Department of Physiological Botany, Evolutionary Biology Centre, Uppsala University, Villavägen 6, Sweden
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although all Nostoc sequences examined shared high similarity, differences were observed in one stem-loop. This stem-loop could be divided into two classes, both built up from two base pairing heptanucleotide repeats. Size variation was primarily caused by different numbers of repeats, but some strains also contained additional sequences in this stem-loop not following the heptanucleotide repeat motif. Several sequences showing similarity with these additional sequences were identified in the Nostoc punctiforme genome. Furthermore, the regions flanking these sequences contained the same, or similar, heptanucleotide repeats as those flanking the corresponding sequences in the intron. It is proposed that both slipped strand mispairing during replication and homologous recombination among different loci in the genome are important processes causing variation between introns.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Group-I and -II introns are two types of RNA enzymes that catalyze their own splicing by different mechanisms (Saldanha et al. 1993
). Although both groups are widely distributed, group-I introns have a broader phylogenetic distribution interrupting a wide variety of mitochondrial and plastid genes, nuclear rRNA genes, bacterial tRNA genes, and protein-coding genes of eukaryotic organisms and viruses. The ability of group-I introns to catalyze their own splicing is dependent on their highly conserved secondary and tertiary structures. Different group-I introns have relatively little sequence similarity, but all share a series of short conserved elements, P, Q, R, and S, known as the catalytic core (Cech 1988
).
The tRNALeu (UAA) intron in cyanobacteria is a group-I intron and has been suggested to be of ancient origin because it interrupts, in all known cases, the tRNA gene at a conserved position in the anticodon triplet (between the second and third base). Moreover, the tRNALeu (UAA) introns from cyanobacteria and chloroplasts show high homology and they are more closely related to each other than to any other known sequences, indicating an early origin. The fact that the intron is not found in all, but in many cyanobacterial and chloroplast lineages is best explained by multiple losses (Paquin et al. 1997
; Besendahl et al. 2000
). However, the evolutionary history of the tRNALeu (UAA) intron is still debated, and it has been suggested that the intron is of more recent origin and has been introduced through lateral transfer (Rudi and Jakobsen 1997
, 1999
). In this study we will discuss the evidence supporting these theories.
In a number of studies, we have used the tRNALeu (UAA) intron as a marker for diversity and specificity of cyanobacteria symbiotically associated with plants and fungi (Paulsrud and Lindblad 1998
; Paulsrud, Rikkinen, and Lindblad 1998
; Costa, Paulsrud, and Lindblad 1999
; Paulsrud, Rikkinen, and Lindblad 2000
; Costa et al. 2001
; Paulsrud, Rikkinen, and Lindblad 2001
). The symbioses we have been interested in all contain cyanobacteria of the Nostoc type as their symbiont. The tRNALeu (UAA) intron has been instrumental in revealing both interesting patterns of diversity and spatial distribution of the cyanobacterial symbionts. Our previous studies provided us with a large number of sequences from closely related Nostoc strains from natural populations. Using these sequences, as well as other tRNALeu (UAA) intron sequences from the databases, it is possible to study evolutionary patterns in this genetic marker and to thus gain interesting insights into its evolution.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Listed in table 1
are all strains and GenBank accession numbers of sequences used in the present study. Strains are arranged according to Bergey's Manual of Systematic Bacteriology (Garrity 2001
, pp. 473599). All sequences were imported to MacVectorTM 7.0 for alignments (using ClustalW) and manual manipulations. The alignment of the tRNALeu (UAA) intron sequences was adjusted manually to align homologous positions using the secondary structure model suggested for the Anabaena sp. PCC 7120 intron (Cech, Damberger, and Gutell 1994
). Alignment of the regions shown as schematic hairpins in figure 1
was not attempted. Type-II intron sequences (Rudi and Jakobsen 1999
), deposited in GenBank as cyanobacterial, were not considered in this study (see Discussion).
|
|
RNA mfold Version 2.3 (http://bioinfo.math.rpi.edu/mfold/rna/form1.cgi) was used to predict the hairpin structure formed by the first variable region of the intron in the two Nostoc symbionts Nos9 and Nos30 (fig. 3
). Default settings were used, except for the temperature parameter, which was set to 20°C (Zuker 1989
).
|
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All 54 Nostoc strains share a highly conserved intron sequence having only few variable positions. With one exception, a base pair in element P9, all these variable positions also show variability when different strains from different subsections are compared. In addition to these positions, the comparison of cyanobacteria from different subsections also contains several additional variable positions, especially in element P2 (fig. 1B
). However, the loop of the element P2 (L2), which is known to be involved in tertiary interactions with the element P8 (Cech, Damberger, and Gutell 1994
), is conserved.
