text:
- Extended genome report
- Open Access
Complete genome sequence and description of Salinispira pacifica gen. nov., sp. nov., a novel spirochaete isolated form a hypersaline microbial mat
© Ben Hania et al.; licensee BioMed Central. 2015
- Received: 28 July 2014
- Accepted: 23 November 2014
- Published: 9 February 2015
During a study of the anaerobic microbial community of a lithifying hypersaline microbial mat of Lake 21 on the Kiritimati atoll (Kiribati Republic, Central Pacific) strain L21-RPul-D2T was isolated. The closest phylogenetic neighbor was Spirochaeta africana Z-7692T that shared a 16S rRNA gene sequence identity value of 90% with the novel strain and thus was only distantly related. A comprehensive polyphasic study including determination of the complete genome sequence was initiated to characterize the novel isolate.
Cells of strain L21-RPul-D2T had a size of 0.2 – 0.25 × 8–9 μm, were helical, motile, stained Gram-negative and produced an orange carotenoid-like pigment. Optimal conditions for growth were 35°C, a salinity of 50 g/l NaCl and a pH around 7.0. Preferred substrates for growth were carbohydrates and a few carboxylic acids. The novel strain had an obligate fermentative metabolism and produced ethanol, acetate, lactate, hydrogen and carbon dioxide during growth on glucose. Strain L21-RPul-D2T was aerotolerant, but oxygen did not stimulate growth. Major cellular fatty acids were C14:0, iso-C15:0, C16:0 and C18:0. The major polar lipids were an unidentified aminolipid, phosphatidylglycerol, an unidentified phospholipid and two unidentified glycolipids. Whole-cell hydrolysates contained L-ornithine as diagnostic diamino acid of the cell wall peptidoglycan. The complete genome sequence was determined and annotated. The genome comprised one circular chromosome with a size of 3.78 Mbp that contained 3450 protein-coding genes and 50 RNA genes, including 2 operons of ribosomal RNA genes. The DNA G + C content was determined from the genome sequence as 51.9 mol%. There were no predicted genes encoding cytochromes or enzymes responsible for the biosynthesis of respiratory lipoquinones.
Based on significant differences to the uncultured type species of the genus Spirochaeta, S. plicatilis, as well as to any other phylogenetically related cultured species it is suggested to place strain L21-RPul-D2T (=DSM 27196T = JCM 18663T) in a novel species and genus, for which the name Salinispira pacifica gen. nov., sp. nov. is proposed.
- Spirochaetes
- Fermentative metabolism
- Oxygen tolerance
- Hypersaline microbial mat
- Kiritimati atoll
In several marine to hypersaline coastal environments cyanobacterial mats can be found that are associated with microbialites. Occasionally, the observed microbialite structures resemble stromatolites formed by thin lithifying biofilms [1]. In contrast, microbial mats covering the hypersaline, evaporitic Lake 21 on the Kiritimati atoll (Kiribati, Central Pacific) are characterized by thick reticulate microbialites that form deep below the mat surface in the dark and anoxic zone [2]. Therefore, an alkalinization of the aqueous milieu by CO2 assimilating photoautotrophic cyanobacteria cannot play a key role in the calcification process as suggested for most fossil and modern stromatolites [3]. Rather, it is assumed that in Lake 21 microbial mats anaerobic bacteria stimulate lithification, for instance by degradation of extracellular polymeric substances (EPS) mainly comprising the mat matrix. Disintegration of the dense and negatively charged mat matrix characterizing cyanobacterial mats could lead to the release of bound calcium ions and the reduction of steric effects inhibiting mineral precipitation [2]. In several comprehensive cultivation-independent studies using 16S ribosomal RNA genes for analyzing the composition of bacterial communities inhabiting hypersaline microbial mats it was revealed that members of the Spirochaetes phylum play a major role in these highly diverse ecosystems and are among the most frequently detected phylotypes [4–6]. The observed stratification of sequences affiliated to Spirochaetes in hypersaline mats suggests that they represent a major part of the anaerobic microbial community with a peak abundance in the suboxic zone of the mat [5, 6] thereby indicating their involvement in anaerobic mineralization processes. The targeted isolation of spirochaetes was therefore a major goal in a cultivation-based survey of the microbial community of Lake 21 microbial mats [7]. In that study Spirochaeta-like morphotypes were frequently detected in enrichment cultures that were inoculated with anoxic mat samples and contained a pullulan derivate (Red Pullulan) as carbon source, which suggests a participation of spirochaetes in polysaccharide degradation. From such enrichments a novel strain with a Spirochaeta-like morphology was isolated and designated L21-Rpul-D2T. Phylogenetic analyses based on 16S rRNA gene sequences placed this strain within a clade of halophilic and/or alkaliphilic species within the genus Spirochaeta. The closest phylogenetic neighbor was the type strain of Spirochaeta africana sharing only a 16S rRNA gene sequence identity value of 90%, which is below the genus level according to current phylogenetic concepts [8, 9]. In this study we present a detailed and comprehensive characterization of the phenotype of this strain along with the determination of the complete genome sequence. Our results suggest to place strain L21-Rpul-D2T in a novel species and genus, for which the name Salinispira pacifica gen. nov., sp. nov. is proposed.
Classification and features
Strains and cultivation conditions
Strain L21-RPul-D2T was isolated from an anaerobic enrichment culture inoculated with slurries of a cyanobacterial mat retrieved from the hypersaline Lake 21 on the Kiritimati atoll (Northern Line Islands, Republic of Kiribati). The location of the sampling site and details of the isolation method were described elsewhere [7].
For the preparation of media and incubation under anoxic conditions the anaerobe cultivation technique of Hungate [10] with the modifications introduced by Bryant [11] was used. The basal medium for the characterization of strain L21-RPul-D2T included per liter: sea salts (50.0 g), yeast extract (2.0 g), Biotrypticase (2.0 g), L-cysteine-HCl × H2O (0.5 g), Balch trace element solution (10.0 ml) [12] and 1.0 ml of a 0.1% (w/v) solution of resazurin. The pH was adjusted to 7.2 with 10 M KOH solution and the medium was boiled under a stream of O2 free N2 gas and cooled to room temperature. Aliquots of 5 ml were dispensed into Hungate-type tubes, degassed under 80% N2 and 20% CO2 gas mixture, and subsequently sterilized by autoclaving at 120°C for 20 min. Before inoculation aliquots of the following sterile anoxic stock solutions were injected into the tubes containing 5 ml of medium: 0.1 ml of 10% (w/v) NaHCO3, 0.1 ml of 2% (w/v) Na2S × 9H2O, and 0.05 ml of 30% (w/v) MgCl2 × 6 H2O. For routine cultivation 20 mM D-glucose was added to the medium from a 1 M sterile anoxic stock solution.
For comparison the following type strains of related alkaliphilic Spirochaeta species were obtained from the DSMZ culture collection (Braunschweig, Germany): S. asiatica DSM 8901T, S. africana DSM 8902T, and S. dissipatitropha DSM 23605T. All of these strains were cultured according to the recommendations given in the DSMZ catalogue of strains [13] except that the DSMZ medium 1263 was used instead of DSMZ medium 700 for growing the strains DSM 8901T and DSM 8902T.
The determination of the almost complete 16S rRNA gene sequence of strain L21-RPul-D2T was already described in [7] and deposited in the GenBank/EMBL/DDBJ databases under the accession number KC665949. Phylogenetic trees based on almost complete 16S rRNA gene sequences with a minimum length of 1300 nucleotides were reconstructed using distance matrix (neighbor-joining), parsimony and maximum-likelihood programs included in the ARB package [14]. The dataset of aligned and almost complete 16S rRNA gene sequences was based on the ARB SILVA database release 111 (July 2012) [15].
Morphology and physiology
Shape and pigmentation
Pigments were extracted for spectroscopic analyses from strain L21-RPul-D2T and related type strains of Spirochaeta with a mixture of acetone/methanol (7:2 v/v) as described previously [24]. Spectra were recorded with a Thermo BioMate 6 UV–VIS split beam spectrophotometer. Pigments were formed under anaerobic and semiaerobic incubation conditions and gave cells a yellow to light orange color. Absorption spectra of the pigments extracted with acetone/methanol were characteristic for carotenoids with maxima at 439 and 468 nm and a shoulder at 487 nm. Carotenoid-like pigments could be also extracted from cell pellets of the type strains of S. africana and S. dissipatitropha, but not of S. asiatica.
Growth conditions
The pH, temperature and NaCl concentration ranges for growth were determined using basal medium supplemented with 20 mM D-glucose. Different pH values (5 to 9) of the medium were adjusted in increments of around 0.5 by injecting aliquots of anoxic stock solutions of 100 mM HCl (acidic pH values), 10% (w/v) NaHCO3 or Na2CO3 (basic pH values) in Hungate-type tubes containing 5 ml of medium. Water baths were used for incubating bacterial cultures from 15 to 55°C. NaCl requirement was determined by directly weighing NaCl in Hungate-type tubes before dispensing modified basal medium containing per liter: Na2SO4 (5.70 g), KCl (1.00 g), KBr (0.04 g), yeast extract (2.00 g), Biotrypticase (2.00 g), L-cysteine-HCl (0.50 g), NaHCO3 (0.30 g), resazurin (1.00 mg) and 10.00 ml of Balch trace element solution. Strain L21-RPul-D2T was moderately halophilic and grew optimally at a salinity of 5% (w/v). It required at least 2% (w/v) NaCl for growth and tolerated salinities up to 15% (w/v), which is the highest known salt tolerance of any known member of the free-living spirochaetes and grew at temperatures ranging from 20 to 45°C, with an optimum at 35°C. The pH range for growth was 6.5–8.4, with an optimum at pH 6.9-7.0.
Substrate utilization
