Phylogeny of Firmicutes with special reference to Mycoplasma (Mollicutes) as inferred from phosphoglycerate kinase amino acid sequence data

Phylogeny of Firmicutes with special reference to Mycoplasma (Mollicutes) as inferred from phosphoglycerate kinase amino acid sequence data

  1. Matthias Wolf1
  2. Tobias Müller1
  3. Thomas Dandekar1 and 
  4. J. Dennis Pollack2

+Author Affiliations

  1. 1University of Würzburg, Department of Bioinformatics, Biocenter, Am Hubland, 97074 Würzburg, Germany
  2. 2Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, 2066 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210, USA
  1. Correspondence
    Thomas Dandekar
    [email protected]
    J. Dennis Pollack
    [email protected]


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The phylogenetic position of the Mollicutes has been re-examined by using phosphoglycerate kinase (Pgk) amino acid sequences. Hitherto unpublished sequences from Mycoplasma mycoides subsp. mycoidesMycoplasma hyopneumoniae and Spiroplasma citri were included in the analysis. Phylogenetic trees based on Pgk data indicated a monophyletic origin for the Mollicutes within the Firmicutes, whereas Bacilli (Firmicutes) and Clostridia (Firmicutes) appeared to be paraphyletic. With two exceptions, i.e. Thermotoga (Thermotogae) and Fusobacterium (Fusobacteria), which clustered within the Firmicutes, comparative analyses show that at a low taxonomic level, the resolved phylogenetic relationships that were inferred from both the Pgk protein and 16S rRNA gene sequence data are congruent.


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  • Published online ahead of print on 12 December 2003 as DOI 10.1099/ijs.0.02868-0.

  • The GenBank/EMBL/DDBJ accession numbers for the Pgk sequences of Mycoplasma mycoides subsp. mycoidesSpiroplasma citri and Mycoplasma hyopneumoniae are BX293980, AJ580006 and AY319328, respectively.

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Morphologically and microbiologically, Mollicutes are classified as Bacteria that were probably derived from lactobacilli, bacilli or streptococci by regressive evolution and genome reduction, to produce the smallest and simplest free-living and self-replicating cells (Razin et al., 1998). Their lifestyle is, in general, parasitic. Structurally, Mollicutes are characterized by the complete lack of cell wall and the presence of an internal cytoskeleton (Balish & Krause, 2002Dandekar et al., 2002).

Based on 16S rRNA data, the taxonomy, as well as the phylogeny and evolution, of Mollicutes have recently been discussed (Johansson & Pettersson, 2002Maniloff, 2002). By phylogenetic analysis, low-G+C, Gram-positive Bacteria (Firmicutes) comprise three groups: BacilliClostridia and Mollicutes. However, based on 16S rRNA gene sequence data, only the Mollicutes are well-supported as being monophyletic (Ludwig & Klenk, 2001).

In this study, we present the results of our analysis that used phosphoglycerate kinase (Pgk) amino acid sequences as a molecular marker, instead of 16S rRNA, to examine the phylogeny of Firmicutes taxa. Pgk is one of the oldest ‘housekeeping’ enzymes; its evolutionary time has been estimated to be about 40 million years, which is about twice as long as was required for 1 % mutation to occur in cytochrome c or glyceraldehyde-3-phosphate dehydrogenase (Ciccarese et al., 1989). Other reports consider that Pgk is evolving at a linear rate of four to six accepted point mutations in 100 million years, i.e. about the same rate as for cytochrome c (Fothergill-Gilmore, 1986). Even for a ‘housekeeping’ enzyme, this is a very conserved sequence (Fothergill-Gilmore, 1986Fothergill-Gilmore & Michels, 1993). The pgk gene may be an example of a ‘core’ household gene (Daubin et al., 2002). The metabolic role of Pgk, especially in Mollicutes, has recently been discussed (Pollack et al., 2002). The role of Pgk is particularly consequential in Mollicutes, as these bacteria lack cytochrome pigments and the citric acid cycle and are thought to synthesize most of their ATP by substrate phosphorylation during glycolysis, mediated by the presumably essential action of Pgk and pyruvate kinase (Pollack, 2002).

