Background
The science of molecular evolution has paved the way for understanding evolutionary processes in which genetic redundancy (the presence of two or more genes capable of serving the same functional role in an organism) is a major source of genetic novelty and robustness. In fact, a recent analysis of 106 bacterial genomes revealed that a significant number of genetic redundancies have persisted in individual genomes 1, and systematic gene deletion experiments have demonstrated that approximately 300 out of 4000 genes are indispensable for two bacterial species, Escherichia coli 2 and Bacillus subtilis 3, suggesting the presence of considerable redundancy in the bacterial genome. However, these findings also raise the question of how genetic redundancy is maintained within a genome, because functionally redundant genes are likely to be eliminated by selective pressure as shown in a large-scale analysis of protein-protein interactions in Saccharomyces cerevisiae, in which the redundant interactions due to duplicated genes generally are not persisted long after gene duplication 4. In order to explain the process, a theoretical model has been developed to provide insight into the retention of redundant genes, which is hypothesized to depend on their degree of functional redundancy 5, there have been no empirical studies of gene evolution to support this theoretical model directly. The study described here aimed to substantiate the model of evolution of genetic redundancy on the basis of the analysis of a ribonuclease family that has contributed to our understanding of some aspects of molecular evolution, such as adaptive evolution 67, positive Darwinian selection 8, and the origin of retroviruses with long terminal repeats (LTR) 9.
Ribonuclease H (RNase H; EC 3.1.26.4), one member of the ribonuclease family, is an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrids 10. Because various studies have revealed the presence of RNase H in eukaryotes, prokaryotes, and retroviruses, this compound is considered to be one of the most widely conserved enzymes 11. Although the physiological functions of RNase H are not fully understood, this enzyme is thought to play several roles in DNA replication 121314, DNA repair 1516, and RNA transcription 1718. In terms of its medical importance, RNase H activity in retroviruses (including HIV-1) is necessary for their replication, and the enzyme has thus been regarded as one of the drug targets for AIDS chemotherapy 19. This enzyme is also suggested to be related to the antiviral immune response in humans, because mutations of the RNase H-encoding gene have been found in individuals affected by a human neurological disease, Aicardi-Goutières syndrome 20. Therefore, the accumulation of experimental data on the secondary structures and enzymatic features of RNase H from many studies of its biological significance has provided us with an opportunity to use this knowledge in the field of molecular evolution.
Unlike retroviruses, which possess a single RNase H gene, most prokaryotic and eukaryotic genomes contain multiple RNase H genes. According to the nomenclature for these enzymes, prokaryotic RNase H is generally classified into three groups: RNase HI, HII, and HIII. Eukaryotic RNase H is divided into RNase H1 and H2 21. Phylogenetic analyses using RNase H sequences have proposed the following classification: Type 1 (prokaryotic RNase HI, eukaryotic RNase H1, and viral RNase H) and Type 2 (prokaryotic RNase HII and HIII, and eukaryotic RNase H2), and it is important to note that no prokaryotic species with a combination of RNase HI and HIII genes has yet been identified 11. Additionally, in contrast to eukaryotes, which tend to contain both RNase H1 and H2 genes, the combination of RNase H genes in prokaryotes varies among species, and the overall nature of this variation is poorly understood. Therefore, further study is required to clarify the presence or absence of RNase H genes in these species.
We conducted a comparative analysis of the complete genomes of 353 prokaryotes (326 bacteria and 27 archaea) and examined the combination of RNase H genes and the potential evolutionary processes that could explain the effects of functional redundancy on gene evolution, as described by a theoretical model of genetic redundancy 5. Our findings suggest that the RNase HI and HIII genes have evolved in a mutually exclusive manner owing to their functional similarities. This molecular evolution of RNase H genes is the first actual example of how the degree of functional redundancy has implications for changes in gene constitution during the course of evolution.
Results
Genome-wide identification of RNase H genes
To identify RNase HI, HII, and HIII coding sequences in a genome, two strategies (a remote homology search and a protein domain search) were applied to ensure maximum coverage of the genes (See Methods). Using the complete genomes for 326 strains from 235 bacterial species and 27 strains from 27 archaeal species, we retrieved 342 RNase HI genes, 333 RNase HII genes, and 76 RNase HIII genes (see Additional file 1). Almost all genomes contained one or more RNase H genes, and there was little difference in the types and numbers of RNase H genes among several strains of a given species, with the exception of Buchnera aphidicola and Xanthomonas campestris. The RNase HI-related gene of B. aphidicola str. APS (Acyrthosiphon pisum) contained a frameshift mutation that resulted in a loss of RNase H activity (Dr. Naoto Ohtani, Keio University, personal communication), whereas non-frameshifted RNase HI genes were identified in B. aphidicola str. Bp (Baizongia pistaciae) and B. aphidicola str. Sg (Schizaphis graminum). A frameshift mutation was also found in the RNase HII-related gene of Xanthomonas campestris pv. vesicatoria str. 85-10. In contrast, other strains of X. campestris pv. campestris (str. 8004 and str. ATCC 33913) had a non-frameshifted RNase HII. Therefore, we assumed that B. aphidicola had an RNase HI gene and X. campestris had an RNase HII gene at the species level. Accordingly, we counted the number of RNase H genes in the 27 archaeal and 235 bacterial species listed in Table 1. The RNase HI gene was present in 33% (9/27) of the archaeal species and 89% (210/235) of the bacterial species, the RNase HII gene was present in all archaeal species and in 94% (220/235) of the bacterial species, and the RNase HIII gene was identified in 4% (1/27) of the archaeal species and in 17% (40/235) of the bacterial species. This result is consistent with a previous report that RNase HII is the more universal gene in prokaryotes 11. Most species had a single copy of a given gene, but multiple genes encoding RNase HI were found in 11% (3/27) of the archaeal species and 16% (37/235) of the bacterial species.
