Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia

Abstract

Background: The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia.

Results: We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C1-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins.

Conclusion: The results of genome analysis of M. infernorum support the monophyly of the PVC superphylum. M. infernorum possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from Proteobacteria.

Reviewers: This article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.

Figure 1

Circular representation of the "Methylacidiphilum infernorum" V4 genome. The first and second circles show coding regions in positive and negative strands classified by COG functional categories. Potential integrative plasmid region is shown as a blue segment. The third circle shows tRNA and rRNA coding genes. The fourth circle shows variations in G+C content of the genome with respect to the mean G+C value. The fifth circle shows GC-skew plot of the genome showing approximate origin of replication and termination sites.

Figure 2

Maximum Likelihood phylogenetic tree of bacteria constructed from concatenated alignment of ribosomal proteins. Phylogenetic tree of 59 selected bacterial species (listed in the Supplementary Table 1 [see Additional file 1]) was constructed from concatenated alignments of 51 ribosomal proteins. Bootstrap values are shown only for members of the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum.

Figure 3

Taxonomic affiliations of "Methylacidiphilum infernorum" V4 proteins. Taxonomic affiliations of the best BLAST hits for each of the M. infernorum proteins to the RefSeq database were analyzed as described in the Methods section.

Figure 4

Analysis of eggNOG phyletic patterns of proteins encoded in various bacterial genomes. The plot shows the position of individual genomes on the plane of the first two principal components. Major bacterial groups are indicated. A. All 59 bacteria (Supplementary Table 1 [see Additional file 1]). B. Cyanobacteria and Chlamydia removed from the dataset. C. Self-organizing map of the genome content.

Figure 5

Reconstruction of methanotrophic and central metabolism pathways of "Methylacidiphilum infernorum" V4. For each predicted reaction, the generic gene name (blue) and M. infernorum V4 gene identifier (red, "Minf" prefix is omitted) are shown. The methylotrophy-related pathways are shown by magenta. Accessory products and metabolites are shown be green. Reactions for which no candidate enzyme was confidently predicted are indicated by dashed arrows. Key metabolites are shown as follows: light blue for amino acids, dark yellow for nucleotides, brown for sugars, pink for cofactors. Abbreviations: THF – tetrahydrofolate, CoA – coenzyme A.

Figure 6

Adaptation of "Methylacidiphilum infernorum" V4 to the acidic environment. Distribution (estimated probability density function) of isoelectric points of proteins encoded in genomes of microorganisms with different pH preferences.