1 0304-5722
Marine
Cyanobacteria
BULLETIN~EINSTITUT
OCÉANOGRAPHIQUE
FONDATION ALBERT 1
erPRINCE DE MONACO
Numéro pécial 19
1999
Le Lude publiée dans le BulletilL de {'Institut ucéwwgrapliiqu sont analy. ·c. uindexée-dam :
The works publishedill tlze Blll1elin de l']n -lÎlUl océanographique {Ire abstracted orilldèxedill :
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ft
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onaco-Ville
98000 0 A
Marine
Cyanobacteria
Editors:
Loïc CHARPY A.W.D. LARKUM
MONACO
MUSÉE OCÉANOGRAPHIQUE 1999
Bulletin de l'Institut océanographique, Monaco Numéro spécia119
Ce volume est publié par François DOUMENGE, Directeur du Musée océanographique,
Avec le concours d'Anne TOULEMONT, Maître de ConférencesàJ'Institut océanographique.
Réalisation: TyPAü Sarl (Paris 11')
(ÔMusée océanographique, Monaco 1999
Toute reproduction ou représentation. intégrale ou partielle. par quelque procédé 411e ce soit.du tex.te ctdc~
imagc~du présent ouvrage,e.\ét'Ul~Csans l'autorisation de l'éditeur,csiillicite et constitue une contrefaçon.
Seules sont autorisées les reproductions strictement réservéesàl'usage privé du copiste et non destinéesà une utilisiJtion co\lc<.:tive, ainsi que les analyses et courtes citations justifiées par le caractère scientifique ou d'information de l'œuvre dans la4uelle ellcs sont incorporées (loidu 11 mars 1957 sur la protection des droits d'auteur.articles"ll et 41. ct Code pénal. article 425).
ISBN 2-7260-0210-2 (numéro spécial 19)
II Bulletin de l'Institut océallogruphiqlle. MO/laeo,n°spécial19 (1999)
Contents
PLANKTON MICROORGANISMS WORKTEAM
Light-induction of the Synechocystis PCC6803 secA gene involves promoter remodelling
S.Bulteau, K. Mazouni, C.Cassier-Chauvat, F.Chauvat . The cytochrome b6
f
complex of ChlamydomonasCatherine De Vitry . . . . 7 Use of molecular tools for the study of genetic relationships
of heterocystous cyanobacteria
Isabelle Iteman, Rosmarie Rippka, Nicole Tandeau de Marsac,
Michael H erdman 13
Flow cytometric analysis of lightldark synchronized cells of Chlamydomonas reinhardtii
S.Lemaire, M. Hours, C. Gèrard-Hirne, A. Trouabal, O. Roche,
J.-P. Jacquot. . . . 21 A new class of genes regulated by light and the circadian rhythm
S.Lemaire, M. Stein, E. Issakidis, E. Keryer, V. Benoit,
C.Gèrard-Hirne, M. Miginiac-Maslow, J.-P. Jacquot. . . . 27 Plasmid shuffling manipulation of essential genes in Synechocystis
PCC6803: mutational analysis of the plant-like ferredoxin
M. Poncelet,C. Cassier-Chauvat, X. Leschelle, H. Bottin,F. Chauvat. 33
SESSION1 - TAXINOMY AND PHYLOGENY
Whole cell fatty acid profile as a tool for clustering Cyanothece strains of saline environments into sub-groups
Roberto De Philippis, Maria Cristina Margheri, Alessandra Bastianini, Massimo Vincenzini . . . . 39 Cyanobacterial sequences retrieved directly from
the Great Sippewissett Salt Marsh, MA, USA
Jesse Dillon, Annick Wilmotte . . . . 47
BuliClin de l'Institut océanographique,MOIli/CO,nOspécial 19 (1999) III
MARINE CYANOBACTERIA
Diversity of Marine Cyanobacteria
Stjepko Golubic, Thérèse Le Campion-Alsumard, Susan E. Campbell 53 Characterization ofrnpB, the gene encoding the RNA component
of RNase P ofProchlorococcus marinus
Wolfgang R. Hess, Astrid Schon 77
A taxonomie revision of the genusArthrospirabased on certain new criteria
N. Jeeji Bai . . . . 83 Marine cyanobacteria from Bahia Concepcion, BCS, Mexico
Alejandro Lopez-Cortes . . . . 87 Molecular biological characterization of unicellular marine
Cyanobacteria from the Baltic Sea
Andrea Mikolajczak, Rainer Soller, Dietmar Blohm, Ulrich Fischer 95 Cyanobacterial diversity in marine ecosystems as seen
by RNA polymerase(rpoe]) gene sequences
Brian Palenik, Gerardo Toledo, Mike Ferris 101
Molecular phylogeny ofProchlorococcusecotypes
Gabrielle Rocap, Lisa R. Moore, SallieW.Chisholm . . . . 107 Molecular phylogenetic relationship betweenSynechocystis trididemni andProchloron didemni
A. Shimada, S. Kanai, R.A. Lewin, T.Maruyama 117
SESSIONII - ENVIRONMENT: ECOLOGY AND GLOBAL CHANGE Recent cyanobacterial stromatolites in the lagoon of Tikehau atoll (Tuamotu Archipelago, French Polynesia): preliminary observations C. Charpy-Roubaud, T. Le Campion,S.Golubic, G. Sarazin 121 Cyanobacteria and associated micro organisms characterize
coarse shoreline carbonates of One Tree Island, Australia
Marcos Gektidis ... . . . . 127 Euendolithic cyanobacteriafcyanophyta and their traces in Earth history Ingrid Glaub, Sinisa-Josef Balog, Martina Bundschuh, Marcos Gektidis, Klaus Hofmann, Gudrun Radtke, Horst Schmidt, Klaus Vogel . . . . 135 Standing crop and organic matter of intertidal microbial mats
fol1owing the 1991 oil spill in the Arabi an Gulf
L. Hoffmann, J. Gérardin 143
The cyanobacteria of coral reefs
A. W.D. Larkum 149
IV Bulletin de /"Institut océanoRraphique, Monaco. n° spécial 19(1999)
CONTENTS
Single-celled cyanobacteria in the first-year sea ice and ice-covered waters of the Northern Hemisphere
Louis Legendre, Brigitte Robineau, Bernard LeBlanc 169 Geophysiology of cyanobacterial biofilms and
the "dyssymmetry" principle
George S. Levit, AnnaA. Gorbushina, Wolfgang E. Krumbein 175 Effect of salinity and light intensity on superoxide dismutase
and ascorbate peroxidase activity fromMicrocoleus chthonoplastes Strain SC7B9002-1
R. Tovar, A. Gonzalez, A. Lopez-Cortés,V.Ascencio, J.L. Ochoa 197 Sedimentary organic matter derived from cyanobacteria:
composition. structure and properties of kopara deposits
Jean Trichet, Christian Defarge ... . . . 203 Analysis of cyanophage diversity and population structure in
a south-north transect of the Atlantic ocean
William H. Wilson, NicholasJ. Fuller, Jan R. Joint, Nicholas H. Mann 209
SESSIONIII - NUTRIENT
Photoacclimation response of Baltic Sea Picocyanobacteria in culture Patrizia Albertano, Emanuela Viaggiu, LucasJ. Stal 217 Distribution of nitrogenase in the marine non-heterocystous
cyanobacteriumTrichodesmiwn: a review
Birgitta Bergman. . . . 223 The peculiarities of bioenergetics coupling in Cyanobacteria
under low L1 mH+ and L1 mNa+
1.1. Brown, Yu. L. Chaban, R.R. Jzhmukhametov, S.G. Karakis,
D.1. Pogorelov 229
Nitrogen fixation by marine cyanobacteria: historical and global perspectives
Douglas G. Capone, EdwardJ.Carpenter . . . . 235 Cyanobacterial community changes in sulfide biotopes of shallow
coastal waters of the Baltic Sea
Ulrich Fischer, Jorg Rethmeier, Andreas Rabenstein, Anja Potthoff . . 257 Dissolved organic nitrogen release and amino acid oxidase activity
by Trichodesmium spp.
