Ribosomal RNA Sequence Comparisons Research Articles

SWPhylo - A Novel Tool for Phylogenomic Inferences by Comparison of Oligonucleotide Patterns and Integration of Genome-Based and Gene-Based Phylogenetic Trees.

Modern phylogenetic studies may benefit from the analysis of complete genome sequences of various microorganisms. Evolutionary inferences based on genome-scale analysis are believed to be more accurate than the gene-based alternative. However, the computational complexity of current phylogenomic procedures, inappropriateness of standard phylogenetic tools to process genome-wide data, and lack of reliable substitution models which correlates with alignment-free phylogenomic approaches deter microbiologists from using these opportunities. For example, the super-matrix and super-tree approaches of phylogenomics use multiple integrated genomic loci or individual gene-based trees to infer an overall consensus tree. However, these approaches potentially multiply errors of gene annotation and sequence alignment not mentioning the computational complexity and laboriousness of the methods. In this article, we demonstrate that the annotation- and alignment-free comparison of genome-wide tetranucleotide frequencies, termed oligonucleotide usage patterns (OUPs), allowed a fast and reliable inference of phylogenetic trees. These were congruent to the corresponding whole genome super-matrix trees in terms of tree topology when compared with other known approaches including 16S ribosomal RNA and GyrA protein sequence comparison, complete genome-based MAUVE, and CVTree methods. A Web-based program to perform the alignment-free OUP-based phylogenomic inferences was implemented at http://swphylo.bi.up.ac.za/. Applicability of the tool was tested on different taxa from subspecies to intergeneric levels. Distinguishing between closely related taxonomic units may be enforced by providing the program with alignments of marker protein sequences, eg, GyrA.

The universal tree of life: an update.

Biologists used to draw schematic “universal” trees of life as metaphors illustrating the history of life. It is indeed a priori possible to construct an organismal tree connecting the three major domains of ribosome encoding organisms: Archaea, Bacteria and Eukarya, since they originated by cell division from LUCA. Several universal trees based on ribosomal RNA sequence comparisons proposed at the end of the last century are still widely used, although some of their main features have been challenged by subsequent analyses. Several authors have proposed to replace the traditional universal tree with a ring of life, whereas others have proposed more recently to include viruses as new domains. These proposals are misleading, suggesting that endosymbiosis can modify the shape of a tree or that viruses originated from the last universal common ancestor (LUCA). I propose here an updated version of Woese’s universal tree that includes several rootings for each domain and internal branching within domains that are supported by recent phylogenomic analyses of domain specific proteins. The tree is rooted between Bacteria and Arkarya, a new name proposed for the clade grouping Archaea and Eukarya. A consensus version, in which each of the three domains is unrooted, and a version in which eukaryotes emerged within archaea are also presented. This last scenario assumes the transformation of a modern domain into another, a controversial evolutionary pathway. Viruses are not indicated in these trees but are intrinsically present because they infect the tree from its roots to its leaves. Finally, I present a detailed tree of the domain Archaea, proposing the sub-phylum neo-Euryarchaeota for the monophyletic group of euryarchaeota containing DNA gyrase. These trees, that will be easily updated as new data become available, could be useful to discuss controversial scenarios regarding early life evolution.

