Unité des Cyanobactéries (CNRS URA 2172), Département de Biochimie et Génétique Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France1
Author for correspondence: Isabelle Iteman. Tel: +33 1 4568 8416. Fax: +33 1 4061 3042. e-mail: iiteman{at}pasteur.fr
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ABSTRACT |
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Keywords: 16S rRNA, internal transcribed spacer, phylogeny, sequence heterogeneity, restriction fragment length polymorphism
Abbreviations: IGS, intergenic spacer region; ITS, internal transcribed spacer of the rRNA operon; PCC, Pasteur Culture Collection of Cyanobacteria; PPFD, photosynthetic photon flux density; RB, relative binding
The GenBank accession numbers of the 16S rDNA gene sequences reported in this paper are AY038032AY038037.
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INTRODUCTION |
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Classically assigned to the Nostocales, many planktonic heterocystous cyanobacteria are characterized by the synthesis of gas vesicles, bouyancy-providing structures that permit the filaments to move vertically in the water column (Walsby, 1981 ). Many also produce potent neuro- or hepatotoxins, or both (reviewed by Sivonen & Jones, 1999
) and, as a consequence of their bouyancy, often accumulate in dense water-blooms that are dangerous for the health of fish, birds and mammals, including man. Detailed studies of phylogenetic relationships amongst these organisms have been mostly based on 16S rRNA gene sequences (Lyra et al., 1997
; Barker et al., 1999
; Beltran & Neilan, 2000
; Lehtimäki et al., 2000
; Li et al., 2000
; Lyra et al., 2001
; Moffitt et al., 2001
; Saker & Neilan, 2001
). In some cases other sequences have been examined, for example those of the DNA-dependent RNA polymerase gene (rpoC1; Fergusson & Saint, 2000
; Wilson et al., 2000
), the non-coding intergenic spacer of the phycocyanin operon (PC-IGS: Neilan et al., 1995
; Barker et al., 1999
, 2000
; Bolch et al., 1999
) and the IGS between two adjacent copies of the gvpA gene encoding the major structural gas vesicle protein (Barker et al., 1999
).
The above studies emphasized individual genera or monophyletic groups of heterocystous cyanobacteria such as Nodularia spp. (Lehtimäki et al., 2000 ; Moffitt et al., 2001
), Anabaena circinalis (Beltran & Neilan, 2000
), Anabaena/Aphanizomenon strains (Lyra et al., 2001
) or Cylindrospermopsis (Saker & Neilan, 2001
), and none defined the relationships between these groups. A comparison of these studies suggests the existence of five apparently monophyletic clusters of planktonic heterocystous cyanobacteria. One of these contains organisms assigned to two different genera, Anabaena and Aphanizomenon. A second cluster regroups only species of Anabaena. The third lineage is mainly composed of strains identified as Anabaena circinalis, but also contains a strain named as Anabaena affinis (Beltran & Neilan, 2000
). However, for members of these three clusters, the strain descriptions were often insufficient to exclude the possibility that representatives of a single species may carry different specific epithets, or that strains of the same morphotype may have different names, as a consequence of the subtle definitions that discriminate between these botanical species. The two remaining clusters are respectively represented by planktonic and non-planktonic members of the genus Nodularia and isolates of the tropical planktonic species Cylindrospermopsis raciborskii. Phylogenetic studies of these latter organisms were accompanied by more detailed descriptions (Barker et al., 1999
; Bolch et al., 1999
; Lehtimäki et al., 2000
; Saker & Neilan, 2001
). The characteristic cell morphology or heterocyst differentiation patterns of members of these two clusters are more easily recognizable than those of strains of the first three clusters and thus facilitate identification, at least at the generic level. Within each of the five clusters, the 16S rDNA sequence similarity is so high as to suggest either that their members may be assignable to a single species, or that the resolving power of the analysis is insufficient to distinguish different species, or even different genera.
In this work, to futher study the taxonomic and phylogenetic relationships of the planktonic heterocystous strains, 11 well-characterized isolates were examined by investigating the 16S rRNA gene and the internal transcribed spacer (ITS) located between the 16S rRNA and 23S rRNA genes. The sequences of the 16S rRNA genes of five isolates were determined. Three strains (Aphanizomenon flos-aquae PCC 7905, Anabaena flos-aquae PCC 9302 and Nodularia sp. PCC 9350) represent three of the five clusters described above. The alkaliphilic planktonic members of the genera Anabaenopsis (PCC 9215) and Cyanospira (PCC 9501), which share a coiled trichome morphology and whose phylogenetic position was previously unknown, were examined for the first time. We also studied the relationships of these five strains to those of six additional isolates by RFLP analyses of their 16S rDNA amplicons. The number and size differences of the ITS region in all 11 strains were examined by PCR amplification. A preliminary account of some aspects of this study was presented by Iteman et al. (1999) .
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METHODS |
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Salinity and temperature maxima for growth.
