Gene Conversion Drives Within Genic Sequences: Concerted Evolution of Ribosomal RNA Genes in Bacteria and Archaea

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1 J Mol Evol (2000) 51: DOI: /s Springer-Verlag New York Inc Gene Conversion Drives Within Genic Sequences: Concerted Evolution of Ribosomal RNA Genes in Bacteria and Archaea Daiqing Liao Department of Microbiology and Infectious Diseases, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, e Avenue Nord, Québec, J1H 5N4 Canada Received: 31 March 2000 / Accepted: 15 June 2000 Abstract. Multiple copies of a given ribosomal RNA gene family undergo concerted evolution such that sequences of all gene copies are virtually identical within a species although they diverge normally between species. In eukaryotes, gene conversion and unequal crossing over are the proposed mechanisms for concerted evolution of tandemly repeated sequences, whereas dispersed genes are homogenized by gene conversion. However, the homogenization mechanisms for multiple-copy, normally dispersed, prokaryotic rrna genes are not well understood. Here we compared the sequences of multiple paralogous rrna genes within a genome in 12 prokaryotic organisms that have multiple copies of the rrna genes. Within a genome, putative sequence conversion tracts were found throughout the entire length of each individual rrna genes and their immediate flanks. Individual conversion events convert only a short sequence tract, and the conversion partners can be any paralogous genes within the genome. Interestingly, the genic sequences undergo much slower divergence than their flanking sequences. Moreover, genomic context and operon organization do not affect rrna gene homogenization. Thus, gene conversion underlies concerted evolution of bacterial rrna genes, which normally occurs within genic sequences, and homogenization of flanking regions may result from co-conversion with the genic sequence. Key words: Concerted evolution Multigene families rrna Gene conversion Bacteria and Archaea Comparative genomics Introduction Multigene families exist in virtually all living organisms. Multigenes usually encode abundant molecules required for housekeeping functions. Genetic analysis reveals that genes of a multigene family exhibit extraordinary sequence homogeneity within a species, despite a normal level of divergence between orthologous genes in different species. These observations indicate that members of a multigene family evolve in a concerted fashion (Elder and Turner 1995; Liao 1999). Concerted evolution has been studied extensively for tandemly repeated multigene families in eukaryotes. In fact, the phenomenon of concerted evolution was first described for the tandemly arrayed ribosomal genes in Xenopus (Brown et al. 1972). Subsequent studies on rdna arrays in various eukaryotes have provided major insights into the mechanisms of concerted evolution (Arnheim et al. 1980; Coen et al. 1982; Elder and Turner 1995; Hillis et al. 1991; Schlötterer and Tautz 1994; Seperack et al. 1988). These studies, along with theoretical modeling, led to three major models that can account for sequence homogenization of tandemly repeated genes: unequal crossing over (Ohta 1976; Smith 1976; Szostak and Wu 1980), gene conversion (Dover 1982; Edelman and Gally 1970; Gangloff et al. 1996; Hillis et al. 1991; Nagylaki and Petes 1982; Ohta and Dover 1983), and gene amplification (Weiner and Denison 1983). The relative roles of these mechanisms in concerted evolution of large tandem arrays, such as rdna and alphoid satellites, are difficult to assess because the experimental analysis of these arrays

2 306 Fig. 1. Genomic distribution of the prokaryotic rrna genes. Four representative prokaryotic genomes are illustrated. The size (kb) of the entire genome of each microorganism is indicated in parenthesis following the name of each species. The nucleotide coordinates of the rrna genes were according to the RNA gene table in Entrez Genomes. The rrna gene cluster is depicted as an arrow, and the distance (kb) between each cluster is indicated. For comparison, the tandem-arrayed organization of the rrna genes (rdna array) in two eukaryotic species is also shown. Note that the figure is not drawn to scale. with essentially identical repeats has proven to be very challenging. Although the eukaryotic rrna genes are the best studied examples of concerted evolution, their high copy number (generally over 100; in some cases >1000) and the large copy size preclude exhaustive study (Elder and Turner 1995; Hillis and Dixon 1991). For example, the human rrna genes have large repeat unit ( 43 kb) that are arranged in large tandem array ( 100 repeats), and the 500 rrna genes are distributed among five nonsyntenic arrays (nucleolus organizers) (Gonzalez and Sylvester 1995; Sakai et al. 1995; Seperack et al. 1988). Even in lower eukaryotic organism yeast Saccharomyces cerevisiae, there are rdna repeats of 9.1 kb organized in a single tandem array (Warner 1989). Therefore, comprehensive analyses of multigene families with smaller gene size and fewer number of repeats would be desirable to gain a clearer understanding of