Whole-Genome Optical Mapping and Finished Genome Sequence of Sphingobacterium deserti sp. nov., a New Species Isolated from the Western Desert of China

A novel Gram-negative bacterium, designated ZW^T, was isolated from a soil sample of the Western Desert of China, and its phenotypic properties and phylogenetic position were investigated using a polyphasic approach. Growth occurred on TGY medium at 5–42°C with an optimum of 30°C, and at pH 7.0–11.0 with an optimum of pH 9.0. The predominant cellular fatty acids were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c or C_16:1 ω6c/C_16:1 ω7c) (39.22%), iso-C15:0 (27.91%), iso-C_17:0 3OH (15.21%), C_16:0 (4.98%), iso-C_15:0 3OH (3.03%), C16:0 3OH (5.39%) and C_14:0 (1.74%). The major polar lipid of strain ZW^T is phosphatidylethanolamine. The only menaquinone observed was MK-7. The GC content of the DNA of strain ZWT is 44.9 mol%. rDNA phylogeny, genome relatedness and chemotaxonomic characteristics all indicate that strain ZW^T represents a novel species of the genus Sphingobacterium. We propose the name S. deserti sp. nov., with ZW^T (= KCTC 32092T = ACCC 05744T) as the type strain. Whole genome optical mapping and next-generation sequencing was used to derive a finished genome sequence for strain ZW^T, consisting of a circular chromosome of 4,615,818 bp in size. The genome of strain ZW^T features 3,391 protein-encoding and 48 tRNA-encoding genes. Comparison of the predicted proteome of ZW^T with those of other sphingobacteria identified 925 species-unique proteins that may contribute to the adaptation of ZW^T to its native, extremely arid and inhospitable environment. As the first finished genome sequence for any Sphingobacterium, our work will serve as a useful reference for subsequent sequencing and mapping efforts for additional strains and species within this genus.     

Marinobacter salarius sp. nov. and Marinobacter similis sp. nov., Isolated from Sea Water

Two non-pigmented, motile, Gram-negative marine bacteria designated R9SW1^T and A3d10^T were isolated from sea water samples collected from Chazhma Bay, Gulf of Peter the Great, Sea of Japan, Pacific Ocean, Russia and St. Kilda Beach, Port Phillip Bay, the Tasman Sea, Pacific Ocean, respectively. Both organisms were found to grow between 4°C and 40°C, between pH 6 to 9, and are moderately halophilic, tolerating up to 20% (w/v) NaCl. Both strains were found to be able to degrade Tween 40 and 80, but only strain R9SW1^T was found to be able to degrade starch. The major fatty acids were characteristic for the genus Marinobacter including C_16:0, C_16:1 ω7c, C_18:1 ω9c and C_18:1 ω7c. The G+C content of the DNA for strains R9SW1^T and A3d10^T were determined to be 57.1 mol% and 57.6 mol%, respectively. The two new strains share 97.6% of their 16S rRNA gene sequences, with 82.3% similarity in the average nucleotide identity (ANI), 19.8% similarity in the in silico genome-to-genome distance (GGD), 68.1% similarity in the average amino acid identity (AAI) of all conserved protein-coding genes, and 31 of the Karlin''s genomic signature dissimilarity. A phylogenetic analysis showed that R9SW1^T clusters with M. algicola DG893^T sharing 99.40%, and A3d10^T clusters with M. sediminum R65^T sharing 99.53% of 16S rRNA gene sequence similarities. The results of the genomic and polyphasic taxonomic study, including genomic, genetic, phenotypic, chemotaxonomic and phylogenetic analyses based on the 16S rRNA, gyrB and rpoD gene sequence similarities, the analysis of the protein profiles generated using MALDI-TOF mass spectrometry, and DNA-DNA relatedness data, indicated that strains R9SW1^T and A3d10^T represent two novel species of the genus Marinobacter. The names Marinobacter salarius sp. nov., with the type strain R9SW1^T ( =  LMG 27497^T  =  JCM 19399^T  =  CIP 110588^T  =  KMM 7502^T) and Marinobacter similis sp. nov., with the type strain A3d10^T ( =  JCM 19398^T  =  CIP 110589^T  =  KMM 7501^T), are proposed.     

