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1.  Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing 
Genomic analysis of the methanotrophic verrucomicrobium “Methylacidiphilum infernorum” strain V4 has shown that most pathways conferring its methanotrophic lifestyle are similar to those found in proteobacterial methanotrophs. However, due to the large sequence divergence of its methane monooxygenase-encoding genes (pmo), “universal” pmoA polymerase chain reaction (PCR) primers do not target these bacteria. Unlike proteobacterial methanotrophs, “Methylacidiphilum” fixes carbon autotrophically, and uses methane only for energy generation. As a result, techniques used to detect methanotrophs in the environment such as 13CH4-stable isotope probing (SIP) and pmoA-targeted PCR do not detect verrucomicrobial methanotrophs, and they may have been overlooked in previous environmental studies. We developed a modified SIP technique to identify active methanotrophic Verrucomicrobia in the environment by labeling with 13CO2 and 13CH4, individually and in combination. Testing the protocol in “M. infernorum” strain V4 resulted in assimilation of 13CO2 but not 13CH4, verifying its autotrophic lifestyle. To specifically detect methanotrophs (as opposed to other autotrophs) via 13CO2-SIP, a quantitative PCR (qPCR) assay specific for verrucomicrobial-pmoA genes was developed and used in combination with SIP. Incubation of an acidic, high-temperature geothermal soil with 13CH4 + 12CO2 caused little shift in the density distribution of verrucomicrobial-pmoA genes relative to controls. However, labeling with 13CO2 in combination with 12CH4 or 13CH4 induced a strong shift in the distribution of verrucomicrobial-pmoA genes towards the heavy DNA fractions. The modified SIP technique demonstrated that the primary methanotrophs active in the soil were autotrophs and belonged to the Verrucomicrobia. This is the first demonstration of autotrophic, non-proteobacterial methanotrophy in situ, and provides a tool to detect verrucomicrobial methanotrophs in other ecosystems.
PMCID: PMC3421453  PMID: 22912630
Verrucomicrobia; methane; methanotroph; stable isotope probing; “Methylacidiphilum”; acidophile
2.  Genomic and Physiological Analysis of Carbon Storage in the Verrucomicrobial Methanotroph “Ca. Methylacidiphilum Fumariolicum” SolV 
“Candidatus Methylacidiphilum fumariolicum” SolV is a verrucomicrobial methanotroph that can grow in extremely acidic environments at high temperature. Strain SolV fixes carbon dioxide (CO2) via the Calvin–Benson–Bassham cycle with methane as energy source, a trait so far very unusual in methanotrophs. In this study, the ability of “Ca. M. fumariolicum” to store carbon was explored by genome analysis, physiological studies, and electron microscopy. When cell cultures were depleted for nitrogen, glycogen storage was clearly observed in cytoplasmic storage vesicles by electron microscopy. After cessation of growth, the dry weight kept increasing and the bacteria were filled up almost entirely by glycogen. This was confirmed by biochemical analysis, which showed that glycogen accumulated to 36% of the total dry weight of the cells. When methane was removed from the culture, this glycogen was consumed within 47 days. During the period of glycogen consumption, the bacteria kept their viability high when compared to bacteria without glycogen (from cultures growing exponentially). The latter bacteria lost viability already after a few days when starved for methane. Analysis of the draft genome of “Ca. M. fumariolicum” SolV demonstrated that all known genes for glycogen storage and degradation were present and also transcribed. Phylogenetic analysis of these genes showed that they form a separate cluster with “Ca. M. infernorum” V4, and the most closely related other sequences only have an identity of 40%. This study presents the first physiological evidence of glycogen storage in the phylum Verrucomicrobia and indicates that carbon storage is important for survival at times of methane starvation.
PMCID: PMC3460235  PMID: 23060867
Methylacidiphilum; methane; Verrucomicrobia; carbon storage; glycogen; survival
3.  The genomic landscape of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV 
BMC Genomics  2014;15(1):914.
Aerobic methanotrophs can grow in hostile volcanic environments and use methane as their sole source of energy. The discovery of three verrucomicrobial Methylacidiphilum strains has revealed diverse metabolic pathways used by these methanotrophs, including mechanisms through which methane is oxidized. The basis of a complete understanding of these processes and of how these bacteria evolved and are able to thrive in such extreme environments partially resides in the complete characterization of their genome and its architecture.
In this study, we present the complete genome sequence of Methylacidiphilum fumariolicum SolV, obtained using Pacific Biosciences single-molecule real-time (SMRT) sequencing technology. The genome assembles to a single 2.5 Mbp chromosome with an average GC content of 41.5%. The genome contains 2,741 annotated genes and 314 functional subsystems including all key metabolic pathways that are associated with Methylacidiphilum strains, including the CBB pathway for CO2 fixation. However, it does not encode the serine cycle and ribulose monophosphate pathways for carbon fixation. Phylogenetic analysis of the particulate methane mono-oxygenase operon separates the Methylacidiphilum strains from other verrucomicrobial methanotrophs. RNA-Seq analysis of cell cultures growing in three different conditions revealed the deregulation of two out of three pmoCAB operons. In addition, genes involved in nitrogen fixation were upregulated in cell cultures growing in nitrogen fixing conditions, indicating the presence of active nitrogenase. Characterization of the global methylation state of M. fumariolicum SolV revealed methylation of adenines and cytosines mainly in the coding regions of the genome. Methylation of adenines was predominantly associated with 5′-m6ACN4GT-3′ and 5′-CCm6AN5CTC-3′ methyltransferase recognition motifs whereas methylated cytosines were not associated with any specific motif.