the mode and extent of concerted evolution. The prokaryotic rrna gene families may represent such a simpler system. There are multicopies (generally <10) of the rrna genes in most species of prokaryotes, probably because of high metabolic demand for protein synthesis in a growing cell, as multigene families are generally rare in prokaryotes (Bacteria and Archaea). The genes for the three rrna molecules (23S, 16S, and 5S) found in the ribosome are generally linked together and cotranscribed in a single operon in prokaryotes. The typical length of these three rrna genes in prokaryotic organisms is 2900 bp (23S), 1500 bp (16S), and 120 bp (5S), and their sizes as well as sequences are generally well conserved between different prokaryotic species. Compared to the tandem-arrayed organization of eukaryotic rrna genes, multiple rrna operons are generally dispersed throughout a prokaryotic genome (Fig. 1). Like some dispersed multigene families in several eukaryotic species (Amstutz et al. 1985; Morzycka-Wroblewska et al. 1985), multiple-copy rrna genes in a prokaryotic organism also undergo concerted evolution. Though there is no a priori reason to believe that different DNA recombination mechanisms might operate for concerted evolution in prokaryotes, a systematic analysis of multigene families within a simple genome might not only reveal the mechanism responsible for sequence homogenization but also provide insights into potential influence of genomic context on concerted evolution. Such systematic analyses have become possible with the advent of complete genome sequences of many species. Up to now, complete genome sequences are available for 25 prokaryotic species as well as several eukaryotic species, and the number is increasing rapidly. The availability of DNA sequences for all members of a given multigene family provides a unique opportunity to analyze the molecular mechanisms underlying concerted

3 307 evolution. In this study, I have analyzed the sequences of 23S, 16S, and 5S rrna genes and their immediate flanking sequences in 19 completely sequenced genomes. Striking sequence homogeneity of each individual rrna gene family within a species is found, and homogenization of rrna genes most probably results from frequent gene conversion. Most interesting, genic sequences undergo much slower divergence than their flanking nongenic sequences, suggesting that homogenization most frequently takes place within the genic regions. Thus, gene sequence may be the sole genetic element responsible for their homogenization. Flanking sequence and genomic context of the rrna genes play little if any role in concerted evolution of the prokaryotic rrna genes. Materials and Methods The DNA sequences for the 16S, 23S, and 5S rrna genes and their flanking sequences were obtained from Entrez Genomes in the Gen- Bank. The rrna gene sequences in these organisms were analyzed in this study: Aquifex aeolicus, Archaeoglobus fulgidus, Bacillus subtilis, Borrelia burgdorferi, Chlamydia trachomatis, Chlamydia pneumonia, Escherichia coli, Haemophilus influenzae, Helicobacter pylori strain 26695, Helicobacter pylori strain J99, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma pneumoniae, Pyrococcus horikoshii, Rickettsia prowazekii, Synechocystis PCC6803, and Treponema pallidum. BLAST search was used to define the gene sequences when annotation does not indicate specific boundaries of the rrna genes, or when the annotated coordinates contain obvious errors. Multiple sequence alignment was carried out using CLUSTAL W as implemented in MacVector 6.1 or at the Web site of the Baylor College of Medicine Search Launcher ( Phylogenetic analyses were performed using the DNAML, NEIGHBOR programs in the PHYLIP 3.5c package (Felsenstein 1993) or the PAUP program (Swofford 1993). Distances were computed using the DNA- DIST program in the PHYLIP 3.5c package (Felsenstein 1993) according to the Kimura two-parameter model with the transition/transversion ratio of 2.0. Insertions/deletions (indels) in multiple sequence alignment were discounted in parsimony analyses and distance calculation. The neighbor joining method was used to construct the phylogenetic tree shown in Fig. 2. Results Striking Within-Species Sequence Homogeneity of the rrna Genes in Bacteria and Archaea Thus far, 25 bacterial and archaeal genomes have been completely sequenced. Among them, 6 are Archaea and 19 are Bacteria. For the 19 genomes (both bacterial and archaeal) we surveyed in this study (see Table 1), most of them contain multiple-copy (paralogous) rrna genes, but 7 species carry only one copy of each rrna gene. The genes for 23S, 16S, and 5S are normally organized in a single operon in the order of 16S-23S-5S (Table 1) and are co-transcribed. Notably, however, that the 16S- 23S-5S operon structure has been broken in several species, where either 16S or 5S rrna genes are no longer linked to the operon, and thus become orphaned in other locales within the genome. Interestingly, no stand-alone 23S genes are found in these genomes. The rrna operons are generally dispersed throughout the genome (see Fig. 1), although tandem arrayed structure was found