The International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka 565-0871, Japan1
Author for correspondence: Hiroko Kawasaki. Tel: +81 6 6879 7455. Fax: +81 6 6879 7454. e-mail: kawasaki{at}icb.osaka-u.ac.jp
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ABSTRACT |
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Keywords: Rhodobacter, Rhodopseudomonas, nodule-forming bacteria, phylogeny, isoprenyl diphosphate synthase
Abbreviations: FPP, farnesyl diphosphate
The GenBank accession numbers for the FPP synthase gene sequences reported in this paper are AB053173AB053178, AB053180 and AB062882AB062884.
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INTRODUCTION |
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Farnesyl diphosphate (FPP) synthase, a key enzyme involved in the synthesis of isoprenoid compounds (Ogura et al., 1997 ), is well conserved among micro-organisms (Fujisaki et al., 1990
; Koyama et al., 1993
; Cunillera et al., 1996
; Song & Poulter, 1994
; Anderson et al., 1989
). This enzyme plays an important role in photosynthesis as well as in the synthesis of other physiologically important compounds. Construction of the FPP synthase gene tree showed similar groupings as in the 16S rRNA tree at the suprageneric level within the eubacterial branch; the FPP synthase gene thus appeared to behave in the manner of the 16S rRNA gene (Cantera et al., 2002
). Here, we extended our study on the phylogenetic relationship of the FPP synthase genes among phototrophic and non-phototrophic bacteria belonging to the
-Proteobacteria. We also compared the phylogeny of the FPP synthase genes with that of the 16S rRNA, and showed that the FPP synthase genes of some species from the
-2 Proteobacteria may have been acquired by gene transfer.
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METHODS |
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DNA preparation and amplification of FPP synthase genes.
Chromosomal DNA was prepared as described by Ausubel et al. (1995) . Degenerate PCR primer combinations, which are bound between nucleotide positions 730 and 1170 (E. coli FPP synthase gene sequence numbering; Fujisaki et al., 1990
), were FPP
F3 (5'-CAY GAC GAY MTG CCC KSV ATG GA-3') and either FPP
R1 (5'-CYK YSR CRT CSA GAA TGT CGT C-3') or FPP
R3 (5'-CYT CGA CRT CSA GAA TRT CRT C-3'). They were designed from the highly conserved regions (regions II and VI) in FPP synthase genes of Rhodobacter sphaeroides ATCC 11167T (accession no. AB028044), Rhodobacter capsulatus ATCC 11166T (AB028046), Rhodovulum sulfidophilum W4 (AB028047) and B. japonicum USDA 110 (U12678) to amplify an approximately 420 bp product. Additional primers with lesser degeneracy and more specific for Rhodobacter and Rhodovulum species, FPPRbaF1 (5'-CAT GAC GAY ATG CCC TGY ATG GA-3') and FPP
R4 (5'-TCG TCS GCG AYC TGG AAS GCM AGS C-3'), were later designed from almost exactly the same conserved regions as more sequences became available. The 25 µl reaction mixture consisted of bacterial DNA as the template (10100 ng), 2·5 mM dNTP mix, 25 pmol of each primer and 1·25 U Taq polymerase (TaKaRa Shuzo). The reaction conditions for the primer pair FPP
F1/FPP
R1 were 94 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, with a final extension at 72 °C for 7 min. For the primer pairs FPP
F3/FPP
R3 and FPPRbaF1/FPP
R4, initial denaturation was followed by either 6 or 10 cycles of 96 °C for 1 min, 37 °C for 2 min and 72 °C for 1 min, followed by 25 cycles of 96 °C for 1 min, 2 min at either 55 or 57 °C, and 72 °C for 1 min before the final elongation step. This step-up PCR modification was done to increase the number of putative FPP synthase gene products at low annealing temperature, followed by a high annealing temperature to specifically amplify the gene. PCR reaction was carried out using the GeneAmp PCR system 9700 (PE Applied Biosystems).
Sequencing of FPP synthase genes.
Purified PCR fragments approximately 400450 bases long were cloned using a TA cloning kit (InVitrogen) and transformed into competent cells according to the manufacturers protocol. DNA sequences were determined by the dideoxy chain-termination method (Sanger et al., 1977 ) with a BigDye Terminator cycle sequencing kit (PE Applied Biosystems) using an ABI PRISM 310 Genetic analyser (PE Applied Biosystems). Sequence data were collected by means of the program ABI PRISM (Perkin-Elmer) and assembled with an ABI Auto Assembler (Perkin-Elmer).
