The genome of M. infernorum strain V4 [GenBank: CP000975] consists of a single circular chromosome of 2,287,145 bp. General features of the genome and a summary of the annotation of protein-coding genes are shown in Table 1 and Figure 1. The origin of replication was identified by GC skew analysis 11 and was mapped 250 nt upstream the dnaA gene. We identified approximately 20 loci that correspond to a single class of insertion sequences of the IS605 family 12. No prophages were detected but there is a region in the genome that comprises a potential integrative plasmid (Minf_1153–Minf_1199). Among the 2473 predicted proteins, 731 had no detectable homologs in the NCBI protein database. This fraction of "ORFans" is similar to those reported for the first sequenced genomes from other bacterial phyla and is consistent with the notion that Verrucomicrobia form a distinct clade that is only distantly related to other bacteria. Most of the proteins that had homologs in the database could be assigned to the Clusters of Orthologous Groups of proteins (COGs, 13), see Table 1.

Analysis of the 16S rRNA sequence of M. infernorum identified it as a member of a new subdivision in the phylum Verrucomicrobia 3, see also 45. Ever since the original description of the Verrucomicrobia as a separate lineage of bacteria 14, analysis of 16S rRNA sequences showed clustering of Verrucomicrobia with Planctomycetes and/or Chlamydiae but the bootstrap support for this grouping was typically less than 50% 1516. Similar results were obtained from the analysis of 23S rRNA sequences 17. Recent phylogenetic analyses of 16S rRNA and ribosomal protein sequences have led to the proposal that four bacterial phyla; namely Planctomycetes, Verrucomicrobia, Chlamydiae and Lentisphaerae, together with two candidate phyla; Poribacteria and OP3, comprise a single superphylum 12. However, analysis of phylogenetic trees and shared inserts in several conserved proteins has confirmed the affinity of Chlamydiae and Verrucomicrobia but failed to support the affiliation between these phyla and the Planctomycetes 18. Having determined the complete genome sequence of M. infernorum and with complete genomes also available for representatives of Chlamydiae and Planctomycetes, we constructed phylogenetic trees for concatenated sequences of ribosomal proteins and subunits of the RNA polymerase. In both trees, M. infernorum confidently grouped with Chlamydiae, and the Verrucomicrobia-Chlamydiae clade grouped with Planctomycetes (Figure 2 and Supplementary Figure 1 [see Additional file 1]).

However, sequence analysis of the complete set of predicted proteins of M. infernorum revealed a complex picture. The largest fraction (~23% of the proteins) had their top BLAST hits among proteobacterial proteins, whereas the fraction of proteins that were most similar to homologs from the Verrucomicrobia/Chlamydiae and Planctomycetes group comprised only ~7% (Figure 3). These observations must be interpreted with caution considering the unequal representation of bacterial phyla in current databases (with considerable over-representation of Proteobacteria) as well as the fact that sequence similarity is not necessarily an accurate reflection of phylogenetic affinity. Nevertheless, with these caveats, and considering the availability of several large (albeit, with the exception of Rhodopirellula baltica, unfinished) planctomycete genomes in the database used for M. infernorum genome analysis, the broad spread of the top hits seems to suggest a complex history of this lineage, with numerous putative horizontal gene exchanges shaping the genome. The abundance of proteins with the greatest similarity to homologs from Proteobacteria is compatible with the dominance of this bacterial phylum among the known methylotrophs.

To compare the overall gene composition in M. infernorum with other bacteria, we performed Correspondence Analysis of the matrix of the phyletic patterns (presence or absence of the given gene in a given genome) of 59 bacterial species (listed in Supplementary Table 1 [see Additional file 1]) in the eggNOG database 19. The results show that M. infernorum groups neither with its closest relatives (Chlamydiae and Verrucomicrobia) nor with any other bacterial clade (Figure 4). This lack of clustering by phyletic pattern together with the position of M. infernorum (Figure 4B) near the origin of coordinates (which, by definition, is the baricenter of the data set) further supports the notion of a complex history of the gene set of M. infernorum, with likely contributions from diverse groups of bacteria. The nearest neighbors of M. infernorum in the genome content space (Figure 4C) are various members of Proteobacteria (Rickettsia, Neisseria, Escherichia, Methylococcus), Thermotogae, Aquificae and a single representative of Actinobacteria (Rubrobacter).

