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1.  Archaeal “Dark Matter” and the Origin of Eukaryotes 
Genome Biology and Evolution  2014;6(3):474-481.
Current hypotheses about the history of cellular life are mainly based on analyses of cultivated organisms, but these represent only a small fraction of extant biodiversity. The sequencing of new environmental lineages therefore provides an opportunity to test, revise, or reject existing ideas about the tree of life and the origin of eukaryotes. According to the textbook three domains hypothesis, the eukaryotes emerge as the sister group to a monophyletic Archaea. However, recent analyses incorporating better phylogenetic models and an improved sampling of the archaeal domain have generally supported the competing eocyte hypothesis, in which core genes of eukaryotic cells originated from within the Archaea, with important implications for eukaryogenesis. Given this trend, it was surprising that a recent analysis incorporating new genomes from uncultivated Archaea recovered a strongly supported three domains tree. Here, we show that this result was due in part to the use of a poorly fitting phylogenetic model and also to the inclusion by an automated pipeline of genes of putative bacterial origin rather than nucleocytosolic versions for some of the eukaryotes analyzed. When these issues were resolved, analyses including the new archaeal lineages placed core eukaryotic genes within the Archaea. These results are consistent with a number of recent studies in which improved archaeal sampling and better phylogenetic models agree in supporting the eocyte tree over the three domains hypothesis.
doi:10.1093/gbe/evu031
PMCID: PMC3971582  PMID: 24532674
eukaryogenesis; phylogenetics; “dark matter”; Tree of Life
3.  Planctomycetes and eukaryotes: a case of analogy not homology 
Summary
Planctomycetes, Verrucomicrobia and Chlamydia are prokaryotic phyla that are sometimes grouped together as the PVC superphylum of eubacteria. Some PVC species possess interesting attributes, in particular, internal membranes that superficially resemble eukaryotic endomembranes. Some biologists now claim that PVC bacteria are nucleus-bearing prokaryotes and that they are evolutionary intermediates in the transition from prokaryote to eukaryote. PVC prokaryotes do not possess a nucleus and are not intermediates in the prokaryote-to-eukaryote transition. All of the PVC traits that are currently cited as evidence for aspiring eukaryoticity are either analogous (the result of convergent evolution), not homologous, to eukaryotic traits; or else they are the result of lateral gene transfers. Here we summarize the evidence that shows why most of the purported similarities between the PVC bacteria and eukaryotes are analogous and the rest are consequence of lateral gene acquisition.
doi:10.1002/bies.201100045
PMCID: PMC3795523  PMID: 21858844
4.  The Genome of the Obligate Intracellular Parasite Trachipleistophora hominis: New Insights into Microsporidian Genome Dynamics and Reductive Evolution 
PLoS Pathogens  2012;8(10):e1002979.
The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome) making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome architecture in some, but not all, lineages.
Author Summary
Microsporidians are enormously successful obligate intracellular parasites of animals, including humans. Despite their economic and medical importance, there are major gaps in our understanding of how microsporidians have made the transition from a free-living organism to one that can only complete its life cycle by living inside another cell. We present the larger genome of Trachipleistophora hominis isolated from a human patient with HIV/AIDS. Our analyses provide insights into the gene content, genome architecture and intergenic regions of a known opportunistic pathogen, and will facilitate the development of T. hominis as a much-needed model species that can also be grown in co-culture. The genome of T. hominis has more genes than other microsporidians, it has diverse regulatory motifs, and it contains a variety of transposable elements coupled with the machinery for RNA interference, which may eventually allow experimental down-regulation of T. hominis genes. Comparison of the genome of T. hominis with other microsporidians allowed us to infer properties of their common ancestor. Our analyses predict an ancestral microsporidian that was already an intracellular parasite with a reduced core proteome but one with a relatively large genome populated with diverse repetitive elements and a complex transcriptional regulatory network.
