A comparison of gene organization in the zwf region of the genomes of the cyanobacteria...

7
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 Sciences, Uniwrsip qf Warwick. Cm,entp CV4 7AL, UK Received 21 August 1995; revised 7 September 1995; accepted I4 September I995 Abstract The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, S~~~choco~u.s sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7 120. Comparison of cyanobacterial zwf q se 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-bi- sphosphatase 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. Kepvordx Glucose-6-phosphate dehydrogenase; ;v$: Fructo se- I ,6-bisphosphatase; Swechococcus; Anabaena 1. Introduction 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- * Corresponding author. Tel.: +44 (1203) 523 526; Fax: f44 (1203) 523 701; E-mail: [email protected]. chococcus sp. PCC 7942 exhibited similar dark res- piratory 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 transi- tion 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 0 1995 Federation of European Microbiological Societies. All rights reserved SSDI 037%1097(95)00369-X

Transcript of A comparison of gene organization in the zwf region of the genomes of the cyanobacteria...

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 Sciences, Uniwrsip qf Warwick. Cm,entp CV4 7AL, UK

Received 21 August 1995; revised 7 September 1995; accepted I4 September I995

Abstract

The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular

cyanobacterium, S~~~choco~u.s sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7 120. Comparison of cyanobacterial zwf q se 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-bi- sphosphatase 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.

Kepvordx Glucose-6-phosphate dehydrogenase; ;v$: Fructo se- I ,6-bisphosphatase; Swechococcus; Anabaena

1. Introduction

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-

* Corresponding author. Tel.: +44 (1203) 523 526; Fax: f44

(1203) 523 701; E-mail: [email protected].

chococcus sp. PCC 7942 exhibited similar dark res-

piratory 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 transi-

tion 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 0 1995 Federation of European Microbiological Societies. All rights reserved

SSDI 037%1097(95)00369-X

suggested the light activation of fructose- I ,6-bis- phosphatase, sedoheptulose- 1,7-bisphosphatase, ribu-

lose-5phosphate kinase and NADP-linked glycer- aldehyde-3-phosphate dehydrogenase, possibly via a

thioredoxin-based mechanism [3]. However, there is

as yet little detailed information as regards the mech-

anism(s) of light/dark enzyme activation/inactiv-

ation. 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 se- quences of the proteins are consistent with thiore-

doxin control of activity.

2. Materials and methods

2. I. Bacterial struins, plasmids and culture condi-

tions

Synechococcus sp. PCC7942 and Anabaena sp. PCC7 120 were grown at 30°C under white fluores-

cent 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 re-

spectively 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 previ-

ously [ 191. Plasmid isolation from E. cd, restriction digestion, ligation using T4 ligase and transforma- tions in E, cdi were performed using standard

molecular biological techniques [ 141. DNA frag-

ments were isolated from agarose gels using the Geneclean kit (Bio 101 Inc). Chromosomal DNA

was restricted using various restriction enzymes, ac- cording to the manufacturers’ instructions. Southern blotting of this DNA was performed using nitrocellu-

lose (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 ran- dom 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 and analysis

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.

3. Results and discussion

3.1. Gene organization in Syzechococcus sp. PCC

7942

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. Com- plete 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 surpris- ingly no other ORFs were detected. Sequence infor- mation downstream of wf was obtained by sub-

.‘_vnechoroccuv sp. PCC 7942

petD petB

fapl

Anahaena sp P(‘(‘ 7 I20

fbP tal 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 ( Adxrrmr hp. PCC

7 120) and U33285 (S~ilec,ho~oc,c,rrs sp. 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 down-

stream 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. unpub- lished results) that the protein encoded by this ORF (designated opcA) is involved in the functional as-

sembly 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 hete- rocyst 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,-f gene 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-frac-

tionated 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,us sp. PCC 7942 exhibited 83% similar- ity (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, heterocys- tous strain. However, in the unicellular strain Swe-

chocwcms sp. PCC 7942 there is no ml gene be-

tween .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 Sync-

chococcus 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

Anabaena Nostoc

Synechococcus Chloroplast

Cytosolic Consensus

Anabaena NOStOC

Synechococcus Chloroplast

Cytosolic consensus

Anabaena Nostoc

Synechococcus Chloroplast

Cytosolic consensus

Anabaena Nostoc

Synechococcus Chloroplast

Cytosolic consensus

Anabaena Nostoc

Synechococcus Chloroplast

Cytosolic consensus

dnabaena Nostoc

Synechococcus Chloroplast

Cytosolic consensu*

Anabaena Nostoc

Synechococcus Chloroplast

Cytosolic consensus

Armbaena Nostoc

Synechococcus Chloroplast

Cytosolic consensus

Anabaena Nostoc

Synechococcus Chloroplast

Cytosolic consensus

1 50

. . . . . . . . . . . . . . . . . . . .