Our attempts to identify a genomic signature for the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum, i.e., a set of genes that would be present in all members of this group but in no other organisms, failed to identify any genes fitting that definition. The closest candidate was a protein of unknown function, Minf_1886, which is present in Verrucomicrobia and Planctomycetes but not in Chlamydiae (Supplementary Figure 2 [see Additional file 1]). Similarly, of the 12 protein families that recently have been reported to be specific for the PVC superphylum 2, only four were identified in M. infernorum (Supplementary Table 2 [see Additional file 1]), and representatives of all of these families could also be found in other bacterial clades (data not shown).

Since the gene content of M. infernorum substantially differs from the gene contents of other member of the PVC superphylum, we used the inferred gene set of the last common ancestor of all bacteria (LCBA) to reconstruct the most parsimonious scenario of gene gain and loss in this branch 20. This approach assigned 1382 COGs (genes) to the LCBA 21. The results of the reconstruction for the M. infernorum lineage suggest considerable gene flux dominated by gene loss (526 genes inferred to have been lost and 262 gained). Approximately 75 genes might have been lost at the level of the last common ancestor of the PVC superphylum, including cell division proteins FtsX, FtsE, MinC, MinD, and MinE. Predicted gene gains additionally encompassed M. infernorum proteins that did not fit into any COGs including most of the genes responsible for methylotrophy. There are approximately 200 such proteins that have homologs in the databases and 731 ORFans. In accord with the lifestyle of M. infernorum, it appears that many genes involved in autotrophy were gained whereas genes related to heterotrophic processes were lost (Supplementary Figure 3 [see Additional file 1]). This dynamic was especially prominent among the genes coding for proteins implicated in energy metabolism, where approximately equal numbers of genes have been lost and gained. In most other metabolism-related categories, gene loss exceeded gene gain.

Notably, many regulatory and even informational genes were apparently lost, a trend that might reflect ongoing genome streamlining, especially considering that M. infernorum has the smallest genome among all representatives of Verrucomicrobia with known or estimated genome sizes. Some of the apparent gains and losses appear quite unexpected. In particular, M. infernorum seems to have acquired genes for several proteins that belong to the gene set that is conserved in archaea and eukaryotes. This group of proteins includes three subunits of the proteasome (Minf_1279, Minf_1281 and Minf_1284), and accessory and regulatory proteins encoded in the same neighborhood, ATP-dependent DNA ligase (Minf_0008, COG1423), and archease, a protein apparently involved in diverse nucleic acid modification reactions (Minf_0305, COG1371). These proteins of M. infernorum are most closely related to orthologs encoded in other bacteria; in particular, the proteasome subunits show the strongest similarity to orthologs from Actinobacteria. These observations suggest extensive horizontal gene transfer among bacteria, conceivably, following the initial transfer from an archaeal source.

The specific gene loss in the M. infernorum branch appears to be another manifestation of genome streamlining. Several highly conserved informational and housekeeping genes are encoded in all other sequenced genomes of the PVC superphylum, but not in M. infernorum. This group of lost genes includes those encoding the house-cleaning protein Maf (COG0424), rRNA methylase SpoU (COG0566); tRNA and rRNA cytosine-C⁵-methylase Sun (COG0144), tRNA-dihydrouridine synthase (COG0042), single-stranded DNA-specific exonuclease RecJ (COG0608), and the type II secretory system PulDFG. Several genes, in particular, those for proteins involved in DNA repair, apparently have been lost independently in both the M. infernorum branch and the Chlamydiae branch (these genes are present in other genomes from the Verrucomicrobia/Lentisphaerae branch). These include SbcC (COG0419) and SbcD (COG0420), respectively, an ATPase and exonuclease involved in repair of stalled replication forks, and RadC (COG2003), implicated in recombinational repair. The absence of these proteins in M. infernorum is unexpected because they are encoded in the genomes of the great majority of free-living bacteria.