Archaea in Past and Present Geobiochemical Processes and Elemental Cycles

For a long time, Archaea have been considered as an ancient prokaryotic group comprising specialists restricted to narrow ecological niches. This opinion might have been supported by the characteristics of the first well-investigated isolates, being strictly anaerobic (methanogens), halophilic (haloarchaea), or thermophilic (various groups). This is, however, just the tip of the iceberg. Archaea are abundant in all ecosystems. Representatives of the whole domain span the widest range of ecological adaptations from psychrophilic to hyperthermophilic. They tolerate the widest range of pH as well as salt concentrations and use all types of substrates comprising all kinds of organic molecules as well as reduced inorganic compounds. Nowadays, a multitude of experimental tools is available allowing researchers to study the important role of Archaea and other microorganisms in environmental processes. Most important, analysis of environmental DNA and RNA opens a direct way to assess diversity in specific habitats. The pioneering findings of Carl R. Woese for assessing microbial taxonomy by ribosomal RNA sequence analysis and comparison, together with recently developed powerful amplification and sequencing techniques, widely opened the doors for detection of new hitherto unknown archaeal groups. In particular, the phyla Crenarchaeota and Thaumarchaeota and their significance in environmental cycles could be determined by these techniques. Other tools gain in importance for the assessment of environmental processes. With the increasing availability of annotated genomes, the analysis of metagenomes and metaproteomes becames possible and now helps us to understand the metabolic processes and networks within microbial communities. The identification of lipids allows bulk quantification of specific prokaryotic groups in recent habitats and even in fossil settings. Finally, microscopic techniques along with functional analysis helps us to understand some of these environmental processes also at structural levels, for example, in microbial mats. This special issue of the journal Archaea addresses several aspects of the metabolic, structural, and ecological diversity of Archaea from a wide range of research fields. The contributions by B. Wemheuer et al. point out the diversity of Bacteria and Archaea in hot springs from the Kamchatka Peninsula, revealing the dominance of Proteobacteria, Thermotogae, and Thaumarchaeota. In a second publication, B. Wemheuer et al. address the diversity in a marine habitat with haloarchaea (Halobacteria) as the predominant archaeal group. One of the key processes in sediments is methanogenesis. M. R. Torres-Alvarado et al. show in their study the changes in the abundance and activity of methanogens during dry and rainy seasons in tropical estuarine sediments. The contribution of L. A. Fernandez-Guelfo et al. addresses the directed processes of methanogenesis in an anaerobic reactor suitable for wastewater treatment. The whole anaerobic food chain at different temperatures was evaluated by A. Siggins et al. in an anaerobic reactor with trichloroethylene as a substrate. X. L. H. Lim et al. used the lipid archaeol in anoxic water saturated soil as a biomarker for the biomass of methanogens. Another marker for methanogens was evaluated by C. Wrede et al. for microbial biofilms. Methyl coenzyme M reductase was detected in situ by respective antibodies and was found to be a suitable indicator for the enzymes catalyzing methanogenesis as well as the reverse pathway anaerobic oxidation methane (AOM). Another research article by C. Wrede et al. addresses iron biomineralization in the course of AOM. Finally, a review article on Archaea in symbiosis briefly summarizes the diverse symbiotic interactions of Archaea with organisms of all other domains. We hope that this issue will support to the statement stressed previously: Archaea, being as diverse as Bacteria, make their contribution to all important global elemental cycles. Michael Hoppert Martin Kruger Joachim Reitner Charles Cockell

Evaluation of synergistic and antibacterial activity of Xylaria curta against drug-resistant Staphylococcus aureus and Pseudomonas aeruginosa

Drug resistance problems are increasing worldwide, and hence, there is a need to find novel sources of antimicrobial drugs. At present, natural products still remain the most important resources for discovering new drugs. The screening of natural bioactive compounds from macrofungi is a promising way to meet the increasing threat of drug-resistant strains of human pathogens. In this study, the macrofungus was collected during spring seasons from the soil in tropical evergreen forest of Courtallam, Western Ghats of South India. It was identified as Xylaria curta based on the morphological characteristics and confirmed by 18S ribosomal RNA (rRNA) sequence comparisons. Extracts of fruiting bodies of X. curta were obtained using hexane, ethyl acetate, and methanol solvents. Among them, the ethyl acetate extracts exhibited an effective antimicrobial activity against bacterial and fungal strains. The extracts were also analyzed for antibacterial activity against drug-resistant Staphylococcus aureus strains (1–10) and Pseudomonas aeruginosa strains (1–8). The extract showed a maximum inhibition zone of 23.2 mm, 22.4 mm, and 20.7 mm against P. aeruginosa strain 8, P. aeruginosa strain 1, and S. aureus strain 8, respectively. Further, the extract had an effective minimum inhibitory concentration (MIC) of 200 μg mL−1 against drug-resistant S. aureus strain 8. The fractional inhibitory concentration indices of fruiting body extract with vancomycin and ciprofloxacin were 0.22 and 0.46, respectively. These results indicate that the extract had significant synergistic action with vancomycin. Xylaria curta is a potential source for the development of new antibiotics.