<p>Additional file 1</p>
List of genes containing the RNase H domain from 27 archaea and 326 bacteria.
Click here for file
<p>Table 1</p>
Number of RNase H genes identified in the archaeal and bacterial species.
Kingdom
Type
No. of species
No. of RNase H genes
1
2
3
Archaea
RNase HI
9
6
3
0
RNase HII
27
27
0
0
RNase HIII
1
1
0
0
Bacteria
RNase HI
210
173
32
5
RNase HII
220
219
1
0
RNase HIII
40
40
0
0
Alteration of RNase H combinations in closely related species
Contrary to the situation in eukaryotes, in which both RNase H1 and RNase H2 tend to coexist, various combinations of RNase H genes have been found in prokaryotes 11. To compare the presence and absence of RNase H genes among prokaryotes, we examined the combinations of RNase H genes in individual genomes. Three types of RNase H genes can theoretically produce eight combinations of genes, as shown in the Venn diagram in Figure 1. Because, in practice, we found no species that lacked all three genes (Group H), all species were classified on the basis of the remaining seven RNase H combinations (Table 2). No prokaryotic genome contained the combination of only RNase HI and HIII (Group D); this supports the results of a previous study 11 at the genome-wide level. Although many archaeal species contained only the RNase HII gene (Group F) or a combination of RNase HI and HII genes (Group B) – a finding in agreement with previous reports 2223 – one of the euryarchaeota, Methanosphaera stadtmanae DSM 3091, combined RNase HII with RNase HIII (Group C) instead. On the other hand, 189 of the 235 bacterial species (80%) had combinations of the RNase HI and HII genes (Group B) and 16 of the 235 species (7%) had combinations of the RNase HII and HIII genes (Group C). At the same time, the RNase H combinations in bacteria exhibited more variety than those in the archaea and seemed to differ even among related species, especially in the firmicutes. Interestingly, species that had all three RNase H genes (Group A) were limited to the firmicutes.
<p>Figure 1</p>
Potential combinations of the three RNase H genes
Potential combinations of the three RNase H genes. The Venn diagram consists of three circles (HI, RNase HI; HII, RNase HII; HIII, RNase HIII) that represent the possible combinations of RNase H genes as Groups A to H. The 353 prokaryotic genomes were classified according to this system, and the results are presented in Additional File 1. The numbers under Groups A to H indicate the number of archaeal (left) and bacterial (right) species identified in each of the groups.
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<p>Table 2</p>
Combinations of the three RNase H genes found in the archaeal and bacterial genomes.
Kingdom
Subdivision
No. of species
Classification of RNase H combination
Group A
Group B
Group C
Group D
Group E
Group F
Group G
Archaea
Crenarchaeota
5
0
3
0
0
0
2
0
Euryarchaeota
21
0
6
1
0
0
14
0
Nanoarchaeota
1
0
0
0
0
0
1
0
Total
27
0
9
1
0
0
17
0
Bacteria
Acidobacteria
1
0
1
0
0
0
0
0
Actinobacteria
20
0
18
1
0
1
0
0
Aquificae
1
0
0
1
0
0
0
0
Bacteroidetes
4
0
4
0
0
0
0
0
Chlamydiae
7
0
0
7
0
0
0
0
Chlorobi
3
0
3
0
0
0
0
0
Chloroflexi
2
0
2
0
0
0
0
0
Cyanobacteria
8
0
8
0
0
0
0
0
Deinococcus-Thermus
3
0
3
0
0
0
0
0
Firmicutes
49
15
18
7
0
0
0
9
Fusobacteria
1
0
1
0
0
0
0
0
Planctomycetes
1
0
0
0
0
1
0
0
Alphaproteobacteria
38
0
38
0
0
0
0
0
Betaproteobacteria
23
0
23
0
0
0
0
0
Deltaproteobacteria
11
0
11
0
0
0
0
0
Epsilonproteobacteria
5
0
5
0
0
0
0
0
Gammaproteobacteria
52
0
49
0
0
3
0
0
Spirochaetes
5
0
4
0
0
1
0
0
Thermotogae
1
0
1
0
0
0
0
0
Total
235
15
189
16
0
6
0
9
All species were classified on the basis of the RNase H combinations shown in the Venn diagram in Figure 1.