Patricia M. Glibert, JudithM.O'neil . . . . 265 Effect of ammonium on nitrate/nitrite uptake andntcA expression
inSynechococcus sp. Strain WH 7803
Debbie Lindell, Etana Padan, AntonF. Post 273
Bulletin de l '/mtitut oeéal1oliraphique. MO/luco. n° spécial 19(1999) V
MARINE CY ANOBACTERIA
Utilization of combined forms of N in cultures and natural populations ofTrichodesmium spp.
M.R. Mulholùmd, C.Shoemaker, K. Ohki, D.G. Capone 279 A possible role of temperate phage in the regulation
ofTrichodesmium biomass
Kaori Ohki 287
Grazer interactions with nitrogen-fixing marine Cyanobacteria:
adaptation for N-acquisition?
Judith M. O'Neil 293
Physical-chemical constraints on cyanobacterial growth in the Oceans
Hanslv. Paerl . . . . 319 Diagnostic protein biomarkers for assessing the nutrient status
of cyanobacterial picoplankton in situ
D.]. Scanlan . . . . 351 Nitrogen fixation in microbial mats and stromatolites
Lucas J. Stal . . . . 357 Iron-Iimited semicontinuous culture studies of marineSynechococcus Yi Yin, William J. Henley . . . . 365
SESSIONIV - PRODUCTIVITY
Synechococcus and Prochlorococcus dominance estimated by ftow cytometry in Tuamotu Atoll lagoons
Loïc Charpy, Jean Blanchot 369
Abundance and distribution of photosynthetic prokaryotic picoplankton in shelf waters of the Great Barrier Reet', Australia
Nicholas D. Crosbie, Miles J. Fumas. . . . 377 In situ growth dynamics of the photosynthetic prokaryotic picoplankters Synechococcus and Prochlorococcus
Miles Fumas, Nicholas D. Crosbie . . . . 387 Cyanobacterial photosynthetic apparatus: an overview
Alexander N. Glazer 419
Distribution ofSynechococcus, Prochlorococcus, and picoeukaryotes in the East China Sea
Nianzhi Jiao, Yanhui Yang 435
Phycoerythrins in the sea: abundance and spectral diversity
François Lantoine, Jacques Neveux . . . . 443 VI Bulletin de "Institut océanogr0l'hique, Monaco, n°spé~ial 19 (l'i'i'i)
CONTENTS
Picophytoplankton dynamics in the equatorial Pacific (OOS 1500W)
C.Navarette, J.-M. André, J. Blanchot, M.-H. Radenac, J. Neveux. . . 451 Differentiai distribution and ecology ofProchlorococcus
andSynechococcus in oceanic waters: a review
F.Partensky, J. Blanchot, D. Vaulot . . . . 457 Flow cytometric analysis of picophytoplankton in the South Pacific
and Antarctic Oceans
A. Shimada, S. Kawaguchi, T.Maruyama, M. Naganobu . . . .. 477
SESSIONV - HARMFUL BLOOMS AND TOXINS
Summary of session 5: "Harmful blooms and toxins"
Maija Balode, SergeY. Maestrini , , . . . 481 Cyanobacterial toxins: their occurrence in aquatic environments
and significance ta health
Geoffrey A. Codd . . . . 483 Blooms of the cyanobacteriumLyngbya majuscula in coastal waters of Queensland. Australia
William C.Dennison, Judith M. O'Neil, Elisabeth J. Duffy,
Peter E. Oliver, Glendon R. Shaw . . . . 501
SESSIONVI - AQUACULTURE AND GENETIC MANIPULATIONS
Commercial-scale culture of Cyanobacteria
Michael A. Borowitzka , ,... 507
Genetic manipulations in Synechococcus spp. of marine cluster A
B. Brahamsha 517
Toxicological evaluations ofSpirulina maxima in rodents
German Chamorro, Maria Salazar , , , . . . 529 Protists as a trophic link between picocyanobacteria and
the filter-feeding bivalve Crassostrea gigas
Christine Dupuy, Malika Bel Hassen, Solange Le Gall 533 Production ofSpirulina biomass rich in gamma-linolenic acid
and sulfolipids
Hubert Durand-Chastel , , , . . . 541 Third millenium aquaculture. Farming the micro-oceans
Ripley D. Fox , , , , , , 547
Osmolyte accumulation and stress protein synthesis in salt-stressed cyanobacteria from different habitats
Martin Hagemann, Sabine Fulda, Norbert Erdmann ... , . . . . 565
Bulletin de "Institut océunogral'hique, Monaco, n° spécial J9 (1999) VII
MARINE CY ANOBACTERIA
Chemical characterization and subcellular localization of the lipids ofAphanothece halophytica from hypersaline environments 1.F. LOpez,M.Herntindez-Mariné, C.L6pez-Iglesias, E. Clavero,
1.0. Grimait ... . . . . 571 Cyanobacteria management in aquaculture ponds: a review
Laurence Massaut . . . . 579 Isolation and purification techniques for benthic marine
cyanobacteria - biotechnology potential
Katarzyna A. Palinska, Christiane Becker, Wolfgang E. Krumbein 585 Etlects caused by cyanobacteria in salt-works
Genoveva Popowski Casan, Magalys Sanchez Lorenzo . . . . 593 The raIe of Cyanobacteria in environmental management
G.Subramanian, L. Uma 599
VIII Bulletin de l'Institut océanographique, Monaco, n° spécial 19 (1999)
Light-induction
of the Synechocystis PCC6803 secA gene involves promoter remodelling
S. BULTEAU(l), K. MAZOUNI(I), C.CASSIER-CHAUVAT(1)(2),F.CHAUVAT(1)
*
(l)SBGM bat. 142,
(2)URA 2096 CNRS, DBCMIDSV,
CEA Saclay F91191 Gif-sur-YvetteCedex, France
*
Corresponding author (chauvat@jonas.saclay.cea.fr)The CUITent prokaryotic paradigm for protein translocation is based on the sec apparatus in the enterobacterium Escherichia coli where the essential secAgene product (Berghofer et al., 1995) recognizes proteins to be exported via an amino-terminal signal peptide. Then the SecA ATPase activity cata- lyzes precursor protein movement across the inner cell membrane (for review see Curtis & Martin, 1994). Under normal, protein export-proficient condi- tions, SecA protein autogenously represses its own translation through bin- ding to the secretion response element which overlaps the secA ribosome- binding site on geneX-secA-mutT polycistronic rnRNA (Dolan & Oliver, 1995; Gray, 1989; Hidalgo & Demple, 1997). In contrast, secA expression is derepressed by approximately lO-fold when protein export is blocked (Hidalgo& Demple, 1997).
