An Ancient Respiratory System in the Widespread Sedimentary Archaea Thermoprofundales.

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A-E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

A novel NhaD-type Na+/H+ antiporter from the moderate halophile and alkaliphile Halomonas alkaliphila.

In this study, a NhaD-type Na+/H+ antiporter gene designated Ha-nhaD was obtained by selection of genomic DNA from the moderate halophile and alkaliphile Halomonas alkaliphila in Escherichia coli KNabc lacking 3 major Na+/H+ antiporters. The presence of Ha-NhaD conferred tolerance of E. coli KNabc to NaCl up to 0.6 mol·L-1 and to LiCl up to 0.2 mol·L-1 and to an alkaline pH. pH-dependent Na+(Li+)/H+ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc/pUC-nhaD but not those of KNabc/pUC18. Ha-NhaD exhibited Na+(Li+)/H+ antiport activity over a wide pH range from 7.0 to 9.5, with the highest activity at pH 9.0. Protein sequence alignment and phylogenetic analysis revealed that Ha-NhaD is significantly different from the 7 known NhaD-type Na+/H+ antiporters, including Dw-NhaD, Dl-NhaD, Vp-NhaD, Vc-NhaD, Aa-NhaD, He-NhaD, and Ha-NhaD1. Although Ha-NhaD showed a closer phylogenetic relationship with Ha-NhaD2, a significant difference in pH-dependent activity profile exists between Ha-NhaD and Ha-NhaD2. Taken together, Ha-nhaD encodes a novel pH-dependent NhaD-type Na+/H+ antiporter.

Novosphingobium oryzae sp. nov., a potential plant-promoting endophytic bacterium isolated from rice roots.

A novel endophytic bacterium, strain ZYY112T, isolated from rice roots, was characterized by a polyphasic approach. In phylogenetic analyses based on 16S rRNA gene sequences, ZYY112T showed highest sequence similarity to Novosphingobium sediminicola HU1-AH51T (97.2 %) and less than 97 % similarity with respect to other Novosphingobium species with validly published names. The DNA G+C content of strain ZYY112T was 60.8 mol%. The level of DNA-DNA relatedness between strain ZYY112T and N. sediminicola DSM 27057T was 33.7 % (reciprocal 5.2 %), which supported the suggestion that ZYY112T represented a novel species of the genus Novosphingobium. Ubiquinone Q-10 was the unique respiratory quinone (100 %). The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, an unknown aminolipid and an unknown phospholipid. The major fatty acids of strain ZYY112T were summed feature 8 (consisting of C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (consisting of C16 : 1ω7c and/or C16 : 1ω6c), C14 : 0 2-OH and C16 : 0. The major polyamine of ZYY112T was spermidine, which is a characteristic trait of the genus Novosphingobium. Characterization by genotypic, chemotaxonomic and phenotypic analysis indicated that strain ZYY112T represents a novel species of the genus Novosphingobium, for which the name Novosphingobium oryzae sp. nov. is proposed. The type strain is ZYY112T ( = ACCC 06131T = JCM 30537T).

Pseudomonas zhaodongensis sp. nov., isolated from saline and alkaline soils.