Antibacterial Activity of Wild Xylaria sp. Strain R005 (Ascomycetes) Against Multidrug-Resistant Staphylococcus aureus and Pseudomonas aeruginosa

There is a growing need for new and effective antibiotic agents due to the recent emergence of life-threatening, multidrug-resistant bacterial infections such as methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. In the present study, the antimicrobial potential of mushroom was investigated against multidrug-resistant bacterial strains. The mushroom was identified as Xylaria sp. strain R005 based on the morphological characteristics and confirmed by 18S ribosomal RNA sequence comparisons. The crude ethyl acetate extracts of culture filtrate and fruiting bodies of Xylaria sp. showed significant antibacterial activity against multidrug-resistant S. aureus strains (1-10) and P. aeruginosa strains (1-8). The minimum inhibitory concentration of the ethyl acetate extracts of culture filtrate and fruiting bodies ranged from 225 µg/mL to 625 µg/mL, and 120 µg/mL to 625 µg/mL, respectively, against clinical strains of S. aurues and P. aeruginosa. The synergistic action of extracts of Xylaria sp. with vancomycin and ciprofloxacin was observed against S. aureus strain 6 and P. aeruginosa strain 3, respectively. The fractional inhibitory concentration indices (FICIs) of culture filtrate extract with vancomycin and ciprofloxacin were 0.5 and 0.18, respectively. The FICI of fruiting body extract with vancomycin and ciprofloxacin were 0.5 and 0.375, respectively. These results clearly indicate that the metabolites of culture filtrate and fruiting bodies of Xylaria sp. are the potential source for production of new antimicrobial compounds.

The 3Cs provide a novel concept of bacterial species: messages from the genome as illustrated by Salmonella

A key issue troubling bacterial taxonomy and systematics is the lack of a biological species definition. Criteria to be used for defining bacterial species on genetic and biological bases should be able to reveal clear-cut boundaries among clusters of bacteria. To date, DNA-DNA re-association assays and ribosomal RNA sequence comparison have been useful in determining relative evolutionary distances among bacteria but the data are continuous and thus cannot define bacterial clusters as taxonomic units to be called species. Using Salmonella as models, we have looked for definite genetic and biologic uniqueness of clusters of bacteria. Based on our findings that each Salmonella lineage has a unique genome structure shared by strains of the same lineage but not overlapping with strains of other Salmonella lineages, we conclude that this is a result of genetic isolation following divergence of the bacteria. We propose that there should be genetic boundaries between different species of bacteria at the genomic level, which awaits further genomic information for validation.

Tensor Decomposition Reveals Concurrent Evolutionary Convergences and Divergences and Correlations with Structural Motifs in Ribosomal RNA

Evolutionary relationships among organisms are commonly described by using a hierarchy derived from comparisons of ribosomal RNA (rRNA) sequences. We propose that even on the level of a single rRNA molecule, an organism's evolution is composed of multiple pathways due to concurrent forces that act independently upon different rRNA degrees of freedom. Relationships among organisms are then compositions of coexisting pathway-dependent similarities and dissimilarities, which cannot be described by a single hierarchy. We computationally test this hypothesis in comparative analyses of 16S and 23S rRNA sequence alignments by using a tensor decomposition, i.e., a framework for modeling composite data. Each alignment is encoded in a cuboid, i.e., a third-order tensor, where nucleotides, positions and organisms, each represent a degree of freedom. A tensor mode-1 higher-order singular value decomposition (HOSVD) is formulated such that it separates each cuboid into combinations of patterns of nucleotide frequency variation across organisms and positions, i.e., “eigenpositions” and corresponding nucleotide-specific segments of “eigenorganisms,” respectively, independent of a-priori knowledge of the taxonomic groups or rRNA structures. We find, in support of our hypothesis that, first, the significant eigenpositions reveal multiple similarities and dissimilarities among the taxonomic groups. Second, the corresponding eigenorganisms identify insertions or deletions of nucleotides exclusively conserved within the corresponding groups, that map out entire substructures and are enriched in adenosines, unpaired in the rRNA secondary structure, that participate in tertiary structure interactions. This demonstrates that structural motifs involved in rRNA folding and function are evolutionary degrees of freedom. Third, two previously unknown coexisting subgenic relationships between Microsporidia and Archaea are revealed in both the 16S and 23S rRNA alignments, a convergence and a divergence, conferred by insertions and deletions of these motifs, which cannot be described by a single hierarchy. This shows that mode-1 HOSVD modeling of rRNA alignments might be used to computationally predict evolutionary mechanisms.

