Department of Biology, University of York, York, England
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Abstract |
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Introduction |
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The molecular-clock hypothesis, using molecular data in conjunction with fossil evidence, has been invoked to interpret phylogenetic relationships and to date speciation events. Iwabe et al. (1989)
argued that gene paralogs, derived from a common ancestor following a gene duplication, are suitable for molecular clock studies, since the resulting phylogeny can be rooted unambiguously at the duplication event. Genes subject to uniform selective pressures are useful for molecular-clock studies, since these genes are most likely to conform to the central assumptions of the theory that the evolutionary process is independent at all sites. Genes that most closely adhere to this model are pseudogenes and genes that are so central to metabolism that they are thought to be under little directional selective pressure due to the functional constraints imposed on their products. Glutamine synthetase (GS) (EC 6.3.1.2) is a key enzyme in nitrogen assimilation that is both duplicated and conserved in function. The gene duplication event appears to have occurred before the split of prokaryotes and eukaryotes (Kumada et al. 1993
). One form, GSI, has been found only in prokaryotes, whereas another form, GSII, is found in all eukaryotes and has been detected in a minority of prokaryotes. A third form of GS, glnT, has been identified only in rhizobia; it is distantly related to the other two forms and may not be a functional homolog (Shatters, Lui, and Kahn 1993
). The adherence or nonadherence (Brown et al. 1994
) of GS genes to the molecular-clock theory has been discussed in detail. However, allowing for a number of ancient gene transfers, GS evolution appears reasonably clocklike when either only nonsynonymous mutations (Kumada et al. 1993
) or only second codon positions (Pesole et al. 1991, 1995
) are used. The X-ray structure of the Salmonella typhimurium GSI protein (GlnA) has been determined: it is a dodecameric homopolymer in which the active site is formed between subunits (Yamashita et al. 1989
). The need for intimate contacts with several other subunits might explain why these sequences are so highly conserved.
Taboada et al. (1996)
investigated the relative mobility of the GSI and GSII proteins from different species of rhizobia using two-dimensional gel electrophoresis. Their results suggest that the GSII enzyme is the more divergent, having a different mobility for each species screened, whereas the GSI enzyme mobilities were identical across all species. The aim of our work was to use GSI and GSII gene sequences from different species of rhizobia to investigate the evolutionary relationships for each gene, with the possibility of dating the major branch points within the family.
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Materials and Methods |
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Sequence Data and Accession Numbers
Sequence data corresponding to residues 1451086 (942 bp) and residues 121948 (828 bp) of the S. meliloti GSI and GSII database sequences, respectively, were determined for both strands. Accession numbers are given in table 1
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Phylogenetic Analyses
The sequences obtained were aligned using CLUSTAL X (Thompson et al. 1997
). The alignments were adjusted to match the alignment of Pesole et al. (1995)
when analyses of both GSI and GSII sequences were undertaken. Neighbor-joining analyses (Saitou and Nei 1987
) were undertaken using PAUP*, version 4.0b2a (Swofford 1998
), and maximum-likelihood analyses using DNAml (which uses the F84 model) and DNAmlk in the PHYLIP 3.57c suite of programs (Felsenstein 1993
). All alignments are available from S.L.T.
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Results |
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All of the PCR products were sequenced on both strands, and the sequence data were used in conjunction with the earlier alignments to design the primers GSI-3/4 and GSII-3/4 for specific amplification of the 5' region of the GSI and the 3' region of the GSII genes, respectively. PCR products of the expected size were obtained for all type strains screened with these primer sets, except for A. caulinodans with the GSII-3/4 primer pair. Bradyrhizobium japonicum generated a weak amplification product with the GSI-3/4 primer pair, and a reliable sequence could not be obtained by direct sequencing. However, from the poor-quality sequence obtained and published partial B. japonicum GSI sequences (accession numbers M26735 and M10926), a new forward primer, GSI-5, was designed that gave reliable amplification and unambiguous sequence data. A long M. huakuii GSI sequence was generated using primers GSI-3 and GSI-2 with a 49°C anneal and a 120-s extension, and this product was sequenced with all four GSI primers. Long GSI and GSII products can be amplified for all of the fast-growing rhizobia using the lower anneal temperature, an increased extension time, and the GSI-3/2 or GSII-1/4 primer pair, respectively.