doi:10.1371/journal.ppat.1002979
PMCID: PMC3486916  PMID: 23133373
5.  A congruent phylogenomic signal places eukaryotes within the Archaea 
Determining the relationships among the major groups of cellular life is important for understanding the evolution of biological diversity, but is difficult given the enormous time spans involved. In the textbook ‘three domains’ tree based on informational genes, eukaryotes and Archaea share a common ancestor to the exclusion of Bacteria. However, some phylogenetic analyses of the same data have placed eukaryotes within the Archaea, as the nearest relatives of different archaeal lineages. We compared the support for these competing hypotheses using sophisticated phylogenetic methods and an improved sampling of archaeal biodiversity. We also employed both new and existing tests of phylogenetic congruence to explore the level of uncertainty and conflict in the data. Our analyses suggested that much of the observed incongruence is weakly supported or associated with poorly fitting evolutionary models. All of our phylogenetic analyses, whether on small subunit and large subunit ribosomal RNA or concatenated protein-coding genes, recovered a monophyletic group containing eukaryotes and the TACK archaeal superphylum comprising the Thaumarchaeota, Aigarchaeota, Crenarchaeota and Korarchaeota. Hence, while our results provide no support for the iconic three-domain tree of life, they are consistent with an extended eocyte hypothesis whereby vital components of the eukaryotic nuclear lineage originated from within the archaeal radiation.
doi:10.1098/rspb.2012.1795
PMCID: PMC3497233  PMID: 23097517
phylogenetics; eukaryotes; evolution; tree of life
6.  The SAR11 Group of Alpha-Proteobacteria Is Not Related to the Origin of Mitochondria 
PLoS ONE  2012;7(1):e30520.
Although free living, members of the successful SAR11 group of marine alpha-proteobacteria contain a very small and A+T rich genome, two features that are typical of mitochondria and related obligate intracellular parasites such as the Rickettsiales. Previous phylogenetic analyses have suggested that Candidatus Pelagibacter ubique, the first cultured member of this group, is related to the Rickettsiales+mitochondria clade whereas others disagree with this conclusion. In order to determine the evolutionary position of the SAR11 group and its relationship to the origin of mitochondria, we have performed phylogenetic analyses on the concatenation of 24 proteins from 5 mitochondria and 71 proteobacteria. Our results support that SAR11 group is not the sistergroup of the Rickettsiales+mitochondria clade and confirm that the position of this group in the alpha-proteobacterial tree is strongly affected by tree reconstruction artefacts due to compositional bias. As a consequence, genome reduction and bias toward a high A+T content may have evolved independently in the SAR11 species, which points to a different direction in the quest for the closest relatives to mitochondria and Rickettsiales. In addition, our analyses raise doubts about the monophyly of the newly proposed Pelagibacteraceae family.
doi:10.1371/journal.pone.0030520
PMCID: PMC3264578  PMID: 22291975
7.  Informational Gene Phylogenies Do Not Support a Fourth Domain of Life for Nucleocytoplasmic Large DNA Viruses 
PLoS ONE  2011;6(6):e21080.
Mimivirus is a nucleocytoplasmic large DNA virus (NCLDV) with a genome size (1.2 Mb) and coding capacity ( 1000 genes) comparable to that of some cellular organisms. Unlike other viruses, Mimivirus and its NCLDV relatives encode homologs of broadly conserved informational genes found in Bacteria, Archaea, and Eukaryotes, raising the possibility that they could be placed on the tree of life. A recent phylogenetic analysis of these genes showed the NCLDVs emerging as a monophyletic group branching between Eukaryotes and Archaea. These trees were interpreted as evidence for an independent “fourth domain” of life that may have contributed DNA processing genes to the ancestral eukaryote. However, the analysis of ancient evolutionary events is challenging, and tree reconstruction is susceptible to bias resulting from non-phylogenetic signals in the data. These include compositional heterogeneity and homoplasy, which can lead to the spurious grouping of compositionally-similar or fast-evolving sequences. Here, we show that these informational gene alignments contain both significant compositional heterogeneity and homoplasy, which were not adequately modelled in the original analysis. When we use more realistic evolutionary models that better fit the data, the resulting trees are unable to reject a simple null hypothesis in which these informational genes, like many other NCLDV genes, were acquired by horizontal transfer from eukaryotic hosts. Our results suggest that a fourth domain is not required to explain the available sequence data.