for several operons in B. subtilis and for the two 23S-5S operons in B. burgdorferi. The sequences of rrna genes are extremely homogenous within a genome. No sequence heterogeneity was detected for multiple-copy 23S, 16S, or 5S genes in A. aeolicus, C. trachomatis, H. influenzae, H. pylori (strain J99), M. thermoautotrophicum, and Synechocystis PCC6803. Five of these six species have only two rrna operons, whereas there are six operons in H. influenzae. Thus, the number of genes in a genome may not affect the degree of homogenization. Indeed, there are 10 and 7 rrna operons in B. subtilis and E. coli, respectively; again, the rrna genes in these two species display remarkable sequence homogeneity. Figure 2 shows a phylogenetic tree based on 16S rrna gene sequences from the 19 completely sequenced genomes listed in Table 1. The horizontal branch lengths reflect the degree of sequence divergence. To facilitate visual observation, all genes within a genome are shaded in the tree, and the average sequence divergence (distance) values within a species is also indicated next to the shaded areas. From the branch lengths within a species as well as the divergence values, we can easily see the extraordinary withinspecies homogeneity. From this tree, we can also easily appreciate that sequence homogeneity within a genome is much greater than that between genomes. For example, the average divergence among the seven E. coli 16S rrna gene is per site, whereas the average divergence between the 16S rrna genes in E. coli and its close relative H. influenzae is , which is 24 times greater than the within-species divergence in E. coli. Essentially identical patterns were observed for 23S and 5S rrna genes (data not shown). These results clearly show that the bacterial and archaeal rrna genes undergo concerted evolution. Intriguingly, obvious sequence heterogeneity was observed for the intergenic spacers between 16S and 23S genes in B. subtilis, E. coli, H. influenzae, and T. pallidum. This is mainly due to the presence or absence of trna genes or the presence of different trna genes in this intergenic region. For examples, in E. coli, trna Ala and trna Ile are present in the 16S 23S intergenic spacers in operons rrna, rrnd, and rrnh, while only trna Glu is found in the corresponding region in operons rrnb, rrnc, rrne, and rrng. In B. subtilis, the 16S 23S region contains trna Ile and trna Ala in both rrno and rrna operons, whereas the other eight operons have no trna genes in the corresponding region. In addition, other types of changes, including substitutions and insertions/ deletions (indels), are also frequent in the intergenic

4 308 Table 1. Organism Ribosomal RNA genes in Bacteria and Archaea Number of operons Operon organization Aquifex aeolicus 2 16S-(tRNA Ala )-23S-5S (both operons) Archaeoglobus fulgidus 1 16S-(tRNA Ala )-23S 5S (orphan) Bacillus subtilis 10 16S-(tRNA Ile -trna Ala )-23S-5S (rrna and rrno) 16S-23S-5S (rrnb, rrnd, rrne, rrnh, rrni, rrnj, rrng and rrnw). rrnj-rrnw, and rrni-rrnh-rrng form two separate tandem clusters Borrelia burgdorferi a 2 16S (orphan) b...23s-5s-23s-5s (tandem duplication of 23S-5S cluster) Chlamydia trachomatis 2 16S-23S-5S (both operons) Chlamydia pneumoniae 1 16S-23S-5S Escherichia coli 7 16S-(tRNA Ile -trna Ala )-23S-5S (rrna and rrnh) 16S-(tRNA Glu )-23S-5S (rrnb, rrnc, rrne, and rrng) 16S-(tRNA Ile -trna Ala )-23S-5S-(tRNA Thr )-5S (rrff) (rrnd) Haemophilus influenzae 6 16S-(tRNA Ile -trna Ala )-23S-5S (rrna, rrnc, and rrnd) 16S-(tRNA Glu )-23S-5S (rrnb, rrne, and rrnf) Helicobacter pylori c strain S-5S (both operons) 16S (orphan, two copies) Helicobacter pylori strain J S-5S (both operons) 16S (orphan, two copies) Methanobacterium thermoautotrophicum 2 16S-(tRNA Ala )-23S-5S (both operons) Methanococcus jannaschii 2 16S-(tRNA Ala )-23S (rrna) 16S-(tRNA Ala )-23S-5S (rrnb) 5S (orphan) Mycobacterium tuberculosis 1 16S-23S-5S Mycoplasma genitalium 1 16S-23S-5S Mycoplasma pneumoniae 1 16S-23S-5S Pyrococcus horikoshii 1 16S-(tRNA Ala )-23S 5S (orphan) Rickettsia prowazekii 1 23S-5S 16S (orphan) Synechocystis PCC S-(tRNA Ile )-23S-5S (both operons) Treponema pallidum 2 16S-(tRNA Ile )-23S-5S (rrna) 16S-(tRNA Ala )-23S-5S (rrnb) a Linear chromosome. b The 16S gene is located 3 kb 5 to the first 23S gene. c A truncated orphan 5S is also present in the genome. GenBank accession numbers for these complete genomes are: AE (A. aeolicus), AE (A. fulgidus), AL (B. subtilis), AE (B. burgdorferi), AE (C. trachomatis), AE (C. pneumoniae), U00096 (E. coli), L42023 (H. influenzae), AE (H. pylori 26695). AE (H. pylori J99), AE (M. thermoautotrophicum), L77117 (M. jannaschii), AL (M. tuberculosis), L43967 (M. genitalium), U00089 (M. pneumoniae), AP to AP (P. horikosshii), AJ (R. prowazekii), AB (Synechocystis PCC6803), AE (T. pallidum). spacers. The remarkable contrast of homogeneity in the genic sequences to heterogeneity in the intergenic spacers implies that concerted evolution does not reflect gross replacement of one operon with another; rather it is a gradual, region-by-region homogenization process (see below). Patchy Gene Conversion Is Responsible for the Homogenization