<Emphasis Type="Italic">Corynebacterium defluvii</Emphasis> sp. nov., isolated from Sewage

A Gram-positive, aerobic, non-motile, rod-shapeds, catalase-positive, and oxidase-negative strain, designated Y49^T, was isolated from sewage collected from Jilin Agricultural University, China. It grew at 20–40°C (optimum at 30°C), at pH 6.0–8.0 (optimum at 7.0) and at 0–1.0% sodium chloride (optimum at 0%). The major isoprenoid quinone was menaquinone-8 (MK-8) and the polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, four unidentified lipids, and two unidentified aminolipids. The peptidoglycan was meso-diaminopimelic acid. The cell-wall sugars were galactose, arabinose, and glucose. The fatty acids were C_9:0, C_16:0, C_16:1 ω9c, C_17:1 ω9c, C_18:3 ω6c (6,9,12), C_18:1 ω9c, and C_18:0. The DNA G+C content was 51.4 mol%. Based on the 16S rRNA gene sequence analysis, the nearest phylogenetic neighbors of strain Y49^T were Corynebacterium efficiens DSM 44549^T (97.5%), Corynebacterium callunae DSM 20147^T (97.2%), Corynebacterium deserti GIMN 1.010^T (96.8%), Corynebacterium glutamicum ATCC 13032^T (96.4%), and other species belonging to this genus (92.3–95.4%). The DNA-DNA relatedness value between strain Y49^T and C. efficiens DSM 44549^T, C. callunae DSM 20147^T, C. deserti GIMN1.010^T, and C. glutamicum ATCC 13032^T was 25.5±2.0%, 21.1±1.0%, 16.5±0.5%, and 13.5±0.9%, respectively. Based on the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain Y49^T represents a novel species of the genus Corynebacterium, for which the name Corynebacterium defluvii sp nov. is proposed. The type strain is Y49^T (= KCTC 39731^T =CGMCC 1.15506^T).     

Flavobacterium plurextorum sp. nov. Isolated from Farmed Rainbow Trout (Oncorhynchus mykiss)

Five strains (1126-1H-08^T, 51B-09, 986-08, 1084B-08 and 424-08) were isolated from diseased rainbow trout. Cells were Gram-negative rods, 0.7 µm wide and 3 µm long, non-endospore-forming, catalase and oxidase positive. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25°C. They grew in a temperature range between 15°C to 30°C, but they did not grow at 37°Cor 42°C. Based on 16S rRNA gene sequence analysis, the isolates belonged to the genus Flavobacterium. Strain 1126-1H-08^T exhibited the highest levels of similarity with Flavobacterium oncorhynchi CECT 7678^T and Flavobacterium pectinovorum DSM 6368^T (98.5% and 97.9% sequence similarity, respectively). DNA–DNA hybridization values were 87 to 99% among the five isolates and ranged from 21 to 48% between strain 1126-1H-08^T, selected as a representative isolate, and the type strains of Flavobacterium oncorhynchi CECT 7678^T and other phylogenetic related Flavobacterium species. The DNA G+C content of strain 1126-1H-08^T was 33.2 mol%. The predominant respiratory quinone was MK-6 and the major fatty acids were iso-C_15∶0 and C_15∶0. These data were similar to those reported for Flavobacterium species. Several physiological and biochemical tests differentiated the novel bacterial strains from related Flavobacterium species. Phylogenetic, genetic and phenotypic data indicate that these strains represent a new species of the genus Flavobacterium, for which the name Flavobacterium plurextorum sp. nov. was proposed. The type strain is 1126-1H-08^T ( = CECT 7844^T = CCUG 60112^T).     