Strain NEAU-ST5-21(T) was isolated from saline and alkaline soils in Zhaodong City, Heilongjiang Province, China. It was aerobic, Gram-stain-negative, rod-shaped and motile with a polar flagellum. It produced yellow-orange colonies with a smooth surface, and grew in the presence of 0-5 % (w/v) NaCl (optimum 0 %, w/v), at temperatures of 20-40 °C (optimum 28 °C) and at pH 7-11 (optimum pH 7). Phylogenetic analyses based on the separate 16S rRNA gene sequences and concatenated 16S rRNA, gyrB and rpoD gene sequences indicated that strain NEAU-ST5-21(T) belongs to the genus Pseudomonas in the class Gammaproteobacteria. The most closely related species is Pseudomonas xanthomarina, whose type strain (KMM 1447(T)) showed gene sequence similarities of 99.0 % for 16S rRNA, 81.8 % for gyrB and 85.0 % for rpoD with strain NEAU-ST5-21(T). DNA-DNA hybridization values between strain NEAU-ST5-21(T) and P. xanthomarina DSM 18231(T), Pseudomonas kunmingensis CGMCC 1.12273(T), Pseudomonas stutzeri DSM 5190(T), Pseudomonas oleovorans subsp. lubricantis DSM 21016(T), Pseudomomas chengduensis CGMCC 2318(T), Pseudomonas alcaliphila DSM 17744(T) and Pseudomonas toyotomiensis DSM 26169(T) were 52±0 % to 25±2 %. The DNA G+C content of strain NEAU-ST5-21(T) was 65 mol%. The major fatty acids (>10 %) were C18 : 1ω7c and/or C18 : 1ω6c, C16 : 1ω7c and/or C16 : 1ω6c and C16 : 0, the predominant respiratory quinone was ubiquinone 9, and polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, one unknown phospholipid, phosphatidylglycerol, one unknown aminolipid, one unknown lipid and a glycolipid. The proposed name is Pseudomonas zhaodongensis sp. nov., NEAU-ST5-21(T) ( = ACCC 06362(T) = DSM 27559(T)) being the type strain.

Halobacillus andaensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soil.

A Gram-stain-positive, endospore-forming, moderately halophilic bacterial strain, NEAU-ST10-40T, was isolated from a saline and alkaline soil in Anda City, China. It was strictly aerobic, rod-shaped and motile by peritrichous flagella. It formed light yellow colonies and grew at NaCl concentrations of 3-15 % (w/v) (optimum, 8 %, w/v), at pH 7.0-9.0 (optimum, pH 8.0) and at 4-60 °C (optimum, 30 °C). It contained meso-diaminopimelic acid in the cell-wall peptidoglycan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that it belonged to the genus Halobacillus. Levels of 16S rRNA gene sequence similarity between strain NEAU-ST10-40T and the type strains of related species of the genus Halobacillus ranged from 98.8 % (Halobacillus alkaliphilus FP5T) to 97.1 % (Halobacillus kuroshimensis IS-Hb7T). DNA-DNA hybridization relatedness values between strain NEAU-ST10-40T and H. alkaliphilus DSM 18525T, Halobacillus campisalis KCTC 13144T, Halobacillus yeomjeoni DSM 17110T, Halobacillus halophilus DSM 2266T, Halobacillus litoralis DSM 10405T, Halobacillus dabanensis DSM 18199T, Halobacillus salinus DSM 18897T, Halobacillus naozhouensis DSM 21183T, Halobacillus trueperi DSM 10404T and Halobacillus salsuginis DSM 21185T were from 43 ± 1 to 19 ± 1 % (mean ± sd). The DNA G+C content was 39.3 mol%. The major fatty acids (>10 %) were anteiso-C15:0, anteiso-C17:0 and iso-C16:0, the only respiratory quinone detected was MK-7, and polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown phospholipids and three unknown lipids. On the basis of the data presented, strain NEAU-ST10-40T is considered to represent a novel species, for which the name Halobacillus andaensis sp. nov. is proposed. The type strain is NEAU-ST10-40T ( = CGMCC 1.12153T = DSM 25866T).

Kocuria dechangensis sp. nov., an actinobacterium isolated from saline and alkaline soils.