Despite the general trend toward gene loss, we identified several lineage-specific expansions of paralogous gene families in the M. infernorum genome. Several of these expanded families encode membrane proteins. There are at least 19 paralogs of a TonB-like outer membrane receptor (COG1629) that is involved in import of essential organometallic micronutrients, including iron-siderophores 22. There are also 10 clusters coding for the outer membrane channel protein TolC (COG1538) and/or the periplasmic (fusion) protein AcrA (COG0845), which might be involved in multidrug or heavy-metal efflux 23. Another expansion includes 6 paralogs of the starvation-inducible outer membrane lipoprotein Slp (COG3065) 24. Expansion of these protein families is typical of proteobacterial methylotrophs and Proteobacteria in general; however, expansion of the Slp family might be related to acid resistance of M. infernorum (see below).

Analysis of the M. infernorum genome allowed us to reconstruct its central metabolic pathways and mechanisms of methane utilization (Figure 5).

Like other members of the PVC superphylum, M. infernorum shows gene loss and alteration of multiple components of the cell-division protein machinery. As indicated above, genes for FtsX, FtsE, MinC, MinD, and MinE have been lost in the M. infernorum lineage, whereas the ftsZ gene apparently has experienced a period of accelerated evolution (data not shown) as previously demonstrated for the ftsZ genes of several Prosthecobacter species, which also belong to the Verrucomicrobia 40. In contrast, the rate of evolution of ftsK and ftsA genes did not seem to be affected (data not shown). Unlike Prosthecobacter dejongeii 4142, M. infernorum does not encode tubulin or homologs of any other distinctive eukaryotic proteins involved in cell division or cytoskeletal functions. Both Chlamydia and Planctomycetes show morphological correlates of the altered cell-division apparatus, a unique condensed form of chromatin in the former and a striking, nucleus-like enclosure containing the chromosome in the latter 4344. The observations on the unusual planctomycete cell morphology, some hints from planctomycete genome analysis, and the finding of tubulin in Prosthecobacter dejongeii have led to speculation on the potential relevance of the cell division mechanism in this group of bacteria to evolution of the eukaryotic nucleus and cytoskeleton 45. Neither detailed analyses of the planctomycete genomes.(4647 nor the present analysis of the first completed verrucomicrobial genome provide any support for this idea. However, it does seem that there was a major alteration of cell division mechanisms at the onset of evolution of the PVC superphylum, with subsequent elaborations in individual lineages 48, and experimental study of division in these bacteria will be of major interest.

M. infernorum encodes a glutamate decarboxylase (Minf_0102) and a potential glutamate/γ-aminobutyrate antiporter (Minf_1788), as well as an arginine decarboxylase (biosynthetic, Minf_2107) and a potential arginine/agmatine antiporter (Minf_1531). These enzyme/transporter pairs could potentially counteract acidification of the cytoplasm by binding excess H⁺ ions and releasing CO₂ 49. Another potential acid-resistance mechanism that could be utilized by M. infernorum is agmatine hydrolysis by agmatine deiminase (Minf_0964), which releases NH₃ that would bind excess H⁺ ions 50. M. infernorum encodes neither urease, which accounts for acid resistance in Helicobacter pylori 51, nor orthologs of E. coli proteins HdeA, HdeD, YhiD, and YhiF that are also implicated in acid resistance. However, M. infernorum carries two paralogs of the Na⁺/H⁺ antiporter NapA 52 and six paralogs of the gene coding for the starvation-inducible outer membrane lipoprotein Slp 24, which has an unknown function but is often co-expressed with acid-resistance genes 49.