The focus of this study is on the phylogenetic position of the amino acid sequences of Mollicutes Pgks and their relationships to the 16S rRNA Mollicutes subgroups that were established by Johansson et al. (1998). We included unpublished Pgk sequences from Mycoplasma mycoides subsp. mycoidesMycoplasma hyopneumoniae and Spiroplasma citri. Furthermore, the utility of Pgk as a phylogenetic marker to analyse the phylogeny and evolution of Firmicutes is discussed.

In initial studies, Pollack (2002) hypothesized that Pgk should be an attractive marker for phylogenetic analyses. Similarly, our own preliminary analyses of the pgk gene were also performed by using a computational neighbour-joining (NJ) method to analyse 100 complete pgk gene sequences from different life forms (BacteriaArchaea and Eukarya). We found relationships and clustering that were similar or identical to those already established by microbiological and phenotypical criteria. Individual and entire groupings, e.g. CrenarchaeotaEuarchaeota, ciliates, fungi, plants, mammals and the αβ and most of the γ divisions of the Eubacteria were entirely separable. Furthermore, the NJ branching order of the Mollicutes exactly followed the predicted 16S rRNA groupings that were described by Johansson et al. (1998). Of additional interest was the closer relationship of the Mollicutes to the low-G+C non-spore-formers Staphylococcus aureus and Lactobacillus delbrueckii subsp. bulgaricus, and their greater distance from the low-G+C spore-formers Bacillus and Clostridium spp. The NJ-derived Pgk tree suggested that Mycoplasma spp. are related more closely to the Streptococcus/Lactobacillus subgroups and less closely to Bacillus and Clostridium spp. The relationship of the Mollicutes to Streptococcus/Lactobacillus was first reported by Neimark (1979), who concluded that the Mollicutes (acholeplasmas) descended from this group. His view was based on both the similarity of their fructose 1,6-bisphosphate-activated lactate dehydrogenases and the immunological homology of their aldolases.

In this study, the Pgk tree was re-examined and tested by more sensitive and rigorous computational means than the NJ techniques. Here, we have used distance, maximum-parsimony (MP) and maximum-likelihood (ML) methods to calculate a Pgk-based phylogenetic tree of the Firmicutes.

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We studied all available complete amino acid Pgk sequences of Firmicutes, plus related sequences (Fusobacterium and Thermotoga), firstly by using BLAST (basic local alignment search tool), i.e. iterative sequence alignment procedures (Altschul et al., 1997). We included three previously unpublished Mollicutes sequences. Alignment and direct comparison of amino acid sequences (GenBank accession numbers are given in Fig. 1) were performed with CLUSTALX (Thompson et al., 1994) and the Windows-based multi-sequence alignment editor of Hepperle (2002).

Fig. 1.View larger version: 

Fig. 1.

ML tree, as derived from PROML analysis of Pgk sequences. The outgroup is forced. Numbers at nodes indicate bootstrap support values (>50 %) for clusters to the right of them, as calculated by MP/ML (TREEPUZZLE)/nj (PHYLIP)/nj (TREECON). Boxed areas enclose the following 16S rRNA subgroups of Mollicutes (Johansson et al., 1998): pneumoniae group III, hominis group IV and spiroplasma group II.

An MP analysis of aligned amino acid sequences was conducted by using PAUP* version 4.0b10 win32 (Swofford, 2002). Heuristic searches with 10 random taxon addition replicates and tree bisection–reconnection swapping were applied. The MulTrees and Collapse options of PAUP* were used and character changes were interpreted with ACCTRAN optimization. Characters were weighted equally and coded as unordered; gaps were treated as missing data. Bootstrap support was estimated, based on 100 replicates.

For an ML analysis that used the WAG model (Whelan & Goldman, 2001), TREEPUZZLE (Strimmer & von Haeseler, 1996) was used with 10 000 quartet-puzzling steps. Further, using the JTT model (Jones et al., 1992), an ML topology was obtained by using PROML, as implemented in PHYLIP 3.6 (Felsenstein, 1993).