Burkholderia denitrificans sp. nov., isolated from the soil of Dokdo Island, Korea

A novel, Gram-negative, bacterial strain KIS30-44^T was identified from wet forest soil collected on the Korean island of Dokdo. Growth of the strain was observed at 15?C30°C, pH 5?C9, 0?C3% NaCl, and 950 mM KNO₃. KIS30-44^T reduced nitrate to nitrogen gas. Analysis of the 16S rRNA gene sequence showed that KIS30-44^T was phylogenetically related to Burkholderia sacchari, Burkholderia mimosarum, and Burkholderia oxyphila (98.1%, 98.0%, and 98.0% sequence similarity, respectively). The genomic G+C content was 63.5 mol%. KIS30-44^T exhibited less than 52% DNA-DNA relatedness with the type strains of 9 closely related Burkholderia species. The major isoprenoid quinone was Q-8. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unknown aminolipids. The major fatty acids in KIS30-44^T were C_16:0, C_18:1 ??7c and summed feature 3 (iso-C_15:0 2-OH and C_16:1 ??7c), and the strain contained half the amount of C_17:0 cyclo found in the 9 closely related Burkholderia species. The results of these phenotypic, 16S rRNA gene sequence, DNA-DNA hybridization, and chemotaxonomic data indicate that KIS30-44^T represents a novel species within the genus Burkholderia, for which the name Burkholderia denitrificans (Type strain KIS30-44^T =KACC 12733^T =DSM 24336^T) is proposed.     

Crystallization and preliminary analysis of crystals of the 24-meric hemocyanin of the emperor scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (M_W = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.     

Recruitment Kinetics of DNA Repair Proteins Mdc1 and Rad52 but Not 53BP1 Depend on Damage Complexity

The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET  = 2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T₀ after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ₁. Mdc1 accumulates faster (T₀ = 17±2 s, τ₁ = 98±11 s) than 53BP1 (T₀ = 77±7 s, τ₁ = 310±60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T₀ = 73±16 s, τ₁ = 1050±270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ₁ of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.     

Methanocella conradii sp. nov., a thermophilic, obligate hydrogenotrophic methanogen, isolated from Chinese rice field soil

Background

Methanocellales contributes significantly to anthropogenic methane emissions that cause global warming, but few pure cultures for Methanocellales are available to permit subsequent laboratory studies (physiology, biochemistry, etc.).

Methodology/Principal Findings

By combining anaerobic culture and molecular techniques, a novel thermophilic methanogen, strain HZ254^T was isolated from a Chinese rice field soil located in Hangzhou, China. The phylogenetic analyses of both the 16S rRNA gene and mcrA gene (encoding the α subunit of methyl-coenzyme M reductase) confirmed its affiliation with Methanocellales, and Methanocella paludicola SANAE^T was the most closely related species. Cells were non-motile rods, albeit with a flagellum, 1.4–2.8 µm long and by 0.2–0.3 µm in width. They grew at 37–60°C (optimally at 55°C) and salinity of 0–5 g NaCl l⁻¹ (optimally at 0–1 g NaCl l⁻¹). The pH range for growth was 6.4–7.2 (optimum 6.8). Under the optimum growth condition, the doubling time was 6.5–7.8 h, which is the shortest ever observed in Methanocellales. Strain HZ254^T utilized H₂/CO₂ but not formate for growth and methane production. The DNA G+C content of this organism was 52.7 mol%. The sequence identities of 16S rRNA gene and mcrA gene between strain HZ254^T and SANAE^T were 95.0 and 87.5% respectively, and the genome based Average Nucleotide Identity value between them was 74.8%. These two strains differed in phenotypic features with regard to substrate utilization, possession of a flagellum, doubling time (under optimal conditions), NaCl and temperature ranges. Taking account of the phenotypic and phylogenetic characteristics, we propose strain HZ254^T as a representative of a novel species, Methanocella conradii sp. nov. The type strain is HZ254^T ( = CGMCC 1.5162^T = JCM 17849^T = DSM 24694^T).

Conclusions/Significance

Strain HZ254^T could potentially serve as an excellent laboratory model for studying Methanocellales due to its fast growth and consistent cultivability.     