Substrates (D-glucose, D-ribose, sucrose, D-fructose, D-xylose, D-arabinose, lactose, D-maltose, D-mannose, D-melibiose, D-cellobiose, D-trehalose, glycerol, ethanol, methanol, formate, acetate, pyruvate, fumarate, DL-lactate, succinate, DL-malate, citrate, butyrate, propionate) were tested at a final concentration of 20 mM in glucose-free basal medium. Casamino acids, peptone, starch and pullulan were tested at a final concentration of 2 g l-1 and 80% H2 and 20% CO2 gas mixture with and without acetate (2 mM) was tested under 2 bars of overpressure. To test for electron acceptors, sodium thiosulfate (20 mM), sodium sulfate (20 mM), sodium sulfite (2 mM), elemental sulfur (10 g l-1), sodium nitrate (20 mM), or sodium nitrite (2 mM) were added to the medium. Cultures were subcultured at least twice under the same experimental conditions before determination of growth rates. H2S production was determined photometrically as colloidal CuS according to Cord-Ruwisch [25]. End-products of metabolism were measured by high pressure liquid chromatography (HPLC) after 2 days of incubation at 35°C [26]. Strain L21-RPul-D2T had a strictly fermentative-type of metabolism and required yeast extract or Trypticase peptone for growth, but no vitamins. It was saccharolytic and used pullulan, starch, N-acetylglucosamine, D-fructose, D-glucose, D-maltose, D-mannose, and D-trehalose for growth, but not peptides or amino acids. In addition the carboxylic acids fumarate and pyruvate were utilized as electron donors. No positive growth response was obtained with chitin, arabinose, cellobiose, D-galactose, lactose, melibiose, D-ribose, sucrose, D-xylose, acetate, butyrate, casamino acids, citrate, formate, lactate, malate, propionate, succinate, ethanol, glycerol, D-mannitol, methanol, and H2/CO2 (8:2 v/v). The end-products resulting from D-glucose fermentation were acetate, lactate, ethanol, CO2 and H2. Sulfate, sulfite, elemental sulfur, nitrate and nitrite were not used as terminal electron acceptors. Thiosulfate and nitrate were not reduced during anaerobic growth. Despite a negative reaction of cells in tests for oxidase and catalase activity, growth did not depend on prereduced media. Oxygen concentrations of up to 10% were tolerated in the headspace gas atmosphere of statically incubated cultures. During semiaerobic cultivation (5 – 10% O2 in the headspace gas atmosphere) the growth yield did not increase compared to anaerobic incubation and the pattern of fermentation products did not change significantly, hence oxygen was not used as electron acceptor for respiratory metabolism.
Resistance to antibiotics
Evidence code
Current classification
Domain Bacteria
Phylum Spirochaetes
Class "Spirochaetia"
Order Spirochaetales
Family Spirochaetaceae
Genus Salinispira
Species Salinispira pacifica SRubellilLLeisingOwenweeksiahongkongensis
Type strain L21-RPul-D2T
Gram stain
Cell shape
Temperature range
Optimum temperature
Salinity range
2-15% (NaCl)
Optimum Salinity
5% (NaCl)
Oxygen requirement
anaerobic, oxygen-tolerant
Carbon source
carbohydrates, carboxylic acids
Energy metabolism
chemoheterotroph, fermentative
hypersaline microbial mat
Biotic relationship
Biosafety level
anoxic mat sample
Geographic location
Lake 21, Kiritimati, Kiribati (Central Pacific)
Sample collection time
March 2011
0.1 m below surface
The cellular fatty acid pattern of strain L21-RPul-D2T was determined from cells grown to early stationary phase in TYG medium [7]. For comparison additional cellular fatty acid patterns were determined from related type strains cultured in DSMZ medium 1263 under the conditions given in the DSMZ catalogue of strains [13]. The preparation and extraction of fatty acid methyl esters from biomass and their subsequent separation and identification by gas chromatography was done as described elsewhere [35]. Polar lipid analyses were carried out by the DSMZ Identification Service according to the published protocols [36]. Diagnostic diamino acids of the cell wall peptidoglycan were detected in hydrolysates (4 N HCl, 100°C, 16 h) of whole cells by using GC/MS as described by Schumann [37]. The cellular fatty acid composition of strain L21-RPul-D2T in comparison with the profiles of phylogenetically related Spirochaeta strains is shown in Additional file 2. The major cellulary fatty acids (>5% of the total abundance) of the novel isolate were C14:0, C16:0, iso-C15:0, and C18:0. Unique characteristics of strain L21-RPul-D2T compared to all related Spirochaeta strains were a predominance of the fatty acids C18:0 (9.7%) and iso-C15:0 (11.7%) combined with the presence of the branched fatty acid ante-C15:0. The determined polar lipid pattern revealed major amounts of phosphatidylglycerol, an unidentified aminolipid, an unidentified phospholipid and two distinct glycolipids (Additional file 3). The cell wall peptidoglycan contained L-ornithine as diagnostic diamino acid (type A1β according to the classification of Schleifer and Kandler [38]), which is a characteristic of the family Spirochaetaceae[30].
Genome sequencing and annotation
Genome project history
The genome of strain L21-RPul-D2T was sequenced within the DFG funded project "Microbial control of mineralization processes in non-marine biofilms and microbial mats". The strain was chosen for genome sequencing according to its low 16S rRNA gene sequence identity value to related type strains.
Genome sequencing project information
Source material identifier
Finishing quality
Libraries used
Three genomic libraries: PacBio SMRTbell™ library (> 10 kb) for draft assembly; Illumina PE library (350 bp insert size) and 454 PE library (3 kb insert size) for error correction
Sequencing platforms
PacBio RSII, Illumina GA IIx, 454
Fold coverage
365 (PacBio)
HGAP 2 (SMRTPortal 2.1.0), BWA
Gene calling method
Locus Tag
GenBank ID
GenBank Date of Release
February 14, 2014
NCBI project ID
Project relevance
Environmental, biodiversity
Growth conditions and DNA isolation
A culture of strain L21-RPul-D2T was grown anaerobically in TYG medium [7] at 35°C. Genomic DNA was isolated using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the standard protocol provided by the manufacturer but modified by an incubation time of 60 min, incubation on ice overnight on a shaker, the use of additional 50 μl proteinase K (21 mg ml-1), and the addition of 200 μl protein precipitation buffer. DNA is available from the Leibniz Institute DSMZ through the DNA Network [39].
Genome sequencing and assembly
The genome was sequenced using a combination of three genomic libraries (Table 2). SMRTbell™ template library was prepared according to the instructions from Pacific Biosciences, Menlo Park, CA, USA, following the Procedure & Checklist Greater than 10 kb Template Preparation and Sequencing. Briefly, for preparation of 10 kb libraries ~10 μg genomic DNA was end-repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P4 from Pacific Biosciences, Menlo Park, CA, USA. Reactions were carried out according to the manufacturer's instructions. SMRTbell™ template was exonuclease treated for removal of incompletely formed reaction products. Conditions for annealing of sequencing primers and binding of polymerase to purified SMRTbell™ template were assessed with the Calculator in RS Remote, Pacific Biosciences, Menlo Park, CA, USA. SMRT sequencing was carried out on the PacBio RSII (Pacific Biosciences, Menlo Park, CA, USA) taking one 120-minutes movie for each SMRT cell. In total 5 SMRT cells were run. Illumina sequencing was performed on a GA IIx platform with 150 cycles. The paired-end library contained inserts of an average insert size of 350 bp and delivered 3.0 million reads. A second Illumina run was performed on a Miseq platform to gain a higher sequencing depth. To achieve longer reads, the MiSeq library was sequenced in one direction for 300 cycles, providing another 2.0 million reads. 454 paired-end jumping library of a 3 kb insert library was performed on a 1/8 lane. Pyrosequencing resulted in 92,601 reads with an average read length of 371 bp assembled in Newbler (Roche Diagnostics).
A draft long read genome assembly named L21-Pul-V2-SP2_HGAP_5SC_std was created using the "RS_HGAP_Assembly.1" protocol included in SMRTPortal version 2.1.0 including all 5 SMRT cells. Standard parameters were applied including: Assembly – Preassembly: Compute Minimum Seed Read Length true, Allow Partial Alignments true, Trim FASTQ Output true, --- Celera Assembler v1 Genome Size (Bp) 5000000, Target Coverage 15, Overlapper Error Rate 0.06, Overlapper Min Length 40, Overlapper K-mer 14. Thus, one final contig could be obtained, which was trimmed, circularized and adjusted to dnaA as first gene. A total coverage of 365× has been calculated within the long read assembly process. Quality check of the final consensus sequences regarding overall coverage as well as SNPs was performed using IGV [40] after mapping of Illumina and 454 short read data onto the draft genome using BWA [41].
Genome annotation
Genome annotation and analysis were done using the RAST server [42]. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [32].
Genome properties
Genome statistics
% of Total*
Genome Size (bp)
DNA coding (bp)
DNA G + C (bp)
DNA scaffolds
Total genes
Protein coding genes
RNA genes
Pseudo genes
Genes with function prediction
Genes in paralog clusters
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
CRISPR repeats
Number of genes associated with general COG functional categories
% of total*
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Defense mechanisms
Signal transduction mechanisms
Cell wall/membrane biogenesis
Cell motility
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Function unknown
Not in COGs
Comparative genomics
Shared content of conserved genes among the genomes of Salinispira pacifica strain L21-RPul-D2 T and related type strains of Spirochaeta
Type strain
1. Salinispira pacifica
2. S. africana
3. S. alkalica
4. S. smaragdinae
5. S. cellobiosiphila
6. S. thermophila
7. S. bajacaliforniensis
Values of the shared gene content were in the range from 26 to 84% and 16S rRNA gene identity values varied between 84 and 99%, which indicates an extensive genetic heterogeneity within this monophyletic group. A recent study proposed to use the percentage of conserved proteins (POCP) among two genomes for genus demarcation [33]. According to this proposal strains having POCP values below 50% and 16S rRNA gene sequence identity values below 93% should belong to different genera. Consequently, only the two species S. smaragdinae and S. bajacaliforniense would belong to the same genus by having a 16S rRNA gene identity value of 99% and sharing 85% of their gene content, all other species and the novel strain L21-RPul-D2T would belong to different genera. This view is corroborated by some recently published whole genome sequence-based phylogenies containing a significant number of genome-sequenced representatives of the Spirochaetaceae[23, 43].