In cases where variation in single nucleotides occurs, the secondary structure of the intron is often retained. This can be seen both in cases where a change in one position is accompanied by a change in the base pairing strand so that different sequences either have a G:C or A:U base pair in this position, as well as where base pairing of G:U type allows changes to occur on one strand without disturbing the base pairing structure. In figure 1
we have labeled these degenerate bases where different sequences among those used to construct the consensus sequence show either different canonical base pairs (that is either A:U or G:C) or differences between sequences involving G:U base pairing, in combination with either A:U or G:C base pairing. As can be seen in figure 1
, most of the degenerate positions in base pairing regions are also labeled to indicate the retained structure among different sequences. There are two possible explanations for this: (1) there may be a requirement for base pairing to retain the structure needed for autocatalysis of the intron, which would give a selection pressure for compensatory changes, and (2) the tendency for a higher mutation rate in unpaired or mispaired bases on structures formed by single-stranded DNA during, for example, transcription may also play an important role (Wright 2000
).
Heptanucleotide Repeats as a Source of Variation
When all available Nostoc tRNALeu (UAA) intron sequences are compared, most differences are found in the first variable region of the intron. This region was found to consist of degenerate heptanucleotide repeats (fig. 2
) that fold into a hairpin structure (fig. 3
) allowing base pairing of the repeats. Figure 2
shows the different heptanucleotide repeats in this region for all Nostoc tRNALeu (UAA) intron sequences. Size variation in the variable region is caused by different numbers of repeats and, in some cases, by the insertion of other genetic elements not having the heptanucleotide repeats. These elements are represented as schematic hairpins in figure 2
. For example, when comparing Nos28 and Nos29, an indel of a single repeat is responsible for their different sizes. On the other hand, when comparing Nos9 and Nos30, the size difference is caused by an additional sequence inserted within one repeat (fig. 3
).
|
Increasing numbers of short-sequence DNA repeats are being identified in prokaryotes as more genomes become available and different algorithms that allow a simple way of detecting repeats are developed. Also, in cyanobacteria, several short-sequence repeats have been found, but the significance of their existence is still unknown (Mazel et al. 1990
; Asayama et al. 1996
; Lindberg, Hansel, and Lindblad 2000
). A possible mechanism involved in their formation is slipped strand mispairing occurring in combination with inadequate DNA mismatch repair pathways (Strand et al. 1993
). Also, the peculiar secondary structure of repetitive DNA allows mismatching of neighboring repeats, and depending on the strand orientation, repeats can be inserted or deleted during DNA duplication mediated by DNA polymerase (van Belkum et al. 1998
). These mechanisms can be responsible for the variation observed in the number of repeats in the variable region of the Nostoc tRNALeu (UAA) intron. The fact that sequences from Nostoc strains are different in this region has some implications for the alignment of sequences based on secondary structure predictions. Although it is often necessary to use secondary structure predictions in order to align homologous positions, as is the case with these intron sequences, one must be cautious when aligning hypervariable regions with little sequence similarity but with shared structural features (Hancock and Vogler 2000
). In this case, the Nostoc sequences could be divided into two classes with very different primary sequence but similar secondary structure in the variable region. To assume that the two classes are homologous based on their structural features would most likely be wrong. Supporting this is the fact that this region, in more distantly related cyanobacteria, such as Anabaena cylindrica and Calothrix desertica PCC7102 (GenBank accession numbers U83251 and U83252, respectively), contains the same heptanucleotide repeat motifs as the Nostoc class-1 sequences.
