Is the 16S–23S rRNA Internal Transcribed Spacer Region a Good Tool for Use in Molecular Systematics and Population Genetics? A Case Study in Cyanobacteria

Sarah L. Boyer, Valerie R. Flechtner and Jeffrey R. Johansen

Department of Biology, John Carroll University


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We amplified, TA-cloned, and sequenced the 16S–23S internal transcribed spacer (ITS) regions from single isolates of several cyanobacterial species, Calothrix parietina, Scytonema hyalinum, Coelodesmium wrangelii, Tolypothrix distorta, and a putative new genus (isolates SRS6 and SRS70), to investigate the potential of this DNA sequence for phylogenetic and population genetic studies. All isolates carried ITS regions containing the sequences coding for two tRNA molecules (tRNA and tRNA). We retrieved additional sequences without tRNA features from both C. parietina and S. hyalinum. Furthermore, in S. hyalinum, we found two of these non-tRNA-encoding regions to be identical in length but different in sequence. This is the first report of ITS regions from a single cyanobacterial isolate not only different in configuration, but also, within one configuration, different in sequence. The potential of the ITS region as a tool for studying molecular systematics and population genetics is significant, but the presence of multiple nonidentical rRNA operons poses problems. Multiple nonidentical rRNA operons may impact both studies that depend on comparisons of phylogenetically homologous sequences and those that employ restriction enzyme digests of PCR products. We review current knowledge of the numbers and kinds of 16S–23S ITS regions present across bacterial groups and plastids, and we discuss broad patterns congruent with higher-level systematics of prokaryotes.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
During the last decade, biologists have employed a variety of molecular techniques to address questions about phylogeny, evolution, and population diversity. Analysis of 16S (small subunit) rRNA and, more recently, the 16S–23S internal transcribed spacer (ITS) has figured heavily in these studies, particularly those involving prokaryotic and eukaryotic microorganisms.

Several phycologists have used the 16S rRNA gene to provide insight into the phylogenetic relationships of cyanobacterial genera within the orders proposed by Komárek and Anagnostidis (1986, 1989)Citation (e.g., Giovannoni et al. 1988Citation ; Wilmotte and Golubic 1991Citation ; Wilmotte et al. 1992Citation ; Wilmotte, Neefs, and De Wachter 1994Citation ; Nelissen et al. 1996Citation ; Nübel et al. 1996Citation ; Turner 1997Citation ). However, Fox, Wisotzkey, and Jurtshuk (1992)Citation concluded that identity in 16S rRNA sequence data was not sufficient grounds for establishing species identity and thus not appropriate for studies at the subgeneric level. As a result, researchers have increasingly turned to the more variable 16S–23S ITS.

Restriction enzyme digestion of the 16S–23S ITS region has been used for phylogenetic analyses of strains of nonphotosynthetic eubacterial genera (Navarro et al. 1992Citation ; Vinuesa et al. 1998Citation ). Restriction digests of this region have also been used to examine variability and phylogenetic relationships within orders of cyanobacteria (Lu et al. 1997Citation ), among genera of heterocystous cyanobacteria (West and Adams 1997Citation ), and among strains in a single filamentous genus (Scheldeman et al. 1999Citation ). Otsuka et al. (1999)Citation were the first and are currently the only research group to use direct sequencing of the ITS to study subgeneric phylogenetic relationships in a cyanobacterial genus, Microcystis. They found that the phylogeny based on the ITS data did not correlate perfectly with established Microcystis morphospecies or phycoerythrin production, although concordance with microcystin production was evident.