The growth responses to different salinities or temperatures of the 11 planktonic strains were examined in test tubes (18 mm diameter) containing 6 ml medium. For the salinity tests, medium a was used for all strains (except for strains PCC 9302 and PCC 9349, for which medium b was employed), with or without a supplement of Turks Island Salts to give the equivalent of 0·25, 0·5, 1 and 2 times the salinity of seawater. This allowed verification of whether the high NaHCO3/Na2CO3 concentration in the maintenance medium c for Anabaenopsis PCC 9420 and the two strains of Cyanospira was necessary in the presence of elevated salt concentrations. Incubation conditions were as described for the stock cultures. Testing for temperature tolerance was performed in medium a (PCC 9215, PCC 9216 and PCC 9608), medium b (PCC 9302 and PCC 9349), medium e (PCC 7905 and PCC 9332) and medium a supplemented with 0·2x Turks Island Salts, found to be optimal for the remaining strains. For lack of incubators providing both different temperatures and a light/dark regime, the cultures were incubated at 23, 30 and 37 °C, receiving an irradiance of 7 µmol quanta m-2 s-1 of continuous white light.
Photomicroscopy.
Filament suspensions were placed onto dry agarose-coated slides (2%, w/v, in sterile H2O) and photographed using Plan-Neofluar objectives (40x/0·75 Ph or 100x/130 Oil Ph3) and an Axioskop 2 microscope (Carl Zeiss Jena). Images were taken with a CCD camera (DEI-750; Optronics Engineering) fitted to the camera port of the microscope, transferred to a PC and further processed with Paint Shop Pro 4 (JASC) and Photoshop 5.0 (Adobe Europe).
DNA extraction and amplification of the 16S rRNA gene and ITS.
PCR amplification was performed on purified DNA or directly on cell lysates as described previously (Iteman et al., 2000 ). Amplification of the 16S rRNA gene was carried out by PCR using primers A2 and S17 (Table 2
). The size of the amplicon was 1451 bp versus 1489 bp for the whole gene. The ITS region was amplified by using primers 322 and 340 (Table 2
). The PCR products were migrated on 2·5% (w/v) agarose gels and visualized on a UV table after staining with ethidium bromide. To study the heterogeneity of the 16S rRNA genes within the genome of Anabaena flos-aquae PCC 9302, an amplification with primers 318 and S11 (Table 2
) was made directly on cells following three washings in sterile H2O. The PCR products (569 bp) of four independent reactions were mixed and cloned into the pGEM-T vector (Promega) after purification with PCR purification kit (Qiagen), as described by Iteman et al. (2000)
. For a random selection of the positive clones, an amplification reaction (four tubes per clone) was performed using oligonucleotides complementary to the flanking M13 primer sites of the vector, and the PCR products of each were pooled prior to sequencing.
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Ribotyping.
The genomic DNA of the 11 planktonic heterocystous strains, together with that of Synechocystis PCC 6803 as control, was prepared from large-volume cultures (400 ml). After centrifugation, the pellet was resuspended in 20 ml NET buffer (1 M NaCl, 100 mM EDTA and 6 mM Tris/HCl, pH 8·0) containing lysozyme (2 mg ml-1). After at least 3 h at 37 °C, during which lysis was verified several times by microscopic observation, the cells were centrifuged (10000 g for 15 min). The DNA released into the supernatant was recovered by precipitation with an equal volume of 2-propanol and centrifugation (10000 g for 15 min). The crude DNA pellet was resuspended in 10 ml 1xTE buffer (100 mM Tris/HCl and EDTA 1 mM, pH 7·4) and deproteinized with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, by vol.), followed by extraction with chloroform/isoamyl alcohol (24:1, v/v). For strains PCC 7905, PCC 9332, PCC 9350 and PCC 9608, whose lysis was less efficient, the cell pellet fractions were also resuspended in 10 ml 1xTE buffer and deproteinized separately. Finally, the purified DNA of all fractions was precipitated by addition of 0·1 vol. sodium acetate solution (3 M; pH 5·2) and 1 vol. 2-propanol. After overnight incubation at 4 °C and centrifugation at 10000 g for 30 min, all DNA precipitates were dried under vacuum and resuspended in 300 µl to 1 ml 0·1xTE buffer. DNA from supernatant and pellet fractions was pooled. Five microlitres of each DNA were restricted using EcoRV or HindIII according to the manufacturers instructions and the restriction products were migrated on 0·8% (w/v) agarose gel (Litex FMC) in 1xTris/borate/EDTA buffer (Sambrook et al., 1989 ). After visualization of the bands by staining with ethidium bromide, the digested DNA was transferred by Southern blotting (Sambrook et al., 1989
) from the agarose gel to a nylon membrane (Amersham Pharmacia Biotech) after alkaline denaturation (0·5 M NaOH and 1·5 M NaCl) and a step of neutralization (0·02 M NaOH and 1 M ammonium acetate). The membranes were washed with 2xSSC (300 mM NaCl and 30 mM sodium citrate, pH 7·0), air-dried and exposed under UV light to fix the DNA fragments. The non-radioactive probe labelling, the step of prehybridization and hybridization of the membrane and the detection reaction were performed using the ECL random prime labelling and detection kit according to the manufacturers instructions (Amersham Pharmacia Biotech). The probe was the PCR product corresponding to the 16S rDNA of Nostoc PCC 7120.
RFLP of PCR products corresponding to the 16S rRNA gene.
The PCR products (10 µl) corresponding to the 16S rRNA genes of the 11 planktonic strains and Nostoc PCC 7120 were digested with each of 12 restriction enzymes (AluI, BanII, BspMI, DdeI, HaeII, NheI, NruI, PstI, RsaI, SacI, SphI and XcmI) according to the manufacturers instructions. The digestions were migrated in 2·5% (w/v) agarose gel (Litex FMC) in 1x Tris/borate/EDTA buffer at 100 V. The gels were stained with ethidium bromide, visualized on a UV table and photographed. The PCR product of the 16S rRNA gene of Nostoc PCC 7120 was used as reference restriction profile. The presence or absence of each restriction band was recorded in a matrix, and the dataset was analysed by distance methods in the software package TREECON (Van de Peer & De Wachter, 1994 ).