In silico and phylogenetic analyses.
Nucleotide sequences obtained in this work were converted to amino acid sequences after removing the gap-containing and the primer-annealing regions using Genetix-Win software (version 3.1.0). Seven FPP synthase gene sequences obtained from the National Center for Biotechnology Information (NCBI) databases (http://www.ncbi.nlm.nih.gov/) were combined with the partial sequences for sequence and phylogenetic analyses. A total of 93 amino acids (excluding gaps) were used to generate phylogenetic trees by the neighbour-joining method with CLUSTAL X (Thompson et al., 1997 ; Jeanmougin et al., 1998
) (with 1000 bootstrap replicates using default parameters) and by the maximum-likelihood method (100 replicates using the Jones, Taylor and Thornton method) with MOLPHY version 2.3 (Adachi & Hasegawa, 1996
). NJPlot (Perrière & Gouy, 1996
) and TreeView (Page, 1996
) were used to analyse the phylogenetic relationships of FPP synthase genes from various species. E. coli ispA (the FPP synthase gene in E. coli) was included in the analyses and used as an outgroup.
Nucleotide sequence accession numbers.
The sequences determined in this study were submitted to the DDBJ/EMBL/GenBank databases and assigned accession numbers AB053173AB053178, AB053180 and AB062882AB062884 (Table 1).
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RESULTS AND DISCUSSION |
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Phylogenetic analysis of the FPP synthase sequence
The results of the phylogenetic tree inference analysis by the neighbour joining method are shown in Fig. 1. The relationship among species in the
-3 Proteobacteria based on the FPP synthase amino acid sequences is consistent with the results obtained from 16S rRNA sequences.
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One notable deviation of the FPP synthase phylogeny from the 16S rRNA phylogeny is the positioning of the non-phototrophic A. ferrugineum IAM 12616T outside the Rhodobacter clade. This organism is closely related to the members of the phototrophic Rhodobacter genus, and its closest neighbours are Rba. azotoformans and Rba. sphaeroides (Uchino et al., 1997 ) based on 16S rRNA phylogeny, although it differs from the species of Rhodobacter in its G+C content, and by the absence of photosynthetic abilities, bacteriochlorophyll a and intracytoplasmic membrane systems, which are defining characteristics of the genus Rhodobacter (Uchino et al., 1998
). The result of the analysis using the maximum-likelihood method, however, does not support the position of this species outside the Rhodobacter clade, instead placing A. ferrugineum closer to Rba. azotoformans and Rba. sphaeroides. This implies that the position of A. ferrugineum IAM 12616T in the FPP synthase gene tree is uncertain. Nevertheless, our data demonstrate the phenotypic diversity of species in the RhodobacterRhodovulum group, as exemplified by the close association of the non-phototrophic A. ferrugineum IAM 12616T with phototrophic species of the Rhodobacter and Rhodovulum genera in the FPP synthase tree. It is imperative that more detailed taxonomic and phylogenetic studies using other molecular genes as markers, or comparison of the genome contents between this species and species of Rhodobacter, be performed in order to determine an adequate taxonomic position for A. ferrugineum IAM 12616T. Our results clearly show that FPP synthase is a useful molecule not only for differentiating Rhodobacter species from Rhodovulum species but also for revealing variations between A. ferrugineum IAM 12616T and the species of Rhodobacter.
Unusual clustering of representative members of the -2 Proteobacteria
The FPP synthase gene of the phototrophic members of the -2 subgroup, namely Rps. palustris ATCC 17001T and Rps. palustris VA2-2, formed a separate cluster from the two strains of the non-phototrophic B. japonicum, i.e. strains IAM 12608T and USDA 110. As supported by the high homology among the FPP synthase protein sequences, the distance analysis placed the two Rps. palustris strains far from these two strains of B. japonicum (Fig. 1a
). This suggests that the FPP synthase genes of these two species are not closely related and probably evolved from different ancestors. Sequence analysis of 16S rRNA placed Rps. palustris and B. japonicum into a single group despite their differing phenotypes: Rps. palustris is phototrophic, whereas B. japonicum is a non-phototrophic, nodule-forming bacterium.