Although secA was found recently in the genome of (i) cyanobacteria i.e.
prokaryotic organisms which possess a plant-like photosynthetic apparatus and are regarded as the progenitor of plastids (Kumar et al., 1993), (ii) algal platids (Link, 1996; Marraccini et al., 1993), and (iii) plants nuclei (McNicho- las et al., 1997; Mermet-Bouvier& Chauvat, 1994; Nakai et al., 1994), very little is known conceming the function and the regulation of secA in photo- synthetic organisms. Therefore, we have undertaken the analysis of the secA gene in the widely-used unicellular cyanobacterium Synechocystis PCC6803
Bulletin de ['Institut océanographique, Monaco,n° spécial 19(1999) 1
PLANKTON MICROORGANISMS WORKTEAM
(Synechocystis) which is amenable ta gene manipulation (Negre et al., 1998;
Poncelet et al., 1998). We report here that the single capy gene secA is essen- tia1 to Synechocystis, as occurs in E.coli (Berghéifer et al., 1995). Hopefully, the recently developed plasmid shuffling procedure (Pugsley, 1993) for in vivo mutational analysis of essential genes in Synechocystis should allow a detailed molecular analysis of the secA function in this host.
We also report that secA is transcribed into a monocistronic mRNA of about 2,8 kb. which is induced up to four-fold by an increase from low to high light fluence. To determine whether the light signais for this response invol- ves photosynthesis, cells were incubated with or without 3-(3,4-dichloro- phenyl)-l,l-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport. DCMU prevented the normallight response by bloc king reaccumu- lation of secA transcripts when dark-adapted cells were returned to the light.
Because NADPH can be obtained either from photosynthetic electron trans- port or from glucose metabolism, and the disappearance of secA transcript under darkness was avoided by using glucose, it was concluded that effect on secAmRNA levels might be due to the availability of reducing power and not to the light per se.
In accordance with the hypothesis that plastids are of endosymbiotic ori- gin, the chloroplast-encoded RNA polymerase is most similar to the cyano- bacterial enzyme in having two subunits that correspond to the E.coli
P'
subunit (Salavati & Oliver, 1995). Furthermore, chloroplast promoters utili- zed by this enzyme in harboring0?üE.coli-like promoters consisting of - 35 and - 10 consensus elements (Salavati& Oliver. 1995) are similar to cyano- bacterial promoters which have yet ta be analysed through mutation (Schmidt et al., 1988). Thus, one of the aim of this study was to analyze in detail the core-promoter elements of the Synechocystis secA gene and to determine whether they were separable from signais that conferred light-responsive expression. Portions of the secA 5' region were transcriptionally fused ta the catreporter gene of the promoter probe plasmid vector pSB2A which replica- tes autonomously in Synechocystis at 10 copies per œil, i.e. at one copy per chromosome equivalent (Negre et al., 1998), and analyzed in vivo. Ail pro- moter fragments assayed were cloned at the same unique restriction site (SnaBI) of the promoter probe vector pSB2A. This approach, and the utiliza- tion of highly stereotyped procedures for culturing œlls prior to CAT assay of promoter activity, yielded precise data with very small fluctuations between experiments. Comparisons of plasmid DNA yields isolated from cyanobacte- rials reporter strains and of the efficiency of back transformation ta E. coli suggested there was no difference in plasmid copy number in Synechocystis.
In addition, primer extension experiments were conducted in parallel with RNA isolated from this reporter strain, and from wild type cells as a control, ta verify that transcription of the secA-cat fusion gene was initiated from the same site as in the native secA gene, as expected.
The 3-fold high-light induction of CAT-activity driven by the whole secA promoter region (- 361 to+187) was of the same magnitude that the four fold accumulation of secA transcript detected by Northern blot, showing that light induction truly occurs at the level of transcription.
2 Bulletin de ['Institut océanographique, Monaco, n° spécial 19 (1999)
LIGHT-INDUCTlON OF THESYNECHOCYSTISPCC6803SEc.AGENE
+1 PE2 -35 ••---_~ 10
~
secALL_---Lc=J_L----'~_~~:
- c = J - - - - ' PEl30 bp spacer
HL
'NT
~
•
. .
~§:
secALL ...c=J---'_
i== ~ :
PEl -35. .-10 +1 PE2
17 bp spacer
HL
mutant
Figure 1. Model for regulation of the secA gene. The DNA motifs are shown as boxes and the transcription start site is indicated as+1. Transcript levels are repre- sented by the number and the thickness of the rightward arrows.
The transcriptional control region of thesecA gene consists of 4 discrete components: the basic promoter (BP, - 71 ta+47) unaffected by light inten- sity and three cis-acting regulatory elements with no intrinsic promoter acti- vitY which ail modulate the strength of BP only in their native position and orientation. The positive element (POl> - 361 to - 71) upstream of BP stimu- lates BP-activity about two fold, irrespectively of light intensity. Similarly, the negative element (NE, +47 to +117) downstream of BP (i.e. in the untranslated leader region) decreases about 6-fold the strength of BP-strength in a light-independent way. In contrast, the stimulatory element (P02, +117, to+187) behind NE (i.e. overlapping with the beginning of the secA coding
Bulletin de ['Institut océanographique, Monaco, n° spécial 19 (1999) 3
PLANKTON MICROORGANISMS WORKTEAM
sequence) can sense light since it does stimulate BP activity by a factor of 1.5 and3.2under low light and high light, respectively. Interestingly, the positive effect of the POz element overcome the negative influence of the NE-element which, in tum, dominates POl-stimulation.
Further deletion analysis of the secA basic promoter indicated that the5' and 3' boundaries mapped to positions -54 and +5. This core promoter contains a TGtTAagAT motif resembling an extendedE. colicr70-type - 10ele- ment of consensus sequence TGnTATAAT, located at the canonical distance (i.e. 7nucleotides) from the transcription start point. InE. coli, all of the con- tacts between promoters lacking a-35box andcr7o-containing RNA polyme- rase holoenzyme occur at this extended-10region (Valentin,1997, 1993).By contrast, theSynechocystis secA promoter also possesses a TTGAat hexamer similar to canonical E. coli - 35 (TTGACA) boxe, which was not located at the correct distance (i.e. 17bp) from the - 10.Site-directed mutagenesis analy- sis of these elements demonstrated that the sequence of both the - 35and the - 10boxes is critical for secA promoter activity whereas the sequence of the spacer region is not important. We have also examined the effect of changing the spacing between the - 35and - 10regions of the promoter, and found that maximum promoter activity was seen with the canonical distance (i.e. 17bp).
Interestingly, the mutant secA promoter with the 17 bp spacer length has gained the ability of being induced by light similarly to the fullsecA promoter region. To our knowledge this is the first report on the mutational analysis of the core promoter of a cyanobacterial gene, and the first evidence that light- responsiveness can be achieved through alteration of the spacing between the - 35and - 10promoter boxes.
Collectively our results suggests (figure 1) that light induction remodels the unusual configuration of the wild-typesecA promoter into a highly active form. probably by compensating for the suboptimal (too long) distance (i.e.
30nucleotides) between the - 10and - 35elements, in a way similar to what occurs in E. coli for the redox control of soxS promoter by activated soxR.
This could be mediated through DNA looping resulting from binding of trans-acting factor(s) onto the cis-acting regulatory elements which flank the secA core promoter.
REFERENCES
BERGHOFER1.,KARNAUCHOVL,HERRMANN R.G., KLOSGEN R.B., 1995. - Isolation and characterization of a cDNA encoding the SecA protein from Spinach chloro- plasts. - J.Biol. Chem.,270 (31), 18341-18346.
CURTIS S.E., MARTIN J.A., 1994. - The transcription apparatus and the regulation of transcription initiation. - In:D.A. Bryant (ed.),The Molecular Biology of Cyano- bacteria.Kluwer Academic Publisher, Dordrecht,613-639.
DOLAN K.M., OLIVER D.B., 1995. - Characterization of Escherichia coli SecA protein binding to a site on ils mRNA involved in autoregulation. - J. Biol.
Chem.,266,23329-23333.
GRAY M.W.,1989. - The evolutionary origin of organelles. - TIG,5,295-299.
