Reyranella terrae sp. nov., isolated from an agricultural soil, and emended description of the genus Reyranella.

A novel strain designated 11G32T was isolated from an agricultural soil cultivated with Chinese cabbage in Korea. The cells were Gram-stain-negative, aerobic, non-motile and rod-shaped. The strain grew at 15-28 °C (optimum, 20 °C), pH 5.0-7.0 (optimum, pH 5.0-6.0) and without NaCl. Phylogenetically, the strain was found to be closely related to members of the genus Reyranella and showed 16S rRNA gene sequence similarities of 97.18, 96.76 and 95.99 % with Reyranella graminifolii Wo-34T, Reyranella massiliensis 521T and Reyranella soli KIS14-15T, respectively. The major fatty acids were C19 : 0 cyclo ω8c, C16 : 0, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and 11-methyl C18 : 1ω7c. The predominant ubiquinone was Q-10. The polar lipids profile revealed the presence of phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, unidentified aminolipids, unidentified phospholipids and unidentified lipids. On the basis of data presented, strain 11G32T is considered to represent a novel species of the genus Reyranella, for which the name Reyranella terrae sp. nov. is proposed. The type strain is 11G32T (=KACC 18486T=NBRC 111476T).

Freeze-thaw revival of rotifers and algae in a desiccated, high-elevation (5500 meters) microbial mat, high Andes, Perú.

This is the first study of the highest elevation cyanobacteria-dominated microbial mat yet described. The desiccated mat was sampled in 2010 from an ephemeral rock pool at 5500 m above sea level in the Cordillera Vilcanota of southern Perú. After being frozen for 6 years at -20 °C in the lab, pieces of the mat were sequenced to fully characterize both the 16 and 18S microbial communities and experiments were conducted to determine if organisms in the mat could revive and become active under the extreme freeze-thaw conditions that these mats experience in the field. Sequencing revealed an unexpectedly diverse, multi-trophic microbial community with 16S OTU richness comparable to similar, seasonally desiccated mats from the Dry Valleys of Antarctica and low elevation sites in the Atacama Desert region. The bacterial community of the mat was dominated by phototrophs in the Cyanobacteria (Nostoc) and the Rhodospirillales, whereas the eukaryotic community was dominated by predators such as bdelloid rotifers (Philodinidae). Microcosm experiments showed that bdelloid rotifers in the mat were able to come out of dormancy and actively forage even under realistic field conditions (diurnal temperature fluctuations of -12 °C at night to + 27 °C during the day), and after being frozen for 6 years. Our results broaden our understanding of the diversity of life in periodically desiccated, high-elevation habitats and demonstrate that extreme freeze-thaw cycles per se are not a major factor limiting the development of at least some members of these unique microbial mat systems.

tRNA signatures reveal a polyphyletic origin of SAR11 strains among alphaproteobacteria.

Molecular phylogenetics and phylogenomics are subject to noise from horizontal gene transfer (HGT) and bias from convergence in macromolecular compositions. Extensive variation in size, structure and base composition of alphaproteobacterial genomes has complicated their phylogenomics, sparking controversy over the origins and closest relatives of the SAR11 strains. SAR11 are highly abundant, cosmopolitan aquatic Alphaproteobacteria with streamlined, A+T-biased genomes. A dominant view holds that SAR11 are monophyletic and related to both Rickettsiales and the ancestor of mitochondria. Other studies dispute this, finding evidence of a polyphyletic origin of SAR11 with most strains distantly related to Rickettsiales. Although careful evolutionary modeling can reduce bias and noise in phylogenomic inference, entirely different approaches may be useful to extract robust phylogenetic signals from genomes. Here we develop simple phyloclassifiers from bioinformatically derived tRNA Class-Informative Features (CIFs), features predicted to target tRNAs for specific interactions within the tRNA interaction network. Our tRNA CIF-based model robustly and accurately classifies alphaproteobacterial genomes into one of seven undisputed monophyletic orders or families, despite great variability in tRNA gene complement sizes and base compositions. Our model robustly rejects monophyly of SAR11, classifying all but one strain as Rhizobiales with strong statistical support. Yet remarkably, conventional phylogenetic analysis of tRNAs classifies all SAR11 strains identically as Rickettsiales. We attribute this discrepancy to convergence of SAR11 and Rickettsiales tRNA base compositions. Thus, tRNA CIFs appear more robust to compositional convergence than tRNA sequences generally. Our results suggest that tRNA-CIF-based phyloclassification is robust to HGT of components of the tRNA interaction network, such as aminoacyl-tRNA synthetases. We explain why tRNAs are especially advantageous for prediction of traits governing macromolecular interactions from genomic data, and why such traits may be advantageous in the search for robust signals to address difficult problems in classification and phylogeny.

