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The Institute for Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850, USA
Correspondence
Jonathan H. Badger
jbadger{at}tigr.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-proteobacterium currently classified as a member of the order ‘Rhodobacterales’. Although this classification is supported by 16S rRNA gene sequence phylogeny, 23S rRNA gene sequence analysis, concatenated ribosomal proteins, HSP70 and EF-Tu phylogenies all support classifying Hyphomonas neptunium as a member of the Caulobacterales instead. The possible reasons why the 16S rRNA gene sequence gives conflicting results in this case are also discussed.
Published online ahead of print on 3 December 2004 as DOI 10.1099/ijs.0.63510-0.
Newick tree files and FASTA-format sequence alignments used to generate the trees are available as supplementary information in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-proteobacterium that was isolated from sea water from the harbour at Barcelona, Spain, and was originally described as Hyphomicrobium neptunium (Liefson, 1964
). This description was later emended to the current Hyphomonas neptunium on the basis of DNA–DNA hybridization information (Moore et al., 1984), which showed a closer relationship with Hyphomonas polymorpha (Pongratz, 1957), a marine prosthecate bacterium isolated from a diver with a severe sinus infection, than with other members of the genus Hyphomicrobium. Hyphomonas neptunium also lacks, as does Hyphomonas polymorpha, the ability to utilize C1 molecules as carbon sources, whereas recognized members of Hyphomicrobium have this ability (Moore et al., 1984).
Members of Hyphomonas have an unusual reproductive cycle for prosthecate bacteria; daughter cells are formed on the distal side of the stalk, indicating that DNA, proteins and other cellular components must traverse the stalk (Hirsch, 1974). This trait is shared with numerous marine bacteria originally classified as members of the genus Caulobacter, and the closer relationship between these caulobacters and Hyphomonas to the exclusion of the freshwater caulobacters is also supported by 16S rRNA gene sequence phylogeny (Strömpl et al., 2003; Abraham et al., 1999; Stahl et al., 1992). However, to our knowledge, there have been no studies suggesting a close relationship between freshwater members of Caulobacter (such as Caulobacter crescentus CB15) and Hyphomonas. Currently, Hyphomonas is classified as a member of the order ‘Rhodobacterales’ (Garrity et al., 2005), whereas the caulobacters are considered members of the eponymous order Caulobacterales (Henrici & Johnson, 1935). In this paper we show that, although 16S rRNA gene sequence analysis supports the current classification, phylogenies based on other markers, such as the 23S rRNA gene and many protein sequences, support grouping Hyphomonas as a member of the Caulobacterales. The implications for the taxonomy of the ‘Rhodobacterales’ and Caulobacterales are discussed, as recent taxonomic recommendations (Stackebrandt et al., 2002) support taking into account phylogenetic analyses from multiple genes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Gene predictions were provided by GLIMMER (Delcher et al., 1999) and functional assignments were produced according to Tettelin et al. (2001). The following complete (or nearly complete) genomes of -proteobacteria were used as sources of sequences for phylogenetic analyses: Agrobacterium tumefaciens C58 (Wood et al., 2001), Anaplasma phagocytophilum HZ (TIGR, unpublished), Bradyrhizobium japonicum USDA 110 (Kaneko et al., 2002), Brucella suis 1330 (Paulsen et al., 2002), C. crescentus CB15 (Nierman et al., 2001), Ehrlichia chaffeensis ArkansasT (TIGR, unpublished), Mesorhizobium loti MAFF303099 (Kaneko et al., 2000), Neorickettsia sennetsu Miyayama (TIGR, unpublished), Novosphingobium aromaticivorans DSM 12444T [Joint Genome Institute (JGI), unpublished], Rhodobacter capsulatus SB1003 (Integrated Genomics, unpublished), Rhodopseudomonas palustris CGA009 (Larimer et al., 2004), Rhodospirillum rubrum ATCC 11170T (JGI, unpublished), Rickettsia conorii Malish 7T (Ogata et al., 2001), Silicibacter pomeroyi DSS-3T (Moran et al., 2004), Sinorhizobium meliloti 1021 (Capela et al., 2001) and Wolbachia pipientis wMel (Wu et al., 2004). Additionally, the genome of Escherichia coli K-12 MG1655 (Blattner et al., 1997) was used as a source of outgroup sequences. The data from the published genomes were obtained from GenBank; the unpublished data can be obtained from TIGR (http://www.tigr.org/tdb/mdb/mdbinprogress.html), JGI (http://genome.jgi-psf.org/microbial/) and Integrated Genomics (http://ergo.integratedgenomics.com/R_capsulatus.html).
Phylogenetic analysis.