A Gram-stain positive, strictly aerobic, non-motile and coccus-shaped actinobacterium, designated strain NEAU-ST5-33(T), was isolated from saline and alkaline soils in Dechang Township, Zhaodong City, PR China. It formed beige-yellow colonies and grew at NaCl concentrations of 0-5% (w/v) (optimum 0%), at pH 6.0-9.0 (optimum pH 7.0) and over a temperature range of 4-50 °C (optimum 35 °C). Based on 16S rRNA gene sequence analysis, strain NEAU-ST5-33(T) was phylogenetically closely related to the type strains of species of the genus Kocuria, Kocuria polaris CMS 76or(T), Kocuria rosea DSM 20447(T), Kocuria turfanensis HO-9042(T), Kocuria aegyptia YIM 70003(T), Kocuria himachalensis K07-05(T) and Kocuria flava HO-9041(T), with respective sequence similarities of 98.8%, 98.8%, 98.3%, 98.1%, 98.1% and 97.9%. DNA-DNA hybridization relatedness values of strain NEAU-ST5-33(T) with type strains of the closely related species ranged from 54 ± 1% to 34 ± 1%. The DNA G+C content was 61.2 mol%. The major fatty acids (>5%) were C15 : 0 anteiso, C15 : 0 iso and C16 : 1ω7c and/or C16 : 1ω6c. The major menaquinone detected was MK-8 (H2), and the polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unknown aminolipid and one unknown lipid. On the basis of the genotypic, chemotaxonomic and phenotypic data, we propose that strain NEAU-ST5-33(T) represents a novel species of the genus Kocuria, with the name Kocuria dechangensis sp. nov. The type strain is NEAU-ST5-33(T) ( = CGMCC 1.12187(T) = DSM 25872(T)).

Pseudomonas songnenensis sp. nov., isolated from saline and alkaline soils in Songnen Plain, China.

The strain NEAU-ST5-5(T) was isolated from the saline and alkaline soil in Songnen Plain, North East of China. The bacterium was found to be aerobic, Gram-stain negative, rod-shaped and motile by means of several polar flagella. It forms yellow-orange colonies with a radial wrinkled surface. Phylogenetic analyses based on the separate 16S rRNA gene sequences and concatenated 16S rRNA, gyrB and rpoD gene sequences indicated that it belongs to the genus Pseudomonas in the class Gammaproteobacteria. Strain NEAU-ST5-5(T) shows gene sequence similarities of 98.8-97.1 % for 16S rRNA, 90.5-78.4 % for gyrB and 90.4-71.1 % for rpoD with type strains of the closely related species of the genus Pseudomonas, respectively. DNA-DNA hybridization relatedness between strain NEAU-ST5-5(T) and type strains of the most closely related species, Pseudomonas stutzeri DSM 5190(T), P. xanthomarina DSM 18231(T), P. kunmingensis CGMCC 1.12273(T), P. alcaliphila DSM 17744(T) and P. oleovorans subsp. lubricantis DSM 21016(T) were 43 ± 1 to 25 ± 2 %. The major fatty acids (>10 %) were determined to be C18:1 ω7c/C18:1 ω6c, C16:1 ω7c/C16:1 ω6c and C16:0, the predominant respiratory quinone was identified as ubiquinone 9 and polar lipids were found to consist of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid, one unidentified aminophospholipid and one unknown lipid. The genotypic, chemotaxonomic and phenotypic analysis indicated that strain NEAU-ST5-5(T) represents a novel species of the genus Pseudomonas, for which the name Pseudomonas songnenensis sp. nov. is proposed. The type strain is NEAU-ST5-5(T) (=ACCC 06361(T) = DSM 27560(T)).

Planococcus dechangensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soils in Dechang Township, Zhaodong City, China.

Strain NEAU-ST10-9(T) is a moderately halophilic, coccoid and non-motile bacterium isolated from saline and alkaline soils in the Dechang Township, Zhaodong City, China. The bacterium was found to be aerobic and Gram-stain positive. It forms orange colonies and grows at NaCl concentrations of 2-10 % (w/v) (optimum, 4 % w/v), at 4-50 °C (optimum, 30 °C) and at pH 6.0-10.0 (optimum, pH 7.0). Phylogenetic analyses based on 16S rRNA gene sequences indicated that it belongs to the genus Planococcus within the family Planococcaceae. The most closely related species was Planococcus maritimus, whose type strain (TF-9(T)) showed gene sequence similarities of 99.1 % for 16S rRNA, 83.7 % for gyrB and 87.0 % for rpoB with those of strain NEAU-ST10-9(T), respectively. DNA-DNA hybridization relatedness values between strain NEAU-ST10-9(T) and type strains P. maritimus DSM 17275(T) , P. rifietoensis DSM 15069(T) , P. plakortidis DSM 23997(T), P. citreus DSM 20549(T), P. maitriensis DSM 15305(T), P. salinarum KCTC 13584(T) and P. columbae DSM 17517(T) were from 55 ± 1 to 32 ± 2 %. The DNA G+C content was found to be 45.2 mol %. The major fatty acids (>5 %) were determined as C15:0 anteiso, C16:1 ω7c alcohol, C17:1 ω9c and C17:0 anteiso. The major menaquinones of strain NEAU-ST10-9(T) were identified as MK-7 and MK-8. The polar lipids were found to contain of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphocholine and two unknown lipids. The genotypic, chemotaxonomic and phenotypic analysis indicated that strain NEAU-ST10-9(T) represents a novel species of the genus Planococcus, for which we proposed the name Planococcus dechangensis sp. nov. The type strain is NEAU-ST10-9(T) (=CGMCC 1.12151(T)=DSM 25871(T)).