Sequences that did not follow the heptanucleotide repeat motif formed an additional hairpin that could be found interrupting a single repeat (e.g., Nos30) or between two consecutive repeats (e.g., Nos20) (fig. 2 ). These additional sequences were of different lengths; five were 24 nucleotides, one was 48 (2 x 24), and the other two were 42 and 45 nucleotides long. Figure 3 shows the hairpin formed by the first variable regions of the tRNALeu (UAA) intron from two Nostocs. As can be seen, the two regions share a high level of similarity and differ only by the extra hairpin formed by the sequence not having the heptanucleotide repeat motif. The genome sequence of N. punctiforme ATCC 29133 (Version 08jun00, last updated on September 1, 2000http://www.jgi.doe.gov/JGI_microbial/html/nostoc/nostoc_homepage.html) was searched using all these additional sequences. Several similar sequences were found but only for the 24-nucleotide-long sequences (fig. 4 ). Most were situated in noncoding regions but some were found interrupting ORFs. The number of the contigs and the positions where these sequences were found are indicated in figure 4 . As the genome sequence is incomplete, sequence annotations may change when a more detailed analysis is made available. It should be pointed out that we are comparing sequences from different symbiotic Nostocs with the genome sequence of N. punctiforme, which does not contain any additional sequences in the variable region of the intron. The sequences found in the genome sequence show great similarity with their intron counterparts. When analyzing the regions flanking these similar sequences, heptanucleotide repeats similar or identical with those found in the intron sequences were identified (fig. 4 ). An explanation for the insertion of these additional sequences, consistent with our observations, is a mechanism allowing the exchange of different DNA sequences between separate regions of the genome containing similar heptanucleotide repeats motifs. Such a mechanism would involve homologous recombination between different loci in the genome containing similar heptanucleotide repeat motifs.
|
Recently however, data was presented that suggested a polyphyletic origin of tRNALeu (UAA) introns within the cyanobacterial radiation (Rudi and Jakobsen 1997
, 1999
). Several authors have also cited these studies when the origin of the tRNALeu (UAA) intron has been discussed (Paquin, Heinfling, and Shub 1999
; Besendahl et al. 2000
). The evidence they presented for a complex origin of cyanobacterial tRNALeu (UAA) introns include the following: (1) presence of two different types of tRNALeu introns sometimes even in the same strain, one being of the type initially reported from cyanobacteria and chloroplasts and a second type more similar to other tRNA introns, and (2) lack of intron in several lineages.
The interpretation that this implies a complex pattern of intron evolution is, however, not unproblematic. The two different types of tRNALeu introns were found to be situated in different types of tRNA genes. The new intron type was inserted into a new type of tRNA gene; this would therefore not be a case of lateral transfer of introns but rather a case of a different tRNA gene containing a different intron. When the conventional tRNALeu (UAA) gene contained introns, they were always of the well-known type. It is also worth noting that all those sequences were obtained as PCR products from cultures from the NIVA collection, which consists of unialgal, but not axenic, cultures (Rudi and Jakobsen 1997
, 1999
). The cyanobacterial origin of this second type of tRNA gene and intron thus remains to be shown by cloning of a greater genomic fragment containing both the new type of intron and clearly cyanobacterial genes. This is especially important because tRNALeu introns recently have been detected in other eubacteria such as Pseudomonas (GenBank accession number AY029760), which is a potential contaminant of cyanobacterial cultures. The many cyanobacterial genomes that are currently being sequenced will undoubtedly soon shed light on this controversy.
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When analyzing size differences between closely related Nostoc sequences, these were found to be caused primarily by different numbers of copies of heptanucleotide repeats and, in some cases, because of additional sequences. Slipped strand mispairing was suggested to cause the difference in number of repeats, and homologous recombination within the genome was suggested to give rise to the additional sequences.
![]() |
Supplementary Material |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
1 Contributed equally to this study
Keywords: cyanobacteria
tRNALeu intron
evolution
heptanucleotide repeat