Operons containing the genes coding for the three rRNAs (16S, 23S, 5S) and their associated ITS regions are normally present in multiple copies in the bacterial genome (7 in Escherichia coli and Salmonella, 10 in Bacillus subtilis). In E. coli, these copies are named rrnA–rrnE, rrnH, and rrnG. Antón, Martínez-Murcia, and Rodríguez-Valera (1998)Citation have demonstrated that there are major heterogeneities among operons in terms of the type and number of tRNA genes present. In E. coli K12, within the 16S–23S ITS, operons rrnB, rrnC, rrnE, and rrnG contain a gene coding for tRNAGlu-2, whereas operons rrnA, rrnD, and rrnH have genes for tRNAIle-1 and tRNAAla-1B. There are other major heterogeneities in various regions of the ITS within each of these two general "types" of ITS region. It has been suggested that the spacer sequence could reflect intraspecies phylogeny (García-Martínez et al. 1996Citation ), but that to target the same operon (rrnA, rrnB, rrnC, rrnD, rrnE, rrnH, or rrnG) in multiple strains, operon-specific primers must be used in PCR (Antón, Martínez-Murcia, and Rodríguez-Valera 1998Citation ).

In this study, we examined variability in the ITS among multiple rRNA operons in five species: Scytonema hyalinum, Tolypothrix distorta, Calothrix parietina, Coelodesmium wrangelii, and a putative new genus (isolates SRS6 and SRS70). These species represent three of the four families currently recognized in the order Nostocales. Most of the 16S rRNA data available for members of this order come from representatives of the fourth family (Nostocaceae), including sequences from Nostoc and Anabaena. ITS sequences have also recently been reported for several species of Nostoc (Lu 1999Citation ; Iteman et al. 2000Citation ; Li 2000Citation ). Very little data exist from the three families that we examined: partial 16S rRNA sequences are available in GenBank from three isolates of Scytonema (in the family Scytonemataceae), but no sequence data exist for members of the families Microchaetaceae (represented in this study by T. distorta and C. wrangelii) or Rivulariaceae (represented by C. parietina). Therefore, the data presented here are of value to anyone interested in understanding relationships within the order Nostocales. In addition, our study is the first comparison of ITS sequence similarities among multiple members of a cyanobacterial order. We review the current knowledge of the numbers and kinds of ITS regions present across bacteria and plastids and discuss the potential utility of the ITS region as a tool for both broad- and fine-scale phylogeny reconstruction.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Collection and Isolation of Cyanobacterial Strains
Cyanobacterial isolates used in this study were all collected, isolated, and identified by V.R.F. and J.R.J. For those cyanobacteria isolated from soils, dry soil samples were crushed, subsampled, and dilution plated as described in Flechtner (1999)Citation . Cyanobacteria were isolated into unialgal culture from the plates and kept on agar slants of Z-8 medium (Carmichael 1986Citation ). Coelodesmium wrangelii was isolated directly from a stream sample onto agar slants of Z-8 medium. All isolates were examined on Olympus photomicroscopes with Nomarski DIC optics. Strains were kept in dim light (<50 µE/cm2/s illuminance) at 7°C on a 12:12 h light : dark cycle.

DNA Extraction
DNA was extracted from 20 mg of fresh unialgal tissue using the Cullings (1992)Citation modification of the Doyle and Doyle (1987)Citation technique. The resultant DNA was suspended in 50 µl TE and stored at -20°C.

Polymerase Chain Reaction
Primers were designed after Wilmotte, Van der Auwera, and De Wachter (1993)Citation , Wilmotte (1994)Citation , and Nübel, Garcia-Pichel, and Muyzer (1997)Citation . They were designated primer 1 (5'-CTC TGT GTG CCT AGG TAT CC-3'; after Wilmotte, Van der Auwera, and De Wachter 1993Citation ), primer 2 (5'-GGG GGA TTT TCC GCA ATG GG-3'; after Nübel, Garcia-Pichel, and Muyzer 1997Citation ), primer 3 (5'-CGC TCT ACC AAC TGA GCT A-3'; after Wilmotte 1994Citation ), primer 4 (5'-ATT AGC TCA GGT GGT TAG-3' after Wilmotte, Van der Auwera, and De Wachter 1993Citation ), and primer 5 (5'-TGT ACA CAC CGG CCC GTC-3'; after Wilmotte, Van der Auwera, and De Wachter 1993Citation ).

The positions of these primers with regard to the 16S RNA gene, the 23S RNA gene, and the transfer RNA genes that had previously been found between them are shown in figure 1 . Primers (Midland Certified Reagent Company) were made up in 100 µM stock solutions. For use in PCR, a mix of 1.2 µl each of two primers and 7.6 µl sterile water was made.