The M. infernorum genome also carries two operons encoding H⁺-translocating F₁F_O-ATPase subunits, the first such case among all sequenced microbial genomes. One of these operons (Minf_2417–Minf_2424) is most similar to the ATPase operons of other members of Verrucomicrobia (data not shown) and probably represents the form of the enzyme that is ancestral to the PVC superphylum. By contrast, the second ATPase operon (Minf_0839–Minf_0846) is most similar to the ATPase genes from gamma-proteobacteria, such as Methylococcus capsulatus (Supplementary Figure 6 [see Additional file 1]), and might have been acquired by a relatively recent lateral gene transfer. These enzymes are reversible, so it remains to be determined whether they synthesize ATP by utilizing proton gradient (allowing influx of protons into the cell) or couple ATP hydrolysis with pumping protons out of the cell.

Adaptations to the extremely acidic environment are also seen in the amino acid composition of M. infernorum proteins. The distribution of isoelectric points of M. infernorum proteins shows a substantial excess of high-pI (basic) proteins (Figure 6) and is much more similar to that of other acidophiles (e.g. Acidiphilium cryptum JF-5) than to that of mesophiles (Escherichia coli K12) or alkaliphiles (Alkalilimnicola ehrlichei MLHE-1).

Like some other extreme acidophiles, M. infernorum encodes a relatively simple signal transduction system that includes 8 sensor histidine kinases and 10 response regulators (with 7 pairs clustered in operons), but no Ser/Thr protein kinases, adenylate or diguanylate cyclases, or chemotaxis receptors. Although M. infernorum might be capable of gliding motility (using Minf_0409–Minf_0411 proteins), it has no chemotaxis genes.

Similarly to most other thermophiles, M. infernorum has the CRISPR-associated system, which is involved in anti-phage defense 5354. The system contains a predicted polymerase cassette (the polymerase itself is encoded by Minf_0882 gene) that seems to be a thermophilic determinant, i.e. is found primarily in thermophiles 5355. Despite the presence of 6 clusters of CRISPRs (altogether 25 repeats), the system might be inactive considering that that cas1 gene (Minf_0870) that is ubiquitous in CRISPR-associated systems is truncated. Another system often found in thermophiles is a pair of proteins containing a minimal nucleotidyltransferase and the HEPN (COG2250) domain that might be a novel toxin-antitoxin system (KSM, YIW, EVK, unpublished observations). The latter proteins also have a role in phage defense and stress response 56, along with the better studied systems of restriction-modification 57. We identified a few toxin-antitoxin components in M. infernorum (Minf_0121, Minf_0349, Minf_1374) and a classic restriction-modification system (Minf_1805 and Minf_1806). There is an additional locus encoding DNA methylases, helicases and a nuclease that might have a similar role (Minf_0316–328).

M. infernorum possesses an elaborate system of heavy-metal resistance. Along with the expansion of heavy metal efflux systems mentioned above, we identified a mercury reduction system (Minf_0449–Minf_0451), arsenate reductase (Minf_1582), putative silver efflux pump (Minf_2037), and a tellurium resistance protein (Minf_2102).

John A. Fuerst, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia (nominated by Mark Ragan, The University of Queensland, Brisbane, Australia)

This study reports and analyses the complete genome sequence of an extremely acidophilic methanotrophic member of the distinctive Bacterial phylum Verrucomicrobia, "Candidatus Methylacidiphilum infernorum" isolate V4, from a New Zealand methane-emitting geothermally heated soil and growing optimally at pH 2.0–2.5 and at 60°C with 25%(v/v) methane as sole source of energy. Related thermoacidophilic organisms have also been isolated recently from other geothermal areas, and these organisms seem to represent a clade of methane-oxidizers in this phylum, a result of significance concerning our understanding of C₁-compound metabolism and evolution of C₁ transfer enzymes, since the only known cultivated methane utilizers are members of the phylum Proteobacteria and the only phylum other than the proteobacteria in which some C₁-transfer enzymes (though not functional C₁ metabolism) have been found is the phylum Planctomycetes. Significantly, phylum Planctomycetes has been proposed on the grounds of 16S rRNA, 23S RNA and ribosomal protein sequence analysis to be related to the phylum Verrucomicrobia members in a single 'PVC" superphylum also proposed to contain phyla Chlamydiae, Lentisphaerae and the candidate phyla Poribacteria and OP3, but the support for the group has been relatively weak, with stronger support for a link between Verrucomicrobia and Chlamydiae than for either of these phyla with Planctomycetes.