Phylogenetic analysis.
The 16S rRNA sequences were aligned in the ARB editor (http://www.mikro.biologie.tu-muenchen.de/pub/ARB/) with a representative dataset of sequences of other heterocystous cyanobacteria. The sequences employed (with GenBank accession numbers) are shown in Table 3. Relationships between the strains were inferred by distance methods in ARB, in TREECON (Van de Peer & De Wachter, 1994
) and by the maximum-likelihood method (fastDNAml v 1.1; Olsen et al., 1994
). Highly variable regions of uncertain alignment and those that had gaps in more than 60% of the sequences were excluded from the analysis, resulting in a total of 1412 comparable positions. The phylogenetic tree (Fig. 2) was midpoint rooted using the non-heterocystous Leptolyngbya boryana PCC 73110 (accession number X84810) as outgroup. A second tree (Fig. 7) was inferred with the same methods but employing all positions of two short (569 bases) sequences of individual clones of the Anabaena flos-aquae PCC 9302 16S rRNA genes and the corresponding region from several close relatives.
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RESULTS |
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Cylindrospermopsis raciborskii (Seenayya & Subba-Raju, 1972 ) is morphologically distinct from all other planktonic cyanobacteria, being distinguished by the thin trichomes (less than 4 µm diameter) that may be loosely coiled and contain cylindrical cells. Heterocysts are conical to spear-shaped and occur exclusively at the ends of the trichome. Although represented by only a single strain in Fig. 2
, organisms assigned to this species are closely related (Saker & Neilan, 2001
), showing at least 99·8% sequence similarity, and are united into cluster VI. The present analysis reveals that this cluster is genetically distant from all other planktonic heterocystous cyanobacteria.
The closest relatives to members of clusters I to V are two strains of Anabaena cylindrica (PCC 7122 and NIES 19) that do not form gas vesicles, are motile and correspond to a species that has never been associated with bloom formation. The former strain shows 95·6% sequence similarity to, for example, Nodularia sp. PCC 7804 in cluster IV. However, it is evident that strains presently assigned to the genus Anabaena are very diverse, since strains carrying the latter generic epithet are dispersed throughout the tree, with Anabaena PCC 7108 being only distantly related both to the planktonic Anabaena spp. and to the A. cylindrica strains (Fig. 2). At the root of the heterocystous cyanobacterial tree lie three Calothrix strains, characterized by filaments containing heterocysts principally in a basal position, with the trichome tapering from base to apex (see Rippka et al., 1979
, 2001
), and the heavily ensheathed Scytonema hofmanni PCC 7110, which differentiates heterocysts generally in intercalary positions. The available sequences of members of the genus Nostoc fall into two different branches of the tree. Three organisms cluster closely with Nostoc punctiforme PCC 73102, which has been proposed as type strain of this species (Rippka & Herdman, 1992
; Rippka et al., 2001
). However, the atypical Nostoc sp. strains PCC 7118 and PCC 7120 (see Rippka et al., 2001
), together with a strain named as Anabaena variabilis (NIES 23), are clearly separated from the N. punctiforme group, being more closely related (96·9%) to Cylindrospermum stagnale PCC 7417. Finally, three strains assigned to the order Stigonematales (Chlorogloeopsis fritschii PCC 6718, Fischerella muscicola PCC 7414 and Mastigocladus HTF PCC 7518) cluster within the heterocystous members of the order Nostocales, as previously observed (Giovannoni et al., 1988
; Turner, 1997
; Wilmotte & Herdman, 2001
).
Multiple rRNA operons
A typical ribotype pattern, obtained following digestion of total DNA with HindIII, is shown in Fig. 3. This enzyme has no recognition site in any of the 16S rDNA sequences available for planktonic heterocystous strains, and resulted in four bands (Anabaenopsis strains PCC 9215, PCC 9216, PCC 9420 and PCC 9608; Cyanospira strains PCC 9501 and PCC 9502; Nodularia PCC 9350) or five bands (Anabaena flos-aquae strains PCC 9302, PCC 9332 and PCC 9349; Aphanizomenon flos-aquae PCC 7905) after Southern hybridization with the 16S rDNA probe. In contrast, restriction of genomic DNA of the unicellular strain Synechocystis PCC 6803 (whose 16S rDNA also lacks HindIII sites) indicated that this isolate contained only two copies of the operon (Fig. 3
), consistent with the published genome sequence (Kaneko et al., 1996
). Since the bands of higher molecular mass in the heterocystous strains are of size sufficient to contain two or more copies of the rrn operon, the total DNA was also restricted with EcoRV, which has a single recognition site within the 16S rDNA of each organism for which this sequence is available. With this enzyme, 8 or 10 hybridizing bands were revealed (data not shown), as expected if only a single copy of the operon was present in each HindIII band. Seven strains were thus shown to possess four copies of the rrn operon, and four strains contained five copies. Within genera for which several strains were examined, each showed a distinct ribotype pattern.