Additionally, by using default settings, two NJ trees (Saitou & Nei, 1987) were generated by using TREECON for Windows (Van de Peer & De Wachter, 1994) and PHYLIP 3.6. Bootstrap support was estimated, based on 500 and 100 replicates, respectively. All trees were rooted by using Nostoc sp. (Q8YPR1) and displayed by using TREEVIEW (Page, 1996).

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From comparison of different substitution models and different tree phylogeny algorithms, molecular phylogenetic analyses of Pgk sequences resulted in identical tree topologies with high bootstrap support values (Fig. 1).

The monophyletic Mollicutes (bootstrap support of up to 95 %) are the sister group of Bacilli II plus Fusobacterium (Fusobacteria) (Fig. 1). This large clade (MollicutesBacilli II and Fusobacterium) is the sister group to Thermotoga (Thermotogae), which, like Fusobacterium, does not belong to the Firmicutes. Because of the relative positions of Fusobacterium and Thermotoga, the Firmicutes appear to be polyphyletic. Bacilli I (Fig. 1), followed by Clostridia I and II (Fig. 1), clustered at basal positions within this polyphyletic assemblage that comprises low-G+C, Gram-positive Bacteria (Firmicutes).

It is of note that, regardless of the algorithm applied, Bacilli and Clostridia appeared to be paraphyletic. Within the Mollicutes, the pneumoniae group, the spiroplasma group and the hominis group (the latter represented by Mycoplasma pulmonis and M. hyopneumoniae) are supported strongly (Fig. 1). The grouping of M. pulmonis within the pneumoniae group and the respective positions of Bacillus subtilisBacillus haloduransStreptococcus pyogenes and Lactococcus lactis also reflect and support genome trees that were established by gene content or gene order (Dandekar et al., 2002). Hitherto unpublished sequences clustered as follows: M. hyopneumoniae, as mentioned above, is the sister group to M. pulmonisM. mycoides subsp. mycoides is the sister group to Mycoplasma capricolum subsp. capricolum and Spiroplasma citri is the sister group to the M. mycoides/M. capricolum cluster.

At a low taxonomic level, especially within the Mollicutes, the resolved phylogenetic relationships inferred from both protein and 16S rRNA gene sequence data are congruent. The nine Mollicutes clustered into three Pgk subclades (Fig. 1). The members of each of these Pgk subclades were, without exception, the same as previously grouped by 16S rRNA analyses (Johansson et al., 1998Johansson & Pettersson, 2002): 16S rRNA group III (Mycoplasma pneumoniaeMycoplasma genitaliumUreaplasma urealyticum and Mycoplasma penetrans), 16S rRNA group IV (M. hyopneumoniae and M. pulmonis) and 16S rRNA group II (M. mycoides subsp. mycoidesM. capricolum subsp. capricolum and S. citri).

Furthermore, the non-spore-forming genera StreptococcusStaphylococcusListeria and Lactobacillus (clustering within Bacilli II) and the spore-forming genera Geobacillus and Bacillus (Bacilli I) are well-supported as being monophyletic (Fig. 1). The genus Mycoplasma appeared to be paraphyletic (because of Ureaplasma and Spiroplasma) and Clostridium is also paraphyletic in this analysis (Fig. 1). A more schematic (unrooted) representation of relationships within the Firmicutes is given in Fig. 2.

Fig. 2.View larger version: 

Fig. 2.

Schematic (unrooted) representation of relationships within the Firmicutes.

We believe that Pgk may be used as an appropriate marker, yielding high bootstrap support values, for phylogenetic analysis of Firmicutes taxa. In most cases, we found that support is even higher than that found for 16S rRNA analyses (data not shown).