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Fig. 1.—Map of primer sites in the 16S–23S rRNA operon that were used in this study. Primers 1 and 2 were used in the first PCR reaction, and that product was reamplified using primers 1 and 5 to obtain the "short" PCR product used in cloning. Primers 3 and 4 were used when it was not possible to obtain an ITS region containing both tRNA genes with primers 1 and 5. ISR-B, ISR-C, and ISR-D are noncoding intergenic spacer regions which surround the tRNA genes for isoleucine (tIle) and alanine (tAla)

 
Initially, each DNA sample was amplified using primers 1 and 2. This resulted in a product approximately 1,600 bp long ("long PCR"), which was then used as a template for a reamplification using primers 1 and 5, resulting in a product approximately 600 bp long ("short PCR"). Each 100-µl reaction contained 86 µl sterile water, 10µl 10 x buffer (Promega), 0.5 µl of each dNTP (G, A, T, C) at 10 mM, 0.5 µl of the primer mixture described above, 0.5 µl Taq polymerase (Promega), and, typically, 1.0 µl template DNA (5–10 ng).

PCR was optimized for each species, with the template DNA amount varying from 0.5 to 2.0 µl and the annealing temperatures varying from 55°C to 57°C. The most commonly used profile for the long-PCR reaction using primers 1 and 2 was 94°C for 1 min, 57°C for 1 min, and 72°C for 4 min (35 cycles), followed by a 10-min extension at 72°C. For the short-PCR reamplifications, the most commonly used profile was 94°C for 1 min, 56°C for 45 s, and 72°C for 2 min (20 cycles). Reactions were carried out using Thermolyne's Amplitron and Temptronic thermocyclers. Results were checked using a 1% agarose gel.

Sequencing
Short PCR product was cloned into plasmids containing the sites for the universal primers M13 forward and reverse on either side of the cloning site using Invitrogen's TOPO TA Cloning Kit for Sequencing, version A. Plasmid DNA was generally obtained from three of the resultant clones using Qiagen's QiaPrep Spin Kit.

Automated sequencing was performed by Cleveland Genomics with the universal primers M13 forward and reverse.

Data Analysis
Forward and reverse primer sequences were checked against each other by generating the reverse complement of the "reverse" sequence using Oxford Molecular Group's Omiga and aligning it with the "forward" sequence using the CLUSTAL W Multiple Sequence Alignment Program, version 1.7 (Thompson, Higgins, and Gibson 1994Citation ) via the Baylor College of Medicine's Search Launcher (Smith et al. 1996Citation ) at http://dot.imgen.bcm.tmc.edu:9331/. This resulted in the longest possible read of the sequence, in addition to acting as a check on the sequencing. Sequences were aligned using CLUSTAL W. These alignments were checked by eye.

GenBank Numbers
ITS sequence data for species examined in this study were deposited with GenBank. Accession numbers are as follows: C. parietina, AF236642 (two tRNAs) and AF236643 (no tRNAs); C. wrangelii, AF236652; S. hyalinum, AF236650 (no tRNAs), AF236651 (two tRNAs), AY007688 (no tRNAs); T. distorta, AY007689; Tolypothrix field sample, AF236644–AF236649 (six sequences); putative new genus, AF2326659.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Agarose Gel Electrophoretic Analysis of PCR Products
Agarose gel electrophoresis of ITS PCR products (short PCR) from five cyanobacterial genera (fig. 2 ) revealed obvious differences among the genera. We consistently obtained a single band from both isolates of the putative new genus (SRS6 and SRS70), C. wrangelii, and T. distorta and two or more bands from S. hyalinum and C. parietina. The presence of multiple bands in PCR reactions suggested that at least some of our isolates possessed multiple rRNA operons.



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Fig. 2.—Digital photograph of an agarose gel with internal transcribed spacer (ITS) (short) PCR products from the following taxa (left to right): Calothrix parietina, Syctonema hyalinum, Coelodesmium wrangelii, putative new genus (isolate SRS70), and Tolypothrix distorta. To the right is {lambda} BstEII digest. Note that S. hyalinum and C. parietina, both of whose short-PCR products show two distinct bands, are the taxa from which we retrieved multiple ITS sequences of different lengths

 
Sequence Data from the 16S–23S ITS Region
To investigate the possibility that multiple rRNA operons existed in the genomes of at least some of our cyanobacterial species, we cloned our short PCR products into competent E. coli cells. At least three individual clones per isolate were selected for automated sequencing.