Attenuation of the Fish Pathogen Francisella sp. by Mutation of the iglC* Gene

Fish francisellosis is an emergent disease caused by gram-negative facultative intracellular bacteria of the genus Francisella. Different strains of the bacterium have caused high mortalities in warmwater and coldwater fish species. Francisella sp. isolates from fish have been found to share more than 97% identity to the human pathogen Francisella tularensis upon 16S ribosomal RNA sequence comparison. Homologue genes of the F. tularensis intracellular growth locus (iglA*, iglB*, iglC*, and iglD*) were identified from LADL 07-285A, a clinical isolate obtained from diseased Nile tilapia Oreochromis niloticus. The iglABCD operon DNA sequence comparison revealed that Francisella LADL 07-285A had 94% identity with F. philomiragia subsp. philomiragia and 83% identity with F. tularensis subsp. novicida U112. The functions of the conserved proteins corresponding to the genes are elusive but appear to be essential for the ability of Francisella sp. to survive within macrophages and cause disease. An insertion mutation was made in the iglC* gene of LADL 07-285A by allelic exchange, and the iglC* mutant was found to be attenuated after intraperitoneal and immersion challenges in Nile tilapia. Laboratory challenge methods for inducing francisellosis in Nile tilapia were evaluated by intraperitoneal injection and immersion with serial dilutions of Francisella LADL 07-285A. The dose lethal to 50% of test fish at 40 d postchallenge was 10(-5.3) (about 1.2 X 10(3) colony-forming units/fish) by intraperitoneal injection and was 10(-1) (2.3 X 10(7) colony-forming units/mL of tank water) by immersion.

Is Mycobacterium tuberculosis a closer relative to Gram-positive or Gram–negative bacterial pathogens?

The phylogenetic position of Mycobacterium tuberculosis relative to other bacteria is controversial. Its cell wall has characteristics of both Gram-positive and Gram-negative bacteria. In the standard reference of bacterial phylogeny based on 16S ribosomal RNA sequence comparison, M. tuberculosis belongs to the high G+C Gram-positive bacteria that form a monophyletic group with the low G+C Gram-positive bacteria such as Bacillus subtilis. Some analyses indicate no particular relationship between these two groups. The availability of the complete genome sequence of M. tuberculosis allows us to reexamine this issue from genomic perspectives, as genome-based phylogenies may be more representative of the evolutionary history of whole organisms than molecular trees. In the genome tree constructed based on conserved gene content, M. tuberculosis is more related to Gram-negative than to Gram-positive bacteria as reflected by the evolutionary distance between nearest ancestral units. This conclusion may be supported by another analysis showing that M. tuberculosis shares relatively more orthologous genes for energy production and conversion with Gram-negative bacteria, in particular, Escherichia coli and Pseudomonas aeruginosa, than with Gram-positive bacteria.

A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont.