of the rrna Genes Occasional sequence heterogeneity is found among the multi-copy rrna genes within the genome in several species; this may prove to be informative to decipher the molecular mechanisms responsible for rrna gene homogenization. Indeed, numerous informative sites are present in the rrna genes in E. coli, B. subtilis, and several other organisms. Figure 3 illustrates the multiple sequence alignment within a few segments of E. coli 16S genes (rrs), 23S genes (rrl), and 16S 23S intergenic spacers. Several sequence conversion events have evidently occurred between rrsc, rrsd, and rrsg in the illustrated regions (Fig. 3A). Because these sequence tracts share multiple substitutions, it must result from sequence conversion and must not be due to multiple independent mutations (convergent evolution). Likewise, apparent conversion events were observed in several regions of 23S rrna genes, and the 16S 23S intergenic spacers, as depicted in the panels B and C of Fig. 3, as well as in the rrna genes and intergenic spacers in B. subtilis (data not shown). Individual conversion tracts appear to be short, apparently <500 bp (short conversion tracts were also observed in other organisms, see Discussion). For example, although the two segments shown in Fig. 3A are very

5 309 Fig. 2. A phylogenetic tree based on all 16S rrna gene sequences from 19 completely sequenced genomes. The DNA sequences of 16S rrna genes were aligned using CLUSTAL W and the distance values (number of substitutions per site) were computed using DNADIST program (Felsenstein 1993) according to the Kimura two-parameter model with the transition to transversion ratio of 2.0. The tree was constructed using neighbor joining method as implemented in program NEIGHBOR in the PHYLIP package (Felsenstein 1993). The paralogous genes within a genome are shaded and the average distance among them is indicated next to the shaded area. The species names are abbreviated: Aae: A. aeolicus, Afu: A. fulgidus, Bsu: B. subtilis, Bbu: B. burgdorferi, Ctr: C. trachomatis, Cpn: C. pneumoniae, Eco: E. coli, Hin: H. influenzae, Hpy: H. pylori 26695, Hpy-J99: H. pylori J99, Mth: M. thermoautotrophicum, Mja: M. jannaschii, Mtu: M. tuberculosis, Mge: M. genitalium, Mpn: M. pneumoniae, Pho: P. horikoshii, Rpr: R. prowazekii, Syn: Synechocystis PCC6803, Tpa: T. pallidum. The multiple sequence alignment used for constructing this tree is available on request. close to each other only 150 bp apart the conversion between rrsd and rrsg in the two regions may not be due to a single event because there are three nucleotide differences between rrsg and rrsd in the sequence between the two illustrated regions (from position 98 to position 245). Moreover, there is no evidence of recent sequence conversion between sequences 5 to the rrsd and rrsg. In fact, rrsd and rrsg differ significantly from each other in the 5 flanking sequences (Fig. 3D). Also, several independent gene conversion events that convert short sequence stretches are evident in other regions between operons rrnd and rrng. One can see a putative conversion tract in the intergenic spacers between 16S and 23S genes of rrnd and rrng around the position 430,

6 310 depicted in Fig. 3C. This short sequence stretch of only 18 bp identifies two deletion tracts of 5 and 4 bps, and two tracts of substitutions of 2 bps that are shared between rrnd and rrng. However, the overall sequence similarity in the intergenic spacers between the two operons are minimal, especially between position 60 and 106, as shown in Fig. 3C. Furthermore, rrnd has two trna genes (trna Ile and trna Ala ), whereas rrng has only one trna gene (trna Glu ) in the 16S 23S intergenic regions. Similarly, gene conversion may have also taken place in between positions 2795 and 2815 of rrld and rrlg (Fig. 3B); again, this tract appears to be short because rrld suffered several rare single nucleotide deletions in the region close to the conversion tract depicted in Fig. 3B (the first deletion at position 2714, just 82 bps 5 to position 2795). Thus, we conclude that sequence conversion between paralogous rrna genes is patchy and occurs in discontinuous tracts throughout the genic regions as well as in the immediate flanking sequences in multiple conversion events. Rapid Divergence of Sequences Flanking rrna Genes Sequences immediately flanking paralogous rrna genes in a genome are also highly homogenous, suggesting that they are also subject to homogenization. For example, the sequences of about 150 bps immediately 5 to the 16S rrna genes among the seven operons in E. coli are virtually identical (with only two transitions). I note, however, that the flanking sequences generally accumulate more substitutions than the genic sequences. Furthermore, the longer the distance between the flanking sequence and the genic region is, the more substitutions we see in the flanking regions. In E. coli, although the 150-bp segments immediately flanking the 16S rrna gene are homogenous in all operons (the black-filled rectangle in Fig. 3D), the sequences further upstream are quite heterogeneous. Operons rrna, -B, -C, and -G share about 210-bp homogenous sequences