We sequenced the ITS regions from two isolates of the putative new genus (SRS6 and SRS70) and one isolate each for C. wrangelii, T. distorta, S. hyalinum, and C. parietina. The sizes of the 16S–23S ITS regions in these genera ranged from 347 in C. parietina to 648 bp in one sequence from S. hyalinum. Two sequence organization patterns were evident: some 16S–23S ITS sequences contained no tRNA molecule sequences (fig. 3 , i) and some contained both tRNAAla and tRNAIle (fig. 3 , ii). We have denoted the intergenic spacer regions (ISRs) located within the ITS and between the various coding sequences using the following designations: ISR-A (16S–23S, without interruption from tRNAs) (fig. 3, i ), ISR-B (16S-tRNAIle), ISR-C (tRNAIle-tRNAAla), and ISR-D (tRNAAla-23S) (fig. 3, ii ).



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Fig. 3.—Stylized map showing two patterns observed in the 16S–23S rRNA ITS region within the cyanobacterial isolates examined in this study. The four intergenic spacer regions (ISRs) are labeled based on their relationship to the tRNA genes present

 
Variability in ITS and ISR Size and Sequence Among Species and Within Isolates
We found striking variability in the size of the ITS region among the various species examined (fig. 4 ). Even when sequences from multiple taxa showed the same overall pattern of configuration, the sizes of the individual 16S–23S ITS regions often differed tremendously. For example, while all species carry at least one copy of the 16S–23S ITS region containing both tRNAIle and tRNAAla, the size of the entire 16S–23S ITS region for this operon is 491 bp in C. wrangelii and 648 bp in S. hyalinum (fig. 4 ). The size difference is particularly noticeable in ISR-C, which contains 83 bp in S. hyalinum but a mere 9 bp in C. parietina.



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Fig. 4.—Map of 16S rRNA internal transcribed spacer regions containing both tRNA genes in five isolates of heterocystous cyanobacteria from soils. Letters in the sequence refer to intergenic spacer region (ISR) designation (B = ISR-B, etc.). Sequence length (in bp) is given below each ISR.

 
We found evidence for multiple nonidentical rRNA operons in S. hyalinum and C. parietina. Calothrix parietina carries at least one two-tRNA-containing and at least one tRNA-lacking operon. Scytonema hyalinum carries at least one two-tRNA-containing and at least two tRNA-lacking operons. The two tRNA-lacking ITS regions of S. hyalinum are both 400 bp long but nonidentical in sequence (fig. 5 ). We noted the presence of all structural features of the ITS identified by Iteman et al. (2000)Citation in each of these three S. hyalinum sequences (fig. 5 ).



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Fig. 5.—Alignment of the 16S–23S rRNA internal transcribed spacer (ITS) region from a single isolate of Scytonema hyalinum. One sequence (clone B) contains both tRNAIle and tRNAAla; the other two (clones A and C) contain neither tRNA and are identical in length but nonidentical in sequence. Note that all three sequences contain all important structural elements of the ITS, as described by Iteman et al. (2000)Citation . Tips of stem structures are indicated between arrowheads. Region D5 lies between V3 and 23S but cannot be specifically identified in the absence of sequence data from the 23S–5S ITS.

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
During the last 15 years, our understanding of evolutionary relationships among microorganisms has expanded dramatically, due in large part to the use of 16S (small ribosomal subunit [SSU]) sequence data. This approach, pioneered by Carl Woese (Woese, Kandler, and Wheelis 1990Citation ), has become so widely used that 16S sequence data will be the basis for defining taxonomic groups in the second edition of Bergey's Manual of Determinative Bacteriology. However, some investigators (e.g., Fox, Wisotzkey, and Jurtshuk 1992Citation ) have questioned whether sufficient variability exists in 16S RNA to allow discrimination among species of a genus or strains of a species. It has been suggested that the ITS region separating 16S and 23S sequences in the rRNA operon might be useful for these fine levels of discrimination. In the discussion that follows, we review current knowledge of the number of rRNA operons and patterns of 16S–23S ITS configurations therein in bacteria and plastids, with special focus on cyanobacteria. We finish by discussing the usefulness of ITS data for phylogenetic studies.