According to small subunit ribosomal RNA (ss rRNA) sequence comparisons all known Archaea belong to the phyla Crenarchaeota, Euryarchaeota, and--indicated only by environmental DNA sequences--to the 'Korarchaeota'. Here we report the cultivation of a new nanosized hyperthermophilic archaeon from a submarine hot vent. This archaeon cannot be attached to one of these groups and therefore must represent an unknown phylum which we name 'Nanoarchaeota' and species, which we name 'Nanoarchaeum equitans'. Cells of 'N. equitans' are spherical, and only about 400 nm in diameter. They grow attached to the surface of a specific archaeal host, a new member of the genus Ignicoccus. The distribution of the 'Nanoarchaeota' is so far unknown. Owing to their unusual ss rRNA sequence, members remained undetectable by commonly used ecological studies based on the polymerase chain reaction. 'N. equitans' harbours the smallest archaeal genome; it is only 0.5 megabases in size. This organism will provide insight into the evolution of thermophily, of tiny genomes and of interspecies communication.

Archaeal phylogeny based on ribosomal proteins.

Until recently, phylogenetic analyses of Archaea have mainly been based on ribosomal RNA (rRNA) sequence comparisons, leading to the distinction of the two major archaeal phyla: the Euryarchaeota and the Crenarchaeota. Here, thanks to the recent sequencing of several archaeal genomes, we have constructed a phylogeny based on the fusion of the sequences of the 53 ribosomal proteins present in most of the archaeal species. This phylogeny was remarkably congruent with the rRNA phylogeny, suggesting that both reflected the actual phylogeny of the domain Archaea even if some nodes remained unresolved. In both cases, the branches leading to hyperthermophilic species were short, suggesting that the evolutionary rate of their genes has been slowed down by structural constraints related to environmental adaptation. In addition, to estimate the impact of lateral gene transfer (LGT) on our tree reconstruction, we used a new method that revealed that 8 genes out of the 53 ribosomal proteins used in our study were likely affected by LGT. This strongly suggested that a core of 45 nontransferred ribosomal protein genes existed in Archaea that can be tentatively used to infer the phylogeny of this domain. Interestingly, the tree obtained using only the eight ribosomal proteins likely affected by LGT was not very different from the consensus tree, indicating that LGT mainly brought random phylogenetic noise. The major difference involves organisms living in similar environments, suggesting that LGTs are mainly directed by the physical proximity of the organisms rather than by their phylogenetic proximity.

Genome comparison of Mycobacterium tuberculosis and other bacteria.

The availability of the complete genome sequence of Mycobacterium tuberculosis allows its phylogenetic analysis based on the whole genome rather than single genes. As a genome-based tree is more representative of whole organisms and less inconsistent than single-gene trees, it could provide a better index for interpretation and inference about the origin and nature of species. The standard bacterial phylogeny based on 16S ribosomal RNA sequence comparison shows that M. tuberculosis is more related to Gram-positive than to Gram-negative bacteria. Our results based on genome comparison in terms of shared orthologous genes challenge this implication. We demonstrate that M. tuberculosis is more related to Gram-negative than to Gram-positive bacteria by a quantitative analysis on the genome tree. The numerical distance data derived from genome comparison and those from 16S rRNA comparison show high significant correlation, implying that conserved gene content carries a strong phylogenetic signature in evolution.

A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron

In the present study, we determined the sequence of group I self-splicing introns found in the large ribosomal RNA subunit of Candida albicans, Candida stellatoidea and the recently-described species Candida dubliniensis. It was found that both the intron and ribosomal RNA nucleotide sequences are almost perfectly identical between different C. albicans strains as well as between C. albicans and C. stellatoidea strains. Comparisons of ribosomal RNA sequences suggest that local isolates of atypical C. albicans from individuals infected with human immunodeficiency virus can be assigned to the C. dubliniensis species. C. dubliniensis strains also harbor a group I intron in their ribosomal RNA, as observed in about 40% of C. albicans strains and all C. stellatoidea strains. This novel C. dubliniensis group I intron is identical to the C. albicans and C. stellatoidea intron, except for two widely divergent stem-loop regions. Despite these differences, the C. dubliniensis intron possesses self-splicing ability in an in vitro assay. Taken together, these data support the idea that C. albicans and C. stellatoidea should be joined together as variants of the same species while C. dubliniensis is a distinct but closely related microorganism. To our knowledge, the C. albicans and C. dubliniensis introns are the first example of a pair of homologous group I introns differing only by the presence of apparently facultative sequences in some stem-loops suspected to be involved in stabilization of tertiary structure.