Energy metabolism
Strain L21-RPul-D2T used sugars and some carboxylic acids as substrates for fermentative growth, whereas peptides or amino acids were not utilized. Monosaccharides are transported into the cell via group translocation using the phosphotransferase system (PTS). Several genes encoding phosphocarrier proteins (L21SP2_2173 and L21SP2_3294), enzyme I (L21SP2_1343 and L21SP2_3295), and enzyme II (L21SP2_0100, L21SP2_0865, L21SP2_3408, and L21SP2_3409) of a putative PTS transport mechanism were detected. In addition, genes encoding a TRAP-type dicarboxylate transporter (L21SP2_1277 and L21SP2_1278) were present. The assimilation of peptides, which had a stimulating effect on growth, is probably catalyzed by transporters of the ABC-type (e.g., L21SP2_0701 - 0706). Presence of genes encoding both subunits of the key enzyme 6-phosphofructokinase (pyrophosphate-dependent) (L21SP2_0225 and L21SP2_2454) indicates that glucose is metabolized to pyruvate through the Embden-Meyerhof-Parnas (EMP) pathway in strain L21-RPul-D2T. According to the genome sequence several alternative reactions are possible for the further oxidation of pyruvate to acetyl-CoA. A pyruvate dehydrogenase complex encoded by the genes L21SP2_2176 - 2179 catalyzes the oxidative decarboxylation of pyruvate with concomitant release of CO2 and NADH. The pyruvate dehydrogenase complex is typically found in aerobic bacteria and its presence in obligate fermentative bacteria is unusual, but maybe explained by the aerotolerant lifestyle of this strain. In contrast to the pyruvate dehydrogenase complex, both alternative enzymes found in L21-Rpul-D2T are very sensitive to oxygen and operate only under anoxic conditions. The enzyme most frequently found in anaerobic bacteria for pyruvate oxidation is pyruvate:ferredoxin oxidoreductase, which is also present in L21-Rpul-D2T (L21SP2_2933) and releases reduced ferredoxin instead of NADH. The third route of pyruvate oxidation is catalyzed by pyruvate:formate lyase, which produces formate and acetyl-CoA and is typically found in Gram-negative facultatively anaerobic bacteria performing a mixed acid fermentation. In L21-RPul-D2T this enzyme is encoded at L21SP2_2006. The intermediate metabolite acetyl-CoA can be further oxidized to CO2 via the citric acid cycle, which appears to be fully functional in this strain, or reduced to ethanol by a combined acetaldehyde and alcohol dehydrogenase, both encoded by one gene at L21SP2_0358. No genes for the synthesis of respiratory lipoquinones, soluble cytochromes or membrane-bound terminal oxidases were present, which indicates that substrate-level phosphorylation is the main mechanism for the generation of ATP under anaerobic conditions. Consequently, the regeneration of NAD+ and oxidized ferredoxin has to be achieved by fermentative reactions, for example by reduction of pyruvate to lactate by a D-lactate dehydrogenase encoded at L21SP2_2897. Furthermore, several genes for distinct [FeFe] hydrogenases were present (e.g., L21SP2_0276, L21SP2_0545). Cytoplasmic iron-only hydrogenases are known to be involved in the regeneration of reduced ferredoxin and the production of hydrogen in some fermentative bacteria [44].
On the other hand, genes for several membrane-bound enzyme complexes were detected that could be involved in the generation or utilization of a chemiosmotic gradient without participation of an electron transport chain. For example it was found that in some fermenting thermophilic archaea a chemiosmotic gradient is generated by the oxidation of reduced ferredoxins at membrane-bound energy-coupling hydrogenases [45, 46]. In strain L21-Rpul-D2T a RNF-like electron transport complex encoded at L21SP2_0447 – 0452 could catalyze the oxidation of ferredoxin with NAD+, which has been shown to be coupled to translocation of protons or sodium ions in several anaerobic prokaryotes [47]. In addition, a multimeric complex with similarity to a sodium translocating NADH:quinone oxidoreductase (L21SP2_2184 – 2188) and a multicomponent sodium/proton antiporter (L21SP2_1997–2003) could be detected. A V-type ATP synthase is encoded by the genes L21SP2_1878–1884. This enzyme complex could function as an ATP driven ion pump as in Streptococcus faecalis[48] or in the utilization of a proton gradient for the synthesis of ATP as in Thermus thermophilus[49], thereby enabling an alternative to substrate-level phosphorylation.
Oxidative stress and carotenoid synthesis
Strain L21-RPul-D2T was isolated from the subsurface layers of a cyanobacterial mat that can be exposed to changing concentrations of oxygen due to the photosynthetic activity of cyanobacteria in the light. Like several other spirochaetes strain L21-RPul-D2T is aerotolerant and can grow fermentatively in the presence of oxygen, which may represent an important feature for the persistence of spirochaetes in cyanobacterial mats. Oxygen in cells of anaerobic microorganisms can have harmful effects by the formation of reactive oxygen species (ROS) at the active site of certain enzymes containing flavins [50]. To prevent oxidative stress by ROS, anaerobic bacteria have to keep intracellular concentrations of oxygen and its partially reduced derivates low. Although, it was observed that strain L21-RPul-D2T actively reduces oxygen-containing cultivation media during growth, no genes encoding a putative terminal oxidase or catalase could be detected in the genome sequence, which represent the main oxygen removal mechanisms of anaerobic respiratory bacteria, e.g. sulfate reducers [51]. In contrast, most aerotolerant fermentative bacteria use a NADH oxidase system for reduction of oxygen. Detoxification with NADH can be catalyzed by a single enzyme as in Brachyspira hyodysenteriae[52] or by a cascade of reactions including the electron carrier proteins rubredoxin and rubrerythrin [53]. Although, no NADH oxidase gene was annotated by the RAST server two candidate genes encoding putative NADH oxidase domains (L21SP2_0477 and L21SP2_0797) were detected in the genome sequence of L21-RPul-D2T by performing a BLASTP search with the NADH oxidase of B. hyodysenteriae (Q59917). However, at least one of both enzymes probably acts as a coenzyme A disulfide reductase that is a highly similar orthologue of NADH oxidase. Based on the absence of genes encoding glutathione oxidoreductase or enzymes for the synthesis of glutathione it can be deduced that in strain L21-RPul-D2T reduced coenzyme A represents the main low-molecular-weight thiol redox buffer. It has been revealed that in Borrelia burgdorferi reduced coenzyme A is oxidized by H2O2 and reduced again by coenzyme A reductase at the expense of NADH [54] thereby protecting cells against oxidative stress. In addition, alternative systems for detoxification of oxygen based on rubrerythrin (L21SP2_2848) and rubredoxins (L21SP2_1281, L21SP2_2197 and L21SP2_2972) seems to be active in strain L21-RPul-D2T, although no genes encoding a putative NADH:rubredoxin oxidoreductase were detected in the genome sequence. Some of the endogenous formed H2O2 may be reduced to water by the alkyl hydroperoxide reductase AhpCF (L21SP2_3374, L21SP2_3375) that also uses organic hydroperoxides as substrates [55]. For removal of the highly reactive superoxide anion a manganese superoxide dismutase (L21SP2_1673) or a superoxide reductase (L21SP2_1734) could be used.
Independent of the enzymatic reduction of ROS at the expense of NAD(P)H as reductant carotenoids seem to be used by certain spirochaetes to protect cellular lipids against oxidative stress. All aerotolerant species in the clade of halophilic/alkaliphilic spirochaetes represented by L21-RPul-D2T produce carotenoids, whereas the strictly anaerobic species S. asiatica is unpigmented (Additional file 1). Furthermore, it was observed that in some spirochaetes the production of carotenoids is induced only in the presence of oxygen [30, 56]. Hydroxylated derivates of β-carotene like zeaxanthin were shown to have a high antioxidant effect by scavenging singlet oxygen [57], and thus are probably used by strain L21-RPul-D2T and related Spirochaeta species for the protection of their cell membranes against oxidative stress. The biosynthesis of carotenoids in L21-RPul-D2T depends on a phytoene synthase (L21SP2_2164) and phytoene desaturase (L21SP2_2163) that transform the precursor geranygeranyl diphosphate in lycopene, which is further converted to β-carotene by a lycopene cyclase (L21SP2_0393). Zeaxanthin could then be synthesized by a putative beta-carotene hydroxylase (L21SP2_3046).
The type of the genus Spirochaeta is represented by a description of a distinct morphotype occurring in sulfidic freshwater or marine mud that was designated S. plicatilis. Characteristics of Gram-negative staining cells identified as S. plicatilis are a helical shape, a size of 50–250 μm in length and 0.5–0.75 μm in width and a bundle of periplasmic flagella. Long filaments may consist of multicellular chains of cells [30, 58]. The conspicuous morphology of S. plicatilis, i.e. Gram-negative staining helical cells with periplasmic flagella, which was used for the definition of the genus Spirochaeta later turned out to be a characteristic of the whole phylum Spirochaetes[30] and thus is of limited value for the definition of a genus. No cultured type strain or 16S rRNA gene sequence of S. plicatilis is available as reference for comparative studies with other strains of this genus, thereby preventing an exact placement of this species within the phylum Spirochaetes. However, the morphological description of S. plicatilis does not fit to strain L21-RPul-D2T or any other cultured species of the genus Spirochaeta, which do not form filaments of 50–250 μm in length, have a smaller cell width and only two periplasmic flagella [30]. Within the phylum Spirochaetes distinctive morphological traits seem to be conserved at genus or family level, so that for example strains that exclusively grow in the form of non motile spherical cells are found only within the genus Sphaerochaeta or cells with hooked ends are typical for Leptospira. We conclude, therefore, that all Spirochaeta species currently available in pure culture are most likely misclassified due to significant morphological differences to the type of this genus. Therefore, it is proposed to rename species of the genus Spirochaeta according to recent taxonomic concepts based on molecular methods. As outlined above, the large phenotypic and phylogenetic divergence to any cultured described strain would also suggest placing strain L21-Rpul-D2T in a novel species and genus within the family Spirochaetaceae. The novel genus can be clearly distinguished from the most closely related species S. africana, S. asiatica and S. dissipatitropha, which are alkaliphilic. According to our taxonomic concept S. asiatica and S. dissipatitropha likely represent a separate genus that could be in turn distinguished from S. africana and the novel strain L21-Rpul-D2T by a different salinity optimum. This classification scheme is also corroborated by the obtained cellular fatty acid data shown in the Additional file 2: Strain L21-Rpul-D2T can be distinguished from all closely related type strains by the presence of the fatty acid ante-C15:0 and absence of C18:1c11, S. africana is characterized by the presence of C16:1c9 DMA and significant amounts of C14:0 DMA, whereas the unique feature of both species S. asiatica and S. dissipatitropha is the absence of the fatty acid iso-C15:0. The reclassification of S. africana, S. asiatica and S. dissipatitropha is however beyond the scope of this study and would require genome-sequencing of additional type strains. Formal descriptions of the proposed novel taxa follow below:
Description of Salinispiragen. nov
Salinispira (Sa.li.ni.spi'ra. L. n. salinum, salt-cellar; L. fem. n. spira, coil, spire; N.L. fem. n. salinispira, a saline spiral)
Free-living, Gram-negative, slender and helical-shaped cells without hooks at ends and a width below 0.3 μm. Multicellular filaments are not observed. Rotation of cells and undulatory motility is conferred by two periplasmic flagella overlapping in the middle of the cell. No spores formed. Coccoid bodies resembling spheroplasts are formed in stationary phase cultures. Biomass has a slightly yellow-orange color due to carotenoid-like pigments. The diagnostic diamino acid of the peptidoglycan is L-ornithine. Major cellular fatty acids are C14:0, C16:0 and iso-C15:0. The polar lipid composition is dominated by phosphatidylglycerol, phospholipids, aminolipids, and glycolipids. No cytochromes or respiratory lipoquinones present. Tests for oxidase and catalase are negative. Aerotolerant, neutrophilic, mesophilic and moderately halophilic. Strictly fermentative metabolism. Amino acids or peptides are not utilized; thiosulfate or nitrate are not reduced. Yeast extract required for growth, but not vitamins. Resistance against rifampicin and kanamycin A. The type species belongs to the family Spirochaetacea within the order Spirochaetales.
Description of Salinispira pacificasp. nov.
Salinispira pacifica (pa.ci'fi.ca. L. fem. adj. pacifica, peaceful, referring to the Pacific Ocean, the origin of the type strain).
The main characteristics are as given for the genus. In addition, optimal conditions for growth are 35°C, pH 7.0 and a salinity of 50 g l-1 NaCl; salinities of up to 15% NaCl are tolerated. The following compounds are used for growth: N-acetylglucosamine, D-fructose, fumarate, D-glucose, D-maltose, D-mannose, pullulan, pyruvate, starch, and D-trehalose. No positive growth response is obtained with acetate, arabinose, butyrate, casamino acids, cellobiose, chitin, citrate, ethanol, formate, D-galactose, glycerol, lactate, lactose, malate, D-mannitol, melibiose, methanol, propionate, D-ribose, succinate, sucrose, D-xylose, and H2/CO2 (8:2 v/v). The end-products resulting from D-glucose fermentation are acetate, lactate, ethanol, CO2 and H2. Sulfate, sulfite, elemental sulfur, nitrate and nitrite were not used as terminal electron acceptors. In addition to the major fatty acids listed above, significant amounts of C18:0, ante-C15:0, C18:1, C16:1, iso-C13:0, C16:0 2OH and iso-C14:0 are present. Susceptible to ampicillin (1000 mg l-1), carbenicillin (1000 mg l-1), penicillin G (1000 mg l-1), D-cycloserin (1000 mg l-1), chloramphenicol (20 mg l-1), gentamicin (1000 mg l-1), and tetracycline (500 mg l-1). The DNA G + C content of the type strain is 51.9 mol%.
The type strain is L21-RPul-D2T (=DSM 27196T =JCM 18663T), isolated from the suboxic zone of a hypersaline microbial mat at the shore of Lake 21, Kiritimati, Republic Kiribati.
The authors gratefully acknowledge the help of Sabine Wellnitz and Regine Fähnrich, DSMZ, for growing cells of strains DSM 8901T, DSM 8902T, and DSM 23605T. Evelyne Brambilla, DSMZ, is acknowledged for DNA extraction and quality control of DNA. We thank Simone Severitt and Nicole Mrotzek for excellent technical assistance regarding SMRTbell™ template preparation and sequencing. We are grateful to the Genome Analytics group (HZI Braunschweig) for providing Illumina sequence data of DSM 27196T. The ID service of the DSMZ is acknowledged for peptidoglycan, cellular fatty acid and polar lipids analyses. This study was funded by the German Research Foundation, project Kl 1000/2-2 of the Research Unit 571 "Geobiology of Organo- and Biofilms".
Authors' Affiliations
- Dupraz C, Visscher PT: Microbial lithification in marine stromatolites and hypersaline mats. Trends Microbiol 2005, 13:429–38. 10.1016/j.tim.2005.07.008View ArticlePubMedGoogle Scholar
- Arp G, Helms G, Karlinska K, Schumann G, Reimer A, Reitner J, Trichet J: Photosynthesis versus exopolymer degradation in the formation of microbialites on the atoll of Kiritimati, Republic of Kiribati, Central Pacific. Geomicrobiol J 2012, 29:29–65. 10.1080/01490451.2010.521436View ArticleGoogle Scholar
- Arp G, Reimer A, Reitner J: Photosynthesis-induced biofilm calcification and calcium concentrations in Phanerozoic oceans. Science 2001, 292:1701–4. 10.1126/science.1057204View ArticlePubMedGoogle Scholar
- Isenbarger T a, Finney M, Ríos-Velázquez C, Handelsman J, Ruvkun G: Miniprimer PCR, a new lens for viewing the microbial world. Appl Environ Microbiol 2008, 74:840–9. 10.1128/AEM.01933-07View ArticlePubMed CentralPubMedGoogle Scholar
- Harris JK, Caporaso JG, Walker JJ, Spear JR, Gold NJ, Robertson CE, Hugenholtz P, Goodrich J, McDonald D, Knights D, Marshall P, Tufo H, Knight R, Pace NR, Kirk Harris J, Gregory Caporaso J: Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat. ISME J 2013, 7:50–60. 10.1038/ismej.2012.79View ArticlePubMedGoogle Scholar
- Schneider D, Arp G, Reimer A, Reitner J, Daniel R: Phylogenetic analysis of a microbialite-forming microbial mat from a hypersaline lake of the Kiritimati Atoll, Central Pacific. PLoS One 2013, 8:e66662. 10.1371/journal.pone.0066662View ArticlePubMed CentralPubMedGoogle Scholar
- Spring S, Brinkmann N, Murrja M, Spröer C, Reitner J, Klenk HP: High diversity of culturable prokaryotes in a lithifying hypersaline microbial mat. Geomicrobiol J 2015, 32:in press.View ArticleGoogle Scholar
- Ludwig W, Strunk O, Klugbauer S, Klugbauer N, Weizenegger M, Neumaier J, Bachleitner M, Schleifer KH: Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 1998, 19:554–68. 10.1002/elps.1150190416View ArticlePubMedGoogle Scholar
- Schloss PD, Handelsman J: Status of the microbial census. Microbiol Mol Biol Rev 2004, 68:686–91. 10.1128/MMBR.68.4.686-691.2004View ArticlePubMed CentralPubMedGoogle Scholar
- Hungate R: The anaerobic mesophilic cellulolytic bacteria. Bacteriol Rev 1950, 14:1–49.PubMed CentralPubMedGoogle Scholar
- Bryant M: Commentary on the Hungate technique for culture of anaerobic bacteria. Am J Clin Nutr 1972, 25:1324–8.PubMedGoogle Scholar
- Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS: Methanogens: reevaluation of a unique biological group. Microbiol Rev 1979, 43:260–96.PubMed CentralPubMedGoogle Scholar
- DSMZ catalogue microorganisms [http://www.dsmz.de/catalogues/catalogue-microorganisms.html]
- Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar , Buchner A, Lai T, Steppi S, Jobb G, Förster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, König A, Liss T, Lüssmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, et al.: ARB: a software environment for sequence data. Nucleic Acids Res 2004, 32:1363–71. 10.1093/nar/gkh293View ArticlePubMed CentralPubMedGoogle Scholar
- Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, Glöckner FO: SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 2007, 35:7188–96. 10.1093/nar/gkm864View ArticlePubMed CentralPubMedGoogle Scholar
- List of prokaryotic names validly published (updated June 2014) [http://www.dsmz.de/bacterial-diversity/prokaryotic-nomenclature-up-to-date/prokariotic-nomenclature-up-to-date.html]
- Miyazaki M, Sakai S, Yamanaka Y, Saito Y, Takai K, Imachi H: Spirochaeta psychrophila sp. nov., a psychrophilic spirochaete isolated from subseafloor sediment offshore Shimokita, Japan, and emended description of the genus Spirochaeta . Int J Syst Evol Microbiol 2014., in press: Google Scholar
- Rosselló-Mora R, Amann R: The species concept for prokaryotes. FEMS Microbiol Rev 2001, 25:39–67. 10.1111/j.1574-6976.2001.tb00571.xView ArticlePubMedGoogle Scholar
- Tindall BJ, Rosselló-Móra R, Busse H-J, Ludwig W, Kämpfer P: Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 2010, 60:249–66. 10.1099/ijs.0.016949-0View ArticlePubMedGoogle Scholar
- Pagani I, Liolios K, Jansson J, Chen I-M a, Smirnova T, Nosrat B, Markowitz VM, Kyrpides NC: The Genomes OnLine Database (GOLD) v. 4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2012,40(Database issue):D571–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Liolos K, Abt B, Scheuner C, Teshima H, Held B, Lapidus A, Nolan M, Lucas S, Deshpande S, Cheng J-F, Tapia R, Goodwin LA, Pitluck S, Pagani I, Ivanova N, Mavromatis K, Mikhailova N, Huntemann M, Pati A, Chen A, Palaniappan K, Land M, Rohde M, Tindall BJ, Detter JC, Göker M, Bristow J, Eisen JA, Markowitz V, Hugenholtz P, et al.: Complete genome sequence of the halophilic bacterium Spirochaeta africana type strain (Z-7692 T ) from the alkaline Lake Magadi in the East African Rift. Stand Genomic Sci 2013, 8:165–76. 10.4056/sigs.3607108View ArticlePubMed CentralPubMedGoogle Scholar
- Mavromatis K, Yasawong M, Chertkov O, Lapidus A, Lucas S, Nolan M, Del Rio TG, Tice H, Cheng J-F, Pitluck S, Liolios K, Ivanova N, Tapia R, Han C, Bruce D, Goodwin L, Pati A, Chen A, Palaniappan K, Land M, Hauser L, Chang Y-J, Jeffries CD, Detter JC, Rohde M, Brambilla E, Spring S, Göker M, Sikorski J, Woyke T, et al.: Complete genome sequence of Spirochaeta smaragdinae type strain (SEBR 4228 T ). Stand Genomic Sci 2010, 3:136–44.PubMed CentralPubMedGoogle Scholar