ITS Patterns of Configuration Reflect Higher-Level Phylogenetic Groupings
Gürtler and Stanisich (1996)Citation compared the sizes and makeups of the 16S–23S ITS regions of 44 species in 27 genera of bacteria. During the last four years, many new sequences have been added to the database; all are presented in table 1 . Collectively, the data show three general patterns of sequence composition in 16S–23S ITS regions. The two most common patterns are the absence of tRNA sequences (as in fig. 3, i ) or the presence of two tRNA sequences (tRNAIle and tRNAAla, as in fig. 3, ii ). In the two tRNA-containing ITS regions, in all but one taxon the tRNAIle is just downstream of the 16S rRNA and the tRNAAla is just upstream of the 23S rRNA; the order is reversed only in the recently sequenced plant pathogen Xylella fastidiosa (Simpson et al. 2000)Citation . A third pattern noted is the presence of a lone tRNA which may be tRNAAla, tRNAGlu, or tRNAIle.


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Table 1 16S–23S rRNA ITS Data Currently Available from Archaea, Eubacteria, and Plastids

 
If one considers the 16S–23S ITS sequence data currently available across phylogenetic groups, certain patterns begin to emerge. In the Archaea, members of the kingdom Crenarchaeota have ITS regions lacking tRNA genes, while members of the Euryarchaeota carry tRNAAla (table 1 ). Among the Eubacteria, members of the primitive Aquaficales and Thermotoga carry both tRNAIle and tRNAAla. Proteobacteria display all varieties of ITS configuration, including tRNAGlu only, which has thus far been found exclusively in the {gamma}-division of this group. Among the gram-positive bacteria (Firmicutes), there is a difference in ITS types observed between organisms of low G-C content and organisms of high G-C content. Those of low G-C content (the Bacillus/Clostridium group) display a variety of ITS configurations. Most taxa in this group lack tRNA sequences, and of those that carry tRNA sequences, two tRNAs are present in five species, tRNAAla alone is present in three species, and tRNAIle alone is present in a single species (Staphylococcus aureus) (table 1 ). In contrast, all of the gram-positive bacteria of high G-C content (Actinobacteria) have ITS regions lacking tRNA sequences. Among the cyanobacteria, by far the most common pattern of configuration is the presence of both tRNAIle and tRNAAla; ITS regions in this group may also lack tRNA sequences or carry tRNAIle alone (table 1 ). In plastids, all ITSs contain both tRNAIle and tRNAAla, except in Toxoplasma gondii, which has a split rRNA operon with no ITS (Kohler et al. 1997Citation ), and in the plastids of the parasitic plants Conopholis americana (Wimpee, Morgan, and Wrobel 1992Citation ) and Epifagus virginiana (Wolfe et al. 1992Citation ), which have both undergone a deletion event eliminating most or all of the tRNA from the ITS region. It therefore appears, on the basis of the limited amount of data available, that 16S–23S ITS composition mirrors higher phylogenetic groupings. It will be interesting to see if these patterns hold up as additional ITS regions are sequenced, and it is possible that the configuration of the ITS region could be a character useful for higher-level phylogeny reconstruction.