To elucidate the relationship between RNase HI and HIII, the evolutionary genomic constitution of the RNase H genes was examined in 49 species of firmicutes, because RNase HIII is especially common in this group (classes A, C, D, and G in Table 2). First, we constructed a Bayesian tree based on the nucleotide sequences of the DNA gyrase subunit B (gyrB) genes of the firmicutes, which have been used to infer phylogenetic relationships among prokaryotes 24, and displayed the RNase H combinations of each species (Figure 2). The results of our phylogenetic analysis indicate that RNase H combinations differed even among closely related species. For example, the species in the mollicutes were classified into Groups B (RNase HI and HII), C (RNase HII and HIII), and G (only RNase HIII), showing that the RNase HIII gene is not found in the mollicutes that retain the RNase HI gene. In addition, species with all three RNase H genes (Group A) were found only in the bacillales and lactobacillales, because this combination is not found in species other than firmicutes (see Table 2).
<p>Figure 2</p>
Bayesian phylogenetic tree of 49 species in the firmicutes
Bayesian phylogenetic tree of 49 species in the firmicutes. A phylogenetic tree was constructed with Bayesian inference on the basis of the gyrB sequence alignments of 49 species in the firmicutes. Numbers at the nodes represent posterior probabilities. The scale bar represents 0.1 substitutions per site. Letters A through G next to the species names represent the combinations of RNase H genes defined in Figure 1; B' indicates the presence of dsRHbd.
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More noteworthy is the fact that the RNase HI genes of the species that also have RNase HII genes (Group B) often encode additional conserved protein domains, as represented by the presence of Group B' in Figure 2. This non-RNase H domain was first identified in the N-terminal portion of eukaryotic RNase H1 25 and was designated as a double-stranded RNA (dsRNA) and an RNA-DNA hybrid-binding domain (dsRHbd) because of its ability to bind to dsRNA as well as RNA-DNA hybrids 262728. In prokaryotes, it has been reported that RNases HI of Bacillus halodurans 29 and of Shewanella sp. SIB1 30 that have dsRHbd in the N-terminus possess RNase H activity. In contrast, no such domain was identified in RNase HI of the species that had all three types of RNase H (Group A). Interestingly, RNase HI of B. subtilis [REFSEQ: NP_390082], a member of Group A, exhibited neither RNase H activity nor other nuclease activity, even though RNase HII and HIII possess RNase H activity 2231. This may indicate a difference of RNase H activity between RNase HI without dsRHbd (Group A) and RNase HI with dsRHbd (Group B').
To identify differences in the primary and secondary structures between RNase HI without dsRHbd (Group A) and RNase HI with dsRHbd (Group B'), multiple alignments were performed using the amino acid sequences of each RNase HI domain in the bacillales and lactobacillales and the E. coli RNase HI domain. If the species had multiple RNase HI genes, one gene that was more similar to E. coli RNase HI than to any other gene was selected; these are described in Additional file 2. As a result, the RNase HI sequences were divided into three groups (Figure 3). The amino acid sequences of RNase HI in Group A were similar to that of B. subtilis RNase HI, which exhibited no nuclease activity. In contrast, the primary structures of RNase HI with dsRHbd formed two groups: Group B'1, in which the primary structures of lactobacillales RNase HI were similar to that of E. coli RNase HI, whose nuclease activity has been demonstrated 32, and Group B'2, in which B. halodurans and B. clausii RNase HI had little similarity to other RNase HI but showed RNase H activity 29. There is also a marked difference in the secondary structure. RNase HI in Group A lacked the basic protrusion handle (alpha-helix 3) involved in substrate binding of E. coli RNase HI 3334. On the other hand, all of the lactobacillales RNase HI with dsRHbd in Group B'1 had the basic protrusion handle. Although the basic protrusion handle is not observed in B. halodurans and B. clausii (Group B'2), it has been proposed that dsRHbd could functionally compensate for this basic protrusion 29. From the relationship between structural similarity and RNase H activity, it can be inferred that RNase HI with dsRHbd in Group B' exhibits RNase H activity but it is unclear whether RNase HI in Group A exhibits RNase H activity or not, because the archaeal RNase HI of Halobacterium sp. NRC-1 22 and Sulfolobus tokodaii 7 23 exhibited weak RNase H activity despite the absence of the basic protrusion handle. However, the fact that a double knockout of RNase HII and HIII genes in B. subtilis yields a lethal phenotype 31 indicates that Group A RNase HI genes encoded in the B. subtilis genome do not have the ability to compensate for functions of RNase HII and HIII. Therefore, our results (that RNase HIII is not present in Group B but is present in Group A) suggest that there is some sort of relationship between protein functions and gene constitutions.