doi:10.1371/journal.pone.0021080
PMCID: PMC3116878  PMID: 21698163
8.  Diversity and reductive evolution of mitochondria among microbial eukaryotes 
All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus. Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.
doi:10.1098/rstb.2009.0224
PMCID: PMC2817227  PMID: 20124340
mitochondria; hydrogenosomes; mitosomes; mitochondrial-like organelles
9.  The primary divisions of life: a phylogenomic approach employing composition-heterogeneous methods 
The three-domains tree, which depicts eukaryotes and archaebacteria as monophyletic sister groups, is the dominant model for early eukaryotic evolution. By contrast, the ‘eocyte hypothesis’, where eukaryotes are proposed to have originated from within the archaebacteria as sister to the Crenarchaeota (also called the eocytes), has been largely neglected in the literature. We have investigated support for these two competing hypotheses from molecular sequence data using methods that attempt to accommodate the across-site compositional heterogeneity and across-tree compositional and rate matrix heterogeneity that are manifest features of these data. When ribosomal RNA genes were analysed using standard methods that do not adequately model these kinds of heterogeneity, the three-domains tree was supported. However, this support was eroded or lost when composition-heterogeneous models were used, with concomitant increase in support for the eocyte tree for eukaryotic origins. Analysis of combined amino acid sequences from 41 protein-coding genes supported the eocyte tree, whether or not composition-heterogeneous models were used. The possible effects of substitutional saturation of our data were examined using simulation; these results suggested that saturation is delayed by among-site rate variation in the sequences, and that phylogenetic signal for ancient relationships is plausibly present in these data.
doi:10.1098/rstb.2009.0034
PMCID: PMC2873002  PMID: 19571240
universal tree of life; eukaryote origins; archaebacteria; eocyte; heterogeneous phylogenetic models
10.  Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis 
PLoS Pathogens  2008;4(12):e1000243.
Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas αMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric α/βMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.
Author Summary
In classic model organisms, cleavage of signals that are required to deliver nuclear-encoded proteins to mitochondria is mediated by an enzyme comprising two different subunits, called α or β, neither of which is functional by itself. Here, we have characterized a novel enzyme that functions in the mitosome, a highly reduced mitochondrion, of the pathogenic protist Giardia intestinalis. The Giardia enzyme is unique among eukaryotes because it has undergone reductive evolution to function efficiently as a single β-subunit monomer. We also show that the recent claim that the equivalent enzyme in the hydrogenosome, another type of reduced mitochondrion of the human parasite Trichomonas vaginalis, functions as a homodimer of two β-subunits, is not supported. The Trichomonas enzyme requires both an α- and a β-subunit to function most efficiently. Computational analysis of the Giardia and Trichomonas enzymes reveals that their structures and surface charge distributions have co-evolved to match the peculiar properties of the targeting signals that they process. The Giardia mitosome is an ideal model for studying the limits of mitochondrial reductive evolution and, because it makes cofactors that are essential for Giardia survival, is a potential therapeutic target for this important human parasite.
doi:10.1371/journal.ppat.1000243
PMCID: PMC2597178  PMID: 19096520
11.  Frataxin, a Conserved Mitochondrial Protein, in the Hydrogenosome of Trichomonas vaginalis▿  
Eukaryotic Cell  2007;6(8):1431-1438.