Our findings provide novel insights into the global methylation state of verrucomicrobial methanotroph M. fumariolicum SolV. However, partial conservation of methyltransferases between M. fumariolicum SolV and M. infernorum V4 indicates potential differences in the global methylation state of Methylacidiphilum strains. Unravelling the M. fumariolicum SolV genome and its epigenetic regulation allow for robust characterization of biological processes that are involved in oxidizing methane. In turn, they offer a better understanding of the evolution, the underlying physiological and ecological properties of SolV and other Methylacidiphilum strains.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-914) contains supplementary material, which is available to authorized users.
PMCID: PMC4210602  PMID: 25331649
Methylacidiphilum fumariolicum SolV; Genome assembly; Single molecule sequencing; Pacific biosciences; Methylation; Gene expression; Verrucomicrobial methanotrophs
4.  Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes 
BMC Microbiology  2009;9:5.
The phylum Verrucomicrobia is a divergent phylum within domain Bacteria including members of the microbial communities of soil and fresh and marine waters; recently extremely acidophilic members from hot springs have been found to oxidize methane. At least one genus, Prosthecobacter, includes species with genes homologous to those encoding eukaryotic tubulins. A significant superphylum relationship of Verrucomicrobia with members of phylum Planctomycetes possessing a unique compartmentalized cell plan, and members of the phylum Chlamydiae including human pathogens with a complex intracellular life cycle, has been proposed. Based on the postulated superphylum relationship, we hypothesized that members of the two separate phyla Planctomycetes and Verrucomicrobia might share a similar ultrastructure plan differing from classical prokaryote organization.
The ultrastructure of cells of four members of phylum Verrucomicrobia – Verrucomicrobium spinosum, Prosthecobacter dejongeii, Chthoniobacter flavus, and strain Ellin514 – was examined using electron microscopy incorporating high-pressure freezing and cryosubstitution. These four members of phylum Verrucomicrobia, representing 3 class-level subdivisions within the phylum, were found to possess a compartmentalized cell plan analogous to that found in phylum Planctomycetes. Like all planctomycetes investigated, they possess a major pirellulosome compartment containing a condensed nucleoid and ribosomes surrounded by an intracytoplasmic membrane (ICM), as well as a ribosome-free paryphoplasm compartment between the ICM and cytoplasmic membrane.
A unique compartmentalized cell plan so far found among Domain Bacteria only within phylum Planctomycetes, and challenging our concept of prokaryote cell plans, has now been found in a second phylum of the Domain Bacteria, in members of phylum Verrucomicrobia. The planctomycete cell plan thus occurs in at least two distinct phyla of the Bacteria, phyla which have been suggested from other evidence to be related phylogenetically in the proposed PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) superphylum. This planctomycete cell plan is present in at least 3 of 6 subdivisions of Verrucomicrobia, suggesting that the common ancestor of the verrucomicrobial phylum was also compartmentalized and possessed such a plan. The presence of this compartmentalized cell plan in both phylum Planctomycetes and phylum Verrucomicrobia suggest that the last common ancestor of these phyla was also compartmentalized.
PMCID: PMC2647929  PMID: 19133117
5.  Molecular Signatures for the PVC Clade (Planctomycetes, Verrucomicrobia, Chlamydiae, and Lentisphaerae) of Bacteria Provide Insights into Their Evolutionary Relationships 
The PVC superphylum is an amalgamation of species from the phyla Planctomycetes, Verrucomicrobia, and Chlamydiae, along with the Lentisphaerae, Poribacteria, and two other candidate divisions. The diverse species of this superphylum lack any significant marker that differentiates them from other bacteria. Recently, genome sequences for 37 species covering all of the main PVC groups of bacteria have become available. We have used these sequences to construct a phylogenetic tree based upon concatenated sequences for 16 proteins and identify molecular signatures in protein sequences that are specific for the species from these phyla or those providing molecular links among them. Of the useful molecular markers identified in the present work, six conserved signature indels (CSIs) in the proteins Cyt c oxidase, UvrD helicase, urease, and a helicase-domain containing protein are specific for the species from the Verrucomicrobia phylum; three other CSIs in an ABC transporter protein, cobyrinic acid ac-diamide synthase, and SpoVG protein are specific for the Planctomycetes species. Additionally, a 3 aa insert in the RpoB protein is uniquely present in all sequenced Chlamydiae, Verrucomicrobia, and Lentisphaerae species, providing evidence for the shared ancestry of the species from these three phyla. Lastly, we have also identified a conserved protein of unknown function that is exclusively found in all sequenced species from the phyla Chlamydiae, Verrucomicrobia, Lentisphaerae, and Planctomycetes suggesting a specific linkage among them. The absence of this protein in Poribacteria, which branches separately from other members of the PVC clade, indicates that it is not specifically related to the PVC clade of bacteria. The molecular markers described here in addition to clarifying the evolutionary relationships among the PVC clade of bacteria also provide novel tools for their identification and for genetic and biochemical studies on these organisms.