Identification of important charged residues for alkali cation exchange or pH regulation of NhaH, a Na(+)/H(+) antiporter of Halobacillus dabanensis.

NhaH is a novel Na(+)/H(+) antiporter identified from the moderate halophile Halobacillus dabanensis. In this study, six conserved charged residues located in the putative transmembrane segments (TMS) including TMSV, TMSVI, TMSVIII and TMSXI of NhaH as well as two His residues in Loop III were replaced by site-directed mutagenesis for the identification of their potential roles in the antiport activity and pH regulation. Substitutions D137A, D166A and R325A caused a complete loss of Na(+)(Li(+))/H(+) antiport activity, revealing that D137, D166 and R325 are indispensable for the antiport activity. Substitution D137E led to a significant increase of the apparent Km values for Na(+) and Li(+) without affecting the changes of pH profile, confirming that D137 plays vital roles in alkali cation binding/translocation. Substitution D166E resulted in not only a significant increase of the apparent Km values for Na(+) and Li(+) but also an alkaline shift of pH profile, suggesting that D166 is involved in alkali cation binding/translocation as well as H(+) binding or pH regulation. Substitutions E161N, D224A and D224E caused a significant increase of Km for Na(+) and Li(+), indicating that E161 and D224 partly contribute to alkali cation binding/translocation. Substitution E229K caused an over 50% elevation of the apparent Km for Li(+), without affecting that for Na(+), suggesting that E229 may be mainly responsible for Li(+) binding/translocation. Substitutions H87A and H88A resulted in an acidic shift of pH profile without an effect on Km for Na(+) and Li(+), indicating that H87 and H88 are involved in H(+) binding or pH regulation.

Halomonas songnenensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soils.

A moderately halophilic bacterium (strain NEAU-ST10-39T) was isolated from saline and alkaline soils in the oilfield of Daqing City, Heilongjiang Province, China. The strain was strictly aerobic, Gram-stain-negative, rod-shaped and motile by peritrichous flagella. Its colonies were yellow. It grew at NaCl concentrations of 0.2-15% (w/v) (optimum 4%, w/v), at temperatures of 4-40 °C (optimum 35 °C) and at pH 5-10 (optimum pH 7). It did not produce acids from sugars or alcohols. Its DNA G+C content was 57.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated 16S rRNA, gyrB and rpoD gene sequences indicated that it belonged to the genus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species were Halomonas axialensis, Halomonas meridiana and Halomonas aquamarina, whose types shared 98.3% (16S rRNA), 82.7% (gyrB) and 83.9-84.5% (rpoD) sequence similarity with strain NEAU-ST10-39T. The results of DNA-DNA hybridization assays showed 20±2%-50±1 % relatedness between strain NEAU-ST10-39T and the most closely related species including Halomonas axialensis DSM 15723T, Halomonas meridiana DSM 5425T, Halomonas aquamarina DSM 30161(T), Halomonas johnsoniae DSM 21197T, Halomonas stevensii DSM 21198T, Halomonas nanhaiensis CCTCC AB 2012911(T), Halomonas hamiltonii DSM 21196T and Halomonas arcis CGMCC 1.6494T. The major fatty acids were C18 : 1ω7c (47.2%), C16:1ω7c and/or C16:1ω6c (18.9%) and C16:0 (16.3%), the only respiratory quinone detected was ubiquinone 9 and polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown phospholipids and three unknown lipids. The new isolate is proposed to represent a novel species with the name Halomonas songnenensis sp. nov., NEAU-ST10-39T (=CGMCC 1.12152T=DSM 25870T) being the type strain.