Origin and early evolution of eukaryotes inferred from the amino acid sequences of translation elongation factors 1α/Tu and 2/G

Phylogenetic placements of archaebacteria and protozoa are important in understanding the origin and early evolution of eukaryotes. These problems have been analyzed mainly by comparisons of small subunit ribosomal RNA (SrRNA) sequences. However, the SrRNA phylogeny may sometimes be unreliable, especially when base compositions are biased among species. Because it is difficult to take full account of the bias in inferring the SrRNA tree, alternative examinations using protein sequence data have been very much desired. We analyzed the phylogenetic relationship among eukaryotes, archaebacteria, and eubacteria by the ML method of protein phylogeny using amino acid sequence data of EF-1α/Tu and 2/G. The unrooted tree analyses of both the EF-1α/Tu and 2/G consistently demonstrated that the ‘eocyte’ tree, in which archaebacteria are not monophyletic but eocytes are closer to eukaryotes than to other archaebacteria, is very likely. Further analysis using a composite tree of EF-1α/Tu and 2/G suggested that archaebacteria are closer to eukaryotes than to eubacteria but are not monophyletic. These results clearly support the hypothesis that eukaryotes have evolved from the eocyte-like organism. We also analyzed a protozoan phylogeny including mitochondrion-lacking species by the ML method using EF-1α and EF-2 data sets, and demonstrated (a) that two mitochondrion-lacking species, G. plecoglossi (Microsporidians) and G. lamblia (Diplomonads) probably represent the first and the second earliest offshoots of eukaryotes, respectively; (b) that Trypanosoma is not likely to have diverged next to Giardia as suggested by the SrRNA tree, but shows high affinity with higher eukaryotes; and (c) that protein phylogeny would give a robust estimation because amino acid compositions of conservative proteins do not differ significantly among species.

Comparisons of ribosomal RNA sequences from amitochondrial protozoa: implications for processing, mRNA binding and paromomycin susceptibility

The amitochondrial (a-mt) protozoa include four groups of organisms that are of interest as important human parasites and as probable descendents of the earliest branches of eukaryotic evolution. These organisms have not been directly compared in terms of structure and function of a specific molecule. We sequenced portions of their rRNA-encoding genes coding for the internal transcribed spacers ( ITS1 and 2) and adjoining small subunit (SS), 5.8S and large subunit (LS) rRNAs. Included are sites for RNA processing, mRNA interaction and aminoglycoside binding, as well as potential protein-encoding genes. The ITS of all a-mt protozoa examined are relatively short, but otherwise diverse. They include one or two predominant nucleotides (A in Entamoeba and Trichomonas, T in Encephalitozoon and C in Giardia) and have minimal potential secondary structure, which may form the basis for the preferential processing of ITS sequences. The mechanism employed by a-mt protozoa to bind mRNA may be unique, since Giardia, Trichomonas and Entamoeba mRNAs have unusually short 5′ non-coding regions. In bacteria, the 3′ terminus of the SS rRNA is involved in mRNA binding; analysis of Entamoeba and Trichomonas mRNA 5′ non-coding sequences suggests an analogous mechanism involving potential base pairing to the loop of the terminal SS rRNA hairpin. Giardia sensitivity to paromomycin was previously correlated with the presence of a C:G by near the decoding region of SS rRNA. This bp is also present in Entamoeba and Trichomonas, consistent with their susceptibility. Its absence in Encephalitozoon and other microsporidia predicts paromomycin resistance, and suggests a distinct evolutionary origin for this group.

Reclassification of “ Propionibacterium rubrum” as P. jensenii

The taxonomic relationship of strains previously designated as “ Propionibacterium rubrum” to P. thoenii and P. jensenii was investigated by use of 16S ribosomal RNA sequence comparison, biochemical characteristics and DNA hybridization. A total of 46 strains representing the species P. jensenii and P. thoenii and the former species “ P. rubrum” and also including 21 reference strains and 25 strains isolated from dairy sources were studied. The 16S rRNA sequence of strain “ P. rubrum” CNRZ 85 (= ATCC 4871) was found to be almost identical to that of the type strain of P. jensenii. DNA hybridization data indicated that “ P. rubrum” should belong to the species P. jensenii rather than P. thoenii, as formerly proposed. The “ P. rubrum” strains should then be reclassified as a β-haemolytic biovar of P. jensenii. The genomic species P. jensenii and P. thoenii could be differentiated by biochemical characteristics such as the production of acid from myo-inoshol and starch.