PMCID: PMC3444138  PMID: 23060863
conserved signature indels; signature proteins; Verrucomicrobia; Planctomycetes; Chlamydia; Lentisphaerae; PVC superphylum; phylogenetic trees
6.  Metabolic Regulation of “Ca. Methylacidiphilum Fumariolicum” SolV Cells Grown Under Different Nitrogen and Oxygen Limitations 
Aerobic methanotrophic bacteria can use methane as their sole energy source. The discovery of “Ca. Methylacidiphilum fumariolicum” strain SolV and other verrucomicrobial methanotrophs has revealed that the ability of bacteria to oxidize CH4 is much more diverse than has previously been assumed in terms of ecology, phylogeny, and physiology. A remarkable characteristic of the methane-oxidizing Verrucomicrobia is their extremely acidophilic phenotype, growing even below pH 1. In this study we used RNA-Seq to analyze the metabolic regulation of “Ca. M. fumariolicum” SolV cells growing at μmax in batch culture or under nitrogen fixing or oxygen limited conditions in chemostats, all at pH 2. The analysis showed that two of the three pmoCAB operons each encoding particulate methane monoxygenases were differentially expressed, probably regulated by the available oxygen. The hydrogen produced during N2 fixation is apparently recycled as demonstrated by the upregulation of the genes encoding a Ni/Fe-dependent hydrogenase. These hydrogenase genes were also upregulated under low oxygen conditions. Handling of nitrosative stress was shown by the expression of the nitric oxide reductase encoding genes norB and norC under all conditions tested, the upregulation of nitrite reductase nirK under oxygen limitation and of hydroxylamine oxidoreductase hao in the presence of ammonium. Unraveling the gene regulation of carbon and nitrogen metabolism helps to understand the underlying physiological adaptations of strain SolV in view of the harsh conditions of its natural ecosystem.
PMCID: PMC3404531  PMID: 22848206
Methylacidiphilum; methane; RNA-Seq; Verrucomicrobia; metabolic regulation; nitrogen; pMMO
7.  Draft Genome Sequence of the Volcano-Inhabiting Thermoacidophilic Methanotroph Methylacidiphilum fumariolicum Strain SolV 
Journal of Bacteriology  2012;194(14):3729-3730.
The draft genome of Methylacidiphilum fumariolicum SolV, a thermoacidophilic methanotroph of the phylum Verrucomicrobia, is presented. Annotation revealed pathways for one-carbon, nitrogen, and hydrogen catabolism and respiration together with central metabolic pathways. The genome encodes three orthologues of particulate methane monooxygenases. Sequencing of this genome will help in the understanding of methane cycling in volcanic environments.
PMCID: PMC3393509  PMID: 22740660
8.  Characterization and Evolution of Cell Division and Cell Wall Synthesis Genes in the Bacterial Phyla Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes and Phylogenetic Comparison with rRNA Genes▿ †  
Journal of Bacteriology  2008;190(9):3192-3202.
In the past, studies on the relationships of the bacterial phyla Planctomycetes, Chlamydiae, Lentisphaerae, and Verrucomicrobia using different phylogenetic markers have been controversial. Investigations based on 16S rRNA sequence analyses suggested a relationship of the four phyla, showing the branching order Planctomycetes, Chlamydiae, Verrucomicrobia/Lentisphaerae. Phylogenetic analyses of 23S rRNA genes in this study also support a monophyletic grouping and their branching order—this grouping is significant for understanding cell division, since the major bacterial cell division protein FtsZ is absent from members of two of the phyla Chlamydiae and Planctomycetes. In Verrucomicrobia, knowledge about cell division is mainly restricted to the recent report of ftsZ in the closely related genera Prosthecobacter and Verrucomicrobium. In this study, genes of the conserved division and cell wall (dcw) cluster (ddl, ftsQ, ftsA, and ftsZ) were characterized in all verrucomicrobial subdivisions (1 to 4) with cultivable representatives (1 to 4). Sequence analyses and transcriptional analyses in Verrucomicrobia and genome data analyses in Lentisphaerae suggested that cell division is based on FtsZ in all verrucomicrobial subdivisions and possibly also in the sister phylum Lentisphaerae. Comprehensive sequence analyses of available genome data for representatives of Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes strongly indicate that their last common ancestor possessed a conserved, ancestral type of dcw gene cluster and an FtsZ-based cell division mechanism. This implies that Planctomycetes and Chlamydiae may have shifted independently to a non-FtsZ-based cell division mechanism after their separate branchings from their last common ancestor with Verrucomicrobia.
PMCID: PMC2347405  PMID: 18310338
9.  Magnetotactic Bacteria from Extreme Environments 
Life : Open Access Journal  2013;3(2):295-307.