Rubrimonas shengliensis sp. nov. and Polymorphum gilvum gen. nov., sp. nov., novel members of Alphaproteobacteria from crude oil contaminated saline soil

Four bacterial strains were isolated from a crude oil contaminated saline soil in Shengli Oilfield, China. Strains SL014B-28A2^T and SL014B-80A1 were most closely related to Rubrimonas cliftonensis OCh 317^T, while strains SL003B-26A1^T and SL003B-26A2 were most closely related to but readily different from the species in the Pannonibacter-Labrenzia-Roseibium-Stappia cluster. The major fatty acids were C_18:1ω7c, C_16:0, C_18:0 and 11-Methyl C_18:1ω7c, and C_18:1ω7c, 11-Methyl C_18:1ω7c and C_18:0, respectively, for these two groups of isolates. Q-10 was the predominant ubiquinone. The G + C contents of genomic DNA of the four isolates were 67.9, 69.7, 65.6 and 65.6 mol%. Based on the polyphasic taxonomic characteristics, strains SL014B-28A2^T and SL014B-80A1 represented a novel species of the genus Rubrimonas, for which the name Rubrimonas shengliensis sp. nov. is proposed, with strain SL014B-28A2^T (=LMG 26072^T = CGMCC 1.9170^T) as the type strain. Isolates SL003B-26A1^T and SL003B-26A2 represented a novel genus and species of the family Rhodobacteraceae, for which the name Polymorphum gilvum gen. nov., sp. nov. is proposed, with strain SL003B-26A1^T (=LMG 25793^T = CGMCC 1.9160^T) as the type strain.     

Hymenobacter tibetensis sp. nov., a UV-resistant bacterium isolated from Qinghai–Tibet plateau

A brick-red-pigmented strain (XTM003^T) isolated from the Qinghai–Tibet plateau was investigated using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequence indicated that the organism belonged to the genus Hymenobacter. The predominant menaquinone was MK7. The major fatty acids included iso-15:0, 16:1w5c and summed feature 3 (C_{16: 1}ω7c and/or C_{16: 1}ω6c). The G+C content of the DNA was 55.8%. In addition, DNA–DNA hybridization studies demonstrated that strain XTM003^T had a relatedness value of 50.7% with the phylogenetically most closely related species Hymenobacter norwichensis DSM 15439^T. Based on the results of phenotypic characteristics and DNA–DNA hybridization studies, strain XTM003^T is considered to represent a novel species, for which the name Hymenobacter tibetensis sp. nov. is proposed. The type strain is XTM003^T (=CCTCC AB 207089^T=NRRL B-51271^T).     

Sphingomonas gimensis sp. nov., a novel Gram-negative bacterium isolated from abandoned lead–zinc ore mine

A novel bacterial strain designated 9PNM-6^T was isolated from an abandoned lead–zinc ore mine site in Meizhou, Guangdong Province, China. The isolate was found to be Gram-negative, rod-shaped, orange-pigmented, strictly aerobic, oxidase- and catalase-positive. Growth occurred at 0–4 % NaCl (w/v, optimum, 0 %), at pH 6.0–8.0 (optimum, pH 7.0) and at 15–32 °C (optimum, 28–30 °C). Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain 9PNM-6^T belongs to the genus Sphingomonas, with the highest sequence similarities with Sphingomonas jejuensis NBRC 107775^T (99.7 %), Sphingomonas koreensis KCTC 2882^T (95.1 %) and Sphingomonas dokdonesis KCTC 12541^T (95.1 %). The chemotaxonomic characteristics of strain 9PNM-6^T were consistent with those of the genus Sphingomonas. The predominant respiratory quinone was identified as ubiquinone Q-10, the major polyamine as sym-homospermidine, and the major cellular fatty acids as C_18:1 ω7c, C_16:0, C_16:1 ω7c and/or C_16:1 ω6c and C_14:0 2-OH. The major polar lipids are sphingoglycolipid, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatideylcholine, an unidentified phospholipid and four unidentified aminolipids. The genomic DNA G+C content of strain 9PNM-6^T was determined to be 69.2 ± 0.6 mol%. Based on comparative analyses of morphological, physiological and chemotaxonomic data, and levels of DNA–DNA relatedness values, strain 9PNM-6^T is considered to represent a novel species of the genus Sphingomonas, for which the name Sphingomonas gimensis sp. nov. (Type strain 9PNM-6^T = GIMCC 1.655^T = CGMCC 1.12671^T = DSM 27569^T) is proposed.     