further upstream (gray-filled rectangle in Fig. 3D), whereas operons rrnd, -E, and -H share a segment of about 150 bps (crosshatched rectangle in Fig. 3D), which differs significantly from the gray-filled areas, although a reliable alignment between these regions can still be established. Similar situations were also observed in the 16S 23S intergenic spacer (Fig. 3C) and 3 flanking sequences of rrf genes (Fig. 3E). To quantitatively compare the sequence divergence between genic sequences and flanking regions, we have computed the distances between paralogous sequences for different regions. To avoid overestimating the distance values, we considered only the portions of the flanking sequences for which a reliable multiple sequence alignment can be obtained, which nonetheless might underestimate the real divergence, as this measure might have excluded additional nucleotide changes in the

7 311 Fig. 3. Gene conversion tracts between paralogous rrna operons in the E. coli genome. Segments from different regions of the seven rrn operons were aligned. Shared nucleotides due to sequence conversion in each segment are depicted in white boxes except between position 60 and 106 in panel C, where accurate alignment is not possible. The positions of the first, last nucleotides or a reference position in between for the illustrated segments are indicated on top. A Selected segments from the 16S rrna genes (rrs). B Selected segments from the 23S rrna genes (rrl). C The intergenic spacers between 16S and 23S rrna genes. Between positions 60 and 106, sequences sharing high level of identities are differently marked: rrnb and rrng, shaded: rrnc and rrne, indicated with asterisk; rrna, rrnd, rrnh, boxed. The sequences between positions 106 and 338 contain different trna genes, and are indicated with dashed line. D Sequences 5 to the rrs genes. The homogenous sequences in all operons are depicted with black rectangles; further upstream sequences are not homogenous, but high identities are found among different operons and marked either with gray rectangles (operons rrna, rrnb, rrnc, and rrng), or hatched rectangles (rrnd, rrne, and rrnh). The approximate length of each segment is indicated. Unrelated flanking sequences are denoted by thick dashed lines. E The 3 flanking sequences of rrf genes. The 5S genes (rrf) are shown as white rectangles. A 5-bp segment immediately 3 to the rrf genes is shared in all operons and shown as black rectangles. Further downstream sequences are not shared by all operons. A segment of about 210 bps is shared by three operons (rrna, rrnb, and rrnf) and depicted with a gray rectangle. Note that rrnf contains rrff only, which lies 3 to the rrnd operon, separated only by 135 bps (also see Table 1). Two operons, rrnc and rrnh, share a segment of about 130 bps as depicted in cross-hatched rectangles. Sizes of each shared fragment are indicated. Unrelated flanking sequences are shown as thick dashed lines.

8 312 distance calculations. Figure 4 shows bar graphs of average distance values for different segments of the paralogous operons in E. coli (Fig. 4A), B. subtilis (Fig. 4B), and H. pylori (Fig. 4C). We can see clearly that the average distances between genic sequences are much smaller than that between flanking sequences in almost all instances. Comparison of rrna operons in two strains of H. pylori indicated that divergence is dramatically accelerated in the region flanking rrna genes. H. pylori strains and J99 were both sequenced completely (Alm et al. 1999; Tomb et al. 1997). In both strains, all three rrna genes exist in two copies, and 16S rrna genes are not linked to 23S 5S rrna operons (see Table 1). The genomes of both strains are highly conserved not only in gene sequences but also in overall genomic structure, consistent with a low level of evolutionary divergence between them (Alm et al. 1999). Indeed, the genic sequences of 5S rrna genes (129 bp long) are identical, and those of 16S (1501 bp) and 23S rrnas (2975 bp) differ only in a few positions between the two strains: there are 12 substitutions (10 transitions, 2 indels) between the 16S genes and 17 substitutions (9 transitions, 7 transversion, and 1 indel) between the 23S rrna genes. We then analyzed the sequences flanking the orphaned 16S genes and those flanking 23S and 5S genes as well as the 23S 5S intergenic spacers. Remarkably, although the flanking sequences are virtually identical within each strain, they have undergone striking divergence, even in the short 23S 5S intergenic spacers (230 bps) (Fig. 4C). The extreme divergence value between the 5 flanking sequences of the 23S rrna genes shown in Fig. 4C is not an exaggeration but might reflect the fact that the homogenous sequences are extremely long in this region within a strain (over 7 kb identical sequences preceding the two 23S rrna genes in strain 26695), and the alignable sequences are also relatively long between the two strains (about 1 kb). Figure 4E shows a multiple sequence alignment of sequences 5 to the 16S rrna genes in both H. pylori strains. The aligned region of 515 bps contains 24 transitions, 12 transversions, and 8 indels. The ratio