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The region covering this hypothetical RsaI site and corresponding to positions 4621030 of Synechocystis PCC 6803 (E. coli positions 5161083) was sequenced in 14 clones of the 16S rDNA of strain PCC 9302. Six clones contained the RsaI site at the anticipated position, the remainder did not. The two sequences of PCC 9302 differ in a total of 6 out of 569 positions. The less frequent sequence (represented by clone 2) contains the base A at position 341 that creates the GTAC site for RsaI. This change is compensated by base T at position 316 (Fig. 6) that maintains the base pairing. Since strain PCC 9302 was shown by ribotyping to possess five rrn operons, the ratio (8:6) of the different sequences among the clones suggests that three operons contain the majority 16S rDNA sequence (clone 15, Fig. 6
) and that two contain the less frequent one (clone 2). The sequences were compared with that of Anabaena flos-aquae NRC525-17, which differs in 21 positions from both sequences of strain PCC 9302. The difference (1·3%) between the two 16S rDNA sequences of Anabaena flos-aquae PCC 9302 is the same as the maximal difference observed within cluster III (Fig. 2
) and is sufficient to make them appear as two different taxonomic units, more distant from each other than is the major sequence from that of Anabaena strain 90 (Fig. 7
).
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DISCUSSION |
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It is therefore difficult at present to define the generic or specific status of the five clusters of planktonic heterocystous cyanobacteria on the basis of 16S rDNA sequence homology, and it appears that the classical morphological definitions of the corresponding taxa may in some cases be inadequate. Turner (1997) suggested that, although cyanobacterial 16S rRNA sequence comparisons are not suitable for resolution below the level of genus, generic assignments appear to correlate with sequence data. However, Lyra et al. (2001)
showed a close phylogenetic relationship between non-toxic Aphanizomenon and neurotoxic Anabaena isolates originating from Finland, Norway, the Baltic Sea, Holland and Japan. The present results additionally show that strains named as Aphanizomenon flos-aquae, Anabaena flos-aquae and Anabaena circinalis, although falling into three clusters, are not entirely resolved according to their specific or generic assignments. The subclade containing the Nodularia, Anabaenopsis and Cyanospira clusters is clearly separated from the three clusters containing Anabaena and Aphanizomenon strains, although the low degree of sequence divergence (particularly for Anabaenopsis and Cyanospira) may again not necessarily be in agreement with their assignment to three different genera.
Cylindrospermopsis raciborskii is the only planktonic, potentially toxic, heterocystous cyanobacterium that does not group within the major clade containing the five clusters described above. Members of this species were originally assigned to the genus Anabaenopsis (see Rippka et al., 2001 ), but the new genus Cylindrospermopsis was created by Seenayya & Subba-Raju (1972)
on the basis of the fundamentally different pattern of heterocyst differentiation in members of the former genus. As indicated by the generic epithet, members of Cylindrospermopsis have also been thought to be close to those of Cylindrospermum, since the differentiation of exclusively terminal heterocysts is common to both (Horecká & Komárek, 1979
). The low degree of sequence similarity between Cylindrospermopsis raciborskii AWT205 and Anabaenopsis PCC 9215 (93·3%) is consistent with their generic separation. In contrast, Cylindrospermum PCC 7417 clusters with various strains of Nostoc and Anabaena (Fig. 2
) and has only 92·8% sequence similarity with Cylindrospermopsis raciborskii AWT205, disputing the postulated close relationship between Cylindrospermum and Cylindrospermopsis.
The toxicity (if known) of the strains compared in Fig. 2 is summarized in Table 3
. Cluster I contains strains that produce anatoxin-a (Anabaena sp. strain 86 and Anabaena flos-aquae NRC44-1) and non-toxic strains (Aphanizomenon sp. strains 202 and TR183). The toxicity of Aphanizomenon sp. strains PCC 7905 and BC9601 has not yet been examined. Strain PCC 7905 originated from a lake in the Netherlands, and Anabaena flos-aquae NRC44-1 was obtained from a lake in Canada, whereas all other members of this cluster were isolated from Scandinavia (Anabaena sp. strain 86 and Aphanizomenon sp. strain 202 from freshwater lakes in Finland; Aphanizomenon sp. strains BC9601 and TR183 from the Baltic Sea). The very close phylogenetic relationship between these six strains demonstrates that neither toxicity nor geographical origin are useful taxonomic markers. The same conclusions can be made for members of clusters II, III and IV. Cluster II regroups four isolates of Anabaena circinalis, three originating from Australia and one from Japan (strain NIES 41), but also includes one strain of Anabaena flos-aquae (AWQC112D) from Australia. Two of these produce saxitoxins (A. circinalis AWQ279B and AWT001), two are saxitoxin-negative (A. circinalis AWQC306A and A. flos-aquae AWQC112D) and the toxicity of strain NIES 41 is unknown. Cluster III contains isolates that produce the hepatotoxin microcystin (Anabaena sp. strains 66A and 90, and A. lemmermannii NIVA-CYA 83/1) and originating from Scandinavia, but also includes Anabaena flos-aquae PCC 9302, which, based on its strain history, was isolated from a lake in Canada and produces the neurotoxin anatoxin-a(s) (Carmichael & Gorham, 1978
). In cluster IV, three strains of Nodularia (NSPI-05, PCC 9350 and PCC 7804) produce the hepatotoxin nodularin, strain PCC 73104 is non-toxic and the toxicity of strain BCNOD9427 is unknown. Again, this cluster regroups isolates from Australia (NSPI-05), Canada (PCC 73104), France (PCC 7804) and the Baltic Sea (BCNOD9427 and PCC 9350). As shown in Fig. 2
, the Australian strain Nodularia sp. NSPI-05 has a very high degree of sequence similarity with one of the Baltic Sea isolates (BCNOD9427). These analyses clearly demonstrate that it is impossible on the basis of 16S rDNA sequences to distinguish toxic or non-toxic cyanobacteria or organisms from different geographical regions, and they thus contradict previous conclusions concerning the value of these markers (Beltran & Neilan, 2000
; Lehtimäki et al., 2000
; Moffitt et al., 2001
).