<p>Additional file 2</p>
RNase HI sequences of the bacillales and lactobacillales used for the multiple alignments.
Click here for file
<p>Figure 3</p>
Amino acid sequence alignments for the RNase HI domains
Amino acid sequence alignments for the RNase HI domains. RNase HI protein sequences were derived from the bacillales and lactobacillales listed in Additional File 2 and were aligned by using the Clustal method. Arrows and rectangles indicate beta-strands and alpha-helices, respectively. The upper and lower secondary structures were generated on the basis of the RNase HI domains of E. coli [33] and B. halodurans [29]. Dark and light shadings indicate highly conserved and similar amino acid residues, respectively. Asterisks denote amino acid residues that are involved in the catalytic function of RNase HI. The boxed region below the label for alpha-helix 3 forms a basic protrusion handle in the E. coli RNase HI structure. The combinations of RNase H genes are represented to the right of the sequences (see the text for details). The symbols † and § indicate active and inactive RNase H, respectively.
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Phylogenetic distribution of dsRHbd sequences
Our results (Table 2) clearly showed that the combination of RNase HI and HIII genes (Group D) was not found in the prokaryotes and that most bacterial species had combinations of RNase HI and HII (Group B) or RNase HII and HIII (Group C). Moreover, the combination of RNase H genes has been altered even among closely related species in such a way that functional RNase HI and HIII genes do not coexist in a single genome; in other words, our results provide evidence that RNase HI and HIII tend to evolve in a mutually exclusive manner. Avoiding the simultaneous inheritance of the RNase HI and HIII genes is remarkable when RNase HI contains dsRHbd in the firmicutes, because dsRHbd sequences were found in 15 out of 18 species that combined the RNase HI and HII genes (Group B) and were not found in any of the 15 species that had all three RNase H genes (group A) (see Figure 2). Therefore, dsRHbd appears to be a key domain in the evolutionary process that has led to the current distribution of RNase H genes. Although the characteristics of dsRHbd, such as its enzymatic features 2527 and its secondary structure, have been compared with those of eukaryotic RNase HI 35, little is known about the number and types of dsRHbd in prokaryotes. Therefore, we searched for dsRHbd sequences in the complete genomes of 326 strains from 235 bacterial species and 27 strains from 27 archaeal species in the same way that we searched for the RNase H sequences (See Methods).
The results revealed that the genomes of 30 bacterial species (one of which had two strains) and 1 archaeal species encoded dsRHbd (Table 3), and that the distribution pattern of dsRHbd in prokaryotes did not appear to be correlated with the phylogenetic pattern. Most dsRHbds are fused with the RNase HI domain, but Lactobacillus delbrueckii has two genes encoding dsRHbd; one is associated with the RNase HI domain and the other is associated with the resolvase domain. In addition, it is interesting that the dsRHbds of Gloeobacter violaceus, Bdellovibrio bacteriovorus, and Myxococcus xanthus were identified in the C-terminus of RNase HI even though many dsRHbds were in the N-terminus, as in the eukaryotes. Multiple alignments of the amino acid sequences of prokaryotic dsRHbds showed that the sequences of dsRHbd located in the C-terminus were similar (Figure 4). The process of dsRHbd acquisition can be inferred from the fact that almost half of the RNase HI with dsRHbd was found in firmicutes that have the abilities to acquire new genes through lateral gene transfer 36. In addition, RNase HIII genes were not found in any genomes of the 31 species that encoded RNase HI with dsRHbd (Additional file 3), supporting the hypothesis of mutually exclusive evolution of RNase HI and HIII.
<p>Additional file 3</p>
List of RNase H genes from the species whose genomes encode dsRHbd sequences.
Click here for file
<p>Table 3</p>
List of genes containing dsRHbd in complete genomes.
Kingdom
Species
Accession No.
