regulatory properties. The cytosolic enzyme is
allosterically regulated by AMP, whereas the chloroplast enzyme is regulated via a thiol/disulfide
exchange mechanism involving thioredoxin acting as a
protein disulfide reductase [7]. The chloroplast enzyme, compared to the cytosolic form, typically has
an insertion of 12-17 amino acids with two adjacent
conserved cysteine residues for the light regulation
of enzyme activity [ 151. In cyanobacteria, fructose1,6-bisphosphatase
is required both for the RPP in
the light and also the OPP in the dark. There are
conflicting reports regarding the regulatory properties of cyanobacterial fructose- 1,6_bisphosphatase activity. Bishop [4] reported the enzyme from Anucystis nidulurz~ to exhibit regulatory characteristics that
were not typical for either form of the enzyme. This
conflicts with the report that the regulatory properties of fructose- I ,6-bisphosphatase
from Anucystis
nidulans resembled that of the chloroplast enzyme
with respect to agents such as oxidized and reduced
glutathione. ascorbic acid and dithionite [2.5]. Comparison of the cyanobacterial
amino acid sequences
reported here, and that of Anabuena sp. ATCC
29133 [23], to the plant enzymes reveals them to be
of the cytosolic type, in that they lack the insertion
and adjacent cysteines typical of the chloroplast form
of the enzyme (Fig. 2). Recently it has been demonstrated that even the cytosolic
fructose- I ,6-bisphosphatase from sugarbeet exhibits a slow light
activation and light-dependent
AMP sensitivity [ 131.
This observation may be explained by the presence
of potential redox-sensitive
cysteine pairs predicted
by tertiary structure modelling in cytosolic forms of
the enzyme [I]. However, these potential redox-sensitive cysteines, apart from one, are not conserved in
the cyanobacterial enzymes (Fig. 2). Consequently, it
seems likely that if the cyanobacterial
fructose- I ,6bisphosphatases are subject to light-dark regulation
it is not via a thiol/disulfide
exchange mechanism.
Several studies have been aimed at elucidating the
mechanisms by which glucose 6-phosphate activity
is regulated
during
light-dark
transitions
in
cyanobacteria. Metabolites including NADPH [2,16]
and ATP [IO] have been implicated in regulation and
thioredoxin control has also been proposed by Cossar et al. [8]. In keeping with the thiol/disulfide
exchange mechanism of regulation, sequence analysis of the Swwchococc~l.s
sp. PCC 7942 ,-rvf gene
revealed the protein to have two cysteine residues
which are not present in the enzyme from other
prokaryotic sources [20]. Comparison of the glucose6-phosphate
dehydrogenase
sequences
from Anubaena sp. PCC 7120 (this study), Nostoc sp. ATCC
29133 [23] with that of the Synechococws sp. PCC
7942 protein reveals these two cysteines at positions
188 and 447 to be absolutely conserved (Fig. 31,
reinforcing the likelihood of their role in the regulation of enzyme activity.
and characterization of a cDNA
Acknowledgements
encoding cytoaolic fructose-
1.6.bisphosphatase from spinach. Plant Mol. Biol. 18, 799X02.
Haydar Karakaya was supported by a studentship
provided by the Turkish Government
through Ondokuz Mayis University. This work benefitted from
the use of the SEQNET facility.
[I.?1 Khayat.
E.. Harn. C. and Daie, J. (1993)
Purification and
light-dependent molecular modulation of the cytosolic fructose- I .h-bisphosphatase in sugarbeet leaves. Plant Physiol.
101. 57-63.
[I41
Maniatis. T., Fritach, E.F. and Sambrook. J. (1981-j Molecular Cloning:
A
Laboratory
Manual.
Cold
Spring
Harbor
Laboratory, Cold Spring Harbor, NY.
[I51
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