Magnetotactic bacteria (MTB) represent a diverse collection of motile prokaryotes that biomineralize intracellular, membrane-bounded, tens-of-nanometer-sized crystals of a magnetic mineral called magnetosomes. Magnetosome minerals consist of either magnetite (Fe3O4) or greigite (Fe3S4) and cause cells to align along the Earth’s geomagnetic field lines as they swim, a trait called magnetotaxis. MTB are known to mainly inhabit the oxic–anoxic interface (OAI) in water columns or sediments of aquatic habitats and it is currently thought that magnetosomes function as a means of making chemotaxis more efficient in locating and maintaining an optimal position for growth and survival at the OAI. Known cultured and uncultured MTB are phylogenetically associated with the Alpha-, Gamma- and Deltaproteobacteria classes of the phylum Proteobacteria, the Nitrospirae phylum and the candidate division OP3, part of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum. MTB are generally thought to be ubiquitous in aquatic environments as they are cosmopolitan in distribution and have been found in every continent although for years MTB were thought to be restricted to habitats with pH values near neutral and at ambient temperature. Recently, however, moderate thermophilic and alkaliphilic MTB have been described including: an uncultured, moderately thermophilic magnetotactic bacterium present in hot springs in northern Nevada with a probable upper growth limit of about 63 °C; and several strains of obligately alkaliphilic MTB isolated in pure culture from different aquatic habitats in California, including the hypersaline, extremely alkaline Mono Lake, with an optimal growth pH of >9.0.
PMCID: PMC4187138  PMID: 25369742
magnetotactic bacteria; biomineralization; magnetite; greigite; biodiversity and ecology; extreme environments; extremophiles; astrobiology
10.  Autotrophic Methanotrophy in Verrucomicrobia: Methylacidiphilum fumariolicumSolV Uses the Calvin-Benson-Bassham Cycle for Carbon Dioxide Fixation ▿ †  
Journal of Bacteriology  2011;193(17):4438-4446.
Genome data of the extreme acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicumstrain SolV indicated the ability of autotrophic growth. This was further validated by transcriptome analysis, which showed that all genes required for a functional Calvin-Benson-Bassham (CBB) cycle were transcribed. Experiments with 13CH4or 13CO2in batch and chemostat cultures demonstrated that CO2is the sole carbon source for growth of strain SolV. In the presence of CH4, CO2concentrations in the headspace below 1% (vol/vol) were growth limiting, and no growth was observed when CO2concentrations were below 0.3% (vol/vol). The activity of the key enzyme of the CBB cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), measured with a 13C stable-isotope method was about 70 nmol CO2fixed · min−1· mg of protein−1. An immune reaction with antibody against the large subunit of RuBisCO on Western blots was found only in the supernatant fractions of cell extracts. The apparent native mass of the RuBisCO complex in strain SolV was about 482 kDa, probably consisting of 8 large (53-kDa) and 8 small (16-kDa) subunits. Based on phylogenetic analysis of the corresponding RuBisCO gene, we postulate that RuBisCO of the verrucomicrobial methanotrophs represents a new type of form I RuBisCO.
PMCID: PMC3165502  PMID: 21725016
11.  Genomic and Physiological Characterization of the Verrucomicrobia Isolate Diplosphaera colitermitum gen. nov., sp. nov., Reveals Microaerophily and Nitrogen Fixation Genes 
Previously we reported the cultivation of novel verrucomicrobia, including strain TAV2 (93% 16S rRNA gene identity to its nearest cultivated representative, Opitutus terreae PB90-1) from the gut of the termite Reticulitermes flavipes. To gain better insight into the Verrucomicrobia as a whole and understand the role of verrucomicrobia within the termite gut ecosystem, we analyzed a draft genome and undertook a physiological characterization of TAV2. Strain TAV2 is an autochthonous member of the R. flavipes gut microbiota and groups phylogenetically among diverse Verrucomicrobia from R. flavipes and other termites that are represented by 16S rRNA gene sequences alone. TAV2 is a microaerophile, possessing a high-affinity cbb3-type terminal oxidase-encoding gene and exhibiting an optimum growth rate between 2 and 8% (vol/vol) oxygen. It has the genetic potential to degrade cellulose, an important function within termite guts, but its in vitro substrate utilization spectrum was limited to starch and a few mono- and disaccharides. Growth occurred on nitrogen-free medium, and genomic screening revealed genes for dinitrogenases, heretofore detected in only a few members of the Verrucomicrobia. This represents the first (i) characterization of a verrucomicrobial species from the termite gut, (ii) report of nif and anf genes in a nonacidophilic verrucomicrobial species, and (iii) description of a microaerophilic genotype and phenotype in this phylum of bacteria. The genetic and physiological distinctiveness of TAV2 supports its recognition as the type strain of a new genus and species, for which the name Diplosphaera colitermitum gen. nov., sp. nov., is proposed.
PMCID: PMC3294482  PMID: 22194293
12.  The Candidate Phylum Poribacteria by Single-Cell Genomics: New Insights into Phylogeny, Cell-Compartmentation, Eukaryote-Like Repeat Proteins, and Other Genomic Features 
PLoS ONE  2014;9(1):e87353.
The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake.
PMCID: PMC3909097  PMID: 24498082
13.  Genome-Wide Influence of Indel Substitutions on Evolution of Bacteria of the PVC Superphylum, Revealed Using a Novel Computational Method 
Whole-genome scans for positive Darwinian selection are widely used to detect evolution of genome novelty. Most approaches are based on evaluation of nonsynonymous to synonymous substitution rate ratio across evolutionary lineages. These methods are sensitive to saturation of synonymous sites and thus cannot be used to study evolution of distantly related organisms. In contrast, indels occur less frequently than amino acid replacements, accumulate more slowly, and can be employed to characterize evolution of diverged organisms. As indels are also subject to the forces of natural selection, they can generate functional changes through positive selection. Here, we present a new computational approach to detect selective constraints on indel substitutions at the whole-genome level for distantly related organisms. Our method is based on ancestral sequence reconstruction, takes into account the varying susceptibility of different types of secondary structure to indels, and according to simulation studies is conservative. We applied this newly developed framework to characterize the evolution of organisms of the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) bacterial superphylum. The superphylum contains organisms with unique cell biology, physiology, and diverse lifestyles. It includes bacteria with simple cell organization and more complex eukaryote-like compartmentalization. Lifestyles range from free-living organisms to obligate pathogens. In this study, we conduct a whole-genome level analysis of indel substitutions specific to evolutionary lineages of the PVC superphylum and found that indels evolved under positive selection on up to 12% of gene tree branches. We also analyzed possible functional consequences for several case studies of predicted indel events.