- Abt B, Han C, Scheuner C, Lu M, Lapidus A, Nolan M, Lucas S, Hammon N, Deshpande S, Cheng J-F, Tapia R, Goodwin LA, Pitluck S, Liolios K, Pagani I, Ivanova N, Mavromatis K, Mikhailova N, Huntemann M, Pati A, Chen A, Palaniappan K, Land M, Hauser L, Brambilla E-M, Rohde M, Spring S, Gronow S, Göker M, Woyke T, et al.: Complete genome sequence of the termite hindgut bacterium Spirochaeta coccoides type strain (SPN1 T ), reclassification in the genus Sphaerochaeta as Sphaerochaeta coccoides comb. nov. and emendations of the family Spirochaetaceae and the genus Sphaerochaeta . Stand Genomic Sci 2012, 6:194–209. 10.4056/sigs.2796069View ArticlePubMed CentralPubMedGoogle Scholar
- Spring S, Lünsdorf H, Fuchs BM, Tindall BJ: The photosynthetic apparatus and its regulation in the aerobic gammaproteobacterium Congregibacter litoralis gen. nov., sp. nov. PLoS ONE 2009, 4:e4866. 10.1371/journal.pone.0004866View ArticlePubMed CentralPubMedGoogle Scholar
- Cord-Ruwisch R: A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria. J Microbiol Methods 1985, 4:33–6. 10.1016/0167-7012(85)90005-3View ArticleGoogle Scholar
- Fardeau ML, Ollivier B, Patel BK, Magot M, Thomas P, Rimbault A, Rocchiccioli F, Garcia JL: Thermotoga hypogea sp. nov., a xylanolytic, thermophilic bacterium from an oil-producing well. Int J Syst Bacteriol 1997, 47:1013–9. 10.1099/00207713-47-4-1013View ArticlePubMedGoogle Scholar
- Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, Ashburner M, Axelrod N, Baldauf S, Ballard S, Boore J, Cochrane G, Cole J, Dawyndt P, De Vos P, DePamphilis C, Edwards R, Faruque N, Feldman R, Gilbert J, Gilna P, Glöckner FO, Goldstein P, Guralnick R, Haft D, Hancock D, et al.: The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008, 26:541–7. 10.1038/nbt1360View ArticlePubMed CentralPubMedGoogle Scholar
- Field D, Amaral-Zettler L, Cochrane G, Cole JR, Dawyndt P, Garrity GM, Gilbert J, Glöckner FO, Hirschman L, Karsch-Mizrachi I, Klenk H-P, Knight R, Kottmann R, Kyrpides N, Meyer F, San Gil I, Sansone S-A, Schriml LM, Sterk P, Tatusova T, Ussery DW, White O, Wooley J: The genomic standards consortium. PLoS Biol 2011, 9:e1001088. 10.1371/journal.pbio.1001088View ArticlePubMed CentralPubMedGoogle Scholar
- Woese CR, Kandler O, Wheelis ML: Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A 1990, 87:4576–9. 10.1073/pnas.87.12.4576View ArticlePubMed CentralPubMedGoogle Scholar
- Paster B: Phylum XV. Spirochaetes Garrity and Holt 2001. In Bergey's Manual® Syst Bacteriol. Edited by: Brenner DJ, Krieg NR, Garrity GM, Staley JT. New York: Springer; 2010:471–566.Google Scholar
- Gupta RS, Mahmood S, Adeolu M: A phylogenomic and molecular signature based approach for characterization of the phylum Spirochaetes and its major clades: proposal for a taxonomic revision of the phylum. Front Microbiol 2013,4(July):217.PubMed CentralPubMedGoogle Scholar
- Markowitz VM, Mavromatis K, Ivanova NN, Chen I-M a, Chu K, Kyrpides NC: IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009, 25:2271–8. 10.1093/bioinformatics/btp393View ArticlePubMedGoogle Scholar
- Qin Q-L, Xie B-B, Zhang X-Y, Chen X-L, Zhou B-C, Zhou J, Oren A, Zhang Y-Z: A proposed genus boundary for the prokaryotes based on genomic insights. J Bacteriol 2014, 196:2210–5. 10.1128/JB.01688-14View ArticlePubMed CentralPubMedGoogle Scholar
- Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000, 25:25–9. 10.1038/75556View ArticlePubMed CentralPubMedGoogle Scholar
- Kaksonen AH, Spring S, Schumann P, Kroppenstedt RM, Puhakka JA: Desulfotomaculum thermosubterraneum sp. nov., a thermophilic sulfate-reducer isolated from an underground mine located in a geothermally active area. Int J Syst Evol Microbiol 2006, 56:2603–8. 10.1099/ijs.0.64439-0View ArticlePubMedGoogle Scholar
- DSMZ services and techniques for the identification of Bacteria and Archaea: analysis of polar lipids [http://www.dsmz.de/services/services-microorganisms/identification/analysis-of-polar-lipids.html]
- Schumann P: Peptidoglycan structure. Methods Microbiol 2011, 38:101–29.Google Scholar
- Schleifer KH, Kandler O: Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 1972, 36:407–77.PubMed CentralPubMedGoogle Scholar
- Gemeinholzer B, Dröge G, Zetzsche H, Haszprunar G, Klenk HP, Güntsch A, Berendsohn WG, Wägele J-W: The DNA bank network: the start from a german initiative. Biopreserv Biobank 2011, 9:51–5. 10.1089/bio.2010.0029View ArticlePubMedGoogle Scholar
- Thorvaldsdóttir H, Robinson JT, Mesirov JP: Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 2013, 14:178–92. 10.1093/bib/bbs017View ArticlePubMed CentralPubMedGoogle Scholar
- Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009, 25:1754–60. 10.1093/bioinformatics/btp324View ArticlePubMed CentralPubMedGoogle Scholar
- Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards R a, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R: The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res 2014,42(Database issue):D206–14.View ArticlePubMed CentralPubMedGoogle Scholar
- Abt B, Göker M, Scheuner C, Han C, Lu M, Misra M, Lapidus A, Nolan M, Lucas S, Hammon N, Deshpande S, Cheng J-F, Tapia R, Goodwin LA, Pitluck S, Liolios K, Pagani I, Ivanova N, Mavromatis K, Mikhailova N, Huntemann M, Pati A, Chen A, Palaniappan K, Land M, Hauser L, Jeffries CD, Rohde M, Spring S, Gronow S, et al.: Genome sequence of the thermophilic fresh-water bacterium Spirochaeta caldaria type strain (H1 T ), reclassification of Spirochaeta caldaria , Spirochaeta stenostrepta , and Spirochaeta zuelzerae in the genus Treponema as Treponema caldaria comb. nov., Treponema stenostrepta comb. nov., and Treponema zuelzerae comb. nov., and emendation of the genus Treponema . Stand Genomic Sci 2013, 8:88–105. 10.4056/sigs.3096473View ArticlePubMed CentralPubMedGoogle Scholar
- Calusinska M, Happe T, Joris B, Wilmotte A: The surprising diversity of clostridial hydrogenases: a comparative genomic perspective. Microbiology 2010, 156:1575–88. 10.1099/mic.0.032771-0View ArticlePubMedGoogle Scholar
- Sapra R, Bagramyan K, Adams MWW: A simple energy-conserving system: proton reduction coupled to proton translocation. Proc Natl Acad Sci U S A 2003, 100:7545–50. 10.1073/pnas.1331436100View ArticlePubMed CentralPubMedGoogle Scholar
- Spring S, Rachel R, Lapidus A, Davenport K, Tice H, Copeland A, Cheng J-F, Lucas S, Chen F, Nolan M, Bruce D, Goodwin L, Pitluck S, Ivanova N, Mavromatis K, Ovchinnikova G, Pati A, Chen A, Palaniappan K, Land M, Hauser L, Chang Y-J, Jeffries CC, Brettin T, Detter JC, Tapia R, Han C, Heimerl T, Weikl F, Brambilla E, et al.: Complete genome sequence of Thermosphaera aggregans type strain (M11TL T ). Stand Genomic Sci 2010, 2:245–59. 10.4056/sigs.821804View ArticlePubMed CentralPubMedGoogle Scholar
- Biegel E, Schmidt S, González JM, Müller V: Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes. Cell Mol Life Sci 2011, 68:613–34. 10.1007/s00018-010-0555-8View ArticlePubMedGoogle Scholar
- Heefner DL, Harold FM: ATP-driven sodium pump in Streptococcus faecalis . Proc Natl Acad Sci U S A 1982, 79:2798–802. 10.1073/pnas.79.9.2798View ArticlePubMed CentralPubMedGoogle Scholar
- Toei M, Gerle C, Nakano M, Tani K, Gyobu N, Tamakoshi M, Sone N, Yoshida M, Fujiyoshi Y, Mitsuoka K, Yokoyama K: Dodecamer rotor ring defines H+/ATP ratio for ATP synthesis of prokaryotic V-ATPase from Thermus thermophilus . Proc Natl Acad Sci U S A 2007, 104:20256–61. 10.1073/pnas.0706914105View ArticlePubMed CentralPubMedGoogle Scholar
- Imlay JA: Pathways of oxidative damage. Annu Rev Microbiol 2003, 57:395–418. 10.1146/annurev.micro.57.030502.090938View ArticlePubMedGoogle Scholar
- Cypionka H: Oxygen respiration by Desulfovibrio species. Annu Rev Microbiol 2000, 54:827–48. 10.1146/annurev.micro.54.1.827View ArticlePubMedGoogle Scholar
- Stanton TB, Rosey EL, Kennedy MJ, Jensen NS, Bosworth BT: Isolation, oxygen sensitivity, and virulence of NADH oxidase mutants of the anaerobic spirochete Brachyspira ( Serpulina ) hyodysenteriae , etiologic agent of swine dysentery. Appl Environ Microbiol 1999, 65:5028–34.PubMed CentralPubMedGoogle Scholar
- Riebe O, Fischer R-J, Wampler D a, Kurtz DM, Bahl H: Pathway for H 2 O 2 and O 2 detoxification in Clostridium acetobutylicum . Microbiology 2009, 155:16–24. 10.1099/mic.0.022756-0View ArticlePubMed CentralPubMedGoogle Scholar
- Boylan J a, Hummel CS, Benoit S, Garcia-Lara J, Treglown-Downey J, Crane EJ, Gherardini FC: Borrelia burgdorferi bb0728 encodes a coenzyme A disulphide reductase whose function suggests a role in intracellular redox and the oxidative stress response. Mol Microbiol 2006, 59:475–86. 10.1111/j.1365-2958.2005.04963.xView ArticlePubMedGoogle Scholar
- Seaver LC, Imlay JA: Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli . J Bacteriol 2001, 183:7173–81. 10.1128/JB.183.24.7173-7181.2001View ArticlePubMed CentralPubMedGoogle Scholar
- Canale-Parola E: Physiology and evolution of spirochetes. Bacteriol Rev 1977, 41:181–204.PubMed CentralPubMedGoogle Scholar
- Terao J: Antioxidant activity of beta-carotene-related carotenoids in solution. Lipids 1989, 24:659–61. 10.1007/BF02535085View ArticlePubMedGoogle Scholar
- Blakemore RP, Canale-Parola E: Morphological and ecological characteristics of Spirochaeta plicatilis . Arch Mikrobiol 1973, 89:273–89. 10.1007/BF00408895View ArticlePubMedGoogle Scholar
- Zhilina TN, Zavarzin GA, Rainey F, Kevbrin VV, Kostrikina NA, Lysenko AM: Spirochaeta alkalica sp. nov., Spirochaeta africana sp. nov., and Spirochaeta asiatica sp. nov., alkaliphilic anaerobes from the Continental Soda Lakes in Central Asia and the East African Rift. Int J Syst Bacteriol 1996, 46:305–12. 10.1099/00207713-46-1-305View ArticlePubMedGoogle Scholar
- Pikuta EV, Hoover RB, Bej AK, Marsic D, Whitman WB, Krader P: Spirochaeta dissipatitropha sp. nov., an alkaliphilic, obligately anaerobic bacterium, and emended description of the genus Spirochaeta Ehrenberg 1835. Int J Syst Evol Microbiol 2009, 59:1798–804. 10.1099/ijs.0.016733-0View ArticlePubMedGoogle Scholar
- Greenberg EP, Canale-Parola E: Spirochaeta halophila sp. n., a facultative anaerobe from a high-salinity pond. Arch Microbiol 1976, 110:185–94. 10.1007/BF00690227View ArticlePubMedGoogle Scholar
- Magot M, Fardeau ML, Arnauld O, Lanau C, Ollivier B, Thomas P, Patel BK: Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field. FEMS Microbiol Lett 1997, 155:185–91. 10.1111/j.1574-6968.1997.tb13876.xView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