.I........ . . . . . . . . ,.........

. . . . . . . . . . . . . . . . . . . . . . . . . . .

maasaattts shlllsssrh vasssqpsil sprslfsnng kraptgvrnh . . . . . . . . . . . . . . . . . . . . . . . . . __________ _____..... _________. __________ __________

51 100 . . . . . . ..MA KAsESLDlSV NEsTDkALDR DCTTLSRHVL QQLQSFSaDA

. . . . . . ..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

101 150

QDLsALNnRI ALAgKIVARR LSRAGLMEGv LGFTGEVNVQ 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 t l

151 200 ANDVFISVFk QSGLVCRLAS BEMDEPYYIP ENCPIGRYTL LYDPiDGSSN

ANDVFISVFk QSGLVCRLAS BEMEnPWIP ENCPIGRYTL LYDPiDGSSN

ANqVFISVFr QSGLVCRLAS EEHEkPYYIP ENCPIGRYTL LYDPLDGSaN sNEVFsncLr sSGrtgiiAS BEeDvPvaV. EesysGnYw vFDPLDGSSN sN!JVFVkaLt SSGrtCiLvS EEdEEatFI. EpslrGkYcv vFDPLDGSSN AN-VFISVF- QSGLVCRLAS EEMEEPYYIP ENCPIGRYTL LYDPLDGSSN

t l

201 250 tDtNLSlGS1 FsIRQQE... ._....._.. .GdDsDGqAK DLLtnGRkQI

tDnNLSlGS1 FaIRQQE... . .._.. .GtDsDGkAt DLLanGRkQl

VDvdLnvGSI FaVRrQE... . . . . . . . . .fyDEsheAK DLLQPGdrQI IDaavStGSI FgIyspnDec ivddsddisa 1GsEEqrciv nvcQPGnnl1 IDcgvSiGtI FgIymvkD.. . ..___.. . ..fEtatle DvLQPGknmV ID-NLS-GSI F-IRQQED-- ---------- -G-DEL%-AK DLLQPGR-QI

7 t 251 300 AAGYILYGpS TMLVYTMGtG VHSFTLDPSL GEFILseENI RIPdHGaVYS AAGYILYGpc TMLVYTiGKG VIiSFvLDPSL GEFILTeENI RIPNIiGsVYS AAGYVLYGaS TLLVYsMGqG VHvFvLDPSL GBFVLaqsdI qlPNsGqIYS AAGYcMYssS viFVlTLGKG VfSFTLDPmy GEFVLTqENI eIPkaGrIYS AAGYcMYGsS CtLVlstGsG VngFTLDPSL GEYILThpdI kIPNkGkIYS AAGYILYG-S TMLVYTMGKG VHSFTLDPSL GBFILT-EN1 RIPNHG-IYS

c l

301 350 VNEGNFWQWE ESMReYIRyv HRtEG....Y tARYSGAMVs DIHRILvQGG VNEGNFWQWE ESiReYIRyv HRtEG....Y SARYSGAMVs DIHRILVQGG VNEGNFNQNp EgyRqYIRem HRrEa....Y SgRYSGALVa DfHRILMQGG

fNEGNYqmWD DkLkkYIddl kdpgptgkPY SARYiGsLVg DfHRtLLyGG VNEGNaknWD gpttkYVekc kfptdgsspk SlRYiGsMVa DVHRtLLyGG VNEGNFWQW- ES-R-YIR-- HR-EG---PY SARYSGAMV- DIHRILLQGG

351 400 VFLYPGTIqN PeGKLRLLYE sAPLAFLIqQ AGGrAtTGLV dILDWPkKL VFLYPGTIqN PeGKLRLLYE tAPIdFLIEQ AGGrAtTGLV nILDWPkKL VFLYPeTVFN PtGKLRLLYE aAPMAFLaEQ AGGkAsdGqk pILl+qPqaL IYgYPrdaKs knGKLRLLYE cAPMsFiVEQ AGGkgsdGhs RVLDIqPtei IFLYPGdkKs PnGKLRvLYE VfPMsFLmEQ AGGqAfTGkq RaLDlIPtKi VFLYPGTIKN P-GKLRLLYE -APMAFLIEQ AGG-A-TGLV RILDVVP-KL

401 429 HQRTPLIIGS KEDVaKVESF iqNGH.... HQRTPLIIGS KEDVaKVEsF iqNGH ._.