ORF
Direction
Domain
Archaea
Methanococcus maripaludis
NC_005791
832383–832988
complement
dsRHbd (4–46), RNH (65–201)
Bacteria
Bacteroides fragilis NCTC 9343
NC_003228
207338–207967
complement
dsRHbd (6–48), RNH (79–197)
Bacteroides fragilis YCH46
NC_006347
253987–254616
complement
dsRHbd (6–48), RNH (79–197)
Bacteroides thetaiotaomicron
NC_004663
4371820–4372455
complement
dsRHbd (6–48), RNH (81–199)
Porphyromonas gingivalis
NC_002950
1292573–1293223
direct
dsRHbd (5–47), RNH (85–199)
Gloeobacter violaceus
NC_005125
3808044–3808685
complement
RNH (1–138), dsRHbd (156–197)
Bacillus clausii
NC_006582
1399495–1400094
direct
dsRHbd (6–48), RNH (74–183)
Bacillus halodurans
NC_002570
933504–934094
direct
dsRHbd (6–48), RNH (69–192)
Clostridium acetobutylicum
NC_003030
2659515–2660237
complement
dsRHbd (6–48), RNH (103–238)
Clostridium perfringens
NC_003366
1707913–1708542
complement
dsRHbd (5–49), RNH (70–207)
Clostridium tetani
NC_004557
2281472–2282092
complement
dsRHbd (5–47), RNH (68–203)
Desulfitobacterium hafniense
NC_007907
2075150–2075770
complement
dsRHbd (8–50), RNH (71–203)
Lactobacillus acidophilus
NC_006814
116459–117205
direct
dsRHbd (3–45), RNH (91–245)
Lactobacillus delbrueckii
NC_008054
146497–147264
direct
dsRHbd (4–45), RNH (93–253)
978609–979283
complement
Resolvase (1–106), dsRHbd (127–168)
Lactobacillus johnsonii
NC_005362
118550–119281
direct
dsRHbd (3–45), RNH (86–240)
Lactobacillus plantarum
NC_004567
2310574–2311470
direct
dsRHbd (5–47), RNH (70–225)
Lactobacillus salivarius
NC_007929
459722–460381
direct
dsRHbd (4–46), RNH (59–216)
Mesoplasma florum
NC_006055
559864–560484
complement
dsRHbd (3–45), RNH (63–199)
Mycoplasma capricolum
NC_007633
382602–383222
direct
dsRHbd (5–47), RNH (63–198)
Mycoplasma mycoides
NC_005364
375791–376408
direct
dsRHbd (5–47), RNH (62–198)
Mycoplasma penetrans
NC_004432
1267458–1268120
complement
dsRHbd (5–47), RNH (73–216)
Fusobacterium nucleatum
NC_003454
1651474–1652124
complement
dsRHbd (6–48), RNH (70–215)
Bdellovibrio bacteriovorus
NC_005363
3036447–3037238
complement
RNH (18–164), dsRHbd (207–248)
Desulfotalea psychrophila
NC_006138
1023218–1024003
direct
dsRHbd (28–70), RNH (105–247)
Myxococcus xanthus
NC_008095
2628106–2628873
complement
RNH (3–154), dsRHbd (199–240)
Colwellia psychrerythraea
NC_003910
1743847–1744665
complement
dsRHbd (5–47), RNH (98–247)
Photobacterium profundum
NC_006370
2161121–2161870
complement
dsRHbd (5–47), RNH (77–225)
Saccharophagus degradans
NC_007912
82187–82945
complement
dsRHbd (5–47), RNH (81–228)
Shewanella denitrificans
NC_007954
880428–881219
direct
dsRHbd (5–47), RNH (91–239)
Borrelia burgdorferi
NC_001318
897096–897740
complement
dsRHbd (19–62), RNH (74–211)
Borrelia garinii
NC_006156
899069–899668
complement
dsRHbd (4–47), RNH (88–196)
Thermotoga maritima
NC_000853
1322788–1323459
complement
dsRHbd (7–49), RNH (63–197)
dsRHbd sequences were found in 32 complete genomes (31 species, one with two strains). Open reading frame (ORF) numbers indicate the genomic positions of the genes that encode dsRHbd. Domain numbers indicate the amino acid positions relative to the start of each protein sequence. RNH represents the RNase H group.
<p>Figure 4</p>
Amino acid sequence alignments of dsRHbds from prokaryotes
Amino acid sequence alignments of dsRHbds from prokaryotes. Arrows and rectangles indicate beta-strands and alpha-helices, respectively. These secondary structures were generated on the basis of the N-terminus domain of S. cerevisiae RNase H1 [35]. Arrows and rectangles indicate beta-strands and alpha-helices, respectively. Dark and light shadings indicate highly conserved and similar amino acid residues, respectively. Asterisk represents an identical amino acid residue. The symbol † indicates that the gene containing the resolvase domain encodes the amino acid sequence of dsRHbd. For detailed information, see Table 3.
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Redundant RNase HI genes in a single genome
We also found that 10 of the 31 species listed in Table 3 had multiple RNase HI genes (see Additional file 3). If RNase HI with a dsRHbd gene influences the existence of the RNase HIII gene in a genome, how is the effect exerted on other RNase HI genes? To address this question, we examined the amino acid sequences of RNase HI without dsRHbd in these 10 species. The RNase HI without dsRHbd that were found in five species in the firmicutes and one species in the deltaproteobacteria, with the exception of B. bacteriovorus, were similar in structure (e.g., lacked the basic protrusion) to that of the Group A RNase HI (see Figure 3). On the other hand, the primary structures of RNase HI without dsRHbd in three species of gammaproteobacteria resembled that of E. coli, and there were few differences in their amino acid sequences. Because the primary structures of RNase HI with dsRHbd in the same species in the gammaproteobacteria were also similar to that of E. coli, it is difficult to distinguish redundant RNase HI genes on the basis of their amino acid similarities.