Members of both clusters IV and V, however, share certain features that may be of significance. Most strains of Nodularia were found in environments of high salinity or high pH (Bolch et al., 1999 ; Rippka et al., 2001
), as were the two strains of Cyanospira and three of the four Anabaenopsis isolates (Rippka et al., 2001
) examined in the present study. Strains of these three genera cluster together in the phylogenetic tree (Figs 2
and 5
), and the PCC strains studied, with the exception of Anabaenopsis PCC 9608, all show a relatively high salt tolerance (Table 1
), distinguishing them from their freshwater relatives and suggesting a relationship between habitat and phylogenetic position. However, this again does not appear to be a general rule, since Aphanizomenon strains BC9601 and TR183, isolated from the Baltic Sea (Barker et al., 2000
; Lyra et al., 2001
) are not separated phylogenetically from freshwater isolates of Aphanizomenon (PCC 7905) or Anabaena (strains NRC44-1 and 86) (Fig. 2
). A second feature of potential importance is the ability of all PCC strains of Anabaenopsis, Cyanospira and Nodularia to support more elevated temperatures than the Anabaena and Aphanizomenon strains examined (Table 1
).
Using a larger set of strains than that studied by 16S rDNA sequence analysis, we have shown by RFLP that cyanobacteria assigned in the PCC to Aphanizomenon flos-aquae, Anabaena flos-aquae, Anabaenopsis, Cyanospira and Nodularia appear to each form a distinct cluster (Fig. 5), consistent with their positions in trees inferred from the 16S rDNA sequences. However, in view of morphological similarities, their close phylogenetic relatedness revealed by sequence analysis and their clustering in the 16S rDNA RFLP analysis, it is not possible at present to justify the generic separation of strains assigned to Anabaena flos-aquae and Aphanizomenon flos-aquae. Lyra et al. (1997
, 2001
) and Lehtimäki et al. (2000)
also found close relationships between strains of Anabaena and Aphanizomenon in a 16S rDNA RFLP study. However, Nodularia PCC 73104 was found to group with strains of the genus Nostoc, including the atypical Nostoc sp. PCC 7120, although sequence analyses (Lehtimäki et al., 2000
; Lyra et al., 2001
) demonstrated that Nodularia strains clustered more closely with members of the genera Anabaena and Aphanizomenon, which is in better agreement with the RFLP studies presented here. It therefore seems essential to employ a suitable set of restriction enzymes, which can be chosen by examination of the large number of 16S rDNA sequences now available, in order to permit a higher level of discrimination.
All planktonic heterocystous cyanobacteria examined contain several ITS regions of different length (Fig. 4), confirming the genetic heterogeneity of their rrn operons. In contrast, the two rrn operons of Synechocystis PCC 6803 contain ITS regions of identical size, consistent with the genome sequence (Kaneko et al., 1996
). Variability in size of the ITS has been examined in detail in Nostoc PCC 7120, where the longer and shorter ITS regions were shown to be similar in sequence except for the presence and absence, respectively, of two tRNA genes (Iteman et al., 2000
). Such differences in length of the multiple ITS regions within individual heterocystous cyanobacterial strains appear to be very common (Lu et al., 1997
; Neilan et al., 1997
; West & Adams, 1997
; Barker et al., 1999
; Boyer et al., 2001
), and have also been observed in other bacteria such as Corynebacterium and Streptomyces (Aubel et al., 1997
; Hain et al., 1997
). The ITS patterns of the planktonic strains examined (Fig. 4
) were useful in permitting discrimination between strains of Anabaena flos-aquae that were identical in RFLP analysis of the 16S rDNA amplicons, and separated Anabaenopsis PCC 9420 from its three relatives (strains PCC 9215, PCC 9216 and PCC 9608). Although the ITS patterns of the two strains of Cyanospira were identical, they were clearly different from those of the four Anabaenopsis strains, their closest relatives revealed by RFLP analysis of the 16S rDNA. The size of the different ITS regions is thus a useful additional taxonomic character but, as in the case of Aphanizomenon PCC 7905 and Nostoc PCC 7120, which shared an almost identical ITS banding pattern, it may in some cases not permit the distinction between unrelated organisms.
We have also demonstrated by ribotyping that all the heterocystous strains studied contain up to five rrn operons (Fig. 3), consistent with the multiple operons observed in Anabaena strain CA and Anabaena cylindrica CCAP 1403/2a (PCC 7122) by Nichols et al. (1982)
and in Nostoc PCC 7120 (Ligon et al., 1991
). The two members of the genus Cyanospira, which were indistinguishable by RFLP analysis of the 16S rDNA and whose ITS patterns were identical, were clearly separated by ribotyping, as were the Anabaena flos-aquae strains, whose ITS patterns differed but were identical in RFLP analyses. The four members of Anabaenopsis, only partially separated by ITS and RFLP studies, were again distinguished by their ribotypes. The ribotype patterns of individual strains of the same genus, revealing sequence differences in the regions outside of the 16S rDNA, are therefore highly discriminative markers.