Wangella harbinensis gen. nov., sp. nov., a new member of the family Micromonosporaceae

A novel endophytic actinomycete, designated strain NEAU-J3^T, was isolated from soybean root (Glycine max (L.) Merr) and characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences suggested that strain NEAU-J3^T fell within the family Micromonosporaceae. The strain was observed to form an extensively branched substrate mycelium, which carried non-motile oval spores with a smooth surface. The cell walls of strain NEAU-J3^T were determined to contain meso-diaminopimelic acid and galactose, ribose and glucose were detected as whole-cell sugars. The major menaquinones were determined to be MK-9(H₄) and MK-9(H₆). The phospholipids detected were phosphatidylcholine and phosphatidylethanolamine. The major cellular fatty acids were determined to be C_16:0, C_18:1 ω9c, C_18:0, C_17:0, C_17:1 ω7c, anteiso-C_17:0, C_16:1 ω7c and C_15:0. The DNA G + C content was 62.5 mol%. On the basis of the morphological and chemotaxonomic characteristics, phylogenetic analysis and characteristic patterns of 16S rRNA gene signature nucleotides, strain NEAU-J3^T is considered to represent a novel species of a new genus within the family Micromonosporaceae, for which the name Wangella harbinensis gen. nov., sp. nov. is proposed. The type strain of Wangella harbinensis is strain NEAU-J3^T (=CGMCC 4.7039^T = DSM 45747^T).     

<Emphasis Type="Italic">Frankia inefficax</Emphasis> sp. nov., an actinobacterial endophyte inducing ineffective,non nitrogen-fixing,root nodules on its actinorhizal host plants

Strain EuI1c^T is the first actinobacterial endophyte isolated from Elaeagnus umbellata that was shown to be infective on members of Elaeagnaceae and Morella but lacking the ability to form effective root nodules on its hosts. The strain can be easily distinguished from strains of other Frankia species based on its inability to produce vesicles, the specialized thick-walled structures where nitrogen fixation occurs. Chemotaxonomically, strain EuI1c^T contains phosphatidylinositol, diphosphatidylglycerol, two glycophospholipids and phosphatidylglycerol as phospholipids. The whole cell sugars were composed of glucose, galactose, mannose, ribose, rhamnose and fucose as diagnostic sugars of the species. Major fatty acids were iso-C_16:0, C_17:1 ω8c and C_15:0 and C_17:0 and the predominant menaquinones were MK-9(H₆), MK-9(H₈) and MK-9(H₄). Analysis of the 16S rRNA gene sequence of strain EuI1c^T showed 97, 97.4 and 97.9% identity with Frankia elaeagni DSM 46783^T, Frankia casuarinae DSM 45818^T and Frankia alni DSM 45986^T, respectively. Digital DNA:DNA hybridizations with type strains of the three Frankia species with validly/effectively published names are significantly below 70%. These results warrant distinction of EuI1c^T (= DSM 45817^T = CECT 9037^T) as the type strain of a novel species designated Frankia inefficax sp. nov.     

M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase

The sliding β-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis β-clamp (Mtbβ-clamp) to 3.0 Å resolution. The protein crystallized in the space group C222₁ with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtbβ-clamp is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbβ-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent K_d of 300 nM. In bacteria like E. coli, the β-clamp is known to interact with subunits of the clamp loader, NAD⁺ -dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-clamp loader, it does not interact with MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria.     

<Emphasis Type="Italic">Burkholderia alba</Emphasis> sp. nov., isolated from a soil sample on Halla mountain in Jeju island

A rod-shaped, round and white colony-forming strain AD18^T was isolated from the soil on Halla mountain in Jeju Island, Republic of Korea. Comparative analysis of 16S rRNA gene sequence revealed that this strain was closely related to Burkholderia oklahomensis C6786^T (98.8%), Burkholderia thailandensis KCTC 23190^T (98.5%). DNA-DNA relatedness (14.6%) indicated that the strain AD18^T represents a distinct species that is separate from B. oklahomensis C6786^T. The isolate grew at pH 5.0–9.0 (optimum, pH 7.0), 0–3% (w/v) NaCl (optimum, 0%), and temperature 10–40°C (optimum 35°C). The sole quinone of the strain was Q-8, and the predominant fatty acids were C_16:0, C_17:0 cyclo, and C_19:0 cyclo ω8c. The genomic DNA G + C content of AD18T was 65.6 mol%. Based on these findings, strain AD18^T is proposed to be a novel species in the genus Burkholderia, for which the name Burkholderia alba sp. nov. is proposed (= KCCM 43268^T = JCM 32403^T). The type strain is AD18^T.     