To identify the differences among redundant RNase HI sequences of the gammaproteobacteria (see Additional file 4), we constructed a Bayesian tree based on the nucleotide sequences of the RNase HI domains from 12 species in the gammaproteobacteria (Figure 5). This analysis divided the RNase HI domains into four gene clusters: orthologous RNase HI, including E. coli RNase HI (Group I); RNase HI with dsRHbd (Group II); and other two groups of additional RNase HI (Groups III and IV). Because RNase HI genes in Group I appear to have been inherited by vertical descent from a common ancestor, we defined them as orthologous RNase HI genes. On the other hand, RNase HI genes of Group II to IV seem to have been provided by gene duplication or lateral gene transfer in addition to the original RNase HI genes. Interestingly, orthologous RNase HI was not found in Saccharophagus degradans that contains RNase HI with dsRHbd (Group II). In contrast, Pseudoalteromonas atlantica contains orthologous RNase HI (Group I) instead of RNase HI with dsRHbd, though the presence of Group III RNase HI is common to S. degradans and P. atlantica. In addition, orthologous RNase HI was not found in the genome of Colwellia psychrerythraea, which contains only RNase HI with a dsRHbd gene (Group II). The same statement applies to 21 other prokaryotic species that have only RNase HI with dsRHbd (see Additional file 3). On the other hand, we also found that orthologous RNase HI (Group I) and RNase HI with dsRHbd (Group II) had both been retained in two genomes of Photobacterium profundum and Shewanella denitrificans. These results suggest that RNase HI with dsRHbd may be capable of replacing the original RNase HI. A lineage-specific characterization such as the mapping of gene trees onto species trees using a soft parsimony algorithm 37 is necessary for more precise analysis of the transition of RNase HI genes during the course of evolution.
<p>Figure 5</p>
Bayesian phylogenetic tree for the RNase HI domains
Bayesian phylogenetic tree for the RNase HI domains. A phylogenetic tree was constructed by using Bayesian inference on the basis of the alignments of RNase HI domain sequences from 12 species in the gammaproteobacteria, as listed in Additional File 4. Species names followed by Arabic numerals are used to distinguish multiple forms of RNase HI in a genome; names not followed by Arabic numerals indicate the presence of a single RNase HI in a genome. Numbers at the nodes represent posterior probabilities. The scale bar equals 0.1 substitutions per site.
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Discussion
Using genome-wide and phylogenetic analyses of RNase H genes, we obtained the following findings: (1) most bacterial species had combinations of RNase HI and HII (80%, 189 out of 235 species) or RNase HII and HIII (7%, 16 out of 235 species); (2) the combination of RNase HI and HIII genes was not found in any species (0% in Group D) unless RNase HII was also present (Group A; 15 species in the firmicutes); (3) the combination of RNase H genes has been altered, even in closely related species, in such a way that the functional RNase HI and HIII genes do not coexist in a single genome; (4) dsRHbd was found in RNase HI in 31 out of 189 species (16%) that contain the RNase HI and HII genes; (5) dsRHbd was not found in the RNase HI in all 15 species that contained all three types of RNase H genes; and (6) RNase HI with dsRHbd may have replaced the orthologous RNase HI without dsRHbd in 21 out of 31 species (68%) that have RNase HI with dsRHbd.
To ascertain the cause of the mutually exclusive evolution of RNase HI and RNase HIII, we focused on their enzymatic properties. Previous reports have indicated that RNase HI and HIII digest the RNA moiety of RNA-DNA hybrids such as Okazaki fragments more effectively than is the case for RNase HII 38, whereas only RNase HII is capable of removing a single ribonucleotide of DNA-RNA-DNA/DNA hybrids such as an RNA that has been misincorporated into DNA 1516. In addition, mutagenesis analyses of B. subtilis RNase H genes have shown that single-gene knockout mutants targeting the RNase HII or HIII genes exhibit normal growth, but that double-knockout mutants for both genes are unable to form viable colonies; this suggests that a functional overlap exists between RNase HII and HIII 31. On the other hand, the existence of functional redundancy between RNase HI and HII is not clear, although double-knockout mutants of E. coli RNase HI and HII exhibit a temperature-sensitive phenotype (Dr. Mitsuhiro Itaya, Keio University, personal communication). We hypothesize that the functional similarities and differences among the three RNase H genes may explain this evolutionary process, because a theoretical model of genetic redundancy suggests that the fates of redundant genes are likely to depend on the extent of their functional redundancy 5.