4 Bulletin de l'Institut océanographique, Monaco, nOspécial 19 (1999)
LIGHT-INDUCTION OF THESYNECHOCYSTISPCC6803SEc.AGENE
HIDALGO E., DEMPLE B., 1997. - Spacing of promoter e1ements regu1ates the basal expression of the soxS gene and converts SoxR from a transcriptiona1 activator into a repressor. - EMBOJ.,16, 1056-1065.
KUMARA.,MALLOCH RA., FUJITA N., SMILLIE D.A., ISHIHAMA A., HAYWARO RS., 1993. - The minus 35-recognition region of Escherichia coli sigma 70 is inessen- tia1 for initiation of transcription at an "extended minus 10" promoter. - J.Mol.
Biol.,232, 406-418.
LiNK G., 1996. - Green 1ife: control of ch1orop1ast gene transcription. - Bioessays, 18,465-471.
MARRACCINI P., BULTEAU S., CASSIER-CHAUVAT
e.,
MERMET-BoUVlER P., CHAUVAT p., 1993. - A conjugative p1asmid vector for promoter ana1ysis in severa1 cyano- bacteria of the genera Synechococcus and Synechocystis. - Plant Mol. Biol.. 23, 905-909.McNICHOLAS P., SAVALATI R, OLIVER D., 1997. - Dual regu1ation of Escherichia coli secAtranslation by distinct upstream e1ements. - J.Mol. Biol.,265, 128-141.
MERMET-BoUVIER P., CHAUVATp., 1994. - A conditiona1 expression vector for the cyanobacteria Synechocystis sp. PCC6803 and PCC6714 or Synechococcus sp.
PCC7942 and PCC6301. - Curr. Microbiol.,28, 145-148.
NAKAI M., NOHARAT.,SUGITA D., ENOOT., 1994. - Identification and characteriza- tion of the SecA protein homo1og in the cyanobacterium Synechococcus PCC7942.
- Biochem. Biophys. Res. Comm., 200, 844-851.
NEGRE D., OUOOT
e.,
PROSTJ.,MURAKAMIK.,ISHIHAMAA.,COZZONEA.J.,CORTAyle.,
1998. - FruR-mediated transcriptional activation at the ppsA promoter of Esche- richia coli. '---J. Mol. Biol.,276, 355-365.
PONCELET M., CASSIER-CHAUVAT
e.,
LESCHELLE X., BOTTIN H., CHAUV AT P., 1998.- Targeted deletion and mutational analysis of the essential (2Fe-2S) plant-like ferredoxin in Synechocystis PCC6803 by p1asmid shuffling. - Mol. Microbiol., 28, 813-821.
PUGSLEY A.P., 1993. - The complete general secretory pathway in Gram-negative bacteria. - Microbiol. Rev., 50-108.
SALAVATI R, OLIVER D., 1995. - Competition between ribosome and SecA binding promotes Escherichia coli secA translationa1 regulation. - RNA,1, 745-753.
SCHMIDT M.G., ROLLO E.E., GROOBERG J., OLIVER D.B., 1988. - Nucleotide sequence of secA gene and secA(Ts) mutations preventing protein export in Esche- richia coli. - J.Bacteriol.,170, 3404-3414.
VALENTINK., J997. - Phy10geny and expression of the secA gene from a chromo- phytic a1ga-implications for the evolution of platids and sec-dependent protein translocation. - Curr. Genet.,32, 300-307.
VALENTINK., 1993. - SecA is plastid-encoded in a red alga: implications for the evolution of plastid genomes and the thylakoid protein import apparatus. - Mol.
Gen. Genet., 236, 245-250.
YUANJ., HENRY R, MCCAFFERY M., CLINEK., 1994. - ' SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting. - Science,266, 796-798.
Bulletin de l'Institut océanographique, Monaco, n° spécial 19 (1999) 5
The cytochrome b
6f complex of Chlamydomonas
Catherine deVITRY
Physiologie Membranaire et Moléculaire du Chloroplaste, CNRS UPR1261,Institut de Biologie Physico-Chimique.
13, rue Pierre-et-Marie-Curie, 75005 Paris, France (devitry@ibpc.fr)
The unicellular green alga Chlamydomonas reinhardtii is a model system for the study of photosynthesis and of chloroplast biogenesis. Chlamydo- monashas one large single chloroplast with a photosynthetic apparatus simi- lar to that in higher plants. Chlamydomonas can grow (doubling time of 10 h) without photosynthesis on acetate as a carbon source by the respiration in the mitochondria. Gene transformation occurs mainly by homologous recombi- nation in the chloroplast and heterologous recombination in the nucleus.
Chlamydomonas offers an interesting situation for the study of the cyto- chromeb6
f
complex because it is not required for its growth, which is not the case for the majority of prokaryotes in which the cytochrome complex is also part of the respiratory chain. The cytochromeb6f
complex plays a central role in the photosynthetic electron transfer chain between photosystem II and1;it couples translocation of protons across the membrane to oxidation of lipophilic electron carriers (quinols) and reduction of small hydrophilic pro- teins (plastocyanin or cytochromec6)'via the cytochromebpathway and the Fe2S2 and cytochromef
pathway.CHARACTERIZATION OF CYTOCHROME
bd
SUBUNITS:3 SMALL HYDROPHOBIe SUBUNITS
The cytochrome b6
f
complex of Chlamydomonas comprises 7 subunits (Fig. 1). The four large subunits are in al: 1: 1: 1 ratio. Cytochrome f, cyto- chromeb6and subunitIVare encoded by chloroplast genes (Büschlen et al.,Bulletin de l'Institut océanographique. Monaco, n° spécial 19(1999) 7
PLANKTON MICROORGANISMS WORKTEAM
1991). The Rieske iron-sulfur protein is encoded by a nuclear gene (de Vitry, 1994). The cytochromeb6
f
complex contains three transmembrane subunits of 10w molecular weight, the products of the chloroplast genespetG(Berthold et al., 1995) and petL(Takahashiet al., 1996) and of the nuclearPetMgene (de Vitryet al., 1996; Ketchner& Malkin, 1996).lumen stroma thylakoid membrane
~~~~~
PetG 4 kDa C
Figure 1. Topology of the cytochromeb6f subunits. Cyl.f and Rieske protein are reproduced from cristallographie data on homologous proteins (Marti nez et al., 1994; Iwata et al., 1996). The N-teroùnal helix of the Rieske protein is perhaps ? placed at the membrane surface.
Active cytochrome b6
f
complex of Chlamydomonas can be purified and shows comigration of these small subunits (Pierreet al., 1995). N-terminal sequencing of the 4 kDa band revealed this comigration and allowed to cha- racterize a new subunit PetM. Anti-PetM recognized a band present in the isolated complex and absent in a bddeficient mutant. This antibody was used to show that PetM is an intrinsic protein. It is not extracted by chaotropic treatments and is always found in the pellet as other intrinsic subunits such as subunit IV. The C-tenninus of PetM is probably located in the stroma as that of PetG, as detected by immunostudies and PetM sequence, was determined (de Vitry et al., 1996). The 3 small hydrophobic subunits have no known cofactors. Deletion of PetG drastical1y destabilized the complex in Chlamy- domonas(Bertholdet al., 1995), allowing only a few percent of accumulation of the otherb6f
subunits, while the deletion of PetL also inChlamydomonas allowed up to 50% of accumulation of the otherbd
subunits (Takahashi et al., 1996).The genome of the cyanobacteriaSynechocystisstrain PCC6803, which has been totally sequenced, contains the PetG (most conserved subunit compared to Chlamydomonas) and PetM subunits but lacks PetL (Table 1), confirming the secondary role of the latter.