Molecular evolutionary analyses of nuclear-encoded small subunit ribosomal RNA identify an independent rhizopod lineage containing the Euglyphina and the Chlorarachniophyta.

The Rhizopoda comprise a diverse assemblage of protists which depend on lobose or filose pseudopodia for locomotion. The biochemical and morphological diversity of rhizopods has led to an uncertain taxonomy. Ribosomal RNA sequence comparisons offer a measure of evolutionary relatedness that is independent of morphology and has been used to demonstrate a polyphyletic origin of the Lobosea. We sequenced complete small subunit ribosomal RNA coding regions from the filose amoebae, Euglypha rotunda and Paulinella chromatophora (Euglyphina) to position these taxa in the eukaryote phylogeny. The neighbor-joining analyses show that E. rotunda and P. chromatophora share a monophyletic origin and are not closely related to any lobose amoebae in our analyses. Instead, the Euglyphina form a robust sister group to the Chlorarachniophyta. These results provide further evidence for the polyphyly of the Rhizopoda and support the creation of a new amoeboid lineage which includes the Euglyphina and the chlorarachniophyte algae; taxa with tubular mitochondrial cristae and filose or reticulate pseudopodia.

The stramenopiles from a molecular perspective: 16S-like rRNA sequences from Labyrinthuloides minuta and Cafeteria roenbergensis

Abstract Comparisons of 16S-like ribosomal RNA sequences from the heterotrophic marine flagellate Cafeteria roenbergensis Fenchel et Patterson, the slime net Labyrinthuloides minuta Watson et Raper and other eukaryotes provide insights into the early branching patterns for protists with tripartite flagellar hairs, the heterokont stramenopiles. Within this monophyletic assemblage, heterotrophic bicosoecids and labyrinthulids diverge before the separation of oomycetes and autotrophic stramenopiles. We infer that stramenopiles evolved from a colourless flagellate. Other taxa with chloroplasts containing chlorophyll a + c (dinoflagellates, cryptomonads and haptophytes) represent independent evolutionary lineages that acquired their chloroplasts separately from the endosymbiotic event leading to the autotrophic stramenopiles.

Spirochaeta caldaria sp. nov., a thermophilic bacterium that enhances cellulose degradation by Clostridium thermocellum

Two strains of obligately anaerobic, thermophilic spirochetes were isolated from cyanobacterial mat samples collected at freshwater hot springs in Oregon and Utah, USA. The isolates grew optimally between 48° and 52°C, and did not grow at 25° or 60°C. Both strains fermented various pentoses, hexoses, and disaccharides. Amino acids or cellulose did not serve as fermentable substrates for growth. H2, CO2, acetate, and lactate were end products of d-glucose fermentation. On the basis of physiological characteristics, guanine + cytosine content of DNA, and comparisons of 16S ribosomal RNA sequences, it was concluded that the two isolates were representatives of a novel species of Spirochaeta for which the name Spirochaeta caldaria is proposed. One of the two strains was grown in coculture with a thermophilic cellulolytic bacterium (Clostridium thermocellum) in a medium containing cellulose as the only fermentable substrate. In the coculture cellulose was broken down at a faster rate than in the clostridial monoculture. The results are consistent with the suggestion that interactions between cellulolytic bacteria and non-cellulolytic spirochetes enhance cellulose breakdown in natural environments in which cellulose-containing plant material is biodegraded.

Phylogeny of 4 ciliated anaerobic rumen protozoa inferred from small subunit ribosomal RNA sequence comparisons

Phylogeny of 4 ciliated anaerobic rumen protozoa inferred from small subunit ribosomal RNA sequence comparisons