Five multiple sequence alignments (see supplementary information available in IJSEM Online) were created for the purpose of phylogenetic inference. These alignments were of: (i) the 16S rRNA gene sequence, (ii) the 23S rRNA gene sequence, (iii) 30 concatenated ribosomal proteins (totalling approximately 4000 amino acids), (iv) HSP70 proteins and (v) EF-Tu proteins. The rRNA sequences were aligned and masked using the ALIGN sequence tool of the Ribosomal Database Project (Cole et al., 2003), and the protein sequences were aligned using MUSCLE (Edgar, 2004). For all the alignments, bootstrapped neighbour-joining (Saitou & Nei, 1987) trees were created using the program QUICKTREE (Howe et al., 2002). For the rRNA alignments, bootstrapped maximum-likelihood (Felsenstein, 1981) trees were created using the DNAML program from PHYLIP 3.6b (Felsenstein, 2004), with a 
-distribution (=0·5) of rates over four categories of variable sites. For the protein alignments, PROML (also from PHYLIP 3.6b) was used to create maximum-likelihood trees, applying the JTT (Jones et al., 1992) model of substitution, again with a -distribution (=0·5) of rates over four categories of variable sites. The resulting consensus trees for the protein and rRNA trees were fed into the appropriate program (PROML or DNAML) as user trees in order to obtain the branch lengths. In addition, APIS (J. H. Badger, unpublished), an automated pipeline for phylogenetic inference, was run on all predicted proteins in the Hyphomonas neptunium genome, generating bootstrapped neighbour-joining trees of each protein and its homologues.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; see Table 1 for the GenBank GI numbers and ranges used from published genomes) supports the current classification of Hyphomonas neptunium as a member of the order ‘Rhodobacterales’, and indeed a similar analysis was probably the reason behind this classification. However, none of the other commonly used phylogenetic markers, including the 23S rRNA gene sequence (Fig. 2a), concatenated ribosomal proteins (Fig. 2b), HSP70 proteins (Fig. 2c) and EF-Tu proteins (Fig. 2d), supports this classification. Instead, they support a relationship between Hyphomonas neptunium and C. crescentus. A similar relationship was seen in the trees generated by APIS, in which over 30 % of the Hyphomonas neptunium proteins had a protein from C. crescentus as their closest relative, as opposed to only 6 % that grouped with a member of the ‘Rhodobacterales’. Most notably, the flagellar and other chemotaxis proteins tend to show a closer relationship to those of Silicibacter pomeroyi than to those of C. crescentus, although this may be because the Hyphomonas neptunium versions of these proteins are quite divergent from even their closest known homologues.
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) shows only weak (52 %) support for the currently accepted grouping of Hyphomonas neptunium among the ‘Rhodobacterales’, and the 23S rRNA gene sequence tree (Fig. 2a) shows only somewhat stronger (64 %) support for the alternative classification among the Caulobacterales. The concatenated ribosomal protein tree (Fig. 2b), however, shows excellent support (100 %) for this alternative classification, and levels of support from the HSP70 (Fig. 2c) and EF-Tu (Fig. 2d) trees for the alternative classification are strong as well (69 and 76 %, respectively).
In order to explore further the degree of support that each tree has for the alternative hypotheses, Kishino–Hasegawa–Templeton tests (Kishino & Hasegawa, 1989; Templeton, 1983) were performed to determine whether each alignment preferred the 16S or the 23S rRNA gene sequence tree. For each alignment, if the mean of the log-likelihood differences between the 16S and 23S tree across the sites was greater than 1·96 standard deviations, then the more likely tree was judged to be significantly preferred. The 23S alignment and all protein alignments except for the EF-Tu alignment significantly preferred the 23S tree; although the 16S alignment preferred the 16S tree and the EF-Tu alignment preferred the 23S tree, they did not do so at a statistically significant level.
Evolutionary implications
Although the discovery of conflict between 16S rRNA gene sequence and protein trees is not in itself a novel finding (e.g. Doolittle, 1999; Gupta & Golding, 1993), in general such studies either try to argue for the superiority over rRNA of a single favourite marker protein [as was done by Gupta & Golding (1993) for HSP70] or claim that rampant horizontal gene transfer has destroyed all phylogenetic signal (as in Doolittle, 1999). To our knowledge, this is the first study in which numerous proteins, together with the 23S rRNA gene, consistently yield a single alternative order-level classification for a bacterial species.
What can be the cause of this difference? One possibility is horizontal gene transfer of the 16S rRNA gene. Horizontal gene transfer of the 16S rRNA gene has been suggested as an explanation for patterns seen at the genus level (e.g. Schouls et al., 2003; Parker et al., 2002), and artificially induced transfer of the 16S and 23S rRNA genes between Escherichia coli and Salmonella typhimurium has been demonstrated experimentally (Asai et al., 1999). The presence of only a single copy of the 16S rRNA gene in Hyphomonas neptunium would also make horizontal gene transfer of the 16S rRNA gene possibly easier than in most bacteria. Another possibility could be long-branch attraction (Felsenstein, 1978) in the tree based on 16S rRNA gene sequence analysis, but, as shown in Figs 1 and 2(a), the branch lengths appear not to be particularly long.
In addition to being supported by all the sequence data except that for the 16S rRNA gene, a classification of Hyphomonas as a member of the Caulobacterales also makes sense from the standpoint of phenotypic characters. Like Caulobacter, members of Hyphomonas are aerobic, dimorphic, prosthecate bacteria. In the current classification scheme, these traits either would have had to evolve independently in the ‘Rhodobacterales’ or would have to have been present in a common ancestor of the ‘Rhodobacterales’ and Caulobacterales and then been lost by the majority of the members of the ‘Rhodobacterales’.
Current guidelines for the rearrangement of higher order taxa preclude the transfer of a genus without analysis of the type species (Sneath, 1992). Given that the type species of Hyphomonas is Hyphomonas polymorpha rather than Hyphomonas neptunium, a transfer of the genus Hyphomonas is not presently possible. However, given the close phylogenetic relationship between these two species [according to the 16S rRNA gene sequence and DNA–DNA hybridization studies in Weiner et al. (2000) they are among the most closely related of the eight recognized Hyphomonas species], we expect that future work on Hyphomonas polymorpha will support such a transfer.
Additionally, there exist several genera of prosthecate budding bacteria (Hirschia, Maricaulis and Oceanicaulis) that are immediate relatives of Hyphomonas according to 16S rRNA gene sequence phylogeny (Strömpl et al., 2003). Assuming that this is not an artefact of 16S rRNA gene sequence phylogeny, these genera would have to be transferred into the Caulobacterales along with Hyphomonas. Further work, including genome sequencing of the type species of representatives of these genera, would provide valuable data that will help to clarify the relationships among the prosthecate -proteobacteria, and possibly support the transfer of Hyphomonas.
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ACKNOWLEDGEMENTS |
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