Paracatenula, an ancient symbiosis between thiotrophic Alphaproteobacteria and catenulid flatworms.

Harnessing chemosynthetic symbionts is a recurring evolutionary strategy. Eukaryotes from six phyla as well as one archaeon have acquired chemoautotrophic sulfur-oxidizing bacteria. In contrast to this broad host diversity, known bacterial partners apparently belong to two classes of bacteria--the Gamma- and Epsilonproteobacteria. Here, we characterize the intracellular endosymbionts of the mouthless catenulid flatworm genus Paracatenula as chemoautotrophic sulfur-oxidizing Alphaproteobacteria. The symbionts of Paracatenula galateia are provisionally classified as "Candidatus Riegeria galateiae" based on 16S ribosomal RNA sequencing confirmed by fluorescence in situ hybridization together with functional gene and sulfur metabolite evidence. 16S rRNA gene phylogenetic analysis shows that all 16 Paracatenula species examined harbor host species-specific intracellular Candidatus Riegeria bacteria that form a monophyletic group within the order Rhodospirillales. Comparing host and symbiont phylogenies reveals strict cocladogenesis and points to vertical transmission of the symbionts. Between 33% and 50% of the body volume of the various worm species is composed of bacterial symbionts, by far the highest proportion among all known endosymbiotic associations between bacteria and metazoans. This symbiosis, which likely originated more than 500 Mya during the early evolution of flatworms, is the oldest known animal-chemoautotrophic bacteria association. The distant phylogenetic position of the symbionts compared with other mutualistic or parasitic Alphaproteobacteria promises to illuminate the common genetic predispositions that have allowed several members of this class to successfully colonize eukaryote cells.

Genome sequence of Reyranella massiliensis, a bacterium associated with amoebae.

Reyranella massiliensis is an Alphaproteobacterium member of the class Rhodospirillaceae, growing in amoebae. We sequenced the genome of type strain 521(T). It is composed of a 5,792,218-bp chromosome and encodes 5,675 protein-coding genes and 53 RNA genes, including 3 rRNA genes.

Isolation and characterization of Reyranella massiliensis gen. nov., sp. nov. from freshwater samples by using an amoeba co-culture procedure.

The analysis of three water samples from two cooling towers and one river allowed us to isolate three strains of a novel species of the class Alphaproteobacteria which is phylogenetically related to uncultured alphaproteobacteria. Based upon 16S rRNA gene sequence analysis and phenotypic characterization, we propose to name this novel species Reyranella massiliensis gen. nov., sp. nov., type strain 521(T) ( = CSUR P115(T)  = DSM 23428(T)). The most closely related cultivable micro-organism to this novel bacterium is a member of the genus Magnetospirillum.

Salix purpurea Stimulates the Expression of Specific Bacterial Xenobiotic Degradation Genes in a Soil Contaminated with Hydrocarbons.

The objectives of this study were to uncover Salix purpurea-microbe xenobiotic degradation systems that could be harnessed in rhizoremediation, and to identify microorganisms that are likely involved in these partnerships. To do so, we tested S. purpurea's ability to stimulate the expression of 10 marker microbial oxygenase genes in a soil contaminated with hydrocarbons. In what appeared to be a detoxification rhizosphere effect, transcripts encoding for alkane 1-monooxygenases, cytochrome P450 monooxygenases, laccase/polyphenol oxidases, and biphenyl 2,3-dioxygenase small subunits were significantly more abundant in the vicinity of the plant's roots than in bulk soil. This gene expression induction is consistent with willows' known rhizoremediation capabilities, and suggests the existence of S. purpurea-microbe systems that target many organic contaminants of interest (i.e. C4-C16 alkanes, fluoranthene, anthracene, benzo(a)pyrene, biphenyl, polychlorinated biphenyls). An enhanced expression of the 4 genes was also observed within the bacterial orders Actinomycetales, Rhodospirillales, Burkholderiales, Alteromonadales, Solirubrobacterales, Caulobacterales, and Rhizobiales, which suggest that members of these taxa are active participants in the exposed partnerships. Although the expression of the other 6 marker genes did not appear to be stimulated by the plant at the community level, signs of additional systems that rest on their expression by members of the orders Solirubrobacterales, Sphingomonadales, Actinomycetales, and Sphingobacteriales were observed. Our study presents the first transcriptomics-based identification of microbes whose xenobiotic degradation activity in soil appears stimulated by a plant. It paints a portrait that contrasts with the current views on these consortia's composition, and opens the door for the development of laboratory test models geared towards the identification of root exudate characteristics that limit the efficiency of current willow-based rhizoremediation applications.

Structure, function and organization of antenna polypeptides and antenna complexes from the three families of Rhodospirillaneae.