Phylogenetic Analysis of GSI Sequences
The sequence data for the GSI-1/2 and GSI-3/4 primer sets were combined for each species, translated, and aligned using CLUSTAL X (fig. 3
). Preliminary analyses included translation products of the R. leguminosarum bv. viciae strain RC1001 (X04880) and S. meliloti strain 2011 (U50385) sequences present in the database. These are the only full-length rhizobial GSI sequences available in the databases: the S. medicae sequence obtained in this work was identical to the S. meliloti 2011 sequence. The species S. medicae has only recently been recognized (Rome et al. 1996
); it was formerly classified as S. meliloti type B, and so this finding is not unexpected. The R. leguminosarum sequence obtained in this work was most closely related to the R. leguminosarum RC1001 sequence. These sequences were not identical; there appears to be a short sequence rearrangement in the published amino acid sequence, in which residues 225232 of figure 2
precede residues 220224. The consensus sequence from all of the rhizobial sequences obtained during this work suggests that this rearrangement is peculiar to the published R. leguminosarum RC1001 sequence or may be a sequencing error: in addition to the rearrangement, there are some point mutations that appear to be unique to the published sequence. These two database sequences were omitted from all subsequent analyses.
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How Do the GSII Phylogenies Compare with the GSI Results?
The results of phylogenetic analysis of the GSII data are presented as radial phylograms (fig. 4
) because they cannot be reliably rooted, since A. caulinodans does not have a GSII. The only other bacteria that have been shown to have GSII genes are the high-GC Gram-positives, and these sequences are too diverged to provide a useful outgroup.
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Discussion |
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When residues that are conserved within the rhizobia but differ from the S. typhimurium sequence are considered, the substitutions are usually conservative. The substitution frequency was calculated by comparison of the S. typhimurium and the S. fredii protein sequences (fig. 2
). Residues 136161 (including ß-8, ß-9, and -4) have 20 substitutions, approximately twice the number expected from the frequency for the full-length fragments. Furthermore, this region has a disproportionately high number of substitutions when all possible pairwise comparisons are made between S. fredii, S. typhimurium, and N. gonorrhoeae. Yet, this 20-aa sequence is totally conserved within the 15 fast-growing rhizobia sampled here and within the 7 species of Neisseria sequenced by Zhou, Bowler, and Spratt (1997)
. The three-dimensional model indicates that the dodecameric homopolymer comprises two opposing hexamer rings. These rings are thought to be held together by a ß-sheet structure formed by ß-8 and ß-9 of opposing monomers in each ring. If this is the case, then the ß-8 and ß-9 might be expected to be functionally constrained and, therefore, to have low levels of sequence variation.
Brown et al. (1994)
proposed that there has been an insertion of approximately 25 amino acids in the GSIß subset of sequences that includes the S. typhimurium and rhizobial proteins (see fig. 5
). This insert would correspond to residues 144166 of figure 2
including all of ß-9 and
-4. Pesole et al. (1995)
proposed that the insert corresponds to residues 138162, including most of ß-8, as well as ß-9 and
-4, and corresponding almost exactly to the region of higher-than-expected substitutions between S. fredii and S. typhimurium. If these functionally important structures are the result of an insertion event, then determining the structure of the ancestral form without the insert, represented by either a GSII or a GSI
polymer, should help to identify the boundaries and nature of the insertion event.
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The GSII relationships show several differences from GSI and 16S: R. galegae strongly groups within the Rhizobium cluster, B. japonicum is more closely related to the fast-growing rhizobia than predicted from the other two data sets, and the Mesorhizobium clade is not supported. The M. huakuii GSII sequence appears to be more closely related to Rhizobium sequences than it is to Mesorhizobium sequences. This finding was checked by sequencing GSI and GSII of the M. huakuii type strain (CCBAU 2609) held in the HAMBI collection. The sequences were identical to those obtained for the USDA 4779 strain. Sequences from other strains independently isolated from the same host plant species (Astragalus sinicus) also share the anomalous position of the M. huakuii sequence (our unpublished results). The B. japonicum GSII DNA sequence obtained in this work is most similar, although not identical, to the B. japonicum sequence in the database. The protein sequences are, however, identical: these results suggest that the unusual position of this type strain sequence is not an artifact.