Cloning and identification of Group 1 mrp operon encoding a novel monovalent cation/proton antiporter system from the moderate halophile Halomonas zhaodongensis.

The novel species Halomonas zhaodongensis NEAU-ST10-25(T) recently identified by our group is a moderate halophile which can grow at the range of 0-2.5 M NaCl (optimum 0.5 M) and pH 6-12 (optimum pH 9). To explore its halo-alkaline tolerant mechanism, genomic DNA was screened from NEAU-ST10-25(T) in this study for Na(+)(Li(+))/H(+) antiporter genes by selection in Escherichia coli KNabc lacking three major Na(+)(Li(+))/H(+) antiporters. One mrp operon could confer tolerance of E. coli KNabc to 0.8 M NaCl and 100 mM LiCl, and an alkaline pH. This operon was previously mainly designated mrp (also mnh, pha or sha) due to its multiple resistance and pH-related activity. Here, we will also use mrp to designate the homolog from H. zhaodongensis (Hz_mrp). Sequence analysis and protein alignment showed that Hz_mrp should belong to Group 1 mrp operons. Further phylogenetic analysis reveals that Hz_Mrp system should represent a novel sub-class of Group 1 Mrp systems. This was confirmed by a significant difference in pH-dependent activity profile or the specificity and affinity for the transported monovalent cations between Hz_Mrp system and all the known Mrp systems. Therefore, we propose that Hz_Mrp should be categorized as a novel Group 1 Mrp system.

Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake.

In this study, metagenomic DNA was screened for the Na(+)/H(+) antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na(+)/H(+) antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na(+)/H(+) antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6%) with a putative NhaD-type Na(+)/H(+) antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na(+)/H(+) antiporters from Halomonas elongata (20.8%), Alkalimonas amylolytica (19.0%), Vibrio parahaemolyticus (18.9%) and Vibrio cholerae (18.7 %). pH-dependent Na(+)(Li(+))/H(+) antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na(+)(Li(+))/H(+) antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na(+)/H(+) antiporters. Also, the apparent K m values of Hb_NhaD for Na(+) and Li(+) at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na(+)/H(+) antiporter.

Mechanisms on salt tolerant of Paenibacillus polymyxa SC2 and its growth-promoting effects on maize seedlings under saline conditions.

Soil salinity negatively affects microbial communities, soil fertility, and agricultural productivity and has become a major agricultural problem worldwide. Plant growth-promoting rhizobacteria (PGPR) with salt tolerance can benefit plant growth under saline conditions and diminish the negative effects of salt stress on plants. In this study, we aimed to understand the salt-tolerance mechanism of Paenibacillus polymyxa at the genetic and metabolic levels and elucidate the mechanism of strain SC2 in promoting maize growth under saline conditions. Under salt stress, we found that strain SC2 promoted maize seedling growth, which was accompanied by a significant upregulation of genes encoding for the biosynthesis of peptidoglycan, polysaccharide, and fatty acid, the metabolism of purine and pyrimidine, and the transport of osmoprotectants such as trehalose, glycine betaine, and K+ in strain SC2. To further enhance the salt resistance of strain SC2, three mutants (SC2-11, SC2-13, and SC2-14) with higher capacities for salt resistance and exopolysaccharide synthesis were obtained via atmospheric and room-temperature plasma mutagenesis. In saline-alkaline soil, the mutants showed better promoting effect on maize seedlings than wild-type SC2. The fresh weight of maize seedlings was increased by 68.10% after treatment with SC2-11 compared with that of the control group. The transcriptome analysis of maize roots demonstrated that SC2 and SC2-11 could induce the upregulation of genes related to the plant hormone signal transduction, starch and sucrose metabolism, reactive oxygen species scavenging, and auxin and ethylene signaling under saline-alkaline stress. In addition, various transcription factors, such as zinc finger proteins, ethylene-responsive-element-binding protein, WRKY, myeloblastosis proteins, basic helix-loop-helix proteins, and NAC proteins, were up-regulated in response to abiotic stress. Moreover, the microbial community composition of maize rhizosphere soil after inoculating with strain SC2 was varied from the one after inoculating with mutant SC2-11. Our results provide new insights into the various genes involved in the salt resistance of strain SC2 and a theoretical basis for utilizing P. polymyxa in saline-alkaline environments.