Possibly the most significant result of this paper is that the PVC superphylum is supported when concatenated sequences of ribosomal proteins and RNA polymerase subunits are analysed, including "Candidatus Methylacidiphilum infernorum". This establishes firmer grounds for more detailed investigation of the relationships and links between members of the constituent phyla, at least for Planctomycetes, Verrucomicrobia and Chlamydiae. It also supports the view that more comprehensive taxon sampling may assist the testing of postulated superphyla, and suggests that genomes of more genera and species representative of all sub-divisions of phylum Verrucomicrobia should be sequenced to make this possible and confirm the present analysis. Of course, the recently explored limitations of some concatenated datasets for phylogenetic analysis of deep prokaryote nodes 73 need to be kept in mind. Although this paper reports the first complete genome sequence for a representative of the Verrucomicrobia, this thermoacidophilic species might not be representative of other soil, aquatic and symbiotic verrucomicrobia living in less extreme habitats, and this makes achieving completion of the genome sequencing and analysis of members of other subdivisions an urgent priority, including at the least Verrucomicrobium spinosum, Prosthecobacter dejongeii, Chthoniobacter flavus, Opitutus terrae, Akkermansia muciniphila, the soil isolate "Ellin514", and the marine verrucomicrobial strain DG1235.

This conclusion concerning a possible superphylum based on analysis of a limited set of informational – i.e. translational and transcriptional – genes is complicated and potentially contradicted by BLAST analysis of the complete set of predicted proteins, which indicated the fraction of proteins most similar to homologs from available genes of the PVC group to be only ~7%, while the largest fraction (~23% of the V4 proteins) had top hits among the phylum Proteobacteria. Although the authors admit to certain qualifications concerning over-representation of databases by the Proteobacteria and possible poor correlation of sequence similarity with phylogenetic affinity, they nevertheless favour a perspective or inference where horizontal gene transfer (HGT) dominates the architecture of the genome, going even further to link this to proteobacterial methylotrophy (despite the previously published result 3 concerning pmo genes for particulate methane monooxygenase needed for methane oxidation indicating that Methylacidiphilum genes are completely divergent from those of methylotrophic proteobacteria). The acceptance of such a conclusion may be dependent on the validity of such BLAST analysis, the problems with which in relation to gene transfer detection have been subject to detailed analysis 747576 and such problems may also apply to this case. There appear to be no other criteria than bioinformatic BLAST or COG analysis to strengthen the conclusion of HGT in this case, and there is then a temptation to consistency with the 'global HGT' dogma without sufficient grounds for high probability that HGT is the only explanation for such results. In other words, there may be other explanations for this apparently contradictory relationship to proteobacteria which need to be considered.

Does phylogenetic analysis of individual genes hypothesized to have been transferred indicate a particular group of proteobacteria from which the transfer may have occurred recently, or is this proposed to be an ancient transfer, in which case how is it to be distinguished from transfers in the progenote? Is the polarity of transfer direction unambiguous? Of course even following such analysis, alternative explanations for phylogenetic misplacement of taxa within an alien clade may also then have to be considered.

To strengthen their conclusions, the authors do include a Correspondence Analysis, a type of gene content analysis, which isolates M. infernorum away from either members of phyla Verrucomicrobia and Chlamydiae or any other bacterial clade. They interpret this as supporting a complex history for the gene set of M. infernorum, reflecting contributions of genes from diverse groups, since nearest neighbours in genome content space are members of phylum Proteobacteria, Thermotogae, Aquificae and one Actinobacteria genus. Is there an explanation for this analysis alternative to gene transfer? The inclusion of the deep-branching Thermotogae and Aquificae is interesting, and suggests that one alternative which might be considered is that Verrucomicrobia, or at least this representative of the phylum, might harbour gene contributions which either occurred during the early radiation of the Bacteria or even earlier when phyla of the domain Bacteria were not distinguishable. More detailed phylogenetic analysis of the genes used in the genome content analysis may be needed to test such an alternative.