HeRcPLIIGS aaDVDfVEac 1Aesvp

HQRvPLyIGS tEEVEKlEkY lA.......

HeRsPvflGS yDDVHdIkaL yAsqekta

HQRTPLIIGS KEDVHKVE-F -ANGH----

Fig. 2. An alignment, produced using the PILEUP programme [ 171, of the amino acid sequences of fructose-1,6-bisphosphatase from

S\n&rococcuu sp. PCC 7942 (this study), Ancrhaenrr sp. PCC 7120 (this study), Nostoc sp. ATCC 29133 [23], the chloroplast enzyme from

Arabidopsis thaliancl [I I] and the cytosolic form from Spinoceu olrrc~ru [12]. Amino acid residues agreeing with the consensus are shown

in upper case. The cysteine residues implicated in activation of the chloroplast enzyme are indicated by vertical arrows and those potential

redox-sensitive cysteines in the cytosolic enzyme are marked (* ).

J. Newman et al./ FEMS Microhiolog~ Letter.~ 133 ClYY5) 1X7-193 191

Anabaena

Nostoc

synechococcus

CO!lSeL¶US

Anabaena

Nostoc Synechococcus

consensus

Anabaena

Nostoc Synechococcus

consensus

Anabaena

Nostoc S'ynechococcus

COIX3HX3U.5

Anabaena

Nostoc Symchococcus

consensus

Anabaena Nostoc Synechococcus

consensus

Anabaena

Nostoc Synechococcus

consensus

Anabaena Nostoc

Synechococcus

CCJllSellsUs

Anabaena Nostoc Synechococcus consensus

Anabaena Nostoc Synechococcus

consensus

Anabaena Nostoc Synechococcus consensus

1 50

.mvsLLENPL RVGLqQqgmP EPQIiVIFGA sGDLTwRKLV PAlYkLrrER

.mvsLLENPL RVGLqQqgmP EPQIiVIFGA sGDLTwRKLV PAlYkLrrER mtpkLLENPL RIGLrQdkvP EPQIlVIFGA tGDLTqRKLV PAiYeMhlER ----LLENPL RVGL-Q---P EPQI-VIFGA -GDLT-RKLV PA-Y-L--ER

51 100

RiPPEtTIVG VARREWShEY FREqMqkGmE eahssVelgE 1WqdFsQGLF

RiPPEtTIVG VARREWShEY FREqMqkGmE eahpdVdlgE 1WqdFsQGLF

RlPPElTIVG VARRDWSdDY FREhLrqGvE qfgggIqaeE vWntFaQGLF R-PPE-TIVG VARREWS-EY FRE-M--G-E -----V---E -W--F-QGLF

101 150

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

151 200

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 l

201 250

GKETVQNLLV FRFANAIFEP LWWRQFVDHV QITVAET'VGv EdRAGYYEkA

GKETVQNLLV FRFANAIFEP LWNRQFVDHV QITVABTVGv EdRAGYYEsA

GKETVQNLLV FRFANAIFEP LWNRQYIDHV QITVAETVGl EgRAGWEtA

GKETVQNLLV FRFANAIFEP LWNRQFVDHV QITVAETVG- E-RAGYYE-A

251 300

GALRDMlQNH LMQLYcLTAM EaPNsMdADs IRtEKVKVlQ ATRLADVhnL GALRDMlQNH LMQLYcLTAM EaPNaMdADs IRtEKVKVlQ ATP.LADVhnL