The possibility that one or all of these anomalies might be the consequence of a recent recombination event was investigated using PLATO (Grassly and Holmes 1997
). This program was designed to identify regions of a sequence alignment that produce phylogenies that are inconsistent with the maximum-likelihood phylogeny for the full-length alignment. PLATO, version 2.01f, did not identify any extended regions of the GSII sequence alignment that would generate anomalous phylogenies under the F81 model when all three codon positions were used. Neither did PhylPro (Weiller 1998
), which uses a sliding-window approach to identify recombination sites. Thus, there is no evidence for a recombination break point within the GSII sequence.
PLATO was also used to assess the GSI data set (all positions, F84 model); an extensive region (72509 bp of the alignment; a Z value of 4.99, where values >3.70 are significant) was highlighted by the program. Visual inspection of the GSI protein and DNA sequence alignments did not reveal any obvious sequence discontinuities. The maximum-likelihood phylogeny of this fragment (72509 bp, not shown) is slightly different from that shown in figure 2 B. The anomalous region can accommodate the full-length tree configuration without a significant reduction in log-likelihood when the user-defined trees option is invoked and branch lengths are allowed to vary. Furthermore, removal of single sequences (B. japonicum, S. meliloti, or R. etli) and reanalysis with PLATO results in loss of the anomalous region, suggesting that the anomaly is not dependent on any one sequence. Again, PhylPro did not produce results consistent with a recent recombination event, and we conclude that there is no clear evidence for a recombination break point within the GSI sequence.
Comparison of the intrageneric species relationships for each of the three gene sequences considered in this paper, 16S rDNA, GSI, and GSII (figs. 1, 3A and 4A
) suggest that the Mesorhizobium species relationships are the least consistent. For example, in the 16S phylogeny, the M. ciceri and M. loti sequences are most closely related (100% bootstrap support), whereas the M. ciceri and M. loti GSI sequences are less closely related, although they are definitely in the same clade. According to the GSII analyses, the M. loti sequence is not strongly grouped with the other mesorhizobia. In contrast, all three gene phylogenies support the Sinorhizobium clade. Within this clade, the S. medicaeS. meliloti association is strongly supported in all three phylogenies, although the relationships of the other three species in this clade are poorly resolved on both the GSI and the GSII trees. The R. leguminosarumR. etli and R. tropici AR. tropici B relationships are well supported in all three trees, and these species consistently group together. Recent evidence suggests exchange of chromosomal genes between Mesorhizobium species, which has not been so clearly documented for the other fast-growing rhizobia. Sullivan et al. (1996)
have identified Mesorhizobium isolates that are different species according to DNA : DNA hybridization analysis but have identical or very similar 16S rDNA sequences. However, more research is required before any general conclusions about the levels and extent of gene exchange within and among the different genera of rhizobia can be made.
Can We Date Speciation Within the Rhizobia?
There is very little fossil evidence for bacteria, so divergence times are almost impossible to estimate. One way to circumvent this problem is to correlate bacterial splits with those of higher organisms that have more complete fossil records (Ochman and Wilson 1987
). For example, the divergence times of aphid species were used to date the divergence of their maternally inherited, obligate intracellular symbiont (Buchnera aphidicola) lineages to between 100250 MYA. However, the extrapolation of these dates to other bacterial lineages is now in doubt, because B. aphidicola appears to have an increased substitution rate (Moran, von Dohlen, and Baumann 1995
; Brynnel et al. 1998
). An alternative approach is to calibrate divergence in a bacterial gene with the homologous gene in eukaryotes for which a fossil record is available. As mentioned earlier, duplicated genes such as GS are ideally suited to molecular-clock studies, since the duplication event allows the resultant phylogenetic tree to be rooted unambiguously. This, in turn, allows the uniformity or nonuniformity of rates to be assessed. The most ancient duplication of GS genes is likely to have occurred before the split of prokaryotes and eukaryotes (Kumada et al. 1993
). A molecular-clock approach should allow the divergence times of rhizobial GSII sequences to be estimated from the fossil record for higher organisms. These dates might then allow the divergence times of rhizobia and other bacterial lineages to be estimated using GSI sequences if the rates appear to be equivalent in the two halves of the tree.