Nostoc
Address for correspondence and reprints: José-Luis Costa, Department of Physiological Botany, Evolutionary Biology Centre, Uppsala University, Villavägen 6, SE-752 36 Uppsala, Sweden. jose.costa{at}ebc.uu.se
.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asayama M., M. Kabasawa, I. Takahashi, T. Aida, M. Shirai, 1996 Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains FEMS Microbiol. Lett 137:175-181[ISI][Medline]
Bell-Pedersen D., S. Quirk, J. Clyman, M. Belfort, 1990 Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications Nucleic Acids Res 18:3763-3770[Abstract]
Besendahl A., Y. L. Qiu, J. H. Lee, J. D. Palmer, D. Bhattacharya, 2000 The cyanobacterial origin and vertical transmission of the plastid tRNALeu group-I intron Curr. Genet 37:12-23[ISI][Medline]
Cech T. R., 1988 Conserved sequences and structures of group I introns: building an active site for RNA catalysisa review Gene 73:259-271[ISI][Medline]
Cech T. R., S. H. Damberger, R. R. Gutell, 1994 Representation of the secondary and tertiary structure of group-I introns Nat. Struct. Biol 1:273-280[ISI][Medline]
Costa J.-L., P. Paulsrud, P. Lindblad, 1999 Cyanobiont diversity within coralloid roots of selected cycad species FEMS Microbiol. Ecol 28:85-91[ISI]
Costa J.-L., P. Paulsrud, J. Rikkinen, P. Lindblad, 2001 Genetic diversity of Nostoc endophytically associated with two bryophyte species Appl. Environ. Microbiol 67:4393-4396
Darnell J. E., W. F. Doolittle, 1986 Speculations on the early course of evolution Proc. Natl. Acad. Sci. USA 83:1271-1275[Abstract]
Garrity G., 2001 Bergey's manual of systematic bacteriology, Vol. 1 Springer-Verlag, New York
Hancock J. M., A. P. Vogler, 2000 How slippage-derived sequences are incorporated into rRNA variable- region secondary structure: implications for phylogeny reconstruction Mol. Phylogenet. Evol 14:366-374[ISI][Medline]
Kuhsel M. G., R. Strickland, J. D. Palmer, 1990 An ancient group I intron shared by eubacteria and chloroplasts Science 250:1570-1573[ISI][Medline]
Lindberg P., A. Hansel, P. Lindblad, 2000 hupS and hupL constitute a transcription unit in the cyanobacterium Nostoc sp. PCC 73102 Arch. Microbiol 174:129-133[ISI][Medline]
Mazel D., J. Houmard, A. M. Castets, N. Tandeau de Marsac, 1990 Highly repetitive DNA sequences in cyanobacterial genomes J. Bacteriol 172:2755-2761[ISI][Medline]
Palmer J. D., J. M. Logsdon Jr., 1991 The recent origins of introns Curr. Opin. Genet. Dev 1:470-477[Medline]
Paquin B., A. Heinfling, D. A. Shub, 1999 Sporadic distribution of tRNAArg (CCU) introns among alpha-purple bacteria: evidence for horizontal transmission and transposition of a group I intron J. Bacteriol 181:1049-1053
Paquin B., S. D. Kathe, S. A. Nierzwicki-Bauer, D. A. Shub, 1997 Origin and evolution of group I introns in cyanobacterial tRNA genes J. Bacteriol 179:6798-6806[Abstract]
Paulsrud P., P. Lindblad, 1998 Sequence variation of the tRNALeu intron as a marker for genetic diversity and specificity of symbiotic cyanobacteria in some lichens Appl. Environ. Microbiol 64:310-315
Paulsrud P., J. Rikkinen, P. Lindblad, 1998 Cyanobiont specificity in some Nostoc-containing lichens and in a Peltigera aphthosa photosymbiodeme New Phytol 139:517-524[ISI]
. 2000 Spatial patterns of photobiont diversity in some Nostoc-containing lichens New Phytol 146:291-299[ISI]
. 2001 Field investigations on cyanobacterial specificity in Peltigera aphthosa (L.) Willd New Phytol 152:117-123[ISI]
Reinhold-Hurek B., D. A. Shub, 1992 Self-splicing introns in tRNA genes of widely divergent bacteria Nature 357:173-176[ISI][Medline]
Rudi K., K. S. Jakobsen, 1997 Cyanobacterial tRNALeu (UAA) group I introns have polyphyletic origin FEMS Microbiol. Lett 156:293-298[ISI][Medline]
. 1999 Complex evolutionary patterns of tRNALeu (UAA) group I introns in cyanobacterial radiation J. Bacteriol 181:3445-3451
Saldanha R., G. Mohr, M. Belfort, A. M. Lambowitz, 1993 Group I and group II introns FASEB J 7:15-24
Shub D. A., 1991 The antiquity of group I introns Curr. Opin. Genet. Dev 1:478-484[Medline]
Strand M., T. A. Prolla, R. M. Liskay, T. D. Petes, 1993 Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair Nature 365:274-276[ISI][Medline]
van Belkum A., S. Scherer, L. van Alphen, H. Verbrugh, 1998 Short-sequence DNA repeats in prokaryotic genomes Microbiol. Mol. Biol. Rev 62:275-293
Wright B. E., 2000 A biochemical mechanism for nonrandom mutations and evolution J. Bacteriol 182:2993-3001
Xu M. Q., S. D. Kathe, H. Goodrichblair, S. A. Nierzwicki-Bauer, D. A. Shub, 1990 Bacterial origin of a chloroplast intronconserved self-splicing group-I introns in cyanobacteria Science 250:1566-1570[ISI][Medline]
Zuker M., 1989 On finding all suboptimal foldings of an RNA molecule Science 244:48-52[ISI][Medline]