Water-relation Parameters of Individual Mesophyll Cells of the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana

Water-relation parameters of leaf mesophyll cells of the CAM plant Kalanchoë daigremontiana have been determined directly in cells of tissue slices using the pressure-probe technique. Turgor pressures measured in cells of the second to fourth layer from the cut surface showed an average of 1.82 ± 0.62 bar (mean ± sd; n = 157 cells). This was lower than expected from measurements of the osmotic pressure of the cell sap. The half-time (T_1/2) for water-flux equilibration of individual cells was 2.5 to 8.8 seconds. This is the fastest T_1/2 found so far for higher-plant cells. The calculated values of the hydraulic conductivity were in the range of 0.20 to 1.6 × 10⁻⁵ centimeters second⁻¹ bar⁻¹, with an average of (0.69 ± 0.46) × 10⁻⁵ centimeters second⁻¹ bar⁻¹ (mean ± sd; n = 8 cells). The T_1/2 values of water exchange of individual cells are consistent with the overall rates of water-flux equilibration measured for tissue slices.The volumetric elastic moduli (∈) of individual cells were in the range 13 to 128 bar for turgor pressures between 0.0 and 3.4 bar; the average ∈ value was 42.4 ± 27.7 bar (mean ± sd; n = 21 cells). This ∈ value is similar to that observed for other higher-plant cells.The water-storage capacity of individual cells, calculated as C_c = V/(∈ + πⁱ) (where V = cell volume and πⁱ = internal osmotic pressure) was 9.1 × 10⁻⁹ cubic centimeters bar⁻¹ per cell, and the capacity for the tissue was 2.2 × 10⁻² cubic centimeters bar⁻¹ gram⁻¹ fresh weight. The significance of the water-relation parameters determined at the cellular level is discussed in terms of the water relations of whole leaves and the high water-use efficiency characteristic of CAM plants.     

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T₉TKE₁₂ sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K_d = 25±6 nM to K_d = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K_d = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν_as(P-O) and ν_s(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν_as(UO₂)²⁺ vibration (from 923 cm⁻¹ to 908 cm⁻¹) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.     

Changes in temperature have opposing effects on current amplitude in α7 and α4β2 nicotinic acetylcholine receptors

We have examined the effect of temperature on the electrophysiological properties of three neuronal nicotinic acetylcholine receptor (nAChR) subtypes: the rapidly desensitizing homomeric α7 nAChR, the more slowly desensitizing heteromeric α4β2 nAChR and on α7 nAChRs containing a transmembrane mutation (L247T) that results in dramatically reduced desensitization. In all cases, the functional properties of receptors expressed in Xenopus oocytes at room temperature (RT; 21°C) were compared to those recorded at either physiological temperature (37°C) or at lower temperature (4°C). Alterations in temperature had dramatically differing effects on the amplitude of whole-cell responses detected with these three nAChR subtypes. Compared to responses at RT, the amplitude of agonist-evoked responses with α4β2 nAChRs was increased at high temperature (125±9%, n = 6, P<0.01) and reduced at low temperature (47±5%, n = 6, P<0.01), whereas the amplitude of α7 responses was reduced at high temperature (27±7%, n = 11, P<0.001) and increased at low temperatures (224±16%, n = 10, P<0.001). In contrast to the effects of temperature on α4β2 and wild type α7 nAChRs, the amplitude of α7 nAChRs containing the L247T mutation was unaffected by changes in temperature. In addition, changes in temperature had little or no effect on current amplitude when α7 nAChRs were activated by the largely non-desensitizing allosteric agonist 4BP-TQS. Despite these differing effects of temperature on the amplitude of agonist-evoked responses in different nAChRs, changes in temperature had a consistent effect on the rate of receptor desensitization on all subtypes examined. In all cases, higher temperature resulted in increased rates of desensitization. Thus, it appears that the differing effects of temperature on the amplitudes of whole-cell responses cannot be explained by temperature-induced changes in receptor desensitization rates.     