Figure 5
shows the phylogeny of GSI and GSII sequences, including reference eukaryote sequences and some bacterial GSI sequences, calculated assuming constant rates. The alignment used was based on that of Pesole et al. (1995)
; only second codon positions were considered, and positions with gaps were ignored (245 sites). Pesole et al. (1995)
showed that second codon positions obeyed stationarity for the taxa used: the plot of %GC of second codon positions against %GC of third codon positions showed that all taxa displayed the same base composition, within statistical variations, for second codon positions. This is also true for the sequences used in this study (data not shown). The likelihood ratio test was used to compare the DNAmlk phylogeny shown (Ln -4,148) with the equivalent, unrooted DNAml phylogeny (Ln -4,127) (not shown): the result of this test (
2 = 42, df = 30) suggests that the DNAmlk phylogeny is not significantly less likely than the DNAml phylogeny (DNAmlk/PHYLIP notes).
Only the type species of each genus (B. japonicum, R. leguminosarum, S. fredii, and M. loti) and R. galegae, because of its uncertain phylogenetic position, were included in the calculations for figure 5 . The phylogeny confirms that the B. japonicum GSII sequence is not a reliable outgroup for the fast-growing rhizobia for this gene. The relative distances between the fast-growing rhizobia and B. japonicum differ greatly in the GSI and GSII halves of the tree, which would make any interpretation involving this species rather tenuous. However, the divergence times between the fast-growing rhizobia are remarkably similar for the GSI and GSII halves of the tree, suggesting that these sequences have behaved in a clocklike manner in the rhizobia. This was confirmed by a plot of GSI second-codon-position distances (F84 model) against those for GSII for the taxa in figure 5 that have both genes (the rhizobia and the high-GC Gram-positives). The results suggest that when B. japonicum is ignored, the rate of substitution is equivalent for GSI and GSII for all taxa (within 95% confidence limits).
Recent reports using many genes to assess the likely divergence times of some higher eukaryotes have produced results that are in good agreement with fossil evidence (Goremykin, Hansmann, and Martin 1997
; Kumar and Hedges 1998
; Wang, Kumar, and Hedges 1999
). Kumar and Hedges (1998)
used fossil evidence that dates the diapsid-synapsid (bird-mammal) split at 310 MYA to estimate the amphibian-bird/mammal (360 ± 14.7 MYA) and rodent-human (112 ± 3.2 MYA) divergence times. Their later work (Wang, Kumar, and Hedges 1999
) places the chordate-arthropod split at 993 ± 46 MYA and suggests that the split between animals, plants, and fungi occurred 1,2001,500 MYA. Goremykin, Hansman, and Martin (1997)
estimated divergence times for the angiosperm-gymnosperm and monocot-dicot splits of 160 and 348 MYA (±10%), using a calibration point of 450 MYA for the Marchantiavascular plant divergence. If the bird-mammal split is used as a calibration point for the GS data, however, the divergence times of other splits do not agree with those estimated using multiple gene sequences (table 2
). When the amphibian-bird/mammal split or the angiosperm-gymnosperm split is used for calibration, the divergence times for splits other than the bird-mammal split are in better agreement with those proposed by Kumar and Hedges (1998)
and Goremykin, Hansman, and Martin (1997)
(table 2
).
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Due to the absence of an informative fossil record, dating bacterial divergence times is very difficult. Some estimates have been made (Ochman and Wilson 1987
; Doolittle et al. 1996
) based on the assumption that eukaryotes and bacteria have comparable substitution rates for all genes. These date the E. coliS. typhimurium split at 120160 MYA based on 16S rDNA (Ochman and Wilson 1987
), or at 100 MYA based on several protein sequences (Doolittle et al. 1996
). The GSI data would place the E. coliS. typhimurium split at 6975 MYA, somewhat later than these estimates, which, unlike the GS data, do not have an internal assessment of clocklike behavior for the bacterial and eukaryotic sequences. The similar branch lengths for the fast-growing rhizobia in both the GSI and GSII halves of figure 5
suggest that these paralogs do behave as good molecular clocks.
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Acknowledgements |
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Footnotes |
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1 Keywords: recombination
molecular clock
bacteria
glutamine synthetase
rhizobia
2 Address for correspondence and reprints: Sarah L. Turner, Department of Biology, University of York, P.O. Box 373, York, YO10 5YW, U.K. E-mail: slt4{at}york.ac.uk
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