Structural mechanism of voltage-gated sodium channel slow inactivation.

Voltage-gated sodium (NaV) channels mediate a plethora of electrical activities. NaV channels govern cellular excitability in response to depolarizing stimuli. Inactivation is an intrinsic property of NaV channels that regulates cellular excitability by controlling the channel availability. The fast inactivation, mediated by the Ile-Phe-Met (IFM) motif and the N-terminal helix (N-helix), has been well-characterized. However, the molecular mechanism underlying NaV channel slow inactivation remains elusive. Here, we demonstrate that the removal of the N-helix of NaVEh (NaVEhΔN) results in a slow-inactivated channel, and present cryo-EM structure of NaVEhΔN in a potential slow-inactivated state. The structure features a closed activation gate and a dilated selectivity filter (SF), indicating that the upper SF and the inner gate could serve as a gate for slow inactivation. In comparison to the NaVEh structure, NaVEhΔN undergoes marked conformational shifts on the intracellular side. Together, our results provide important mechanistic insights into NaV channel slow inactivation.

Halomonas zhaodongensis sp. nov., a slightly halophilic bacterium isolated from saline-alkaline soils in Zhaodong, China.

A slightly halophilic bacterium (strain NEAU-ST10-25(T)) was isolated from saline-alkaline soils in Zhaodong City, Heilongjiang Province, China. The strain is a Gram-negative, aerobic motile rod. It accumulates poly-ß-hydroxyalkanoate and produces exopolysaccharide. It produces beige-yellow colonies. Growth occurs at NaCl concentrations (w/v) of 0-15 % (optimum 3 %), at temperatures of 4-60 °C (optimum 35 °C) and at pH 6-12 (optimum pH 9). Its G+C content is 53.8 mol%. Phylogenetic analyses based on the separate 16S rRNA gene and concatenation of the 16S rRNA, gyrB and rpoD genes indicate that it belongs to the genus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species is Halomonas alkaliphila DSM 16354(T), with which strain NEAU-ST10-25(T) showed 16S rRNA, gyrB and rpoD gene sequence similarities of 99.2, 82.3 and 88.2 %, respectively. The results of DNA-DNA hybridization assays showed 60.47 ± 0.69 % DNA relatedness between strain NEAU-ST10-25(T) and H. alkaliphila DSM 16354(T), 42.43 ± 0.37 % between strain NEAU-ST10-25(T) and Halomonas venusta DSM 4743(T) and 30.62 ± 0.43 % between strain NEAU-ST10-25(T) and Halomonas hydrothermalis DSM 15725(T). The major fatty acids are C18:1 ω7c (62.3 %), C16:0 (17.6 %), C16:1 ω7c/C16:1 ω6c (7.7 %), C14:0 (2.9 %), C12:0 3-OH (2.8 %), C10:0 (2.1 %) and C18:1 ω9c (1.6 %) and the predominant respiratory quinone is ubiquinone 9 (Q-9). The proposed name is Halomonas zhaodongensis, NEAU-ST10-25(T) (=CGMCC 1.12286(T) = DSM 25869(T)) being the type strain.

Metabolism of [13C5]hydroxyproline in vitro and in vivo: implications for primary hyperoxaluria.