The authors do note the caveats which have to be applied to at least their BLAST analysis, but various methods for confirming the hypothesis of lateral gene transfer such as GC composition, codon usage, or association with possible mechanisms for transfer 767778 have not yet apparently been applied to the Methylacidiphilum genome problem, and these might potentially reinforce the gene transfer explanation derived so far from BLAST and Correspondence Analysis, though it is also possible that ancient transfer would not be detected by such methods.

The situation of Methylacidiphilum infernorum bears some similarity to that noted for Cenarchaeum symbiosum by Forterre in his review of the analysis of archaeal COGs by Makarova et al. 70 where a gene content analysis indicated possible acquisition of euryarchaeal genes via LGT, in an organism already postulated to have acquired 'lots of bacterial genes' 70. As in the C. symbiosum case where the possibility remains that it represents an early branching archaeal lineage containing bacterial and archaeal homologs lost in other archaea, the alternative hypothesis should be considered that Methylacidiphilum and perhaps also other Verrucomicrobia (and perhaps even also other members of the PVC superphylum) represent members of an early branching lineage containing ancient homologs of genes in other Bacterial phyla which have been subject to wide (and perhaps even unparsimonious!) loss.

Further tests to estimate the relative timing of the proposed HGT 79 might lead to insights about this possibility. Evidence regarding potential gene loss is presented supporting the interpretation that M. infernorum may have acquired genes for several proteins belonging to the gene set conserved in archaea and eukaryotes, but again one asks whether this may alternatively be interpreted not as suggesting HGT among bacteria following initial transfer from archaea but rather as an indication of retention of an ancient signal from an organism close to the LUCA or LCA. Perhaps such an ancient signal could even stem from a lineage analogous to the uncharacterized archaeal lineage recently proposed as a root from which eucaryal archaea-like genes may have originated 80.

The distribution of C₁ transfer enzymes is intriguing, since the tetrahydromethanopterin-dependent enzymes found in some members of phylum Planctomycetes do not seem to have been detected, and Methylacidiphilum is clearly capable of methanotrophy unlike any planctomycete so far isolated. The existing controversy 8182 over the origin of the archaea-like C₁-transfer enzymes of planctomycetes relating to potential gene transfer versus ancient divergence suggests however that it may be productive to examine the phylogenetic relationships of the C₁-transfer enzymes of Methylacidiphilum, especially since they are clearly functional.

The metabolomic pathway analysis of gene content may have limitations for evolutionary insights in this case. The phylogenetic analysis of the pmo genes in this organism in another publication 3 suggested a divergent evolutionary history from methylotrophic proteobacteria, one which might even be consistent with a relatively deep branching core identity for this species, and this line of investigation might be usefully pursued with other genes involved in Methylacidiphilum methylotrophy.

M. infernorum appears to display gene loss in the cell division system, and what is interpreted as accelerated evolution is claimed to have occurred in the ftsZ gene. Considering the non-functional nature of the ftsZ homolog in the PVC member Lentisphaera, it would be highly relevant for the data and analysis of the data relating to this claim to be described in the paper – what reasons are there for interpretation of presumably divergent sequence as accelerated evolution? Why isn't a deep branching of this and other verrucomicrobial ftsZ relative to homologs in other phyla an equally plausible explanation for its divergent sequence? Have long branch attraction artefacts been definitively demonstrated as a most probable alternative explanation for such a deep branch? What is the explanation of accelerated evolution in the apparently retained ftsZ but not in ftsK and ftsA, presumed components of the same ftsZ-dependent divisome?

