Department of Biology, John Carroll University
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Abstract |
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Introduction |
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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)
(e.g., Giovannoni et al. 1988
; Wilmotte and Golubic 1991
; Wilmotte et al. 1992
; Wilmotte, Neefs, and De Wachter 1994
; Nelissen et al. 1996
; Nübel et al. 1996
; Turner 1997
). However, Fox, Wisotzkey, and Jurtshuk (1992)
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 16S23S ITS.
Restriction enzyme digestion of the 16S23S ITS region has been used for phylogenetic analyses of strains of nonphotosynthetic eubacterial genera (Navarro et al. 1992
; Vinuesa et al. 1998
). Restriction digests of this region have also been used to examine variability and phylogenetic relationships within orders of cyanobacteria (Lu et al. 1997
), among genera of heterocystous cyanobacteria (West and Adams 1997
), and among strains in a single filamentous genus (Scheldeman et al. 1999
). Otsuka et al. (1999)
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 rrnArrnE, rrnH, and rrnG. Antón, Martínez-Murcia, and Rodríguez-Valera (1998)
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 16S23S 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. 1996
), 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 1998
).
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 1999
; Iteman et al. 2000
; Li 2000
). 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.
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Materials and Methods |
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DNA Extraction
DNA was extracted from 20 mg of fresh unialgal tissue using the Cullings (1992)
modification of the Doyle and Doyle (1987)
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)
, Wilmotte (1994)
, and Nübel, Garcia-Pichel, and Muyzer (1997)
. They were designated primer 1 (5'-CTC TGT GTG CCT AGG TAT CC-3'; after Wilmotte, Van der Auwera, and De Wachter 1993
), primer 2 (5'-GGG GGA TTT TCC GCA ATG GG-3'; after Nübel, Garcia-Pichel, and Muyzer 1997
), primer 3 (5'-CGC TCT ACC AAC TGA GCT A-3'; after Wilmotte 1994
), primer 4 (5'-ATT AGC TCA GGT GGT TAG-3' after Wilmotte, Van der Auwera, and De Wachter 1993
), and primer 5 (5'-TGT ACA CAC CGG CCC GTC-3'; after Wilmotte, Van der Auwera, and De Wachter 1993
).
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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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 1994
) via the Baylor College of Medicine's Search Launcher (Smith et al. 1996
) 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, AF236644AF236649 (six sequences); putative new genus, AF2326659.
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Results |
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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 16S23S 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 16S23S 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 (16S23S, 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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Discussion |
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ITS Patterns of Configuration Reflect Higher-Level Phylogenetic Groupings
Gürtler and Stanisich (1996)
compared the sizes and makeups of the 16S23S 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 16S23S 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)
. A third pattern noted is the presence of a lone tRNA which may be tRNAAla, tRNAGlu, or tRNAIle.
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ITS Configurations in Cyanobacteria and Plastids
As a group, cyanobacteria show three types of 16S23S 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 1999
), Arthrospira (Nelissen et al. 1994
), Nostoc (Lu 1999
; Iteman et al. 2000
), Synechococcus ("Anacystis") (Tomioka and Sugiura 1984
), and Trichodesmium (Wilmotte, Neefs, and De Wachter 1994
). 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. 1999
), Spirulina (Nelissen et al. 1994
), and Synechocystis (Kaneko et al. 1996
). 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)
and Nodularia (Hayes and Barker 1997
); we found similar regions in Calothrix and Scytonema.
Sequence data from rRNA-encoding genes (Giovannoni et al. 1988
) 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 16S23S 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 (6501,050-bp) inserts in each of the two tRNA sequences in the 16S23S ITS. The identification of similar inserts in the tRNA sequences of plastids from the charophycean alga Coleochaete led Manhart and Palmer (1990)
to propose this alga to be the sister group of land plants.
What kind of 16S23S 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 1997
). 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)
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 23S5S 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 5S23S 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. 1996
); 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 610 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)
, who used Southern hybridization to detect rRNA genes in three species of Anabaena and one species of Nostoc. Chen and Widger (1993)
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 1981
; Williamson and Doolittle 1983
), and one of which contains a tRNAIle pseudogene but has not been completely sequenced (Williamson and Doolittle 1983
). Genome sequencing of Synechocystis (Kaneko et al. 1996
) 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)
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)
, 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)
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)
, 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)
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)
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)
determined that both the tRNA-lacking and the tRNA-containing 16S23S 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 16S23S ITS Sequence Data in Fine-Scale Phylogenetic Studies
The use of the 16S23S ITS region in studies of phylogeny (reviewed Gürtler and Stanisich 1996
; Lu 1999
; Li 2000
), molecular evolution (Antón, Martinez-Murcia, and Rodríguez-Valera 1998
), or population genetics (Navarro et al. 1992
; Otsuka et al. 1999
; Scheldeman et al. 1999
) 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 16S23S ITS regions depends on cyanobacterial-specific primers developed by Wilmotte, Van der Auwera, and De Wachter (1993)
, Nelissen et al. (1994)
, Wilmotte (1994)
, and Nübel, Garcia-Pichel, and Muyzer (1997)
. One of these primers sits at the 5' end of the 23S rRNA molecule. Gürtler and Barrie (1995)
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. 1992
; Lu et al. 1997
; West and Adams 1997
; Vinuesa et al. 1998
; Scheldeman et al. 1999
). 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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Acknowledgements |
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Footnotes |
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1 Keywords: Internal transcribed spacer
cyanobacteria
intergenic spacer region
rRNA
phylogenetics
population genetics
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
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