According to computer simulations using a genetic redundancy model 5, redundant genes do not persist when they are equally effective at performing their functions (Model 1). On the other hand, redundant genes are evolutionarily stable in two situations: when both genes perform the same function but one is less efficient than the other gene (Model 2), and when the main functions of the two genes differ but one of the genes functions similarly to the other gene, but with lower efficiency (Model 3). The insights from this simulation can be applied to the molecular evolution of RNase H genes in prokaryotes. At first glance, the reason why most bacterial species have combinations of RNase HI with RNase HII or RNase HII with RNase HIII can be explained by Model 3; that is, these combinations are evolutionarily stable because both genes in each combination have independent functions but with an unknown degree of functional overlap. Likewise, the combination of RNase HI and HIII genes is evolutionarily unstable owing to their functional redundancy (Model 1), and this may explain why no species has both functional genes in its genome and why the combination of RNase H genes has been altered even in closely related species in such a way that RNase HI and HIII genes will not coexist in a single genome.
It seems that the effect of functional redundancy is more severe for RNase HI with dsRHbd in firmicutes, because RNase HIII was found in all 15 species whose genomes encode RNase HI without dsRHbd but was not found in any of the 31 species containing RNase HI with dsRHbd. Given the distribution pattern of RNase HI with dsRHbd in prokaryotes, we proposed the following process: once RNase HI with dsRHbd is acquired (for example, by lateral gene transfer 39), the combination of RNase HI with dsRHbd and RNase HIII may become evolutionarily unstable owing to their functional redundancy, and one of them is subsequently removed during the course of evolution. We propose this evolutionary process as Model A in Figure 6A. In particular, it is interesting that the RNase H combinations of two species that are regarded as the deepest branching organisms are different: Thermotoga maritima has a combination of RNase HI with dsRHbd and RNase HII, whereas Aquifex aeolicus retains a combination of RNase HII and HIII 31. This may reflect the ancient status of these RNase H combinations in bacteria, and RNase HIII might have been altered along with RNase HI with dsRHbd in T. maritima in accordance with our model. Also, the fact that RNase HIII genes are less abundant than those of RNase HI and HII (Table 1) suggests the possibility that RNase HIII genes have been replaced in genomes by other RNase H genes during the course of evolution.
<p>Figure 6</p>
Possible evolutionary models for the three RNase H genes
Possible evolutionary models for the three RNase H genes. Two models that can explain the mutually exclusive evolution of RNase HIII and RNase HI with dsRHbd (Model A) and of RNase HI with and without dsRHbd (Model B). Fa and Fb indicate the different functions of the RNase H enzymes.
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A similar scenario might have occurred in the case of redundant RNase HI genes, because our findings also suggest that RNase HI with dsRHbd may have replaced the existing RNase HI without dsRHbd in 21 species. As shown in Model B (Figure 6B), once RNase HI with dsRHbd is obtained, it competes with RNase HI without dsRHbd because of their functional redundancy, and one of them is excluded. It is also noteworthy that RNase HI with dsRHbd is encoded as a single-copy gene in prokaryotic genomes (Table 3). Interestingly, human genome contains one RNase H1 with dsRHbd and at least three pseudogenes related to RNase H1 with dsRHbd 40. Although we previously showed that four RNase H1-encoding genes in Caenorhabditis elegans exhibited gene-specific expression patterns during development; one gene encodes RNase H1 with dsRHbd and other three gene encode RNase H1 without dsRHbd 41, it was also found that most of the eukaryotic genomes contained single-copy genes encoding RNase H1 with dsRHbd but RNase H1 without dsRHbd had rarely been identified in the same genome (data not shown). Functional characteristic of RNase H1 with dsRHbd seems to depend on eukaryotic species because disruptions of RNase H1 with dsRHbd resulted in lethal phenotype of fly 42 and mice 13 but showed normal growth in yeast 43 and trypanosoma 44. However, it appears that prokaryotic and eukaryotic genomes have single-copy RNase H1 with dsRHbd and this tendency might be explained by functional redundancy within individual genomes. In a future work, more detailed analysis of eukaryotic RNase H1 genes is required to show the effect of functional redundancy on the evolution of redundant genes in eukaryotes.
We also discovered that several RNase HI genes could exist in a single genome (Table 1) and that there are some cases in which orthologous RNase HI is retained in the presence of RNase HI with dsRHbd (Figure 5). This raises the question of how multiple RNase HI genes can be retained in a single genome. This is difficult to explain using the genetic evolution models described in Figure 6, because multiple RNase HI genes should also have the same function and should be subject to the same mechanisms that govern the fate of redundant genes. In the case of duplicated genes, the usual fate of redundant genes is that one is silenced through a strong purifying selection after a brief period of relaxed selection 45. The number of RNase HI genes differed among species even within the same lineages, suggesting that gene duplication or gene transfer might have occurred relatively recently and that redundant genes may have arisen during a period of relaxed selection. An alternative possible explanation for multiple RNase HI is that neofunctionalization and subfunctionalization have been shown by computer simulation to increase the retention rate of duplicated genes 4647. Although it is not known whether the retention of multiple RNase HI genes resulted from subfunctionalization or neofunctionalization, RNase HI appears to represent the acquisition of a new function based on the example of Streptomyces coelicolor A3(2), which encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain 48. In addition to subfunctionalization, it is also possible that some of the RNase HI genes identified in this study might be in the process of nonfunctionalization (pseudogenization) and can be expected to become pseudogenes 49. Actually, genomic sequences encoding truncated RNase HI domains have been found in some species during genome-wide identification of RNase H genes (data not shown), suggesting the existence of one or more nonfunctionalized RNase HI gene in our dataset. Moreover, even if the coding sequences seem not to have been nonfunctionalized, the regulatory regions might have mutations because duplicated genes are considered to be under active selection pressure owing to energy constraints on gene expression 50. Further investigations will be necessary to reveal the effect of each functionalization on multiple RNase HI genes in prokaryotes.