PMCID: PMC3000692  PMID: 21048002
selection; indel substitutions; PVC superphylum
14.  Analysis of Genome Content Evolution in PVC Bacterial Super-Phylum: Assessment of Candidate Genes Associated with Cellular Organization and Lifestyle 
Genome Biology and Evolution  2012;4(12):1375-1390.
The Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) super-phylum contains bacteria with either complex cellular organization or simple cell structure; it also includes organisms of different lifestyles (pathogens, mutualists, commensal, and free-living). Genome content evolution of this group has not been studied in a systematic fashion, which would reveal genes underlying the emergence of PVC-specific phenotypes. Here, we analyzed the evolutionary dynamics of 26 PVC genomes and several outgroup species. We inferred HGT, duplications, and losses by reconciliation of 27,123 gene trees with the species phylogeny. We showed that genome expansion and contraction have driven evolution within Planctomycetes and Chlamydiae, respectively, and balanced each other in Verrucomicrobia and Lentisphaerae. We also found that for a large number of genes in PVC genomes the most similar sequences are present in Acidobacteria, suggesting past and/or current ecological interaction between organisms from these groups. We also found evidence of shared ancestry between carbohydrate degradation genes in the mucin-degrading human intestinal commensal Akkermansia muciniphila and sequences from Acidobacteria and Bacteroidetes, suggesting that glycoside hydrolases are transferred laterally between gut microbes and that the process of carbohydrate degradation is crucial for microbial survival within the human digestive system. Further, we identified a highly conserved genetic module preferentially present in compartmentalized PVC species and possibly associated with the complex cell plan in these organisms. This conserved machinery is likely to be membrane targeted and involved in electron transport, although its exact function is unknown. These genes represent good candidates for future functional studies.
PMCID: PMC3542564  PMID: 23221607
genome evolution in PVC super-phylum; cellular compartmentalization; DUF1501 and Planctomycetes-specific cytochromes; mucin-degradation by Akkermansia muciniphila
15.  Signature Protein of the PVC Superphylum 
The phyla Planctomycetes, Verrucomicrobia, Chlamydiae, Lentisphaerae, and “Candidatus Omnitrophica (OP3)” comprise bacteria that share an ancestor but show highly diverse biological and ecological features. Together, they constitute the PVC superphylum. Using large-scale comparative genome sequence analysis, we identified a protein uniquely shared among all of the known members of the PVC superphylum. We provide evidence that this signature protein is expressed by representative members of the PVC superphylum. Its predicted structure, physicochemical characteristics, and overexpression in Escherichia coli and gel retardation assays with purified signature protein suggest a housekeeping function with unspecific DNA/RNA binding activity. Phylogenetic analysis demonstrated that the signature protein is a suitable phylogenetic marker for members of the PVC superphylum, and the screening of published metagenome data indicated the existence of additional PVC members. This study provides further evidence of a common evolutionary history of the PVC superphylum and presents a unique case in which a single protein serves as an evolutionary link among otherwise highly diverse members of major bacterial groups.
PMCID: PMC3911108  PMID: 24185849
16.  Chthoniobacter flavus gen. nov., sp. nov., the First Pure-Culture Representative of Subdivision Two, Spartobacteria classis nov., of the Phylum Verrucomicrobia 
Applied and Environmental Microbiology  2004;70(10):5875-5881.
The phylum Verrucomicrobia is increasingly recognized as an environmentally significant group of bacteria, particularly in soil habitats. At least six subdivisions of the Verrucomicrobia are resolved by comparative analysis of 16S rRNA genes, mostly obtained directly from environmental samples. To date, only two of these subdivisions (1 and 4) have characterized pure-culture representatives. We have isolated and characterized the first known pure-culture representative of subdivision 2. Strain Ellin428 is an aerobic heterotrophic bacterium that is able to grow with many of the saccharide components of plant biomass but does not grow with amino acids or organic acids other than pyruvate. Cells are yellow, rod-shaped, nonmotile, and gram-stain negative, and they contain peptidoglycan with direct cross-linkages of the A1γ meso-Dpm type. The isolate grows well at 25°C on a variety of standard biological media, including some used in the routine cultivation of bacteria from soil. The pH range for growth is 4.0 to 7.0. Low levels of menaquinones MK-10 and MK-11 were detected. The major cellular fatty acids are C14:0, a-C15:0, C16:1ω7c, and/or 2OH i-C15:0, and C16:0. The G+C content of the genomic DNA is 61 mol%. We propose a new genus and species, Chthoniobacter flavus gen. nov., sp. nov., with isolate Ellin428 as the type strain, and a new class for the subdivision to which it belongs, Spartobacteria classis nov. Environmental sequences indicate that the class Spartobacteria is largely represented by globally distributed, abundant, and active soil bacteria.