text:
Yes, today is Happy Birthday for a pair of twins.
On May 19, 2014, both how-to books for empaths are officially being published in their first ebook editions.
To start reading, purchase each ebook as a Nook edition. The links are below.
Unfortunately, Nook is not available for sale in most countries outside the U.S. Other editions — like Amazon Kindle — WILL be for sale internationally but take more fiddling with technically. Soon as more ebook editions are available, I will let you know for sure, Blog-Buddies.
If you can purchase a Nook edition from Barnes & Noble, as an ebook, you're good to go. Especially at the end of todays post, you will find some easy ways to read these Nook editions on your computer, smartphone, or tablet. Easy-peasy.
I'm also working on formatting for the paperback editions, which will have the easiest accessibility yet of any of my books, being Print-on-Demand editions through both Ingram and Amazon. More on all that as soon as it happens. Rest assured, I'm using every available minute on these other editions… every available minute and then some.
Introductions, first, to this new pair of books to help every empath enjoy a much better life.
Empath Empowerment Book #2 is The Empowered Empath
And what's the full title of this easiest, most powerful, first book for every kind of empath?
The Empowered Empath:
Owning, Embracing, and Managing Your Special Gifts
Buy it here! This is the first Empath Empowerment® Book to add to your personal first-aid kit — I mean, set of reading materials.
It is a big advancement over Empowered by Empathy, the first book in English for empaths. (Now out of print.) The Empowered Empath is far more sophisticated, with depth, compared to Become The Most Important Person in the Room.
BECOME is still such a practical, helpful book for developing Empath Empowerment. But wait until you get a load of this new one, deeper and more comprehensive, and by far the best start for new empaths (in my opinion).
The Empowered Empath: includes detailed descriptions of 15 different empath gifts. And so much practical information. Just look at all these techniques:
Techniques Designed for Empath Empowerment(R)
- Hey, You. To Become Conscious of Your Consciousness. Chapter 36
- I Like. Breaks the Power of Identification with Other People. Chapter 37
- Install an Automatic Subconscious Alert. Use Breath and Eyes To Jog You Out Of Prolonged Unskilled Empath Merge. Chapter 38
- Introduction to Vibe-Raising Breaths. Become More Present, Awake, Relaxed. Chapter 39
- Introduction to Grounding Breaths. Release Stress. And Maybe You Will Also Become More Physically Aware. Chapter 40
- A Human-Reality App for Grounding Breaths. Transition Wisely from Spiritual Practices, Preventing Unskilled Empath Merges. Chapter 41
- Get Big. Learn How to Make Divine Connection Effective and Effortless. Chapter 43
- Empaths First Aid. How to Remove Imported STUFF. Chapter 44
- Coming Home. The Single Most Important Way to Improve Life for Any Empath. Chapter 46
The detail page for The Empowered Empath, with ordering information and a full Annotated Table of Contents, is here at the Official Rose Rosetree Website. Yes, you can be one of the first in the world to enjoy this new book. 🙂
Because the info is still so new, here is how to see it display properly at www.rose-rosetree.com — open that up and then REFRESH the page. (It's a browser sorting-things-out process, apparently.)
Empath Empowerment Book #2 is The Master Empath
Or, to use the full name…
The Master Empath:
Turning On Your Empath Gifts At Will In Love, Business And Friendship
(Includes Training In Skilled Empath Merge)
Actually, that last part is an understatement, Blog-Buddies, saying that this book for empaths Includes Training in Skilled Empath Merge. Ha!
Nobody, anywhere, has ever presented so many detailed for purposely, powerfully moving into a depth experience of Otherness.
It is very important that first you read The Empowered Empath. Then have yourself a ball doing the most advanced techniques ever — and loads of them — with so many practical applications.
Buy it here.
The detail page for The Master Empath, with ordering information and a full Annotated Table of Contents, has just gone live at the Official Rose Rosetree Website. Yes, you can be one of the first in the world to enjoy this new book.
Here is a summary of the techniques and energetic workarounds you will find (exclusively) in The Master Empath.
Table of Energetic Workarounds and Techniques in The Master Empath
What is an energetic workaround? It is a significant way to overcome problems.
By comparison, tips and coping methods are more superficial. A fully realized energetic workaround will re-orient your awareness, causing you to use your empath gifts more effectively. An energetic workaround can even change your life.
Admittedly an energetic workaround designed to help empaths is less powerful than a dedicated technique designed to help empaths, bringing the power to transport and transform your consciousness. Below you will find both!
- 20 Daily Minutes of Far-Out Technique Time, Tops. The simplest way to avoid — or heal — spiritual addiction. Chapter 3.
- Re-Insert Yourself into Reality. An energetic workaround that could save your job. Chapter 3.
- Conflict Solving without Empath Merge. Prevent unskilled empath merge triggered by conflict. Chapter 5
- Set An Intention. Position your consciousness in a productive direction. Chapter 7
- I Want To Hold Your Hand. Roses favorite technique for Skilled Empath Merge. Chapter 11.
- Deep Listening. Gain insight by exploring your partners Heart Chakra. Chapter 12
- Emergency Disconnect from Skilled Empath Merge. Energy hygiene on demand. Chapter 12.
- Moccasins. Depth experience of someone elses life — of course, you get to choose which someone you will be researching. Chapter 13.
- Taste Someone Elses Reality. Amazing insights await you about primal trust. Chapter 14.
- Find Your Truth Sight Research Tool. Youll never need glasses for 20-20 wisdom, thanks to this technique. Chapter 15.
- Shallow Up Quickie. Such an easy energetic workaround for positioning consciousness back at regular, human frequencies! Chapter 15.
- Self-Discovery with Truth Sight. You dont need a magic mirror from Hogwarts to have fun with this technique. Chapter 16.
- Another Super-Easy Way to Turn Empath Gifts OFF. Heres a fabulously effective energy workaround you can use just about anywhere in the world. Chapter 16.
- Truth Sight For Advanced Empath Merge. This visual technique provides quick entry into the experience of being your chosen Discovery Person. Chapter 17.
- Incognito Truth Sight. Leap into skilled empath merge with just about anyone in the room with you. Chapter 18.
- Truth Sight for TV. An amazing form of Remote Empath Merge that piggybacks on your prior experience at watching television. Chapter 20.
- Locate A Handy Tool For Remote Empath Merge. Learn one more technical skill to help you become a Master Empath. Chapter 21.
- Truth-Touching Empath Merge with TV. And you thought that, previously, you saw something on TV that touched you? Ha, surfacey by comparison! Chapter 22.
- Remote Empath Merge through a Photograph. Even a muggle can make photos move in this way, provided that you're the kind of non-wizard who is a skilled empath. Chapter 23.
- Simple Use Of Your Consciousness Elevator. Ancient yogis would have loved to learn a technique like this one. Chapter 24.
- Even More Amazing Access to your Consciousness Elevator. Now thats really big freedom to fly in spirit! Chapter 25.
- Locate and Ask. Discover how a process-oriented thought is enough to rock an empaths world. Chapter 27.
- Consumer Research Through Empath Merge. Do a right-sized job with energetic literacy, rather than some crazy-making perfectionism. Chapter 31.
- Darshan Merges With Professional Athletes. If you enjoy watching sports, youre going to love this technique. Chapter 32.
- Professional-Level Skilled Empath Merge. How you can, literally, do empath merges like a pro. Chapter 33.
- When a Master Empath Turns Movie Critic. The best actors in the world change in ways that only a skilled empath can fully savor. Chapter 34.
Why Start with Just an Ebook Format?
Blog-Buddies, after so many months of writing and editing, golly! I wanted to get these books published already. So many of you are hungry for this leading-edge information. Ebooks do have the advantage of speedy "printing."
The Nook edition came first because Barnes & Noble is the only truly publisher-friendly ebook provider Ive found. The Empowered Empath did go live today and soon The Master Empath will go live as well. I'll update this blog post just as soon as I find out.
The other ebook platforms will follow as soon as I can get them going, including Amazons Kindle.
Then I will make paperback editions available, as well. More details to follow. For now, Ill just say that plans are to make these new empath books available way more conveniently for you readers in Europe and Australia.
How You Can Start Reading a Nook Edition of My Books on Your Computer, Smartphone, or Tablet
All Nook editions by Rose Rosetree can be found through this empath's ebook link.
To read a Nook book without having to get a Nook device, you can download a free app to your smartphone, tablet, or PC.
Here's the link to download Nook on your Mac: http://www.barnesandnoble.com/u/nook-for-mac/379003592/
Nook for PC: http://www.barnesandnoble.com/u/nook-for-pc/379003591/
Nook for Windows 8: http://www.barnesandnoble.com/u/nook-for-windows-8/379003757/
On your smartphone or tablet, just look up "Nook" in the app store.
Some Nook books are also available to read right in your browser, without having to download an app. That's even easier — you just buy the book through the Barnes & Noble website and start reading. I guess we'll see if that's an option today!!!