We have demonstrated sequence heterogeneity within the 16S rRNA genes of the multiple ribosomal operons of Anabaena flos-aquae PCC 9302. This was first suggested by RFLP analysis, where restriction with RsaI produced bands whose total size exceeded the length of the amplicon, and was confirmed by sequencing a short region of 14 individual 16S rDNA clones. Two distinct sequences were determined, the differences between them corresponding to five transitions (AG or C
T) and one transversion (A
T) (Fig. 6
). This high frequency of transitional substitution suggests that the origin of the intragenomic heterogeneity in the 16S rRNA genes is due to mutation rather than to horizontal gene transfer (Gojobori et al., 1982
; Ueda et al., 1999
). Sequence heterogeneity within the 16S rRNA genes of individual planktonic heterocystous cyanobacteria appears to be widespread, since the RFLP data (Table 4
) indicate its probable occurrence in 10 of the 11 strains examined. Interestingly, the RFLP patterns (Lyra et al., 1997
) of Anabaena sp. strain 90, the nearest relative of strain PCC 9302 (Fig. 2
), did not reveal such heterogeneity following digestion with RsaI, whereas digestion of Anabaena sp. strain 66A with MspI produced bands (approximate total of 1750 bp) in excess of the expected 1500 bp (see Fig. 2
in Lyra et al., 1997
), although the authors did not comment on this discrepancy. Such heterogeneity has been observed only rarely in other prokaryotes, such as Haloarcula marismortui (Mylvaganam & Dennis, 1992
; Dennis et al., 1998
), E. coli (Cilia et al., 1996
; Martinez-Murcia et al., 1999
), Paenibacillus polymyxa (Nübel et al., 1996
), Thermobispora bispora (Wang et al., 1997
) and Phormium yellow leaf phytoplasma (Liefting et al., 1996
) but in fact is probably more common, as reported by Clayton et al. (1995)
. As previously shown for other organisms (Clayton et al., 1995
; Yap et al., 1999
), intra-genomic 16S rDNA sequence heterogeneity has implications for the inference of phylogenetic relationships of planktonic heterocystous cyanobacteria (Fig. 7
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Barker, G. L. A., Hayes, P. K., OMahony, S. L., Vacharapiyasophon, P. & Walsby, A. E. (1999). A molecular and phenotypic analysis of Nodularia (cyanobacteria) from the Baltic Sea. J Phycol 35, 931-937.
Barker, G. L. A., Konopka, A., Handley, B. A. & Hayes, P. K. (2000). Genetic variation in Aphanizomenon (cyanobacteria) colonies from the Baltic Sea and North America. J Phycol 36, 947-950.
Beltran, E. C. & Neilan, B. A. (2000). Geographical segregation of the neurotoxin-producing cyanobacterium Anabaena circinalis. Appl Environ Microbiol 66, 4468-4474.
Bolch, C. J. S., Orr, T. P., Jones, G. J. & Blackburn, S. I. (1999). Genetic, morphological, and toxicological variation among globally distributed strains of Nodularia (Cyanobacteria). J Phycol 35, 339-355.
Booker, M. J. & Walsby, A. E. (1979). The relative form resistance of straight and helical blue-green algal filaments. Br Phycol J 14, 141-150.
Boyer, S. L., Flechtner, V. R. & Johansen, J. R. (2001). Is the 16S23S internal transcribed spacer region a good tool for use in molecular systematics and population genetics? A case study in cyanobacteria. Mol Biol Evol 18, 1057-1069.
Carmichael, W. W. & Gorham, P. R. (1978). Anatoxins from clones of Anabaena flos-aquae isolated from lakes of Western Canada. Mitt Int Ver Limnol 21, 285-295.
Carmichael, W. W. & Gorham, P. R. (1980). Freshwater cyanophyte toxins: types and their effects on the use of microalgal biomass. In Algal Biomass: Production and Use , pp. 437-448. Edited by G. Shelef & C. J. Soeder. Amsterdam:Elsevier.
Cilia, V., Lafay, B. & Christen, R. (1996). Sequence heterogeneity among 16S rRNA sequences, and their effect on phylogenetic analyses at the species level. Mol Biol Evol 13, 451-461.[Abstract]
Clayton, R., Sutton, G., Hinkle, P. J., Bult, C. & Fields, C. (1995). Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent prokaryotic taxa. Int J Syst Bacteriol 45, 595-599.[Abstract]
Dennis, P. P., Ziesche, S. & Mylvaganam, S. (1998). Transcription analysis of two disparate rRNA operons in the halophilic archaeon Haloarcula marismortui. J Bacteriol 180, 4804-4813.
Fergusson, K. M. & Saint, C. P. (2000). Molecular phylogeny of Anabaena circinalis and its identification in environmental samples by PCR. Appl Environ Microbiol 66, 4145-4148.
Florenzano, G., Sili, C., Pelosi, E. & Vincenzini, M. (1985). Cyanospira rippkae and Cyanospira capsulata (gen. nov. and spp. nov): new filamentous heterocystous cyanobacteria from Magadi lake (Kenya). Arch Microbiol 140, 301-306.
Giovannoni, S. J., Turner, S., Olsen, G. J., Barns, S., Lane, D. J. & Pace, N. R. (1988). Evolutionary relationships among cyanobacteria and green chloroplasts. J Bacteriol 170, 3584-3592.[Medline]
Gojobori, T., Ishii, K. & Nei, M. (1982). Estimation of average number of nucleotide substitutions when the rate of substitution varies with nucleotide. J Mol Evol 18, 414-423.[Medline]
Hain, T., Ward-Rainey, N., Kroppenstedt, R. M., Stackebrandt, E. & Rainey, F. A. (1997). Discrimination of Streptomyces albidoflavus strains based on the size and number of 16S23S rDNA intergenic spacers. Int J Syst Bacteriol 47, 202-206.