of transitions to transversion appears normal. However, the frequent indel events are unexpected, as indels are generally considered very rare events. This pattern of mutations could result from gene conversion between two significantly divergent flanking sequences, as it was shown that recombination between divergent DNA molecules can cause dramatic deletion, insertion, and extension of conversion tract into nonhomologous flanking region (Bailis et al. 1992; Belmaaza et al. 1994). Regardless of the mechanisms that generated the remarkable flanking sequence change, the high divergence values of the flanking sequences compared with low divergence of the genic sequences between the two H. pylori strains must reflect that gene conversion responsible for homogenization occurs much more frequently within the genic region, as spontaneous mutations arise at approximately equal frequency for both coding and noncoding sequences. To further illustrate the differential divergence rates for genic and flanking sequences, I compared the flanking sequences of the rrna genes between E. coli and H. influenzae. I note that the sequences flanking rrna genes are rarely conserved between species. Nevertheless, short stretches of sequences immediately flanking the rrna genic regions can still be reliably aligned between E. coli and H. influenzae. The divergence of various regions in the ribosomal operons between the two bacterial genomes is shown in Fig. 4D. Again, the flanking sequences exhibit much higher divergence than the genic regions. Discussion Concerted evolution of multigene families is a universal phenomenon. It operates not only in complex genomes where multigene families are abundant but also in relatively simple genomes of prokaryotes where multigene families are rare. Here I have analyzed the rrna gene families in 19 completely sequenced genomes of Bacteria and Archaea. The availability and analysis of the DNA sequences of all members of a multigene family in multiple species are unprecedented and have provided a picture of the extent and mode of concerted evolution of multigene families. This study suggests that gene conversion initiated within the genic regions of the rrna genes is likely to play the major role in sequence homogenization and hence concerted evolution of dispersed rrna gene families. Gene Conversion in Concerted Evolution Several molecular mechanisms can lead to the apparent sequence conversion observed in the paralogous rrna genes, including (1) sequence conversion via reverse transcription (RT) of rrna and subsequent homologous recombination of cdna of one particular rrna molecule with nonallelic rrna genes in the genome; (2) recombination between nonallelic rrna genes (via reciprocal or copy choice mechanism); and (3) singlestranded DNA invasion of nonallelic genes and subsequent heteroduplex formation. Sequence conversion can ensue from a heteroduplex that might be resolved via DNA repair or through segregation of unrepaired heteroduplex in the next round of replication. The first two mechanisms are unlikely to be responsible for homogenization of the rrna genes for a number of reasons. First, although RT-mediated gene conversion appears to occur in S. cerevisiae (Derr and Strathern 1993), reverse transcriptase activity cannot be detected in many differ-

9 313 Fig. 4. Rapid divergence of sequences flanking rrna genes. Distance values (number of substitutions per site) were computed as described in Materials and Methods for different segments of the paralogous operons in a genome. For nongenic flanking sequences, only the portions for which a reliable multiple sequence alignment can be obtained were considered for distance calculation. This measure may nonetheless underestimate the real divergence. The vertical bars represent an average of all pairwise distances among paralogous segments, solid bars, divergence among genic sequences; gray bars, divergence of flanking sequences. The prefix pre- and post- indicate 5 or 3 flanking sequences of an rrna gene. A Sequence divergence among seven rrn operons in the E. coli K12 genome. The divergence of 3 flanking sequences of rrf (post- 5S) was based on the alignment of the homologous flanks of rrfa, rrfb, and rrff (also see Figure 3E). B Sequence divergence among 10 rrn operons in the B. subtilis genome. The divergence of 3 flanking sequences of rrf (post-5s) was based on the alignment of the homologous flanks of rrfa, rrfg and rrfo, and rrfw. C Sequence divergence between rrn operons in two H. pylori strains (J99 and 26695). The genic and flanking sequences analyzed here are identical within the genome of each strain (see panel E). No divergence was detected in four 5S genes in both strains. The 16S rrna genes are not linked to 23S 5S operons, so both flanks of 16S gene and the 5 flank of 23S gene were examined. D Comparison of rrn operons between E. coli and H. influenzae. The 3 flanking sequences of rrf genes cannot be aligned between the two species; thus, they were not included in the graph. E Alignment of 5 flanking sequences of the 16S rrna genes of H. pylori strains J99 and Divergent nucleotides are highlighted with gray shade.