8 Bulletin de/'InslilUiocéanographique, Monaco. n° spécial 19(1999)
THE CYTOCHROMEb6f COMPLEX OFCHLAMYDOMONAS
Table1.Cornparison of cytochrorneb6fsrnall subunits ofChlamydomonasandSynechocystis.
PetGChlamydomonas reinhardtii
MVEPLLCGIVLGLVPVTIAGLFVTAYLQYRRGDLATY PetGSynechocystisPCC6803
VIEPLLLGIVLGLIPVTLAGLFVAAYLQYKRGNQFNLD PetLChlamydomonas reinhardtii
MLTITSYVGLLIGALVFTLGIYLGLLKVVKLI PetMChlamydomonas reinhardtii
GEAEFIAGTALTMVGMTLVGLAIGFVLLRVESLVEEGKI PetMSynechocystisPCC6803
MTAESMLANGAFIMIGLTLLGLAWGFVIIKLQGSEE HEME-BINDING TO CYTOCHROMEb6
IS ASSISTED BY NUCLEAR FACTORS
The cytochrorneb6
f
cornplex comprises two nuclear-encoded subunits the Rieske protein and PetM; in addition, several nuclear factors affect expres- sion of a single chloroplast gene and rnainly in a post-transcriptional way (Table II). 13 nuclear factors have been characterized in the expression of chloroplast genes of the cytochrorneb6f
cornplex. Four of thern are irnpli- cated in the c-herne-binding to cytochrornef
and cytochrorne c6, a cyto- chrome which is expressed in absence of copper. Cytochrornef
and cytochrorne c6 each bind covalently one herne of type c by thioether linkages between the sulfhydryl groups of two cysteine residues of the protein and the vinyl groups of the tetrapyrrole ring of the herne. Il was expected that as in bacteria or rnitochondria several factors would catalyze this covalent herne binding. In contrast, it was unexpected that cytochrorneb6(PetE) which binds two b hernes considered as non-covalent bound would irnplicate several nuclear factors in its herne-binding (Kuraset al., 1997).Table II. Nuclear loci participating in the expression of the chloroplastpetgene (Girard-Bascouet al., 1995; Gumpelet al., 1995; Kuraset al., 1997;
Inoueetal., 1997; XieetaI., 1998).
Nuclear loci Function
CCSl,CCS2, CCS3, CCS4 c-heme attachment CCBl, CCB2, CCB3, CCB4 b-herne attachrnent
MCAl stability/rnaturation ofpetArnRNA MCBl stability/rnaturation ofpetBrnRNA
MCDl stability/rnaturation ofpetDrnRNA
MCCl stability/rnaturation ofpetCrnRNA
TCAl translation ofpetArnRNA
Bulletin de l'Institut océanographique, Monaco, n° spécial 19(1999) 9
PLANKTON MICROORGANISMS WORKTEAM
Cytochromeb6is more resistant than cytochromebto denaturating condi- tions. Therefore we tried more drastic conditions of denaturation. We were surprised to find that part of the heme-binding to cytochromeb6was resistant to an acetone acid treatment which is classically used to extract b hemes (Ascoli et al., 1981), suggesting that one of two b-hemes of holocytochrome b6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holo-cytochrome b6 , and to identify mutants that are blocked at one step of this pathway.
Chlamydomonas reinhardtii strains carrying substitutions in either one of the four histidines which coordinate the bh or blhemes to the apoprotein were created. These mutations resulted in the appearance of distinct immunoreac- tive species of cytochrome b6 ,which allowed us to specifically identify cyto- chrome b6with altered bh or bl Iigation. In gabaculine-treated wild type and site-directed mutant strains(i.e. heme-depleted, gabaculine is an inhibitor of the tetrapyrrole biosynthetic pathway), we established that (i) the single immunoreactive band, observed in strains carrying the bi-site directed muta- tions, corresponds to apocytochromeb6and (ii) the additional band present in strains carrying bh-site directed mutations corresponds to a b,-heme-depend- ent interrnediate in the formation of holocytochromeb6.
Five nuclear mutants(ccb strains) which are defective in holocytochrome b6 formation display a phenotype which is indistinguishable from that of strains carrying site-directed bh-ligand mutants. The defect is specific for cytochrome b6assembly because theccb strains can synthesize other b-cyto- chromes and ail c-type cytochromes. The ccb strains, which define four nuclear loci (CCBI, CCB2, CCB3,andCCB4), provide the first evidence that ab-type cytochrome requires trans-acting factors for its herne-association.
A schema tic view of the conversion of the apo- to holocytochrome b6is presented in Fig. 2 with the following steps: A) membrane integration of
A
~
lower~heme
~B
gaba- culine
upper lower
ŒJheme
l.-C
CCB1 CCB2 CCB3 CCB4
diffuse
---
asso-ciationD with other be ( subunitsstable
Figure 2. Schematic pathway of the conversion of apo- to hoJo- cytochrome b6and cytochromeb6patterns after urea/SDS-PAGE. A) membrane insertion occurs even in the absence of heme binding. B) formation of a bl-heme-dependent intermediate is prevented by gabaculine.C)formation of bh- and bl-binding holocytochrome b6 requires CCB factors. D) hoJocytochrome b6 accumulates in a concerted process with other bdsubunits (Kuras etal.. 1997).
10 Bulletin de ['Institut océanographique, Monaco, n° spécial 19(1999)
THE CYTOCHROMEb6fcOMPLEXOFCHLAMYDOMONAS
apocytochrome b6occurs even in absence of heme association, B) formation of a b]-dependent intermediate can be prevented by gabaculine treatment, C) bhheme binding to blintermediate requires Ccb-encoded trans-acting factors, D) holocytochrome b6accumulates in a protease resistant form, upon association with the other b6
f
subunits (Kuras et al., 1997).NUCLEAR MUTANTS OF THE FE2S2RIESKE PROTEIN After random mutagenesis of Chlamydomonas, mutants deficient in the Rieske protein were screened. The deficience of Rieske iron-sulfur protein prevented electron transfer to cytochromef, but allowed assembly and accu- mulation of 50 to 75% of the other subunits in a cytochrome b6
f
subcomplex.Rieske deficient mutants affected directly in the PetC gene were identified, as indicated by the WT phenotype after transformation with the PetC gene and the genetic analysis by recombination and complementation tests. Such strains will be used as recipient strains for site-directed mutagenesis of the Rieske protein. Their complementation will allow us to overcome the very low frequency of homologous transformation in the nucleus of Chlamydomonas.
In conclusion, the example of the cytochrome b6
f
complex illustrates the contribution ofC. reinhardtiito the understanding of the genetic expression and functioning of the chloroplast.REFERENCES
ASCOLI P., FANELLI M.R., ANTONINIE., 1981. - Preparation and properties of apo hemoglobin and reconstituted hemoglobin. - Methods in Enzymol., 76,72-87.
BERTHOLD D.A., SCHMIDT c.L., MALKIN R., 1995. - The deletion of petG in Chlamydomonas reinhardtii disrupts the cytochrome bf complex. - J. Biol.
Chem.,270,29293-29298.
BÜSCHLEN S., CHOQUET Y., KURAS R., WOLLMAN P.A., 1991. - Nucleotide sequences of the continuous and separated petA, petB and petD chloroplast genes in Chlamydomonas reinhardtii. - FEBSLeu..284, 257-262.
DE VITRY c., 1994. - Characterization of the gene of the chloroplast Rieske iron- sulfur protein in Chlamydomonas reinhardtii. - J. Biol. Chem., 269,7603-7609.