Horecká, M. & Komárek, J. (1979). Taxonomic position of three planktonic blue-green algae from the genera Aphanizomenon and Cylindrospermopsis. Preslia 51, 289-312.
Iteman, I., Rippka, R., Tandeau de Marsac, N. & Herdman, M. (1999). Use of molecular tools for the study of genetic relationships of heterocystous cyanobacteria. In Marine Cyanobacteria (Bulletin de lInstitut Océanographique, Monaco, special issue 19) , pp. 13-20. Edited by L. Charpy & A. Larkum. Monaco:Institut Océanographique.
Iteman, I., Rippka, R., Tandeau de Marsac, N. & Herdman, M. (2000). Conserved structural and regulatory domains within divergent 16S rRNA23S rRNA spacer sequences of cyanobacteria. Microbiology 146, 1275-1286.
Jeeji-Bai, N., Hegewald, E. & Soeder, C. J. (1977). Revision and taxonomic analysis of the genus Anabaenopsis. Arch Hydrobiol Suppl 51, 3-24.
Jensen, M. A., Webster, J. A. & Straus, N. (1993). Effect of PCR conditions on the formation of the heteroduplex and single-stranded DNA products in the amplification of bacterial rDNA spacers regions. PCR Methods Appl 3, 186-194.[Medline]
Kaneko, T., Sato, S., Kotani, H. & 21 other authors (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3, 109136.[Medline]
Keswani, J. & Whitman, W. B. (2001). Relationship of 16S rRNA sequence similarity to DNA hybridization in prokaryotes. Int J Syst Evol Microbiol 51, 667-678.[Abstract]
Lachance, M. A. (1981). Genetic relatedness of heterocystous cyanobacteria by deoxyribonucleic aciddeoxyribonucleic acid reassociation. Int J Syst Bacteriol 31, 139-147.
Lehtimäki, J., Lyra, C., Suomalainen, S., Sundman, P., Rouhiainen, L., Paulin, L., Salkinoja-Salonen, M. & Sivonen, K. (2000). Characterization of Nodularia strains, cyanobacteria from brackish waters, by genotypic and phenotypic methods. Int J Syst Evol Microbiol 50, 1043-1053.[Abstract]
Li, R., Carmichael, W. W., Liu, Y. & Watanabe, M. M. (2000). Taxonomic re-evaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA gene sequences. Hydrobiologia 438, 99-105.
Liefting, L., Andersen, M., Beever, R., Gardner, R. & Forster, R. (1996). Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma. Appl Environ Microbiol 62, 3133-3139.[Abstract]
Ligon, P. J. B., Meyer, K. G., Martin, J. A. & Curtis, S. E. (1991). Nucleotide sequence of a 16S rRNA gene from Anabaena sp. strain PCC 7120. Nucleic Acids Res 19, 4553.[Medline]
Lu, W., Evans, H. E., McColl, M. & Saunders, V. A. (1997). Identification of cyanobacteria by polymorphisms of PCR-amplified rDNA spacer region. FEMS Microbiol Lett 153, 141-149.
Ludwig, W., Strunk, O., Klugbauer, S., Klugbauer, N., Weizenegger, M., Neumaier, J., Bachleitner, M. & Schleifer, K.-H. (1998). Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19, 554-568.[Medline]
Lyra, C., Hantula, J., Vainio, E., Rapala, J., Rouhiainen, L. & Sivonen, K. (1997). Characterization of cyanobacteria by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene. Arch Microbiol 168, 176-184.[Medline]
Lyra, C., Suomalainen, S., Gugger, M., Vezie, C., Sundman, P., Paulin, L. & Sivonen, K. (2001). Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera. Int J Syst Evol Microbiol 51, 513-526.[Abstract]
Mahmood, N. A. & Carmichael, W. W. (1986). The pharmacology of anatoxin-a(s), a neurotoxin produced by the freshwater cyanobacterium Anabaena flos-aquae NRC 525-17. Toxicon 24, 425-434.[Medline]
Martin, C., Sivonen, K., Matern, U., Dierstein, R. & Weckesser, J. (1990). Rapid purification of the peptide toxins microcystin-LR and nodularin. FEMS Microbiol Lett 68, 1-6.
Martinez-Murcia, A. J., Anton, A. I. & Rodriguez-Valera, F. (1999). Patterns of sequence variation in two regions of the 16S rRNA multigene family of Escherichia coli. Int J Syst Bacteriol 49, 601-610.[Abstract]
Miao, V. P. W., Rabenau, A. & Lee, A. (1997). Cultural and molecular characterization of photobionts of Peltigera membranacea. Lichenologist 29, 571-586.
Moffitt, M. C., Blackburn, S. I. & Neilan, B. A. (2001). rRNA sequences reflect the ecophysiology and define the toxic cyanobacteria of the genus Nodularia. Int J Syst Evol Microbiol 51, 505-512.[Abstract]
Mylvaganam, S. & Dennis, P. P. (1992). Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic archaebacterium Haloarcula marismortui. Genetics 130, 399-410.