PMCID: PMC522106  PMID: 15466527
17.  Verrucomicrobia Are Candidates for Polysaccharide-Degrading Bacterioplankton in an Arctic Fjord of Svalbard 
Applied and Environmental Microbiology  2014;80(12):3749-3756.
In Arctic marine bacterial communities, members of the phylum Verrucomicrobia are consistently detected, although not typically abundant, in 16S rRNA gene clone libraries and pyrotag surveys of the marine water column and in sediments. In an Arctic fjord (Smeerenburgfjord) of Svalbard, members of the Verrucomicrobia, together with Flavobacteria and smaller proportions of Alpha- and Gammaproteobacteria, constituted the most frequently detected bacterioplankton community members in 16S rRNA gene-based clone library analyses of the water column. Parallel measurements in the water column of the activities of six endo-acting polysaccharide hydrolases showed that chondroitin sulfate, laminarin, and xylan hydrolysis accounted for most of the activity. Several Verrucomicrobia water column phylotypes were affiliated with previously sequenced, glycoside hydrolase-rich genomes of individual Verrucomicrobia cells that bound fluorescently labeled laminarin and xylan and therefore constituted candidates for laminarin and xylan hydrolysis. In sediments, the bacterial community was dominated by different lineages of Verrucomicrobia, Bacteroidetes, and Proteobacteria but also included members of multiple phylum-level lineages not observed in the water column. This community hydrolyzed laminarin, xylan, chondroitin sulfate, and three additional polysaccharide substrates at high rates. Comparisons with data from the same fjord in the previous summer showed that the bacterial community in Smeerenburgfjord changed in composition, most conspicuously in the changing detection frequency of Verrucomicrobia in the water column. Nonetheless, in both years the community hydrolyzed the same polysaccharide substrates.
PMCID: PMC4054139  PMID: 24727271
18.  Metagenomic De Novo Assembly of an Aquatic Representative of the Verrucomicrobial Class Spartobacteria 
mBio  2013;4(3):e00569-12.
The verrucomicrobial subdivision 2 class Spartobacteria is one of the most abundant bacterial lineages in soil and has recently also been found to be ubiquitous in aquatic environments. A 16S rRNA gene study from samples spanning the entire salinity range of the Baltic Sea indicated that, in the pelagic brackish water, a phylotype of the Spartobacteria is one of the dominating bacteria during summer. Phylogenetic analyses of related 16S rRNA genes indicate that a purely aquatic lineage within the Spartobacteria exists. Since no aquatic representative from the Spartobacteria has been cultured or sequenced, the metabolic capacity and ecological role of this lineage are yet unknown. In this study, we reconstructed the genome and metabolic potential of the abundant Baltic Sea Spartobacteria phylotype by metagenomics. Binning of genome fragments by nucleotide composition and a self-organizing map recovered the near-complete genome of the organism, the gene content of which suggests an aerobic heterotrophic metabolism. Notably, we found 23 glycoside hydrolases that likely allow the use of a variety of carbohydrates, like cellulose, mannan, xylan, chitin, and starch, as carbon sources. In addition, a complete pathway for sulfate utilization was found, indicating catabolic processing of sulfated polysaccharides, commonly found in aquatic phytoplankton. The high frequency of glycoside hydrolase genes implies an important role of this organism in the aquatic carbon cycle. Spatiotemporal data of the phylotype’s distribution within the Baltic Sea indicate a connection to Cyanobacteria that may be the main source of the polysaccharide substrates.
The ecosystem roles of many phylogenetic lineages are not yet well understood. One such lineage is the class Spartobacteria within the Verrucomicrobia that, despite being abundant in soil and aquatic systems, is relatively poorly studied. Here we circumvented the difficulties of growing aquatic Verrucomicrobia by applying shotgun metagenomic sequencing on a water sample from the Baltic Sea. By using a method based on sequence signatures, we were able to in silico isolate genome fragments belonging to a phylotype of the Spartobacteria. The genome, which represents the first aquatic representative of this clade, encodes a diversity of glycoside hydrolases that likely allow degradation of various complex carbohydrates. Since the phylotype cooccurs with Cyanobacteria, these may be the primary producers of the carbohydrate substrates. The phylotype, which is highly abundant in the Baltic Sea during summer, may thus play an important role in the carbon cycle of this ecosystem.
PMCID: PMC3663571  PMID: 23716574
19.  Evaluating the Evolutionary Origins of Unexpected Character Distributions within the Bacterial Planctomycetes-Verrucomicrobia-Chlamydiae Superphylum 
Recently, several characters that are absent from most bacteria, but which are found in many eukaryotes or archaea, have been identified within the bacterial Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum. Hypotheses of the evolutionary history of such characters are commonly based on the inference of phylogenies of gene or protein families associated with the traits, estimated from multiple sequence alignments (MSAs). So far, studies of this kind have focused on the distribution of (i) two genes involved in the synthesis of sterol, (ii) tubulin genes, and (iii) c1 transfer genes. In many cases, these analyses have concluded that horizontal gene transfer (HGT) is likely to have played a role in shaping the taxonomic distribution of these gene families. In this article, we describe several issues with the inference of HGT from such analyses, in particular concerning the considerable uncertainty associated with our estimation of both gene family phylogenies (especially those containing ancient lineage divergences) and the Tree of Life (ToL), and the need for wider use and further development of explicit probabilistic models to compare hypotheses of vertical and horizontal genetic transmission. We suggest that data which is often taken as evidence for the occurrence of ancient HGT events may not be as convincing as is commonly described, and consideration of alternative theories is recommended. While focusing on analyses including PVCs, this discussion is also relevant for inferences of HGT involving other groups of organisms.