GALRDMvQNH LMQLFsLTAM SpPNsLgADg IRnEKVKVvQ ATRLADIddL GALRDM-QNH LMQLY-LTAM E-PN-M-AD- IR-EKVKV-Q ATRLAD'J-L

301 350

SrSAIRGQYs AGWMkGqqVP gYRtEpGvDP nSsTPTWgM KFLVDNWRWq

SrSAVRGQYs AGWMkGqaVP gYRtEpGvDP nStTPTYVaM KFLVDNWRWk

SlSAVRGQYk AGWMnGrsVP aYRdEeGaDP qSfTPTYVaM KLLVDNWRWq S-SAVRGQY- AGWM-G--VP -YR-E-G-DP -S-TPTYV-M KFLVDNWRW-

351 400

GVPFYLRTGK RMPKKVsEIs IhFrdVPsrM FQSAaQqrN. aNILaMRIQP

GVPFYLRTGK RMPKKVsEIa IhFreVPsrM FQSAaQqtN. aNILtMRIQP GVPFYLRTGK RMPKKVtEIa IqFktVPhlM FQSAtQkvNs pNVLvLRIQP GVPFYLRTGK RMPKKV-EI- I-F--VP--M FQSA-Q--N- -NIL-MRIQP

401 450

NEGISLRFDV KmPGaefRsR SVDMDFsYgs fgieaTsDAY dRLFlDCMMG NEGISLRFDV KmPGaefRtR SVDKDFsYgs fgiqaTsDAY dRLFlDCMMG

NEGVSLRFEV KtPGssqRtR SVDMDFrYdt afgspTqEAY sRLLvDCMLG NEGISLRFDV K-PG---R-R SVDMDF-Y-- -----T-DAY -RLF-DCMMG

l

451 500

DQTLFTRADE VEAaWqWTP aLsvWDsPad patIpqYEAG TWEPaeAEfL DQTLFTRADE VEAaWqWTP aLsvWDaPad pttIpqYEAG TWJZPeqAElL DQTLFTRADE VSAsWrVVTP 1LesWDdPrq aagIsfYEAG TWEPaeAEqL DQTLFTRADE VEA-W-WTP -L--WD-P-- ---I--YEAG TWEP--AS-L

501 525 INqDG..rrw rRl....... _.... INqDG..rrw rR1 _._... . . . . .

INrDGavgw sRipatqlns sgdv IN_DG_____ _R________ _____

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 S yechococcus 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 infor- mation, 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 6- phosphate, which can re-enter the cycle, and glycer-

aldehyde 3-phosphate. Fructose- I ,6_bisphosphatase

ensures that glyceraldehyde 3-phosphate via aldolase

will also re-enter the cycle rather than be metabo-

lized 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 gly- colytic degradation of hexose appears to be of minor importance and that aldolase and fructose- I ,6-bi-

sphosphatase function to provide the oxidative pen- tose phosphate pathway with additional hexose phos-

phates.

3.4. Implications for regulation of enzyme actir!ity

Cyanobacteria, like plants. regulate certain en- zyme activities in response to light-dark transitions.

The light activation of several enzymes including

fructose- I ,6-bisphosphatase, sedoheptulose- I ,7-bi- sphosphatase, ribulose-5-phosphate kinase and

NADP-linked glyceraldehyde-3-phosphate dehydro- genase, possibly via a thioredoxin based mechanism,

has been reported for permeabilized cells of Nostoc

sp. [3]. In plants there are two distinct fructose-l,6-

bisphosphatases, chloroplast and cytosolic, with dif- ferent regulatory properties. The cytosolic enzyme is

allosterically regulated by AMP, whereas the chloro- plast enzyme is regulated via a thiol/disulfide ex- change mechanism involving thioredoxin acting as a protein disulfide reductase [7]. The chloroplast en- zyme, 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, fructose- 1,6-bisphosphatase is required both for the RPP in

the light and also the OPP in the dark. There are

conflicting reports regarding the regulatory proper- ties of cyanobacterial fructose- 1,6_bisphosphatase ac-

tivity. Bishop [4] reported the enzyme from Anucys-

tis nidulurz~ to exhibit regulatory characteristics that

were not typical for either form of the enzyme. This

conflicts with the report that the regulatory proper- ties 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]. Com-

parison 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 demon-

strated that even the cytosolic fructose- I ,6-bi-

sphosphatase 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-sen-

sitive cysteines, apart from one, are not conserved in

the cyanobacterial enzymes (Fig. 2). Consequently, it seems likely that if the cyanobacterial fructose- I ,6-

bisphosphatases 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 Cos- sar et al. [8]. In keeping with the thiol/disulfide

exchange mechanism of regulation, sequence analy- sis 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 glucose- 6-phosphate dehydrogenase sequences from An-

ubaena 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 regula- tion of enzyme activity.

Acknowledgements

Haydar Karakaya was supported by a studentship

provided by the Turkish Government through On-

dokuz Mayis University. This work benefitted from

the use of the SEQNET facility.

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