subset: commoncrawl


text: Just had the news that the six willow arches we installed on the Long Walk outside the gates of Windsor Castle will remain onsite so that the Queen...Elizabeth 2, can see them when she gets home from Balmoral. Apparently Windsor is her "home" she says and Buck Palace is "the Office". So they will be there until 14th October. Hopefully all the added publicity will help to promote the RWRHS Legacy Fund which is to help educate local Windsor students of horticulture. The arches will now go up for auction from the 14th October along with the plants and lovely willow crowns created by Spencer Jenkins. .


text:
David Gandy was an easy choice for this week's Flirty Friday!
If you've never heard of him, he is an absolutely gorgeous and successful model, who has posed in Vogue, GQ, and done campaigns for both Lucky and Dolce & Gabbana. Not only is he extremely attractive, but he's also quite articulate if you watch any of his interviews (you'll also be able to listen to his dreamy British accent).
He's quite simply perfection!
He looks amazing both with and without clothes on, don't you agree?
And because I felt like we needed just one more…
Your welcome!
You can find David Gandy on:
Twitter , FaceBook and Instagram
*Pink Lace & Silver Buckles does not make any claims to these photographs. They were pulled from Google Images.


text:
At CrossFit Five Cities, we offer several different class options as well as limited "Open Gym" time throughout the week.
As a friendly reminder to our community, an athlete who "chooses" to attend Open Gym, should meet the following requirements;
- A CLEAR plan in place on what exactly it is you will be doing for the hour.
- A CLEAR understanding of all the movements/skills you will be executing for the day.
- If you will be executing the C5 class WOD, please see #1 & #2 above.
- A CLEAR concept of how you should be warming yourself up; Aerobically, dynamically, specific movement prep, setup, layout, proper equipment.......etc. As most of you know, proper warmup and preparation is something we take VERY serious here at C5. I'd be willing to argue that if you haven't been following our LIFE program 3x per week for a minimum of 6mo, you most likely have no business coming to an Open Gym time to execute the class workout. Coming to a scheduled group class so you can get Coached by a C5 Fitness Coach would benefit you MUCH more. If you have to ask a Coach what a power clean or thruster is, you need to be attending group classes so you can get educated and Coached in a safe fashion.
- The ability to be self sufficient with your own timing device (clock, wrist watch, smart phone app....etc) to utilize during your session.
- If you are coming to Open Gym to execute skill work or I.D., you MUST meet with the Head Coach onsite to let us know what you will be executing so we can help you organize your session in an efficient manner based upon how many other athletes we have in the room during that time and what exactly everyone else will be doing. This will help everyone involved with the efficiency of the gym layout and space.
In conclusion, your safety, progress, and #FitnessGainz are our main priority for you! Help us to help you by ensuring you attend the services at C5 that best suit your current needs and Fitness levels.
If you need to ask the question; "should I be attending Open Gym?", please talk to a C5 Coach so we can help you best determine what options are best for you.
Make it a great Friday friends!!
A1. 3x60'/db death march (moderate)
*rest 30sec
A2. 3x12 alt. renegade rows
*rest 90sec
B. Every 90sec x 8:
-6 jumping pullups
-5 db hang cleans
-4 ub db thrusters
*focus on good positions and controlled breathing
C. 10-15min cooldown
(row/AB/light jog/walk)
A1. 3x60'/db death march (moderate)
*rest 30sec
A2. 3x12 alt. renegade rows
*rest 90sec
B. Every 90sec x 8:
-6 pullups
-5 hang power cleans (95/68)
-4 ub thrusters
*focus on smooth barbell cycling and controlled breathing
C. 10-15min cooldown
(row/AB/light jog/walk)


text:
Lynne McTaggart, one of the preeminent spokespersons on consciousness, the new physics and the science of spirituality, is the award-winning journalist and author of five books, including the worldwide bestsellers The Field and The Intention Experiment. As editorial director of What Doctors Don't Tell You (www.wddty.com), she also publishes one of the world's most highly praised health newsletters and runs highly popular health and spirituality teleconferences and workshops.
Lynne is also the architect of the Intention Experiments, a web-based 'global laboratory', involving an international consortium of prestigious scientists and thousands of people in countries around the world testing the power of intention to heal the world. Most recently her book The Intention Experiment and its website were prominently featured in the plotline of Dan Brown's latest blockbuster The Lost Symbol. Of Lynne's latest book The Bond: Connecting Through the Space Between Us, Deepak Chopra says, "Once in a very great while, a book comes along that shifts our sense of reality, or extols a new way of living. Lynne McTaggart's extraordinary new book, 'The Bond,' does both."
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text:
Brady's record day
Hang on for a minute...we're trying to find some more stories you might like.
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Marshall wide receiver Tyre Brady put himself in rare air during the Thundering Herd football team's 37-20 loss at NC State in Raleigh, North Carolina Saturday night.
The redshirt junior shattered a Carter-Finley Stadium record for a visiting player and even broke the Wolfpack record after he hauled in 11 receptions and 248 yards.
"I think (he's) pretty damn good," Herd head coach Doc Holliday said. "He's a hell of a player, great kid. He didn't practice much during the week and he went out there and played extremely well. We need him to do that, that's the kind of player he is."
Brady hauled in 11 catches on 16 targets from Litton in the game.
The game plan for the Herd was to get him the ball more in week two, after he caught just three passes for 54 yards and a score against Miami (Ohio).
"We made a concerted effort to get him the ball today," Holliday said during his postgame press conference. "I thought Chase did a good job throwing the ball, and he did a good job of making plays."
The concerted effort by the coaching staff to get the ball to Brady Saturday was prevalent from the start of the game.
On the Herd's second drive of the game, Litton found Brady four times in five tries and three of those completions were for 15 or more yards.
"They count on me to make plays and I just want to be there for them in any way," Brady said. "Every time the ball is thrown my way I feel like I'm supposed to make that play, so I go all out in making the play."
The Miami (Fla.) transfer finished the first half with six catches for 188 yards and a score, but the Wolfpack defense still could not stop him.
In attempt to stop Brady's ability, NC State tried double and triple-teaming him on the outside.
But that was unfruitful as well, and Brady relished in the opportunity.
"Going in, moments like that, that's what a player wants," Brady said. "The great ones want to be in positions like that."
Brady said he had no clue he broke the stadium record for yards until after the game. He and the Herd were trying to win a game still.
While he did not know his stats during the game, Herd fans realized Brady was on his way toward a Marshall program record in addition to the stadium record.
Brady finished just 40 yards shy of tying former Heisman Trophy finalist Randy Moss' school record of 288 yards in Marshall's 1996 Division I-AA playoff first round 59-14 win against Delaware.
"That's a great blessing for me to be up there by him, with the greats," Brady said. "I didn't even know when I was out there (on the field), I was just playing. I watched Moss when I was growing up. I always wanted to make a Moss catch going over the defender."
One thing that was evident when Litton threw Brady's direction was that they had the confidence in each other that if the ball was thrown in the vicinity, a play could be made.
That confidence in each other was formed during Brady's transfer year and in the offseason.
"We did a lot of stuff. We went to work (in the offseason)," Brady said. "Even when I was sitting out last year, sometimes I would pull Chase aside and we'd throw to each other. I always went in there and watched film with him. When he was in there breaking down films against other teams, I'd go in and watch with him so I could see what he sees out there so we could be on one accord."
Brady said seeing the hard work he and Litton put in with each other payoff is the greatest feeling.
"It makes you want to go even harder," Brady said. "Seeing all your hard work pay off puts you on another level."
The previous NC State receiving yards record at Carter-Finley Stadium was set by former wide out Jerricho Cotchery with 217 yards against the North Carolina Tar Heels in 2003.
The visitor's stadium record was set by Wake Forest's James Brim who had 194 receiving yards against the Wolfpack defense in 1986.
Marshall now must ready itself for the Kent State Golden Flashes in week three. Kickoff is scheduled for 6:30 p.m. ET at Joan C. Edwards Stadium with Hall of Fame Weekend festivities planned before kickoff.
Adam Rogers can be contacted at [email protected]


text:
By Mr. Gregory Ripps (INSCOM)March 26, 2013
Kristi and Chief Warrant Officer 3 Scott Hinson, of the 201st Military Intelligence Battalion, 470th MI Brigade, were spotlighted on NBC's "The Today Show," March 26. The couple was featured on the popular morning show to share the unique way Scott Hinson observed their 20th wedding anniversary as he returned to San Antonio from Afghanistan for a brief rest and relaxation trip.
With his daughters in on the surprise, the United Services Organizations of Georgia helped Hinson acquire roses during his layover at Hartsfield-Jackson International Airport in Atlanta. On his connecting flight to San Antonio, Hinson asked 19 fellow airline passengers to hand a red rose to Kristi, who was standing next to the "Welcome Home" sign in the luggage area. While one daughter held the sign their second daughter recorded the rose handoff and their parents' ultimate embrace with the 20th rose for YouTube.


text:
E-mail: email@example.com
Quanzhou TESUNHO Electronics Co.,Ltd
Tel:86 595 2208 2266
Fax:86 595 2208 2266
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1. SOS & APRS(Emergency button)
2. GPS Function
3. Display the users/calling/last caller information
4. Can receive dispatcher message
5. Support 4G/3G/2G Mobile network
6. Support Group Call, P2P Call, Quick Call, Emergency Call
7. Large Capacity (3.63V 5200mAh)
8. Quick charger – 3 hours fully charged
Unique for: Security Guard, Construction site, Industry Production site, Commercial use like: Hotel, Hospital, University, Commercial Event; Sports
Popular for: Airport, Railway, Army, Government, Rescue, Police, Outdoor adventure, Logistics, Taxi, Truck, Camping, Travelling, Big shopping mall, Big Hotel, Outdoor Events, etc.
Main Function: SOS& APRS ,Last call
SOS Function
Long press orange key to turn on/off the emergency call. The transceiver will send the alarm signal and the user name to the dispatcher & the other members in your group.
|Quick Details
|Weight (Body)
|38 ( H ) *58( W ) *135 ( L ) mm
|LCD display
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|Battery capacity
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|Earphone jack
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|GSM900/1800MHz UMTS900/2100MHz LTE OR GSM850/1900MHz UMTS850/1900MHz LTE
|Sim card type
|Normal sim card
|Data consumption
|2.88 Mega Byte per hour
|Audio output power
|Equal or more than 0.5w
|Waterproof rate
|Group call
|Private call
|Friends call
|Emergency Call
|Yes (Panic button)
|Quick Call
|Update Software
More parameters, please contact with us!
Packaging & Shipping
Standard Package for one piece:
Standard Packing :
Payment Method:
Trade Assurance / Bank transfer / Western Union / MoneyGram / Paypal
Our Services
Tesunho is aim to be the leading industrial walkie talkie solution supplier. So our service consists of 3 important parts:
1. Basic Warranty:
√ Replacement: Free new walkie talkie if it can not work at once it arrives.
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365 days
Battery and Charger
90 days
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√ Paid maintenance and parts: for artificial faulty
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√ complete OEM service
3. Technical ODM service: we have abundant of experience on industrial needs customization
√ Rail way station communication device
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Why Us:
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A: We are an R&D based creative and productive manufacturer specialized in CE FCC approved walkie talkie.
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A:1 piece for normal stock; different quantities for OEM or ODM, which is determined by different task workload. Kindly contact us for details.
3.What is your payment terms?
A:30% deposit in advance and 70% balance before shipment.
4.Can you send free samples?
A: You can get free sample by means of returning sample fund after regular order.
Quanzhou TESUNHO Electronics Co.,Ltd
Tel:86 595 2208 2266
Fax:86 595 2208 2266
Mob:86 18959807366


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text:
Two Buffalo won the bid to supply the new roof project on the Salvation Army Harbor Lights building in Minneapolis.
The roof will be installed in the next few weeks, weather permitting.
The Minneapolis Harbor Light Center provides emergency assistance, shelter, housing, chemical dependency treatment, transitional housing, health care services and spiritual support to its clients.
Two Buffalo is proud to be a part of this important project because it will benefit the community.


text:
Mind Map - это интелектуальные карты, которые позволяют ...
Process in your business -- a system that has already produced enormous profit opportunities for businesses of all sizes and varieties nationwide. The book takes you through the process step-by-step. You'll learn how to develop a realistic profit plan for your business and to get it working * provide a forum for exciting new profit ideas from your key managers and employees all down the line * put the best of these ideas into a profit plan the entire company is committed to * put that "game plan" to work generating some immediate, some longer-term bottom line results * and, best of all, how to keep the profits rolling in -- in good times and bad.


text: I've been into this clay stuff. It's a little trickier to work with than I initially thought, but then again I assume that I should be good at everything immediately. This is far from perfect, but it was fun and I might be able to sell something like this and make a million dollars. Score!