Although Fusobacterium nucleatum is an anaerobic, Gram-negative bacterium that is not classified among the low-G+C, Gram-positive Firmicutes, it was studied because of sequence similarities obtained by BLAST and because several of its core metabolic features have been reported to be similar to those of Clostridium spp., Enterococcus spp. and Lactococcus spp. (Kapatral et al., 2002). However, like other members of the Firmicutes, it has a low DNA G+C content of 27 %. Its cell-wall structure results in its Gram-negative character. Our analyses placed it in the Bacilli II group. As reported by Kapatral et al. (2002), it possesses distinguishing genomic features that are found in some members of the Bacilli II group: clustering of ORFs, 16S rRNA gene sequence similarities, uracil monophosphate biosynthesis, Rho factor, elongation factor EF-G, subunits of glutamine tRNA, its peptidases, absence of fatty acid desaturase and certain acyltransferases and transposase content. Interestingly, F. nucleatum lacks nucleoside diphosphate kinase (NdK), a supposedly ubiquitous gene that expresses an essential metabolic activity. The gene was not found in any Clostridium spp. (Firmicutes) nor any Mollicutes (Mycoplasma or Ureaplasma species) sequenced so far (Pollack et al., 2002).

The phylogenetic position of Thermotoga within the Firmicutes was unexpected. The Pgk from Thermotoga was included in the analysis only because of its high sequence similarity, obtained by BLAST search, to Firmicutes Pgk sequences. Thermotoga has an affinity to the low-G+C, Gram-positive Bacteria (Nelson et al., 1999). Daubin et al. (2002) suggested that the phylogenetic position of hyperthermophilic and radioresistant species is close to that of mesophilic species, like Bacilli. We believe that these opinions tend to minimize the concept of the ‘hyperthermophilic origin of life’.

The exact position of Thermotoga within the ‘tree of life’ still remains an open question, as different markers have yielded varying results; they either place Thermotoga close to the root of the ‘tree of life’ (e.g. Brown et al., 2001) or further ‘up’ from the root (Brochier & Philippe, 2002Daubin et al., 2002). Also, the position of Thermotoga with respect to other species is in part compromised by an unknown but important degree of horizontal transfer of genes from other, in particular archaean, species. Similarly, adaptation to a hyperthermophilic environment is a confounding possibility. These factors complicate predictions that involve the genomic content of molecules from organisms that are adapted to high temperature (there are specific adaptations in enzymes such as RNA polymerases, as well as to increase the stability of nucleic acids at higher temperatures).

The value of Pgk as a phylogenetic marker was investigated by Chattopadhyay & Chakrabarti (2003). In that study, evolutionary conclusions were derived solely from a basic statistic (the mean second moment of the codon base distribution) of the coding sequence of Pgk. This statistic was used to position taxonomic groups or organisms in the ‘vertical position’ on the evolutionary tree. The results for Pgk were convincing, whereas those for other genes (e.g. the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase) could not be used as acceptable phylogenetic markers in these computational analyses. Therefore, these and our results strengthen the usefulness of Pgk as a phylogenetic marker.

The Pgk tree of the Mollicutes is in agreement with Neimark (1979), in suggesting that Mycoplasma spp. are related more closely to the Streptococcus/Lactobacillus subgroup than to Bacillus and Clostridium spp.

Pgk shows several advantages for use as a marker enzyme, because of its widespread distribution in central metabolic activity. Further research will extend this analysis to include enzyme-structure data and more biochemical data, to study in more detail the relationships that have been examined here, including other clades and species.

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We gratefully acknowledge receipt of the unpublished Mollicutes Pgk gene sequences of Mycoplasma mycoides subsp. mycoides SC from Joakim Westberg and Karl-Erik Johansson (National Veterinary Institute, Uppsala, Sweden), Mycoplasma hyopneumoniae from F. Chris Minion (Iowa State University, Ames, Iowa, USA) and Steven P. Djordjevic (Elizabeth McArthur Agricultural Institute, Cambden, NSW, Australia) and Spiroplasma citri from Frédéric Laigret and colleagues (INRA, Villenave d'Ornon, France). We thank the students of the Bioinformatics/Phylogenetics courses at the University of Würzburg for valuable discussions.

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