DE VITRY c., BREYTON
c.,
PIERRE Y., POPOT J.L., 1996. - The 4-kDa nuclear- encoded PetM polypeptide of the chloroplast cytochromeb6f complex. - J.Biol.Chem.,271, 10667-10671.
GIRARD-BASCOUJ.,CHOQUETY.,GUMPEL N., CULLER D., PURTON S., MERCHANT S., LAQUERRtÈRE P., WOLLMAN P.A., 1995. - Nuclear control of the expression of the chloroplast pet genes in Chlamydomonas reinhardtii. - In: Mathis P. (ed.), Photosynthesis: from light tobio~phere.Kluwer,3,683-696.
GUMPEL N.J., GIRARD-BASCOUJ.,WOLLMAN P.A., NUGENTJ.H.,PURTON S., 1995.
- Nuclear mutants of Chlamydomonas reinhardtii defective in the biogenesis of the cytochromeb6fcomplex. - Plant Mol. Biol., 29,921-932.
INOUE K., DREYFUSS B.W., KINDLE K.L., STERN D.B., MERCHANT S., SODEINDE a.A.,1997. - Ces], a nuclear gene required for the post-translational assembly of chloroplast e-type cytochromes. - J. Biol. Chem., 272, 31747-31754.
Bulletin de ['Institut océanographique, Monaco. n° spécial 19 (1999) 11
PLANKTON MICROORGANISMS WORKTEAM
IWATA S., SAYNOVITS M., LINK T.A., MICHEL H., 1996. - Structure of a water solu- ble fragment of the bovine heart mitochondrial cytochrome bel complex deter- mined by MAD phasing at 1.5A resolution. - Structure,4, 567-579.
KETCHNER S.L., MALKINR., 1996. - Nucleotide sequence of the PetMgene encod- ing a 4 kDa subunit of cytochromeb6fcomplexfromClzlamydomonas reinhardtii.
- Biochim. Biophys. Acta,1273, 195-197.
KURASR.,DE VITRY
c.,
CHOQUETY.,GIRARD-BASCOU J., CULLER D., BÜSCHLEN S., MERCHANT S., WOLLMAN F.A., 1997. - Molecular genetic identification of a pathway for heme binding to cytochromeb6 . - J.Biol. Chem.,272, 32427-32435.MARTINEZ S.E., HUANG D., SZCZEPANIAK A., CRAMER W.A., SMITH lL., 1994. - Crystal structure of the chloroplast cytochromef reveals a novel cytochrome fold and unexpected heme Iigation. - Structure,2,95-105.
PIERREY.,BREYTON
c.,
KRAMER D., POPOT JL, 1995. - Purification and character- ization of thebJ
complex from Chlamydomonas reinhardtii. - J. Biol. Chem., 270, 29342-29349.TAKAHASHIY.,RAHIRE M., BREYTON
c.,
POPOT JL, JOUOT P., ROCHAIX lD., 1996.- The chloroplast ycf7(petL)openreading frame ofChlamydomonas reinhardtii encodes a small functionally important subunit of the cytochrome
bd -
EMBO J., 15, 3498-3506.XIE Z.,CULLER D., DREYFUSS B.W., KURASR.,WOLLMAN F.A., GIRARD-BASCOU J., MERCHANT S., 1998. - Genetic analysis of chloroplast c-type cytochrome as sem- bly inClzlamydomonas reinhardtii: one chloroplast locus and at least four nuclear loci are required for heme attachment. - Genetics,148, 681-692.
12 Bulletin de J'Institut océanographique, Monaco, n° spécial 19 (1999)
Use of mo1ecu1ar too1s for the study of genetic re1ationships of heterocystous cyanobacteria
Isabelle ITEMAN *, Rosmarie RIPPKA, Nicole TANDEAU de MARSAC, Michael HERDMAN
Physiologie Microbienne (CNRS URA 1129),
Institut Pasteur,28, rue du Dr Roux, 75724Paris Cedex15, France
*
Corresponding author (iiteman@pasteur.fr)INTRODUCTION
Cyanobacteria are oxyphototrophic prokaryotes, originally considered as a class of algae, the blue-green algae. They can be classitied on the basis of mor- phology, cellular differentiation, biochemical, physiological and genetic crite- ria. However, assignment to one of the numerous genera (I50) or species (>1000) created under the Botanical Code of Nomenclature (which even today has priority over the Bacteriological Code of Nomenclature for the naming of validated taxa) is, for lack of living type strains, generally highly subjective.
Morphology, developmental and biochemical parameters may vary with envi- ronmental or culture conditions. Furthermore, morphological resemblance may not necessarily reflect genetic relatedness. It is thus necessary to comple- ment the existing botanical taxonomy by a polyphasic approach that incor- porates genetic evidence for the validity of the traditional genera or species.
Coding for molecules essential to the life of a cell, the ribosomal operon (rmoperon) is universal (except in viruses) and horizontal transfer of this tran- scriptional unit has never been shown.Itis thus a stable genetic marker, having among others served to infer the phylogenetic position of cyanobacteria among the eubacteria (Woese & Fox, 1977). Analyses of the 16S rRNA (encoded by thermSgene) also revealed that, in contrast to both unicellular and filamentous non-heterocystous cyanobacteria, tilamentous heterocystous cyanobacteria represent a monophyletic cluster (Giovannoni et al., 1988;
Ligon 1991; Robinson et al., 1996; Wilmotte et al., 1992, 1993; Coursin, unpublished). We investigated for the tirst time two areas of the ribosomal operon, the rmS gene and the Intergenic Transcribed Spacer (ITS) located
Bulletin de l'lnstitut océanographique, Monaco, n° spécial 19 (1999) 13
PLANKTON MICROORGANISMS WORKTEAM
TableI.Cyanobacterial strains used in this study.
Strain Taxonomie assignment Heteroeysts Propertiesg Source of Isolate PCC 7905" Aphanizomenon fIos-aquae single F/GV+ Brielse Meer, The Netherlands(1)
PCC 9302b Anabaena f1os-aquae lackingf F/GV+/NH Lake, near Saskatoon,Canada (2) PCC 9332" Anabaena f1os-aquae single F/GV+ Lake Windermere, England (3) PCC 9349b Anabaena f1os-aquae lackingf F/GV+/N BeaverhiU Lake, Canada(4)
PCC 9215 a Anabaenopsis sp. paired F/GV+ Coastallagoon, Valencia, Spain(1)
PCC 9216 a Anabaenopsis sp. paired F/GV+ Lake Santa OUala, Spain(1)
PCC 9420c Anabaenopsis elenkinii paired F/GV+ Lake Nakuru, Kenya(5)
PCC 9608 a Anabaenopsis sp. paired F/GV+ Okesund dam, Sweden(8)
PCC 9501c Cyanospira ripkkae single F/GV+ Soda lake Magadi, Kenya(6)
PCC 9502c Cyanospira capsulata single F/GV+ Soda lake Magadi, Kenya(fi)
PCC 9350d Nodularia spumigena single FlGV+/H Baltic Sea, Sweden(7)
PCC 7120" Nostoc/Anabaena Single F/GV- Unknown(l)
PCC 6803< Synechocystis Non hetero- UlGV- Fresh water, Califomia, USA(1)
cystous
aMedium BGlio+ NaHC03(10 mM) - bMedium(a)+ NaN03 (2 mM)~cMedium BGllo+ NaHC03 (65 mM)+ Na2C03 (15 mM) - dMedium(a)+ artificial seawater (20% v/v, Merck Index 9954) - <Medium BG II
- fAbility to form heterocysts lost - gF: cyanobacteria with filamentous morphology and U: cyanobacteria with unicellular morphology; GV+: gas vesicles permanent and GV-: strains without gas vesicles; N: neurotoxin and H: hepatotoxin produced.