10 314 ent types of cells (Inouye and Inouye 1991), including E. coli K12, from which the complete E. coli genome sequence was derived. Yet in this strain gene conversion among the paralogous rrna genes examined in this study is quite evident. Thus, RT-mediated recombination may not be involved in homogenization of multigene families. Second, unequal reciprocal recombination can, in principle, account for homogenization of tandemly repeated genes. However, nonconversion recombination mechanisms (reciprocal or via copy-choice) could not satisfactorily explain the remarkable heterogeneity of sequences flanking rrna genes (see Fig. 3) because one would expect sharp and homogenous junctions between homogenized sequences and flanking chromosomal DNA if homogenization is primarily achieved through repeated rounds of such recombinations. Furthermore, ectopic recombination between repetitive sequences can result in sequence deletion, inversion, or translocation, and such drastic genomic changes lead to genome instability and are often deleterious to the cells. Several lines of evidence suggest that gene conversion via heteroduplex formation plays a major role in homogenization of dispersed multigene families. First, homogenization of the two paralogous tuf genes in Salmonella typhimurium was suggested to be the result of gene conversion via a RecBCD-dependent mechanism (Abdulkarim and Hughes 1996). In E. coli and Salmonella, recombination hotspot Chi (5 -GCTGGTGG-3 ) isimplicated in RecBCD-mediated recombination. The Chi element is one of the most abundant repetitive oligomers in the E. coli genome (Blattner et al. 1997). Interestingly, Chi-like sequences are frequently found within the 16S and 23S rrna genes and their vicinities. For example, the sequence stretch GCTGGCGG near the 5 end of the 16S rrna gene differs from Chi by only one nucleotide at the fifth position, and this change does not appear to affect Chi function (Schultz et al. 1981). Moreover, this sequence stretch is conserved in all bacterial 16S rrna genes. Thus, it appears likely that RecBCD-mediated gene conversion might also be involved in sequence homogenization at the rrna loci. It should be noted that RecBCD/Chi system may not operate in all of these species. Nonetheless, a similar recombination machinery may be responsible for concerted evolution in other bacterial species. Second, in S. cerevisiae, gene conversion via heteroduplex formation also occurs between dispersed multigenes (Nag and Petes 1990). Similarly, genetic exchange between tandem arrayed rrna genes in S. cerevisiae is Rad52-dependent and thus may proceed via gene conversion-like mechanism (Gangloff et al. 1996). Third, analysis of the RNU2 locus in various human populations reveals that repeats within an individual U2 tandem array are more homogenous than between different arrays, which could be explained by frequent intrachromosomal recombination, such as unequal sister chromatid exchange (USCE) and/or intrachromatid gene conversion. Interestingly, concerted evolution does not lead to exchange of markers flanking the U2 tandem arrays in homologous chromosomes (Liao et al. 1997). Thus, gene conversion, not unequal crossing over, is responsible for interchromosomal recombination. In addition, detailed sequence analyses of the junction regions flanking the primate RNU2 locus have documented dramatic rearrangements and divergence of the primate RNU2 junctions (Pavelitz et al. 1999). Simple reciprocal exchanges between homologous sequences could not readily explain faster evolution of junction regions of a multigene family. Indeed, junction rearrangement and divergence of the primate RNU2 locus can be viewed as a consequence of homogenization of U2 tandem arrays by gene conversion (Pavelitz et al. 1999). Likewise, junction sequence heterogeneity and rapid divergence observed here in the bacterial rrna loci can also be explained as a consequence of gene conversion. Gene conversion near the boundaries of the rrna operons can be viewed as one-sided homologous recombination (Belmaaza and Chartrand 1994). It was shown that exogenous linear DNA carrying one homologous and one nonhomologous segment invades chromosomal DNA, and strand invasion and subsequent homologous pairing occur within the homologous segment. Sequence divergence as high as 15% between donor and recipient does not seem to interfere with initiation of recombination, as the initial stage of homologous recombination requires only a minimum length of homology (Shen and Huang 1986). Resolution of the ensuing recombination intermediates at the nonhomologous end, however, leads to illegitimate recombination, which frequently causes significant deletion, duplication, and extension of conversion tract into adjacent, nonhomologous region in the recipient DNA (Belmaaza et al. 1994; Dellaire et al. 1997; Pavelitz et al. 1999). As flanking sequences of the rrna genes diverge at a much higher rate than the genic sequences (Fig. 4), significant heterogeneity might have already been accumulated in the flanks at the time of gene conversion. Thus, one could envision that recombination in the flanking regions could further accelerate flanking sequence divergence. The gradual loss of homogenous flanking sequences as well as accelerated mutations in the junction regions at the rrna loci (see Fig. 3D and E) are consistent with the one-sided homologous recombination model. Fourth, the observed short and noncontiguous conversion tracts at the prokaryotic rrna loci are also consistent with a role for gene conversion in concerted evolution of paralogous rrna genes, as such patterns of conversion were detected in sequence transfer between the tuf genes in S. typhimurium (Abdulkarim and Hughes 1996). Discontinuous and short tracts of sequence conversion might reflect the intrinsic cellular mechanisms that may limit the length of conversion tracts. Mismatch