ITS Configurations in Cyanobacteria and Plastids
As a group, cyanobacteria show three types of 16S–23S ITS region sequence composition. ITS regions containing both tRNAIle and tRNAAla are most common and have been previously reported in members of the genera Anabaena (Lu 1999Citation ), Arthrospira (Nelissen et al. 1994Citation ), Nostoc (Lu 1999Citation ; Iteman et al. 2000Citation ), Synechococcus ("Anacystis") (Tomioka and Sugiura 1984Citation ), and Trichodesmium (Wilmotte, Neefs, and De Wachter 1994Citation ). Our study adds the genera Calothrix, Coelodesmium, Scytonema, Tolypothrix, and a putative new genus (isolates SRS6 and SRS70) to this group (table 1 ). ITS regions with tRNAIle only have been reported in the genera Microcystis (Otsuka et al. 1999Citation ), Spirulina (Nelissen et al. 1994Citation ), and Synechocystis (Kaneko et al. 1996Citation ). It appears that this latter configuration is more frequently seen in the cyanobacteria than in other groups of eubacteria (table 1 ). Spacer regions lacking tRNA sequences altogether had been previously reported in Nostoc (Iteman et al. 2000)Citation and Nodularia (Hayes and Barker 1997Citation ); we found similar regions in Calothrix and Scytonema.

Sequence data from rRNA-encoding genes (Giovannoni et al. 1988Citation ) provided early support for the theory that all plastids arose in an endosymbiotic event from a common cyanobacterial ancestor. All plastids except for those found in two parasitic plants have 16S–23S ITS regions containing both tRNAIle and tRNAAla (table 1 ). There are two rRNA operons in most cases; four taxa have one operon, and Euglena gracilis has three operons (table 1 ). In many algae and the plastids of the liverwort Marchantia polymorpha and nonparasitic angiosperms, the operons appear as inverted repeats. In all land plants, the operon has two unique features: an additional 4.5S rRNA molecule and large (650–1,050-bp) inserts in each of the two tRNA sequences in the 16S–23S ITS. The identification of similar inserts in the tRNA sequences of plastids from the charophycean alga Coleochaete led Manhart and Palmer (1990)Citation to propose this alga to be the sister group of land plants.

What kind of 16S–23S ITS region might have existed in the rRNA operon of the cyanobacterial ancestor of chloroplasts? It seems likely that the order of the components of the rRNA operon(s) of the ancestor was 16S-tRNAIle-tRNAAla-23S-5S, with the two tRNA sequences separated by an ISR of only a few (<10) nucleotides. Evidence supporting this supposition is that this arrangement exists (1) in Aquifex aeolicus and Thermotoga maritima, the two oldest extant bacteria; (2) in most of the extant cyanobacteria for which we currently have data; (3) in the cyanelle of Cyanophora paradoxa; (4) in the chloroplasts of all algae in the Charophyta, Chlorophyta, Rhodophyta, and Bacillariophyta; and (5) in the chloroplast of E. gracilis (table 1 ). The oldest extant cyanobacteria are thought to be members of the genera Gloeobacter and Pseudanabaena (Turner 1997Citation ). No information is currently available on the number or configuration of rRNA operons of these organisms; such information could prove valuable in reconstructing the phylogeny of cyanobacteria and plastids.

Split rRNA Operons
The presence of split rRNA operons has been detected primarily when an entire genome has been sequenced. In most of the mitochondrial genomes for which sequences are available in GenBank, 16S- and 23S-encoding genes exist as separate, intact single copies. Boer and Gray (1988)Citation reported a bizarre organization of rRNA genes in the mitochondrial DNA of Chlamydomonas reinhardtii where each gene is discontinuous and dispersed throughout the genome. More recently, split rRNA operons have also been identified in Eubacteria and Archaea (table 1 ). In the most common pattern, the 23S and 5S genes form an operon, and the 16S rRNA gene is separate; there may be one or more than one copy of each of the genes. In two Archaea (Archaeoglobus fulgidus and Pyrococcus horikoshii OT3), the 16S and 23S genes are linked and the 5S gene is separate. In the Eubacteria, Helicobacter pylori 26695 carries two 23S–5S operons, two single 16S genes, and one separate 5S gene. Rickettsia prowazekii Madrid E and Wolbachia pipientis, three species of Borrelia, and Deinococcus radiadurans carry one or more 5S–23S operons and one or more single 16S genes (table 1 ). Split rRNA operons are also present in the chloroplast of T. gondii, which carries single, unlinked 16S and 23S genes and no 5S gene. To date, the complete genome of only one cyanobacterium, Synechocystis PCC6803, has been determined (Kaneko et al. 1996Citation ); no split operons exist in this taxon. Perhaps as more complete genome sequences from this group of eubacteria are determined, more split operons will be identified.