Comparative primary structural analysis of polypeptides from antenna complexes from species of the three families of Rhodospirillaneae indicates the structural principles responsible for the formation of spectrally distinct light-harvesting complexes. In many of the characterized antenna systems the basic structural minimal unit is an alpha/beta polypeptide pair. Specific clusters of amino acid residues, in particular aromatic residues in the C-terminal domain, identify the antenna polypeptides to specific types of antenna systems, such as B880 (strong circular dichroism (CD)), B870 (weak CD), B800-850 (high), B800-850 (low) or B800-820. The core complex B880 (B1020) of species from Ectothiorhodospiraceae and Chromatiaceae apparently consists of four (alpha 1 alpha 2 beta 1 beta 2) or three (2 alpha beta 1 beta 2) chemically dissimilar antenna polypeptides respectively. There is good evidence that the so-called variable antenna complexes, such as the B800-850 (high), B800-850 (low) or B800-820 of Rp. acidophila, Rp. palustris and Cr. vinosum, are comprised of multiple forms of peripheral light-harvesting polypeptides. Structural similarities between prokaryotic and eukaryotic antenna polypeptides are discussed in terms of similar pigment organization. The structural basis for the strict organization of pigment molecules (bacteriochlorophyll (BChl) cluster) in the antenna system of purple bacteria is the hierarchical organization of the alpha- and beta-antenna polypeptides within and between the antenna complexes. On the basis of the three-domain structure of the antenna polypeptides with the central hydrophobic domain, forming a transmembrane alpha helix, possible arrangements of the antenna polypeptides in the three-dimensional structure of core and peripheral antenna complexes are discussed. Important structural and functional features of these polypeptides and therefore of the BChl cluster are the alpha/beta heterodimers, the alpha 2 beta 2 basic units and cyclic arrangements of these basic units. Equally important for the formation of the antenna complexes or the entire antenna are polypeptide-polypeptide, pigment-pigment and pigment-polypeptide interactions.

Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities.

Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon ((12)C) or stable-isotope-labeled ((13)C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the (13)C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. Importance: The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This is arguably the most powerful application of metagenomics for the recovery of novel genes and a natural partner of the stable-isotope-probing approach for targeting active-yet-uncultured microorganisms. We expanded on previous efforts to combine stable-isotope probing and metagenomics, enriching microorganisms from multiple soils that were active in degrading plant-derived carbohydrates, followed by construction of a cellulose-based metagenomic library and recovery of glycoside hydrolases through functional metagenomics. The major advance of our study was the discovery of active-yet-uncultivated soil microorganisms and enrichment of their glycoside hydrolases. We recovered positive cosmid clones in a higher frequency than would be expected with direct metagenomic analysis of soil DNA. This study has generated an invaluable metagenomic resource that future research will exploit for genetic and enzymatic potential.

Going deeper: metagenome of a hadopelagic microbial community.

The paucity of sequence data from pelagic deep-ocean microbial assemblages has severely restricted molecular exploration of the largest biome on Earth. In this study, an analysis is presented of a large-scale 454-pyrosequencing metagenomic dataset from a hadopelagic environment from 6,000 m depth within the Puerto Rico Trench (PRT). A total of 145 Mbp of assembled sequence data was generated and compared to two pelagic deep ocean metagenomes and two representative surface seawater datasets from the Sargasso Sea. In a number of instances, all three deep metagenomes displayed similar trends, but were most magnified in the PRT, including enrichment in functions for two-component signal transduction mechanisms and transcriptional regulation. Overrepresented transporters in the PRT metagenome included outer membrane porins, diverse cation transporters, and di- and tri-carboxylate transporters that matched well with the prevailing catabolic processes such as butanoate, glyoxylate and dicarboxylate metabolism. A surprisingly high abundance of sulfatases for the degradation of sulfated polysaccharides were also present in the PRT. The most dramatic adaptational feature of the PRT microbes appears to be heavy metal resistance, as reflected in the large numbers of transporters present for their removal. As a complement to the metagenome approach, single-cell genomic techniques were utilized to generate partial whole-genome sequence data from four uncultivated cells from members of the dominant phyla within the PRT, Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Planctomycetes. The single-cell sequence data provided genomic context for many of the highly abundant functional attributes identified from the PRT metagenome, as well as recruiting heavily the PRT metagenomic sequence data compared to 172 available reference marine genomes. Through these multifaceted sequence approaches, new insights have been provided into the unique functional attributes present in microbes residing in a deeper layer of the ocean far removed from the more productive sun-drenched zones above.

Reduction of perchlorate and nitrate by microbial communities in vadose soil.