Neilan, B. A., Jacobs, D. & Goodman, A. E. (1995). Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus. Appl Environ Microbiol 61, 3875-3883.[Abstract]
Neilan, B. A., Stuart, J. L., Goodman, A. E., Cox, P. T. & Hawkins, P. R. (1997). Specific amplification and restriction polymorphisms of the cyanobacterial rRNA operon spacer region. Syst Appl Microbiol 20, 612-621.
Nichols, J. M., Foulds, I. J., Crouch, D. H. & Carr, N. G. (1982). The diversity of cyanobacterial genomes with respect to rRNA cistrons. J Gen Microbiol 128, 2739-2746.
Nübel, U., Engelen, B., Felske, A., Snaidr, J., Wieshuber, A., Amann, R. I., Ludwig, W. & Backhaus, H. (1996). Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178, 5636-5643.
Olsen, G. J., Matsuda, H., Hagstrom, R. & Overbeek, R. (1994). fastDNAml: a tool for construction of phylogenetic trees of DNA sequences using maximum-likelihood. Comput Appl Biosci 10, 41-48.[Abstract]
Rippka, R. & Herdman, M. (1992). Catalogue of Strains. Pasteur Culture Collection of Cyanobacterial Strains in Axenic Culture. Paris: Institut Pasteur.
Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. & Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111, 1-61.
Rippka, R., Castenholz, R. W. & Herdman, M. (2001). Subsection IV. (Formerly Nostocales Castenholz 1989b sensu Rippka, Deruelles, Waterbury, Herdman and Stanier 1979). In Bergeys Manual of Systematic Bacteriology, 2nd edn, vol. 1, The Archaea and the Deeply Branching and Phototrophic Bacteria, pp. 562589. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York: Springler-Verlag.
Rosselló-Mora, R. & Amann, R. (2001). The species concept for prokaryotes. FEMS Microbiol Rev 25, 39-67.[Medline]
Saker, M. L. & Neilan, B. A. (2001). Varied diazotrophies, morphologies, and toxicities of genetically similar isolates of Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) from Northern Australia. Appl Environ Microbiol 67, 1839-1845.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schlösser, U. G. (1994). Sammlung von Algenkulturen at the University of Göttingen. Catalogue of strains. Bot Acta 107, 111-186.
Seenayya, G. & Subba-Raju, N. (1972). On the ecology and systematic position of the alga known as Anabaenopsis raciborskii (Wolosz.) Elenk. and a critical evaluation of the forms described under the genus Anabaenopsis. In Taxonomy and Biology of Blue-green Algae , pp. 52-57. Edited by T. V. Desikachary. Madras:University of Madras.
Sivonen, K. & Jones, G. (1999). Cyanobacterial toxins. In Toxic Cyanobacteria in Water. A Guide to Their Public Health Consequences, Monitoring and Management , pp. 41-111. Edited by I. Chorus & J. Bartram. London:E. & F. N. Spon on behalf of the WHO.
Turner, S. (1997). Molecular systematics of oxygenic photosynthetic bacteria. Plant Syst Evol (Suppl) 11, 13-52.
Ueda, K., Seki, T., Kudo, T., Yoshida, T. & Kataoka, M. (1999). Two distinct mechanisms cause heterogeneity of 16S rRNA. J Bacteriol 181, 78-82.
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569-570.[Medline]
Walsby, A. E. (1981). Cyanobacteria: planktonic gas-vacuolate forms. In The Prokaryotes , pp. 224-235. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. G. Schlegel. Berlin, Heidelberg, New York:Springer-Verlag.
Wang, Y., Zhang, Z. & Ramanan, N. (1997). The actinomycete Thermobispora bispora contains two distinct types of transcriptionally active 16S rRNA genes. J Bacteriol 179, 3270-3276.[Abstract]
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Report of the Ad Hoc Committee on reconciliation of approaches to Bacterial Systematics. Int J Syst Bacteriol 37, 463464.
West, N. J. & Adams, D. G. (1997). Phenotypic and genotypic comparison of symbiotic and free-living cyanobacteria from a single field site. Appl Environ Microbiol 63, 4479-4484.[Abstract]
Wilmotte, A. & Herdman, M. (2001). Phylogenetic relationships among the cyanobacteria based on 16S rRNA sequences. Bergeys Manual of Systematic Bacteriology, 2nd edn, vol. 1, The Archaea and the Deeply Branching and Phototrophic Bacteria, pp. 487493. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York: Springler-Verlag.
Wilmotte, A., Van der Auwera, G. & De Wachter, R. (1993). Structure of the 16S rRNA of the thermophilic cyanobacterium Chlorogloeopsis HTF (Mastigocladus laminosus HTF) strain PCC 7518, and phylogenetic analysis. FEBS Lett 317, 96-100.[Medline]
Wilson, K. M., Schembri, M. A., Baker, P. D. & Saint, C. P. (2000). Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR. Appl Env Microbiol 66, 332-338.
Yap, W. H., Zhang, Z. & Wang, Y. (1999). Distinct types of rRNA operons exist in the genome of the Actinomycete Thermomonospora chromogena and evidence for horizontal transfer of an entire rRNA operon. J Bacteriol 181, 5201-5209.
Zevenboom, W., Van Der Does, J., Bruning, K. & Mur, L. R. (1981). A non-heterocystous mutant of Aphanizomenon flos-aquae, selected by competition in light-limited continuous culture. FEMS Microbiol Lett 10, 11-16.
Received 30 July 2001;
revised 12 October 2001;
accepted 17 October 2001.