Marinobacter persicus sp. nov., a moderately halophilic bacterium from a saline lake in Iran

A Gram-negative, non-endospore-forming, rod shaped, strictly aerobic, moderately halophilic bacterium, designated strain M9B^T, was isolated from the hypersaline lake Aran-Bidgol in Iran. Cells of strain M9B^T were found to be motile and produce colonies with an orange-yellow pigment. Growth was determined to occur between 5 and 20 % (w/v) NaCl and the isolate grew optimally at 7.5–10 % (v/w) NaCl. The optimum pH and temperature for growth of the strain were determined to be pH 7.0 and 35 °C, respectively, while it was able to grow over pH and temperature ranges of 6–8 and 25–45 °C, respectively. Phylogenetic analysis based on the comparison of 16S rRNA gene sequences revealed that strain M9B^T is a member of the genus Marinobacter. The closest relative to this strain was found to be Marinobacter hydrocarbonoclasticus MBIC 1303^T with a similarity level of 97.7 %. DNA–DNA hybridization between the novel isolate and this phylogenetically related species was 13 ± 2 %. The major cellular fatty acids of the isolate were identified as C_16:0, C_19:1 ω6c, C_18:1 ω9c and C_16:1 ω9c. The polar lipid pattern of strain M9B^T was determined to consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and three phospholipids. Ubiquinone 9 (Q-9) was the only lipoquinone detected. The G+C content of the genomic DNA of this strain was determined to be 58.6 mol%. Phenotypic characteristics, phylogenetic analysis and DNA–DNA relatedness data suggest that this strain represents a novel species of the genus Marinobacter, for which the name Marinobacter persicus sp. nov. is proposed. The type strain of Marinobacter persicus is strain M9B^T (=IBRC-M 10445^T = CCM 7970^T = CECT 7991^T = KCTC 23561^T).     

Albirhodobacter marinus gen. nov., sp. nov., a member of the family Rhodobacteraceae isolated from sea shore water of Visakhapatnam, India

A novel marine, Gram-negative, rod-shaped bacterium, designated strain N9^T, was isolated from a water sample of the sea shore at Visakhapatnam, Andhra Pradesh (India). Strain N9^T was found to be positive for oxidase and catalase activities. The fatty acids were found to be dominated by C_16:0, C_18:1 ω7c and summed in feature 3 (C_16:1 ω7c and/or C_16:1 ω6c). Strain N9^T was determined to contain Q-10 as the major respiratory quinone and phosphatidylethanolamine, phosphatidylglycerol, two aminophospholipids, two phospholipids and four unidentified lipids as polar lipids. The DNA G+C content of the strain N9^T was found to be 63 mol%. 16S rRNA gene sequence analysis indicated that Rhodobacter sphaeroides, Rhodobacter johrii, Pseudorhodobacter ferrugineus, Rhodobacter azotoformans, Rhodobacter ovatus and Pseudorhodobacter aquimaris were the nearest phylogenetic neighbours, with pair-wise sequence similarities of 95.43, 95.36, 94.24, 95.31, 95.60 and 94.74 %, respectively. Phylogenetic analysis showed that strain N9^T formed a distinct branch within the family Rhodobacteraceae and clustered with the clade comprising species of the genus Pseudorhodobacter, together with species of the genera Roseicitreum, Roseinatronobacter, Roseibaca and Rhodobaca. Species of the genus Pseudorhodobacter are phylogenetically close with a 16S rRNA gene sequence dissimilarity of 5.9–7.3 % (92.7–94.1 % similarity). Based on the above-mentioned phenotypic characteristics and on phylogenetic inference, strain N9^T is proposed as a representative of a new genus and a novel species of the family Rhodobacteraceae as Albirhodobacter marinus gen. nov., sp. nov. The type strain of Albirhodobacter marinus is N9 (= MTCC 11277^T = JCM 17680^T).