Perchlorate contamination is a concern because of the increasing frequency of its detection in soils and groundwater and its presumed inhibitory effect on human thyroid hormone production. Although significant perchlorate contamination occurs in the vadose (unsaturated) zone, little is known about perchlorate biodegradation potential by indigenous microorganisms in these soils. We measured the effects of electron donor (acetate and hydrogen) and nitrate addition on perchlorate reduction rates and microbial community composition in microcosm incubations of vadose soil. Acetate and hydrogen addition enhanced perchlorate reduction, and a longer lag period was observed for hydrogen (41 days) than for acetate (14 days). Initially, nitrate suppressed perchlorate reduction, but once perchlorate started to be degraded, the process was stimulated by nitrate. Changes in the bacterial community composition were observed in microcosms enriched with perchlorate and either acetate or hydrogen. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes recovered from these microcosms indicated that formerly reported perchlorate-reducing bacteria were present in the soil and that microbial community compositions were different between acetate- and hydrogen-amended microcosms. These results indicate that there is potential for perchlorate bioremediation by native microbial communities in vadose soil.

Horizontal transfer of genes coding for the photosynthetic reaction centers of purple bacteria.

Phylogenetic trees were drawn and analyzed based on the nucleotide sequences of the 1.5-kb gene fragment coding for the L and M subunits of the photochemical reaction center of various purple photosynthetic bacteria. These trees are mostly consistent with phylogenetic trees based on 16S rRNA and soluble cytochrome c, but differ in some significant details. This inconsistency implies horizontal transfer of the genes that code for the photosynthetic apparatus in purple bacteria. Possibilities of similar transfers of photosynthesis genes during the evolution of photosynthesis are discussed especially for the establishment of oxygenic photosynthesis.

Nucleotide sequence of 16S rRNA and phylogenetic position of the green sulfur bacterium Clathrochloris sulfurica.

The almost complete primary structure of the 16S rRNA from the green sulfur bacterium "Clathrochloris sulfurica" was determined by reverse transcriptase sequencing. Comparison of defined invariable parts of the molecule from representatives of 9 major lines of descent from the eubacterial kingdom shows C. sulfurica to be highly related to Chlorobium vibrioforme. The relationship between "Clathrochloris" and Chlorobium is in accord with the present allocation of these two genera into the family Chlorobiaceae.

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus.

The structural genes (hup) of the H2 uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68,108 dalton) and by alignment with the NH2 amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34,256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe-4S] feredoxins.

Sequence evidence for strong conservation of the photoactive yellow proteins from the halophilic phototrophic bacteria Chromatium salexigens and Rhodospirillum salexigens.

The photoactive yellow proteins (PYP) have been found to date only in three species of halophilic purple phototrophic bacteria. They have photochemical activity remarkably similar to that of the bacteria rhodopsins. In contrast to rhodopsins, however, the PYPs are small water-soluble proteins. We now report the complete amino acid sequences of Rhodospirillum salexigens and Chromatium salexigens PYP which allow comparison with the known sequence and three-dimensional structure of the prototypic protein from Ectothiorhodospira halophila. Although isolated from three different families of bacteria, the PYP sequences are 70-76% identical. All three contain 125 amino acid residues, and no insertions or deletions are necessary for alignment. This is a remarkable result when it is considered that electron transfer proteins from these purple bacterial species are only 25-40% identical and that insertions and deletions are needed for their proper alignment. It thus appears that PYP has the same important function in each of the purple bacteria and that most of the amino acid residues are necessary to maintain structure and function. By most standards, PYP would be called a "slowly evolving protein". R. salexigens PYP is uniquely degraded by proteolysis at low ionic strength, probably as a consequence of unfolding due to electrostatic repulsion of the excess negative charge. Therefore it may also be classified as a "halophilic protein".

Isolation of mak1 from Actinoplanes missouriensis and evidence that Pep2 from Streptomyces coelicolor is a maltokinase.

The gene mak1FN coding for maltokinase from Actinoplanes missouriensis is located in a cluster similar to glycogen metabolism clusters identified in Streptomyces coelicolor. Sequence comparisons demonstrate that mak1-related genes coding for homologous proteins are present in many bacterial genomes including taxonomic distantly related groups such as Rhodospirillales or green sulfur bacteria. More than 50% of the aligned sequences are longer than the mak1 gene from A. missouriensis, and the N-terminal portion of these putative maltokinases exhibit high sequence homologies with trehalose synthases. A more detailed sequence comparison indicates a relationship of maltokinases to aminoglycoside phospho-transferases and protein kinases. Transformation of S. lividans with plasmid vectors containing either the mak1 gene from A. missouriensis or the pep2 gene from S. coelicolor resulted in recombinant strains, which produced measurable amounts of maltokinase activity. The proteins Pep2 and Mak1 were over expressed with Streptomyces lividans 66 as a heterologous host and further characterized. The possible physiological function of maltokinases is discussed.