Multiple Nonidentical rRNA Operons in Cyanobacteria
The number of rRNA operons present is known for relatively few bacterial species and ranges from 1 or 2 operons in the members of Archaea and several nonoxygenic eubacterial genera (Mycobacteria, Mycoplasma, Rhodothermus, and Thiobacillus) to 6–10 in some members of the proteobacteria and gram-positive eubacteria (table 1 ). Until recently, the presence of multiple rRNA operons in cyanobacteria has received little attention, so data relating to the number and sequence diversity of rRNA operons in cyanobacterial species are sparse.

The existence of multiple rRNA operons in cyanobacteria was first reported by Nichols et al. (1982)Citation , who used Southern hybridization to detect rRNA genes in three species of Anabaena and one species of Nostoc. Chen and Widger (1993)Citation identified two operons on the physical map of Synechococcus strain PCC 7002. Two groups reported that Synechococcus PCC6301 ("Anacystis nidulans") carries two rRNA operons, one of whose ITS regions contain both tRNAIle and tRNAAla (Tomioka, Shinozaki, and Sugiura 1981Citation ; Williamson and Doolittle 1983Citation ), and one of which contains a tRNAIle pseudogene but has not been completely sequenced (Williamson and Doolittle 1983Citation ). Genome sequencing of Synechocystis (Kaneko et al. 1996Citation ) has shown two rRNA operons, both containing tRNAIle only, present in inverted repeats. In this taxon, sequence data for the ISR regions flanking the tRNA are not available, so information about the sequence identity of the operons is unavailable at this time. In a brief note, Ligon et al. (1991)Citation reported they had identified four rRNA operons in Nostoc (Anabaena) strain PCC 7120 but provided no methods or results to support their statement.

The presence of single rRNA operons or multiple identical operons in some cyanobacterial taxa was suggested by Scheldeman et al. (1999)Citation , who reported finding only one band of a consistent length when they amplified the 16S plus ITS region of multiple isolates of Arthrospira. Otsuka et al. (1999)Citation recovered unambiguous ITS sequences from each of 47 clonal isolates of Microcystis without using a cloning step. Similarly, we sequenced the ITS regions from nine separate clones of the PCR product from two different isolates (SRS 6 and SRS 70) of the putative new genus and always obtained sequences identical in structure (containing both tRNAIle and tRNAAla, fig. 3, ii ), length, and nucleotide sequence.

Evidence for multiple nonidentical rRNA operons has been presented by Wilmotte, Neefs, and De Wachter (1994)Citation , who described problems in obtaining unambiguous rDNA sequences using direct sequencing of ITS PCR products in Trichodesmium NIBB 1067. After cloning these PCR products, they discovered differences in ITS sequences (but not configurations) among clones. More recently, Iteman et al. (2000)Citation found two ITS sequences with completely different configurations in Nostoc PCC 7120.

We identified, based on sequence analysis of individual clones of PCR-amplified ITS regions, two types of ITS regions in a unialgal isolate of C. parietina and three different types of ITS regions from a unialgal isolate of S. hyalinum. This means that C. parietina carries at least two rRNA operons and that S. hyalinum carries at least three different rRNA operons, two of which are identical in size (400 bp) but nonidentical in sequence (fig. 4 ). One significant difference between our results and the findings of Iteman et al. (2000)Citation concerns the number and/or identity of the operons lacking tRNA. While the former investigators found sequence identity between the tRNA-lacking regions of all operons in a single species, we identified sequence differences between two 400-bp tRNA-lacking operons in S. hyalinum. We also identified rRNA operons of similar configurations but different sequence compositions in individual clones of Microcoleus and Nostoc (unpublished data).

Iteman et al. (2000)Citation determined that both the tRNA-lacking and the tRNA-containing 16S–23S ITS regions of Nostoc PCC 7120 contain the sequences necessary for proper folding and processing of this region to release functional rRNA and tRNA. These regions included the D1, D1', D2, and D3 short consensus sequences and the antiterminator box A. A box-B loop-and-stem structure just upstream of box A and a V3 stem-loop structure have variable primary structures but consistent secondary structures. We identified these same regions in the five species examined in this study (e.g., in Scytonema; fig. 4 ). Their presence suggests that all of the ITS regions we sequenced are involved in the folding and processing of rRNA.