(1)Rippka&Herdman, 1992; (2) Mahmood&Carmichael, 1986; (3) Booker&Walsby, 1979; (4)Carmichael&
Gorham, 1980;(5)SchlOsser, 1994;(6)Florenzanoet al., 1985;(7)Martinet al..1990;(8)Rippka, Unpublished.
between the 16S and 23S rRNA genes, in Il planktonic heterocystous isolates (Table 1), sorne of which are known to form toxic water-b100ms (Mahmood&
Carmichael, 1986; Carmichael & Gorham, 1980; Martin et al., 1990). We detennined the 16S rRNA sequences of 5 strains, 3 of which represent genera for which data were previous1y unavai1ab1e. We also demonstrated that RFLP (Restriction Fragment Length Po1ymorphism) of the rrnS amplicons has a resolution power equivalent to that of sequence analysis of this molecule and that the strains examined contain multiple operons with ITS of variable size.
MATERIALS AND METHODS Cyanobacterial strains
AlI strains studied were axenic; their properties and growth media are indi- cated in TableI. The non-planktonic filamentous Nostoc PCC 7120 and the unicelIular Synechocystis PCC 6803 were used as reference strains.
DNA extraction and amplification of the 16S rRNA gene and ITS The DNA used for PCR amplification was isolated by the mini extraction method described previously (Cai& Wo1k, 1990). Amplification of the rrnS gene was carried out by polymerase chain reaction (PCR) using primers A2 and S17 (Table II). The size of the amplicon was 1451 bp versus 1489 bp for 14 Bulletin de l'Institut océanographique, Monaco,n° spécial 19(1999)
GENETIC RELAT10NSHIPS OF HETEROCYSTOUS CY ANOBACTERIA
Table II. Primers used for PCR (16S rRNA genes and ITS amplification) and sequence determination.
Positions in the Location in the Primers Sequences (5'~3')a,b rRNAoperon rRNA genes
ofSynechocystis ofSynechocystis
PCC 6803 PCC 6803
A2 AGAGTTTGATCCTGGCTCAG 8-27
S3 TACCCCACCAACTAGCTAATC 231-211
317 AGGCAGCAGTGGGGAAT 318-334
S4 CTGCTGCCTCCCGTA 326-311
318 GCCAGCAGCCGCGGTAA 463-479
S6 GTATTACCGCGGCTGCTGG 482-464
S8 TCTACGCATTTCACCGCTAC 650-631
319 RGGATTAGATACCCC 730-744
S9 CTACTGGGGTATCTAATC 749-732
320 GAATTGACGGGGRCCC 863-879 rrnSgene
SlO CCGTCAATTCCTTTGAGTT 873-854 (16S)
SIl TCTCACGACACGAGCTGACG 1029-1010 1489 bp
321 GYAACGAGCGCAACCC 1046-1061
S12 AGGGTTGCGCTCGTTG 1062-1047
S14 CATTGTAGTACGTGTGT 1187-1171
322 TGTACACACCGCCCGTC 1332-1348
S15 CGGTGTGTACAAGGCCC 1354-1338
S17 GGCTACCTTGTTACGAC 1457-1441
t
323 ATTAGCTCAGGTGGTTAG 1637-1655 tRNAiie gene in
373 CTAACCACCTGAGCTAAT 1654-1637 the ITS region
465 bp
+ t
340 CTCTGTGTGCCTAGGTATCC 1999-1979 rrnLgene
(23S) 2883 bp
aR (A or G) and Y (C or T) indicate the simultaneous presence of two bases at the same position. - bPrimers in bold correspond to the RNA-like strand.
the whole gene. The part of the rRNA operon containing the ITS region was amplified by using the primers 322 and 340. The PCR mixture contained 10 !lI of Taq commercial buffer, 150 J..lM of each dNTP, 500 ng of each primer and 2.5 U Taq polymerase (Appligène). The total reaction volume was 100 J..lI.
After an initial cycle consisting of 3 min at 95°C, 2 min at 55°C and 30 sec at nT,30 cycles of amplification were started (1 min 30 sec at 95°C, 2 min 30 sec
Bulletin de /"lnstitut océanographique. Monaco, n°spécial 19 (1999) 15
PLANKTON MICROORGANISMS WORKTEAM
at 55°C and 3 min at
neC).
The amplification was finished by 7 min atn°c.
The PCR products were migrated in 2.5% w/v agarose gel (Litex FMC) in Tris Borate buffer at 100V.The gels were stained with ethidium bromide, visua- lized under UV light and photographed.
Sequencing of the 16S rRNA gene and ITS region
Three or more independent PCR products were mixed to avoid sequence errors introduced by the Taq polymerase. After purification by Wizard PCR kit (Promega), the PCR products were sequenced directly or cloned in the pGEM-T vector (Promega). CompetentE. coliJM109 were transformed and recombinant plasmid were purified from white colonies by the alkaline method (Birnboim & Doly, 1979). The two M13 primer sites on the vector and 21 primers as listed in Table II were used with the T7 sequencing kit (Pharmacia) to sequence both strands of therrnSgene and the ITS region.
Restriction Fragment Length Polymorphism of PCR product corresponding to the rrnS gene
10 III of each PCR product were digested with 10 restriction enzymes (BanII, BspMI, DdeI, HaeII, NheI, NruI, PstI, Sad, SphIandXcmI) accord- ing to the manufacturer's instructions. The DNA samples were migrated under the conditions described above.
RESULTS AND DISCUSSION
Phylogenetic relationships between the heterocystous cyanobacteria Five new rrnS sequences were determined (PCC 7905, PCC 9215, PCC 9302, PCC 9350 and PCC 9501) and data were aligned in the Arb editor with complete or partial sequences of 16s rRNA genes previously determined in this laboratory or obtained from data banks. To infer relationships between the strains, a tree was constructed by the maximum likelihood method (fast- DNAml V1.0; Olsen et al., 1994) and was midpoint rooted(MicrocystisPCC 7806 and Leptolyngbya PCC 7376 as outgroup) by the Retree programme (PHYLIP V3.5; Felsenstein, 1993). The planktonic strains studied form a cluster among the monophyletic branch of heterocystous cyanobacteria (Fig. lA) and are divided into 3 subgroups. One is composed ofAnabaena andAphanizomenon,a second containedAnabaenopsisandCyanospira and the third corresponding to the genus Nodularia. Anabaena PCC 7122 is closely related to this cluster but is not a planktonic organism. These phylo- genetic relationships determined with the 16S rRNA sequences are in agree- ment with the morphological characteristics of the strains.
The second study was based on fingerprinting or Restriction Fragment Length Polymorphism (RFLP) of the PCR product corresponding to the rrnSgene.