Recent data suggest that frataxin plays a key role in eukaryote cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (FeS) cluster biosynthesis. We have now identified a frataxin homologue (T. vaginalis frataxin) from the human parasite Trichomonas vaginalis. Instead of mitochondria, this unicellular eukaryote possesses hydrogenosomes, peculiar organelles that produce hydrogen but nevertheless share common ancestry with mitochondria. T. vaginalis frataxin contains conserved residues implicated in iron binding, and in silico, it is predicted to form a typical α-β sandwich motif. The short N-terminal extension of T. vaginalis frataxin resembles presequences that target proteins to hydrogenosomes, a prediction confirmed by the results of overexpression of T. vaginalis frataxin in T. vaginalis. When expressed in the mitochondria of a frataxin-deficient Saccharomyces cerevisiae strain, T. vaginalis frataxin partially restored defects in heme and FeS cluster biosynthesis. Although components of heme synthesis or heme-containing proteins have not been found in T. vaginalis to date, T. vaginalis frataxin was also shown to interact with S. cerevisiae ferrochelatase by using a Biacore assay. The discovery of conserved iron-metabolizing pathways in mitochondria and hydrogenosomes provides additional evidence not only of their common evolutionary history, but also of the fundamental importance of this pathway for eukaryotes.
doi:10.1128/EC.00027-07
PMCID: PMC1951141  PMID: 17573543
12.  Introduction: how and when did microbes change the world? 
doi:10.1098/rstb.2006.1847
PMCID: PMC1626534  PMID: 16754602
early life; palaeoclimes; fossils; cell evolution; atmospheric oxygenation; phylogeny
13.  Mitochondria and hydrogenosomes are two forms of the same fundamental organelle. 
Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria. As hydrogenosomes generally lack a genome, the conversion is probably one way. The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes. There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis. Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis. It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted. It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention.
doi:10.1098/rstb.2002.1190
PMCID: PMC1693103  PMID: 12594927
14.  Grassland Management Regimens Reduce Small-Scale Heterogeneity and Species Diversity of β-Proteobacterial Ammonia Oxidizer Populations 
The impact of soil management practices on ammonia oxidizer diversity and spatial heterogeneity was determined in improved (addition of N fertilizer), unimproved (no additions), and semi-improved (intermediate management) grassland pastures at the Sourhope Research Station in Scotland. Ammonia oxidizer diversity within each grassland soil was assessed by PCR amplification of microbial community DNA with both ammonia oxidizer-specific, 16S rRNA gene (rDNA) and functional, amoA, gene primers. PCR products were analysed by denaturing gradient gel electrophoresis, phylogenetic analysis of partial 16S rDNA and amoA sequences, and hybridization with ammonia oxidizer-specific oligonucleotide probes. Ammonia oxidizer populations in unimproved soils were more diverse than those in improved soils and were dominated by organisms representing Nitrosospira clusters 1 and 3 and Nitrosomonas cluster 7 (closely related phylogenetically to Nitrosomonas europaea). Improved soils were only dominated by Nitrosospira cluster 3 and Nitrosomonas cluster 7. These differences were also reflected in functional gene (amoA) diversity, with amoA gene sequences of both Nitrosomonas and Nitrosospira species detected. Replicate 0.5-g samples of unimproved soil demonstrated significant spatial heterogeneity in 16S rDNA-defined ammonia oxidizer clusters, which was reflected in heterogeneity in ammonium concentration and pH. Heterogeneity in soil characteristics and ammonia oxidizer diversity were lower in improved soils. The results therefore demonstrate significant effects of soil management on diversity and heterogeneity of ammonia oxidizer populations that are related to similar changes in relevant soil characteristics.