PMCID: PMC3505017  PMID: 23189077
PVC superphylum; lateral gene transfer; LGT; gene loss; gene duplication; phylogenetic estimation errors
20.  Laboratory Cultivation of Widespread and Previously Uncultured Soil Bacteria 
Applied and Environmental Microbiology  2003;69(12):7210-7215.
Most soil bacteria belong to family-level phylogenetic groups with few or no known cultivated representatives. We cultured a collection of 350 isolates from soil by using simple solid media in petri dishes. These isolates were assigned to 60 family-level groupings in nine bacterial phyla on the basis of a comparative analysis of their 16S rRNA genes. Ninety-three (27%) of the isolates belonged to 20 as-yet-unnamed family-level groupings, many from poorly studied bacterial classes and phyla. They included members of subdivisions 1, 2, 3, and 4 of the phylum Acidobacteria, subdivision 3 of the phylum Verrucomicrobia, subdivision 1 of the phylum Gemmatimonadetes, and subclasses Acidimicrobidae and Rubrobacteridae of the phylum Actinobacteria. In addition, members of 10 new family-level groupings of subclass Actinobacteridae of the phylum Actinobacteria and classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria of the phylum Proteobacteria were obtained. The high degree of phylogenetic novelty and the number of isolates affiliated with so-called unculturable groups show that simple cultivation methods can still be developed further to obtain laboratory cultures of many phylogenetically novel soil bacteria.
PMCID: PMC309996  PMID: 14660368
21.  The Compartmentalized Bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae Superphylum Have Membrane Coat-Like Proteins 
PLoS Biology  2010;8(1):e1000281.
Compartmentalized bacteria have proteins that are structurally related to eukaryotic membrane coats, and one of these proteins localizes at the membrane of vesicles formed inside bacterial cells.
The development of the endomembrane system was a major step in eukaryotic evolution. Membrane coats, which exhibit a unique arrangement of β-propeller and α-helical repeat domains, play key roles in shaping eukaryotic membranes. Such proteins are likely to have been present in the ancestral eukaryote but cannot be detected in prokaryotes using sequence-only searches. We have used a structure-based detection protocol to search all proteomes for proteins with this domain architecture. Apart from the eukaryotes, we identified this protein architecture only in the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum, many members of which share a compartmentalized cell plan. We determined that one such protein is partly localized at the membranes of vesicles formed inside the cells in the planctomycete Gemmata obscuriglobus. Our results demonstrate similarities between bacterial and eukaryotic compartmentalization machinery, suggesting that the bacterial PVC superphylum contributed significantly to eukaryogenesis.
Author Summary
Despite decades of research, the origin of eukaryotic cells remains an unsolved issue. The endomembrane system defines the eukaryotic cell, and its origin is linked to that of eukaryotes. A search was conducted within all known sequences for proteins that are characteristic of the eukaryotic endomembrane system, using a combination of fold types that is uniquely found in the membrane coat proteins. Outside eukaryotes, such proteins were solely found in the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum. By immuno-electron microscopy, one of these bacterial proteins was found to localize adjacent to the membranes of vesicles found within the cells of one member of the PVC superphylum. Thus, there appear to be similarities between bacterial and eukaryotic compartmentalization systems, suggesting that the bacterial PVC superphylum may have contributed significantly to eukaryogenesis.
PMCID: PMC2799638  PMID: 20087413
22.  Detection and Cultivation of Soil Verrucomicrobia 
Applied and Environmental Microbiology  2005;71(12):8402-8410.
Only one isolate each of the class “Spartobacteria” (subdivision 2 of the phylum Verrucomicrobia) and of subdivision 3 of Verrucomicrobia have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class “Spartobacteria” and were related to “Chthoniobacter flavus.” Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class “Spartobacteria” were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class “Spartobacteria” were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class “Spartobacteria” was inhibited by other culturable bacteria.