AlI Il strains were examined and Nostoc PCC 7120 was used as control of restriction profiles. Depending on the restriction enzyme employed, the patterns were composed of 1 to 8 bands between 1451 bp to less than 50 bp 16 Bulletin de l'Institut océanographique. Monaco,n° spécial 19(1999)
a
b
_1000 bp 900 bp 800 bp 700 bp 600 bp 500 bp 400 bp 300 bp 200 bp
L 1 2 3 4 5 6 7 8 9 10111213 L Anabaena PCCne2
Nadu/aria PCC73lQd Nodufaria PCC 9350 Anabaena PCC 7122
Anabaenopsis PCC92T5 Gyanosplfa PCC 9501 Tolypolhnx PCC 7415 TolYl'orhrixPCC 7/01
Tolyporhnx PCC 760/
Cylindrospermum PCC74, 7 Nostoc PCC 7120
1 , - - - - -Nostoc PCC 73/02 l - Calolhrix PCC 7507
, - - - SC'jtonema PCC71/0 , - - - Catolhnx PCC 6303
Catolhnx PCC 7709
l - Aivularia PCC 1116 , - - - Fischerella PCC 7414
NOS(OCPCC 7120
At1abaenopsisPCC 9215 Anab3enopsisPCC 9216 AnabaenopsisPCC 9<:120 AnabaenopsisPCC 9608
Cyanosplfd rippkaePCC 9501 Cyanosplracapsulata PCC 3502 Nadu/aria spumigenaPCC 9350
Anabaena 'tas ilQlJaePCC 9302 Anabaena 110$ aquae PCC 9332 Anabaena fias aqusePCC 9349
AphanlzomenOfl fias aqu3ePCC 7905
Chlofogloeopsis PCC 67T8
Mastigocladus HTF PCC 7518 0.01
, - - - MfCfOCYStiSPCC7806 ' - - - Leptolyngbya PCC 7376
0.01
Figure 1. a. Phylogenetic tree based on the cyanobacterial 16S rRNA sequences.
b. Dendrogram based on the RFLP profiles of therrnSgene PCR products.
PLANKTON MICROORGANISMS WORKTEAM
(data not shown). To evaluate the discriminatory power of this method, we constructed a matrix recording the presence or absence of every restriction band. The data set was analysed by both distance (RAPDistance V 1.04) and parsimony (PAUPV 3.0); (Swofford, 1992) methods. The phylogenetic tree derived from the analysis (Fig. lB) is largely consistent with that obtained with the 16S rRNA sequences. Anabaenopsis and Cyanospira are resolved as two distinct genetic entities but are closely related. Anabaena fios-aquae and Aphanizomenon fios-aquae are separated on a different branch. Nodularia spumigena PCC 9350 and Nostoc PCC 7120 also represent distinct genetic units, but their position in the tree differs from that obtained by sequence analysis. RFLP with the 10 restriction enzymes used thus confirms the taxo- nomie assignments of the Il planktonic strains to 5 different genera. Lyra et al. (1997) have also demonstrated the value of this technique in a study based on 7 enzymes which separated a number of hepatotoxic heterocystous strains.
However, they were unable to separate Aphanizomenon sp. from the bloom- forming neurotoxic Anabaena strains examined. The judicious choice and number of restriction enzymes is therefore essential to achieve the desired level of taxonomie resolution.
Study of the number and size of the Intergenic Transcribed Spacer (ITS) The ribosomal operon organization is variable between organisms but is generally composed of 5 successive domains: rmS gene (16S rRNA) , ITS region, rmL gene (23S rRNA) , ITS region, 5S rRNA gene. The ITS region may be of variable size, as shown for Corynebacterium (Aubel et al., 1997), Streptomyces (Hain et al., 1997) and certain heterocystous cyanobacteria (Lu et al., 1997). The set of primers 322-340 (located respectively around 150 bp before the 3' end of the rmS gene and at the beginning of the 23S rRNA gene) used to amplify the ITS gave 3 or 4 bands of different intensities ranging in size from approximately 460 to 890 bp for ail 12 heterocystous strains studied (Fig. 1b). These observations indicate a size variation among the ITS of a single cyanobacterial strain whose ribosomal operons therefore have a differ- ent organization. In contrast, only one product was observed with the unicel- lular strain Synechocystis PCC 6803. The heterogeneity of the bands (size and number) confirms the groupings obtained by RFLP analysis, allows to further subdivide the genus Anabaenopsis into 2 groups and clearly separates ail 3 isolates of Anabaenafios-aquae.
CONCLUSION
The planktonic strains examined, together with the non-planktonic Ana- baena cylindrica PCC 7122, form a distinct cluster within the clade of fila- mentous heterocystous cyanobacteria. Interestingly, this cluster harbours only strains that do not exhibit a developmental cycle involving hormogonium for- mation. In addition, the subcluster composed of Anabaena fios-aquae, Apha- nizomenon fios-aquae and Nodularia spumigena regroups representatives of species often known to be toxic (producing either neurotoxins, hepatotoxins, or both). Our results will therefore be important not only for the taxonomy of 18 Bulletin de ['Institut océanographique, Monaco, nO spécial 19 (1999)
GENETIC RELATlONSHIPS OF HETEROCYSTOUS CYANOBACTERIA
planktonic heterocystous cyanobacteria but also for the development of mole- cular tools for the rapid identification of potentially toxic members of this group.
ACKNOWLEDGEMENTS
This work was supported in part by the contract Blü4-CT96-0256 (BASIC) of the European programme BIOTECH (Life Sciences and Tech- nologies, Biotechnology Programme, 1994-1998) and by the Institut Pasteur and CNRS (URA 1129). We thank T. Coursin for supplying unpublished
16S rRNA sequences.
REFERENCES
AUBEL D., RENAUD EN., FRENEY l, 1997. - Genomic diversity of severalCoryne- bacteriumspecies identified by amplification of the 16S-23S gene spacer regions.
- [nt. J.Syst. Bacteriol.,47, 767-772.
BlRNBOIM H.C., DOLY J., 1979. - A rapid alkaline extraction procedure for screening recombinant plasmid DNA. - Nucl. Acids Res.,7, 1513-1523.
BOOKER M.l, W ALSBY A.E., 1979. - The relative form resistance of straight and helical blue-green algal filaments. - Br. Phycol.J.,14, 141-150.
CAlY.,WOLK c.P., 1990. - Use of a conditionally lethal gene inAnabaenasp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. - J.Bacteriol.,172,3138-3145.
CARMICHAEL W.W, GORHAM PR., 1980. - Fresh water cyanophyte toxins: types and their effects on the use of microalgal biomass. - [n: Shelef G. & Soeder C.J., Algal biomass: production and use. Elsevier, North Holland Press, Amsterdam, 437-448.
FELSENSTEIN l, 1993. - PHYLfP (Phylogeny [nference Package) version 3.5c. - University of Washington, Seattle.
FLORENZANO G., SIL! c., PELOSIE.,VINCENZINI M., 1985. - Cyanospira rippkaeand Cyanospira cap.l'ulata (gen.nov. and spp. nov.): new filamentous heterocystous cyanobacteria from Magadi lake (Kenya). - Arch. Microbiol., 140,301-306.
GIOVANNONI S.I., TURNER S.. OLSEN G.I., BARNS S., LANE D.I., PACE N.R., 1988.- Evolutionary relationships among cyanobacteria and green chloroplasts. - J.Bacteriol.,170,3584-3592.
HAINT.,W ARD-RAINEY N., KROPPENSTEDT R.M., STACKEBRANDT E., RAINEY EA., 1997. - Discrimination ofStreptomyces albidoflavusstrains based on the size and number of 16S-23S ribosomal DNA intergenic spacers. - [nt. J.Syst. Bacteriol., 47. 202-206.
LIOON P.J.B., MEYER K.G., MARTIN J.A., CURTIS S.E., 1991. - Nucleotide sequence of a 16S rRNA gene fromAnabaenasp. strain PCC 7120. - Nucl. Acids Res., 19, 4553.
Lu W., EVANS H.E., McCoLL M., SAUNDERS V.A., 1997. - Identification of cyano- bacteria by polymorsphisms of PCR-amplified ribosomal DNA spacer region. - FEMS Microbiol. LeU..153, 141-149.
LYRA c., HANTULA l, VAINIO E., RAPAL J., ROUHIAINENL., SIVONEN K., 1997.- Characterization of cyanobacteria by SDS-PAGE of whole-cell proteins andPCRI RFLP of 16S rRNA gene. - Arch. Microbiol., 168, 176-184.
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