In this study, two possible models were provided to explain the evolution of RNase H combinations in prokaryotic genomes. We believe that our models are the first example of the effects of functional redundancy on changes in gene constitution during the course of gene evolution. Experimental evolution of bacterial species constructed to have mutually exclusive genes by means of genetic engineering may be effective in verification of our models. For example, RNase HI and RNase HIII genes tagged with different drug resistances are inserted into the RNase HI-knockout mutants of E. coli and repeated subcultures of the recombinants allow us to detect the mutated RNase H gene using specific drug resistances as markers. This experimental approach would certainly be worthwhile to explore the fate of redundant RNase H genes in future research.
Methods
Genome-wide identification of genes encoding RNase H and dsRHbd
Complete genomes of 326 strains from 235 bacterial species and 27 strains from 27 archaeal species and the corresponding GenBank files were downloaded from the National Center for Biotechnology Information (NCBI) GenBank FTP site 51; their accession numbers are summarized in Additional file 1. Two strategies were applied to identify sequences of RNase H and double-stranded RNA and RNA-DNA hybrid-binding domains (dsRHbd) in the complete genomes. One was a remote homology search with the PSI-BLAST software 52 and the other was a protein domain search based on Hidden Markov Model (HMM) profiles 53.
For the PSI-BLAST search, a non-redundant peptide sequence database was downloaded from the NCBI BLAST FTP site 54. From this database, peptide sequences of prokaryotes and eukaryotes were extracted by using taxonomy information obtained from the NCBI Taxonomy FTP site 55. To construct a position-specific scoring matrix, a PGP-BLAST search was carried out against 3 506 454 extracted peptide sequences, with an E-value threshold of 0.002 and four iterations. The amino acid and nucleotide sequences corresponding to the RNase HI domain of E. coli K12 [GenBank: AAC73319], the RNase HII domain of E. coli K12 [GenBank: AAC73294], the RNase HIII domain of B. subtilis subsp. subtilis str. 168 [Swissprot: P94541], and the dsRHbd of B. halodurans C-125 [Swissprot: Q9KEI9] were used as queries. Using the resulting matrix, PSI-TBLASTN searches were conducted against the 353 complete genomes by using an E-value threshold of 0.2.
For the HMM profile analysis, the profiles of RNase HI and RNase HII were downloaded from the Sanger Institute's Pfam Web site 56 and the HMM profile of dsRHbd was newly built by using the hmmbuild module of the HMMER 2.3.2 software 53 on the basis of the results of the PSI-BLAST search. The 353 complete genomes were translated into six-frame amino acid sequences. Using these HMM profiles as queries, protein domain searches were performed with the hmmpfam module of the HMMER 2.3.2 software against translated complete genomes with an E-value threshold of 1× 10-6.
On the basis of the outputs of the PSI-BLAST and HMM searches, coding sequences including homologous regions of RNase H or dsRHbd were obtained from GenBank files by using G-language Perl modules 57. When the search revealed unannotated genomic regions, we manually checked for the existence of an open reading frame (ORF) near the genomic region. In order to distinguish genes encoding RNase HII and RNase HIII in the datasets, a PGPBLAST search was conducted against the Conserved Domain Database (a subset of domains from SMART, Pfam, COG, and CD) 58 downloaded from the NCBI CDD FTP site 59.
Phylogenetic analysis
The amino acid and nucleotide sequences of the DNA gyrase subunit B gene (gyrB) were retrieved in a similar way. The CodonAlign 2.0 software (Barry G. Hall, Rochester, NY, USA) was used to align the nucleotide sequences on the basis of alignments of the corresponding amino acid sequences performed with the ClustalW 1.8.3 software 60. The Modeltest 3.7 software 61 was applied to select an appropriate model from the output of the PAUP* Version 4.0 software 62 by using hierarchical likelihood-ratio tests and the Akaike Information Criterion 63. Phylogenetic trees were estimated by Bayesian methods with MRBAYES Version 3.1.2 software 64 under the General Time Reversible model with gamma correction and a proportion of invariable sites 65. In the Bayesian analysis, the Markov chain Monte Carlo search used 1 000 000 generations run with four chains, with trees being sampled every 100 generations, and a consensus tree was estimated by a burn-in of 2500 trees. TreeView software for Power Macintosh 66 was used for viewing and editing the tree.