doi:10.1128/AEM.68.1.20-30.2002
PMCID: PMC126539  PMID: 11772604
15.  Phylogenetic Differences between Particle-Associated and Planktonic Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in the Northwestern Mediterranean Sea 
The aim of this study was to determine if there were differences between the types of ammonia-oxidizing bacteria of the β subdivision of the class Proteobacteria associated with particulate material and planktonic samples obtained from the northwestern Mediterranean Sea. A nested PCR procedure performed with ammonia oxidizer-selective primers was used to amplify 16S rRNA genes from extracted DNA. The results of partial and full-length sequence analyses of 16S rRNA genes suggested that different groups of ammonia-oxidizing bacteria were associated with the two sample types. The particle-associated sequences were predominantly related to Nitrosomonas eutropha, while the sequences obtained from the planktonic samples were related to a novel marine Nitrosospira group (cluster 1) for which there is no cultured representative yet. A number of oligonucleotide probes specific for different groups of ammonia oxidizers were used to estimate the relative abundance of sequence types in samples of clone libraries. The planktonic libraries contained lower proportions of ammonia oxidizer clones (0 to 26%) than the particulate material libraries (9 to 83%). Samples of the planktonic and particle-associated libraries showed that there were depth-related differences in the ammonia oxidizer populations, with the highest number of positive clones in the particle-associated sample occurring at a depth of 700 m. The greatest difference between planktonic and particle-associated populations occurred at a depth of 400 m, where only 4% of the clones in the planktonic library were identified as Nitrosomonas clones, while 96% of these clones were identified as clones that were related to the marine Nitrosospira species. Conversely, all ammonia oxidizer-positive clones obtained from the particle-associated library were members of the Nitrosomonas group. This is the first indication that Nitrosomonas species and Nitrosospira species may occupy at least two distinct environmental niches in marine environments. The occurrence of these groups in different niches may result from differences in physiological properties and, coupled with the different environmental conditions associated with these niches, may lead to significant differences in the nature and rates of nitrogen cycling in these environments.
PMCID: PMC91095  PMID: 9925616
16.  Nitrogen Cycling and Community Structure of Proteobacterial β-Subgroup Ammonia-Oxidizing Bacteria within Polluted Marine Fish Farm Sediments 
A multidisciplinary approach was used to study the effects of pollution from a marine fish farm on nitrification rates and on the community structure of ammonia-oxidizing bacteria in the underlying sediment. Organic content, ammonium concentrations, nitrification rates, and ammonia oxidizer most-probable-number counts were determined in samples of sediment collected from beneath a fish cage and on a transect at 20 and 40 m from the cage. The data suggest that nitrogen cycling was significantly disrupted directly beneath the fish cage, with inhibition of nitrification and denitrification. Although visual examination indicated some slight changes in sediment appearance at 20 m, all other measurements were similar to those obtained at 40 m, where the sediment was considered pristine. The community structures of proteobacterial β-subgroup ammonia-oxidizing bacteria at the sampling sites were compared by PCR amplification of 16S ribosomal DNA (rDNA), using primers which target this group. PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE) and with oligonucleotide hybridization probes specific for different ammonia oxidizers. A DGGE doublet observed in PCR products from the highly polluted fish cage sediment sample was present at a lower intensity in the 20-m sample but was absent from the pristine 40-m sample station. Band migration, hybridization, and sequencing demonstrated that the doublet corresponded to a marine Nitrosomonas group which was originally observed in 16S rDNA clone libraries prepared from the same sediment samples but with different PCR primers. Our data suggest that this novel Nitrosomonas subgroup was selected for within polluted fish farm sediments and that the relative abundance of this group was influenced by the extent of pollution.
PMCID: PMC91005  PMID: 9872782
17.  Analysis of β-Subgroup Proteobacterial Ammonia Oxidizer Populations in Soil by Denaturing Gradient Gel Electrophoresis Analysis and Hierarchical Phylogenetic Probing 
A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic β-subgroup proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DNA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE. Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers. A sequence specific to all β-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147–4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters. DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results were ambiguous because of overlapping banding patterns. Unambiguous band identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes. The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol 62:4147–4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor. The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of β-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.
PMCID: PMC106799  PMID: 9687457

Results 1-17 (17)