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ROBERT Carlyle attended the international premiere of his new film in Edinburgh yesterday.
The actor, accompanied by wife Anastasia Shirley, walked the red carpet at the city's Cineworld cinema to promote his latest movie California Solo. He had previously been expected to miss the Edinburgh International Film Festival screening after cancelling a live interview last week.


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New art gallery opens up in Kampala
01:33 by Kaggwa Andrew
A new gallery has opened in Kampala prominently featuring mostly art collections by new or little known artists.
Located along Kira road in Kamwokya, the Acacia Art Gallery is looking at providing space to artists that may not get a chance to showcase their work in renowned art galleries since they may not have built a following yet.
Ronnie Tindi, an artist and one of the brains behind Acacia Gallery notes that they've created a space where a person doesn't have to be an affiliate of theirs to exhibit and as you could imagine, he notes that they arew also open to genres ranging from abstract, realism and sculpture among others.
Although Kamwokya is known for different galleries such as Umoja or Afriart, there is something very new about Acacia Gallery, for instance, where in other places it's the art and a space, work here was curated in an inclusive way.
They allow a day to day person to interact with the different paintings hanging on the wall, especially with a fact that the space doubles as a restaurant for Ethiopian food, Tindi says with the traffic that follows into the place for food, they hope they start tickling Ugandan minds about art.
The inaugural showcase featured work by Tindi himself, Brian Kavuma, Paul Kintu and many artists from Angavu art studio.
Tindi notes that he's always loved art and thus the vision behind the gallery is showcasing people's ambitions and work in a great space.


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|Tess with her cousin behind her playing dodge ball, a favorite summer past time for the boys.
I watched them play dodge ball and (in a world that isn't allowing dodge ball any more) 2 things struck me.
The first one was this young lady. I love that Tess isn't hesitant to play dodge ball with the boys. Not in the least. I don't think she even noticed that she was the only girl playing amongst several older and very hard -throwing boys. I love that about her. Her strength. Her bravery and tenacity to just go for it, all wrapped up in her petite 45lb and 45" frame. And yet she's so sensitive and vulnerable on the inside. Then again aren't we all a bit like that.
I smile every time I run across the phrase strong is the new pretty. This wonderfully-made young lady seems to epitomize that. I'm secretly hoping more of what she has inside of her rubs off on me. She is so crazy gorgeous.


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German equipment supplier Singulus Technologies AG narrowed its operating loss while boosting overall revenues in its growing solar division last year.
The company posted an operating loss (before interest and tax) of between 33 and 35 million, down from a 49.1 million loss the previous year, according to its preliminary 2015 results. Sales amounted to some 84 million, up from 66.8 million a year ago. Singulus order intake grew from 60 million to 96 million year over year, while its order backlog rose from 14 million to 26 million.
Write-offs and other restructuring costs had squeezed the companys operating result. While EBIT, sales and order intake in the groups solar division increased year over year, the companys optical disc segment performed weakly in view of falling demand, particularly in the case of its Blu-ray Disc Bluline II production system. Waning sales led Singulus to write down replication lines of the Bluline II type for Blu-ray Discs. The company expects only a few new orders for these production systems in the future as disc manufacturers are focusing on the new Ultra HD Blu-ray Disc. As a result, Singulus expects future investment to be mainly in production equipment for Ultra HD Blu-ray Discs.
In the past few years, Singulus has sharpened its focus on the solar segment, developing new production solutions for the industry, including production equipment for thin-film solar cells, like the vacuum machines Vistaris and Cisaris, as well as for crystalline high efficiency cells, such as the Silex II machines. The company said it received successful orders last year.


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Alan Favell
Alan has been working in the financial services industry since 1987, including advising clients as a financial planner since 1996. He has been involved in all aspects of financial planning; including superannuation, investment and retirement planning, wealth creation and risk insurance.
Alan understands the need to provide a value oriented holistic financial planning service to all his clients to ensure their lifestyle financial goals are met. He particularly specialises in preretirement and retirement strategies.
Outside of his professional career Alan is a family man with 3 children and enjoys most sports.
All recommendations will be based upon an analysis of your needs, objectives and personal circumstances.
Our Advisers


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Latest on Sleeping Beauty (2011)
'Sex Doll' On Netflix: European Prostitute Turns Some Tricks, Skips The Treats
For a moment, I thought, hey, this is going to be a movie about an actual sex doll.
Sex On Netflix: Did You Know That Netflix Has A "Steamy" Section?
Whether you're in the mood for a Steamy Foreign Drama, a Steamy Political Thriller or a Steamy Independent Crime movie, we've got your steamy selections covered.
WCW: 'Legend's' Emily Browning
This week, we're head over heels for Legend star, Emily Browning.


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The CSIR -STEPRI has hosted a two day workshop fom the 13th -14th September, 2017 as part of activities of the Ghana National Science-Policy Dialogue Platform.
Background and Rational
The Ghana Science-Policy Dialogue Platform and CCAFS Programme, West Africa based at ICRISAT, Bamako in Mali have over the past five years consistently and successfully collaborated with MoFA and the CSIR in climate change policy research and related climate smart agriculture bringing invaluable benefits to end users. For example Ghana Science-Policy Dialogue Platform after the establishment of sub-national platforms has undertaken a number of initiatives and interventions to contribute to the ultimate policy influence goal of CCAFS, both at national and sub-national levels via top-down – bottom-up approaches. Key among the initiatives at the sub-national level is scenario visioning leading to district-specific scenarios plans to forecast the future of agriculture in the midst of climate change for planning and investment. These plans are necessary for the purpose of mainstreaming CSA into various districts' agriculture development plans. Another marked achievement by the platform is the collaborative development of the CSA Action Plan which seeks to drive the National Climate change Policy document launched by the former President of Ghana few years ago. All these point to the fact that Ghana as a nation is doing its best to reduce the impact of climate change and climate variability and its effects on agriculture.
While a number of policy documents have been developed such as the National Action Programme to Combat Drought and Desertification 2003, Ghana Livestock Development Policy and Strategy 2016 – 2025, Ghana National Climate Change Master Plan Action Programmes for Implementation, National Climate Change Adaptation Strategy (2010) to mentioned few, none of these policy document has in place a strategic financial plan to assist with its implementation making it difficult for harnessing required investment in these thematic areas. It is against this background that Ghana CCAFS Science Policy Dialogue collaborated with the Ministry of Food and Agriculture to develop National Climate Smart Agriculture and Food Security Action Plan (2016-2020). It has also been realized that there is the need as a country to also have in place, a financial machinery to guide investment decisions and implementation of the CSA programme areas in the action. The propose project therefore seeks to develop a CSA Financial Policy Plan (2018-2022) that will serve as an investment and CSA implementation guide. The targeted beneficiaries of this financial plan are firstly national high policy makers (e.g. Parliament) who may enact a financial law based on the bill and implemented by relevant state institutions (particularly, Ministry of Local Government). This will drive CSA Investment hubs for the smooth implementation of the CSA Action Plan at the subnational levels.
General objective: The broad objective of the proposal is to design a comprehensive CSA Financial Plan for the implementation of the CSA Action Plan.
Specific objectives:
- To develop a CSA Financial Plan guided by the CSA action plan document and sensitize high level policy makers, management and financial decision makers and legislators to support the enactment of financial regulation based on the bill.
Exected Results
CSA Financial Plan Policy guided by the CSA action plan developed.
Financial plan document and various policy recommendations shared with high level policy, management, financial decision makers and legislators.
Visibility of platform enhanced locally and internationally
- Project Progress report
- Draft CSA Financial Policy Plan developed
- Final Copies of published CSA Financial Policy Plan Produced by CCAFS West Africa.


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The press reports that the top executives in the federal intelligence community have launched an inquiry into the events leading up to the Marathon Bombings. As the passage quoted below indicates, it will be undertaken by the Intelligence Community Inspector General. In my opinion this is precisely 180 degrees opposite the approach they should undertake. Here's the passage from yesterday's Boston Globe:
The review, sought by James R. Clapper, the director of national intelligence, will be overseen by the Intelligence Community Inspector General, which investigates waste, fraud, abuse, and misconduct within the nation's 17 intelligence agencies.
We need an after-action reflection in which we make it safe for personnel at every level, from the operational people on the street to the analysts to the executives to examine their experiences and honestly share their view on what worked and what could have been done better. But look at the brief of the IG: "investigates waste, fraud, abuse, and misconduct."
What street agent or analyst, with a mortgage and a reputation to protect, is going to be willing to share what she learned? In cubicles, cars and offices in 17 agencies you can hear the brains going into high gear figuring out how to survive the investigation. Not because they will have done anything "wrong." But because it's in the IG's organizational DNA to find and assign blame, not learning. The questions they ask and the way they ask them will elicit defensive responses. It's been this way too long. Questions in the law enforcement setting are techniques for extraction instead of prompts for self-examination and learning. Instead of catalyzing learning the questions become threats.
It's a badly wasted opportunity. We need these that candlepower illuminating what we learned, so we can be better the next time.
The "investigation" is a comfortable default reaction by political leaders. It's probably the best one can expect from the White House, any White House. Congress has already begun its "investigations."
By the time they issue reports and assign blame the agencies have moved on as they must. Time, tide and terrorism wait for no commission.