PMCID: PMC1317444  PMID: 16332828
23.  Genomic Analysis Indicates the Presence of an Asymmetric Bilayer Outer Membrane in Planctomycetes and Verrucomicrobia 
Bacteria of the phylum Planctomycetes are of special interest for the study of compartmental cellular organization. Members of this phylum share a very unusual prokaryotic cell plan, featuring several membrane-bound compartments. Recently, it was shown that this cellular organization might extend to certain members of the phylum Verrucomicrobia. The Planctomycete cell plan has been defined as featuring a proteinaceous cell wall, a cytoplasmic membrane surrounding the paryphoplasm, and an intracytoplasmic membrane defining the riboplasm. So far it was presumed that Planctomycetes did not have an asymmetric bilayer outer membrane as observed in Gram-negative bacteria. However, recent work on outer membrane biogenesis has provided several marker genes in the outer membrane protein (OMP) assembly and the lipopolysaccharide (LPS) insertion complexes. Additionally, advances in computational prediction of OMPs provided new tools to perform more accurate genomic screening for such proteins. Here we searched all 22 Planctomycetes and Verrucomicrobia genomes available in GenBank, plus the recently published genome of “Candidatus Scalindua profunda,” for markers of outer membrane biogenesis and OMPs. We were able to identify the key components of LPS insertion, OMP assembly and at least eight OMPs in all genomes tested. Additionally, we have analyzed the transcriptome and proteome data of the Planctomycetes “Candidatus Kuenenia stuttgartiensis” and “Ca. S. profunda” and could confirm high expression of several predicted OMPs, including the biomarkers of outer membrane biogenesis. These analyses provide a strong indication that an asymmetrical outer membrane may be present in bacteria of both phyla. However, previous experiments have made obvious that the cell envelope of Planctomycetes is clearly divergent from both the Gram-negative and Gram-positive cell types. Thus, the functional implications of the presence of an outer membrane for the Planctomycete cell plan and compartmentalization are discussed and a revised model including an outer membrane is proposed. Although this model agrees with most experimental data, we do note that the presence, location, and role of an outer membrane within the Planctomycetes and Verrucomicrobia awaits further experimental validation.
PMCID: PMC3422733  PMID: 22934092
Planctomycetes; Verrucomicrobia; cellular organization; outer membrane; paryphoplasm; periplasm; compartmentalization
24.  The Rhizosphere Selects for Particular Groups of Acidobacteria and Verrucomicrobia 
PLoS ONE  2013;8(12):e82443.
There is a lack in our current understanding on the putative interactions of species of the phyla of Acidobacteria and Verrucomicrobia with plants. Moreover, progress in this area is seriously hampered by the recalcitrance of members of these phyla to grow as pure cultures. The purpose of this study was to investigate whether particular members of Acidobacteria and Verrucomicrobia are avid colonizers of the rhizosphere. Based on previous work, rhizosphere competence was demonstrated for the Verrucomicrobia subdivision 1 groups of Luteolibacter and Candidatus genus Rhizospheria and it was hypothesized that the rhizosphere is a common habitat for Acidobacteria subdivision 8 (class Holophagae). We assessed the population densities of Bacteria, Verrucomicrobia subdivision 1 groups Luteolibacter and Candidatus genus Rhizospheria and Acidobacteria subdivisions 1, 3, 4, 6 and Holophagae in bulk soil and in the rhizospheres of grass, potato and leek in the same field at different points in time using real-time quantitative PCR. Primers of all seven verrucomicrobial, acidobacterial and holophagal PCR systems were based on 16S rRNA gene sequences of cultivable representatives of the different groups. Luteolibacter, Candidatus genus Rhizospheria, subdivision 6 acidobacteria and Holophaga showed preferences for one or more rhizospheres. In particular, the Holophaga 16S rRNA gene number were more abundant in the leek rhizosphere than in bulk soil and the rhizospheres of grass and potato. Attraction to, and colonization of, leek roots by Holophagae strain CHC25 was further shown in an experimental microcosm set-up. In the light of this remarkable capacity, we propose to coin strain CHC25 Candidatus Porrumbacterium oxyphilus (class Holophagae, Phylum Acidobacteria), the first cultured representative with rhizosphere competence.
PMCID: PMC3862674  PMID: 24349285
25.  Genome Analysis Coupled with Physiological Studies Reveals a Diverse Nitrogen Metabolism in Methylocystis sp. Strain SC2 
PLoS ONE  2013;8(10):e74767.
Methylocystis sp. strain SC2 can adapt to a wide range of methane concentrations. This is due to the presence of two isozymes of particulate methane monooxygenase exhibiting different methane oxidation kinetics. To gain insight into the underlying genetic information, its genome was sequenced and found to comprise a 3.77 Mb chromosome and two large plasmids.
Principal Findings
We report important features of the strain SC2 genome. Its sequence is compared with those of seven other methanotroph genomes, comprising members of the Alphaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. While the pan-genome of all eight methanotroph genomes totals 19,358 CDS, only 154 CDS are shared. The number of core genes increased with phylogenetic relatedness: 328 CDS for proteobacterial methanotrophs and 1,853 CDS for the three alphaproteobacterial Methylocystaceae members, Methylocystis sp. strain SC2 and strain Rockwell, and Methylosinus trichosporium OB3b. The comparative study was coupled with physiological experiments to verify that strain SC2 has diverse nitrogen metabolism capabilities. In correspondence to a full complement of 34 genes involved in N2 fixation, strain SC2 was found to grow with atmospheric N2 as the sole nitrogen source, preferably at low oxygen concentrations. Denitrification-mediated accumulation of 0.7 nmol 30N2/hr/mg dry weight of cells under anoxic conditions was detected by tracer analysis. N2 production is related to the activities of plasmid-borne nitric oxide and nitrous oxide reductases.
Presence of a complete denitrification pathway in strain SC2, including the plasmid-encoded nosRZDFYX operon, is unique among known methanotrophs. However, the exact ecophysiological role of this pathway still needs to be elucidated. Detoxification of toxic nitrogen compounds and energy conservation under oxygen-limiting conditions are among the possible roles. Relevant features that may stimulate further research are, for example, absence of CRISPR/Cas systems in strain SC2, high number of iron acquisition systems in strain OB3b, and large number of transposases in strain Rockwell.
PMCID: PMC3794950  PMID: 24130670

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