The view that previous speculation interpreted as relating cell division in the PVC superphylum to evolution of the eukaryotic nucleus and cytoskeleton has not so far been supported may be a warranted view at this point. However, such conclusions rejecting PVC superphylum relevance to eukaryote evolution are not only based on limited studies but also on studies based on a very limited sampling of taxa and limited types of bioinformatic analysis. That this may be important is indicated by the occurrence of structures reactive with anti-tubulin antibodies in the 'epixenosome' verrucomicrobial symbionts of ciliate protozoa 8384 as well as the demonstrated tubulin homologs in Prosthecobacter species 41, indicating a potentially wider occurrence of this eukaryote cytoskeletal protein among verrucomicrobia than has been detected and one not as easily explained by lateral gene transfer as by an evolutionary retention of a deep signal. Concerning detection of potentially eukaryote-homologous features within PVC group members, analysis at the level of secondary structure may be needed to reveal unsuspected relationships to eukaryote signature proteins such as nuclear pore complex proteins 85. Detection of homologs of PVC proteins among eukaryote proteins may be expected to be difficult, since even within eukaryotes detection of important eukaryote signature proteins such as homologs to nuclear pore complexes may not be trivial, since exceptional heterogeneity occurs between species, and BLAST and even PSI-BLAST approaches may fail due to variation in evolutionary rate alone 86. This question does not appear to be as resolved as the authors have suggested, and relevance of any PVC member to eukaryotes is certainly not refuted by the analysis presented. One might in a lighter mood suggest that it is not over until the sterol-synthesizing nucleated Gemmata planctomycete sings.

This paper is important for suggesting and perhaps stimulating a number of lines of investigation for future genomic analysis of the phylum Verrucomicrobia and the PVC superphylum, but this future analysis should not be constrained by assumptions concerning easy interpretation of the paradoxes posed by Methylacidiphilum and verrucomicrobia, which still seem unsatisfactorily resolved at this point.

Ludmila Chistoserdova, Department of Chemical Engineering, University of Washington, Box 355014, Seattle, WA 98195-5014, USA (nominated by Janet Siefert, Rice University, Houston, TX, United States)

This paper describes analysis of the genome of strain V4, an acidophilic methanotroph belonging to Verrucomicrobia. Not only this is the first genome representing this phylum to be formally described, but this is also a rare precedent of a very fast progress from the isolation and description of a novel strain to genome sequencing and analysis, all within one year! This is very exciting.

Radhey S. Gupta, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada (nominated by Jonathan Eisen, University of California Davis Genome Center, Davis, California, USA)

In this manuscript Hou et al. report the genome sequence for "Candidatus Methylacidiphilum infernorum", which is the first representative from the phylum Verrucomicrobia to be completely sequenced. The organisms from this clade of bacteria are in general very poorly characterized and only few of them have been isolated in pure culture. Hence, the availability of genome sequence for a member of this group should prove very useful for a variety of studies, particularly in clarifying its phylogenetic placement relative to other bacterial phyla. The authors have carried out detailed analyses of M. infernorum genome to reconstruct its central metabolic pathway and have identified many genes/proteins responsible for its ability to utilize methane and adaptation to the acidic environment. Several differences were noted from other methylotrophs, which are mainly alpha- and gamma-proteobacteria. The work on the annotation of various genes involved in different cellular functions and the differences seen in these regards for M. infernorum has been competently carried out and I have no questions or concerns.

Another important aspect of this manuscript relates to the phylogenetic placement of Verrucomicrobia with respect to other bacterial phyla. Recent studies by a number of authors, primarily based on 16S and 23S rRNA 1248, have indicated that species from three bacterial phyla viz.Planctomycetes, Verrucomicrobia-Lentisphaerae and Chlamydiae (as well as Poribacteria and OP3), group together in phylogenetic trees. This has led to the proposal that these groups or phyla should be recognized as part of a single superphylum (PVC). In this work the authors have constructed phylogenetic trees based on concatenated sequences for 51 ribosomal proteins and also the three subunits for RNAP. The trees based on these sequences also support the grouping of these species in a single clade. Other analyses reported here to determine the closest relatives of M. infernorum (e.g. top BLAST hits, Correspondence analysis) have provided no clarification in these regards and these results have been interpreted to suggest a complex evolutionary history of the Verrucomicrobia.