11 315 repair enzymes, such as MutL and MutS, are the major barrier for recombination between divergent sequences (Matic et al. 1995). They may block heteroduplex extension, as gene conversion tracts in a mismatch repairdefective strain of S. cerevisiae are significantly longer than those in an isogenic wild-type strain (Chen and Jinks-Robertson 1998, 1999). Additionally, the conversion tracts induced by DSBs in mouse embryonic stem cells are very short; most of them are less than 58 bps (Elliott et al. 1998). Further support for gene conversion in concerted evolution comes from studies on rdna sequences in triploid parthenogenetic lines of lizards. It was found that homogenization of the rdna arrays always proceeded in the same direction. Thus, biased gene conversion is likely to be responsible for concerted evolution of the rdna arrays in hybrid lines of lizards (Hillis et al. 1991). Collectively, these observations strongly suggest that gene conversion underlies concerted evolution of the paralogous rrna genes in prokaryotes. One might be concerned that the observed sequence conversion tracts in these microbial genomes may be due to errors in computer assignments of repetitive sequences within a sequenced genome, as it is possible that one sequenced segment might be assigned to more than one locus. Though the possibility of misassignments of highly repeated sequences cannot be excluded in any whole genome sequence, this is unlikely to compromise the conclusion because the most informative conversion tracts are found in the E. coli and B. subtilis genomes, whose rrna operons were already sequenced using conventional protocols before the two genomes were completely sequenced. Furthermore, the observed conversion tracts are much shorter than a single sequence read from a typical automatic sequencing run; thus, they could not be the results of sequence assembly mistakes. Differential Conversion Domains This study found that the flanking sequences of the rrna operons undergo much faster divergence than the genic regions. This is unexpected if mechanisms leading to homogenization of all members of a multigene family within a genome operate indiscriminately on both genic sequence and flanking sequences. One can invoke functional constraints or purifying selection on rrna molecules in order to explain why genic regions evolve more slowly than the flanking sequences, because mutations in flanking sequences may be inconsequential. The flanking sequences of bacterial ribosomal operons might indeed provide no biological functions because there is apparently no sequence conservation between flanking sequences of the rrna genes in different species. However, functional constraints alone cannot explain why the genic sequences of the paralogous genes are virtually identical within a genome, because there are obviously regions within the rrna molecules that can tolerate mutations, otherwise there would be little divergence between the rrna genes in different species. In this regard, it is noteworthy that foreign rrna genes with substantial sequence divergence suffice to support growth of an E. coli strain in which all resident rrna operons were inactivated (Asai et al. 1999). Thus, small variations or heterogeneity in the rrna genes do not affect the fitness of an organism, further indicating that purifying selection alone cannot account for the degree of sequence homogeneity of the rrna genes within a species. One possible explanation is that gene conversion responsible for homogenization occurs normally within the genic sequence. In this way, concerted evolution can quickly purge or spread mutations arising within the genic regions. The homogenized flanking sequences may merely be the consequence of gene conversion initiated in the genic sequence. Likewise, this could explain why sequences immediately flanking the genic sequence are more homogenous than distal flanking sequences of the rrna genes among the paralogous loci within a genome. It is unknown why gene conversion primarily occurs in the genic sequences. Active transcription at the rrna operons could potentially increase gene conversion at these loci, as it is well known that transcription can stimulate recombination (Gangloff et al. 1994). However, although the 16S 23S intergenic spacers are cotranscribed with the rrna genes, remarkable sequence heterogeneity exists in the spacer regions (Fig. 3C), suggesting that factors other than transcription may favor the genic regions as donor or recipient in gene conversion. Whatever mechanism is responsible for the differential conversion between genic sequences and their flanks, these results indicate that the genic regions of the rrna genes may be the sole cis elements that determine their concerted evolution, and flanking sequences and genomic context play little, if any, roles in this process. Gene conversion between the paralogous rrna genes is the most likely mechanism for concerted evolution of multiple-copy prokaryotic rrna genes in a genome, but one less likely alternative mechanism to explain the sequence homogeneity of rrna genes within a genome is gene duplication and ectopic reintegration into the genome, as suggested in the gene amplification model (Weiner and Denison 1983). Indeed, the six rrna operons in H. influenzae reside in nonequivalent loci when compared to the locations of the seven E. coli operons (see Fig. 1). Similarly, the relative positions of rrna operons in the genomes of archaeal species, such as M. jannaschii and M. thermoautotrophicum, are not conserved. However, genomic organizations or long-range gene orders are rarely conserved in prokaryotic organisms. For example, although a majority of the genes in H. influenzae (85% of the total genome) share significant sequence identity with their homologues in E. coli, there is no evidence for conservation of gene positions within

12 316 the genome between the two species (de Rosa and Labedan 1998; Tatusov et al. 1996). In fact, extensive chromosomal rearrangement events have shuffled the order of many genes. Interestingly, although the overall genomic organization is almost completely conserved between H. pylori strains and J99, and the position of one 23S 5S gene cluster is the same in both strains, the DNA fragment following the 5S gene in the other rrna operon appears to have suffered rearrangement, as the surrounding genes are different between the two species. As there is apparent evidence of concerted evolution between the rrna genes within each strain (see Results), DNA fragment rearrangement may be a consequence of recent gene conversion during concerted evolution, as discussed above. In summary, analysis of all paralogous rrna genes in 19 completely sequenced genomes of Bacteria and Archaea revealed striking patterns of concerted evolution within a genome. 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