The Use of 16S–23S ITS Sequence Data in Fine-Scale Phylogenetic Studies
The use of the 16S–23S ITS region in studies of phylogeny (reviewed Gürtler and Stanisich 1996Citation ; Lu 1999Citation ; Li 2000Citation ), molecular evolution (Antón, Martinez-Murcia, and Rodríguez-Valera 1998Citation ), or population genetics (Navarro et al. 1992Citation ; Otsuka et al. 1999Citation ; Scheldeman et al. 1999Citation ) is a potentially powerful tool. However, investigators need to be aware of potential problems imposed by the possibility of multiple nonidentical rRNA operons. Any study that depends on PCR amplification of the ITS region may run into the problem of preferential amplification of some operons. Amplification of cyanobacterial 16S–23S ITS regions depends on cyanobacterial-specific primers developed by Wilmotte, Van der Auwera, and De Wachter (1993)Citation , Nelissen et al. (1994)Citation , Wilmotte (1994)Citation , and Nübel, Garcia-Pichel, and Muyzer (1997)Citation . One of these primers sits at the 5' end of the 23S rRNA molecule. Gürtler and Barrie (1995)Citation found significant variation in this region of the 23S rRNA gene of the gram-positive bacterium S. aureus and cautioned that this primer site variability might mean that some rRNA operons might go unamplified or be underamplified in this species. In cyanobacteria, the existing data on 23S sequence composition are insufficient to ascertain whether a similar situation exists. Still, our experience with one field isolate of Tolypothrix suggests that such heterogeneity may be present. Attempts to amplify the ITS region of this organism with primers 1 and 5, which lie in the flanking 16S and 23S sequences (fig. 1 ), consistently produced one very bright band and one or two faint bands. When we sequenced three different clones of that PCR product, we observed only one sequence, which consistently contained no tRNA sequence (fig. 3, i ). However, when we amplified the region with primers 1 and 4 or primers 3 and 5, primer sets that target tRNAIle and tRNAAla sequences (fig. 1 ), we obtained multiple nonidentical sequences containing both tRNA sequences. We interpret these results to indicate that the organism contains both tRNA-containing and tRNA-lacking rRNA operons and that the operon without tRNAs was preferentially amplified during PCR reactions using primers 1 and 5. This phenomenon may impact analyses employing restriction enzyme digests of ITS PCR products (e.g., Navarro et al. 1992Citation ; Lu et al. 1997Citation ; West and Adams 1997Citation ; Vinuesa et al. 1998Citation ; Scheldeman et al. 1999Citation ). That is, if the ITS from one operon is amplified preferentially in one isolate, while the ITS from a different operon is preferentially amplified in another isolate, comparisons of digests of PCR product from those two isolates may be flawed. Experimental designs should therefore contain safeguards against complications imposed by the presence of multiple nonidentical rRNA operons.


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Table 1 Continued

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We thank Alison K. Dillon for developing our molecular protocols and Marisa DeNoble for helping with extractions and initial PCR. Jim Lissemore provided continual advice and support. Louise Lewis provided helpful criticism of early drafts of the manuscript. This work was supported in part by contract DACA88-98-Q-0181 from the U.S. Army Construction Engineering Research Laboratory in Champaign, Ill., and in part by funds from John Carroll University, including a Faculty Summer Research Fellowship to V.R.F. Publication costs were covered by a grant from the University Committee for Research, Service, and Faculty Development.


    Footnotes
 
Pamela Soltis, Reviewing Editor

1 Keywords: Internal transcribed spacer cyanobacteria intergenic spacer region rRNA phylogenetics population genetics Back

2 Address for correspondence and reprints: Valerie R. Flechtner, Department of Biology, John Carroll University, 20700 North Park Boulevard, University Heights, Ohio 44118. flechtner{at}jcu.edu . Back


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Accepted for publication February 13, 2001.