The two B. japonicum strains, on the other hand, tended to cluster and formed a monophyletic group together with the other non-phototrophic bacteria M. loti MAFF 303099 and Sinorhizobium sp. NGR 234, and this cluster was supported by a high bootstrap value of 100%. These organisms, collectively referred to as rhizobia (Van Rhijn & Vanderleyden, 1995 ), are capable of eliciting nodules on their leguminous hosts. Although these rhizobia shared several morphological and biochemical properties, particularly the ability to form nodules in their hosts, they differed from each other in terms of host specificity and did not form a monophyletic group in the 16S rRNA phylogeny, but rather were placed in several well-separated genera. Reconstruction of the evolutionary history from 16S rRNA gene sequence divergence showed that B. japonicum IAM 12608T and B. japonicum USDA 110 are more related to Rps. palustris strains than to either Sinorhizobium sp. NGR234 or M. loti MAFF 303099 (Fig. 1b
). In the FPP synthase tree, however, the positioning of M. loti MAFF 303099 closer to Sinorhizobium sp. NGR 234 than to the B. japonicum strains was similar to the positioning in the 16S rDNA phylogeny. Hence, it is interesting to note that in the FPP synthase gene tree, the non-phototrophic rhizobia (B. japonicum IAM 12608T, B. japonicum USDA 110, Sinorhizobium sp. NGR234 and M. loti MAFF 303099) were all included in a single cluster supported by a significant bootstrap value of 100%, suggesting that their FPP synthase genes share a common ancestor. This was further supported by the high mean sequence similarity value of their FPP synthases. The longer branch length in the phylogenetic tree that supports the cluster of rhizobia, and the higher homology values of the FPP synthase genes as compared to the 16S rDNAs of rhizobia, are suggestive of a recent lateral gene transfer event before their diversification into their present genera. The lateral transfer of symbiotic genes mediated by plasmids and phages in rhizobia has been reported to be common (Sullivan et al., 1995
; Sullivan & Ronson, 1998
), and it is not impossible that FPP synthase gene transfer occurred among these rhizobia. Analysis of the M. loti MAFF 303099 chromosome and the plasmid of Sinorhizobium sp. NGR 234 showed that the FPP synthase genes of these rhizobia are located in the symbiotic island region, flanked by highly conserved genes/ORFs (>90% similarity), and included in a cluster containing the nitrogen fixation and nodulation genes (data not shown). Based on the nucleotide sequence analyses, the FPP synthase gene of B. japonicum strains would have been transferred before the divergence of M. loti MAFF 303099 and Sinorhizobium sp. NGR 234. However, the mechanism of FPP synthase gene transfer in this case remains to be clarified. Alternatively, since the FPP synthase gene is within the symbiotic island region proximal to the nitrogen fixation and nodulation genes, the gene might have played a major role in nodulation; the high similarity of the FPP synthase gene could be due to high selection pressure restricting its divergence.
In summary, the overall FPP synthase tree topology is in good agreement with the 16S rRNA phylogeny, although the FPP synthase gene was more divergent than the 16S rRNA. The greater sequence variations of the FPP synthase gene than the 16S rRNA make this gene useful for indicating the phylogenetic relationships among species belonging to a genus, and relationships among several genera. The use of the FPP synthase gene to clarify phylogenetic relationships at the inter- and intrageneric levels may not be appropriate for rhizobial strains due to the high homology among FPP synthases of rhizobia. Nevertheless, the topology of the FPP synthase tree is useful for identifying unusual evolutionary processes that probably occurred during the diversification of the Proteobacteria, and particularly in the case of the -2 Proteobacteria, as observed from the FPP synthase genes of Rps. palustris and B. japonicum. The discrepancies detected between the FPP synthase phylogeny and the 16S rRNA phylogeny suggest that lateral gene transfer had occurred. FPP synthase sequences therefore offer one of the best opportunities to establish the relationships among the phototrophic Rps. palustris and the nodule-forming bacteria.
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ACKNOWLEDGEMENTS |
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This paper represents a portion of the dissertation submitted by J. J. L. Cantera to Osaka University in partial fulfilment of the requirements for a PhD degree.
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Received 16 January 2002;
revised 5 March 2002;
accepted 11 March 2002.