Hydroxyproline (Hyp) metabolism is a key source of glyoxylate production in the body and may be a major contributor to excessive oxalate production in the primary hyperoxalurias where glyoxylate metabolism is impaired. Important gaps in our knowledge include identification of the tissues with the capacity to degrade Hyp and the development of model systems to study this metabolism and how to suppress it. The expression of mRNA for enzymes in the pathway was examined in 15 different human tissues. Expression of the complete pathway was identified in liver, kidney, pancreas, and small intestine. HepG2 cells also expressed these mRNAs and enzymes and were shown to metabolize Hyp in the culture medium to glycolate, glycine, and oxalate. [(18)O]- and [(13)C(5)]Hyp were synthesized and evaluated for their use with in vitro and in vivo models. [(18)O]Hyp was not suitable because of an apparent tautomerism of [(18)O]glyoxylate between enol and hydrated forms, which resulted in a loss of isotope. [(13)C(5)]Hyp, however, was metabolized to [(13)C(2)]glycolate, [(13)C(2)]glycine, and [(13)C(2)]oxalate in vitro in HepG2 cells and in vivo in mice infused with [(13)C(5)]Hyp. These model systems should be valuable tools for exploring various aspects of Hyp metabolism and will be useful in determining whether blocking Hyp catabolism is an effective therapy in the treatment of primary hyperoxaluria.

[Biodiversity of culturable halotolerant and halophilic bacteria isolated from saline-alkaline soils in Songnen Plain].

OBJECTIVE: To analyze the biodiversity of halotolerant and halophilic bacteria the bacteria in saline-alkaline soils in Songnen Plain, we isolated and purified bacteria samples in the area. METHODS: Halotolerant and halophilic bacteria were isolated from the enriched cultures of the saline-alkline soil samples through the traditional culture method, and 16S rRNA sequences were amplified and analyzed for the determination of phylogenetic relationships. RESULTS: Forty strains were obtained and classified into 34 species, 16 genera, 8 families, 3 phylum (Firmicutes, gamma-Proteobacteria and Actinobacteria) in the Domain Bacteria. The genus Staphylococcus is the predominant group, followed by Halomonas, Oceanbacillus, Bacillus, Kocuria and Pseudomonas. The 16S rRNA sequences of 9 strains showed 97.2 to 99.0% similarities with their closest type strains, suggesting that they may be the potential novel species. The salt tolerance of the strains is mainly concentrated on the 5% - 10% NaCl while alkaline pH resistance of the strains is between pH 9 and 12. Based on the NaCl dependence, 62.5% of the strains were grouped into the halotolerant bacteria, and the rest into the moderate halophiles. CONCLUSION: The halotolerant and halophilic bacteria are very rich in the saline-alkaline soils in Songnen Plain, and the genera Staphylococcus and Halomonas are the predominant groups. Almost all the bacteria from this area could tolerate not only at the high concentration of NaCl, but also at high alkaline pH. More importantly, a number of novel species may be included in this area.

N-type fast inactivation of a eukaryotic voltage-gated sodium channel.

Voltage-gated sodium (NaV) channels initiate action potentials. Fast inactivation of NaV channels, mediated by an Ile-Phe-Met motif, is crucial for preventing hyperexcitability and regulating firing frequency. Here we present cryo-electron microscopy structure of NaVEh from the coccolithophore Emiliania huxleyi, which reveals an unexpected molecular gating mechanism for NaV channel fast inactivation independent of the Ile-Phe-Met motif. An N-terminal helix of NaVEh plugs into the open activation gate and blocks it. The binding pose of the helix is stabilized by multiple electrostatic interactions. Deletion of the helix or mutations blocking the electrostatic interactions completely abolished the fast inactivation. These strong interactions enable rapid inactivation, but also delay recovery from fast inactivation, which is ~160-fold slower than human NaV channels. Together, our results provide mechanistic insights into fast inactivation of NaVEh that fundamentally differs from the conventional local allosteric inhibition, revealing both surprising structural diversity and functional conservation of ion channel inactivation.

Structural basis of ligand binding modes of human EAAT2.

In the central nervous system (CNS), excitatory amino acid transporters (EAATs) mediate the uptake of excitatory neurotransmitter glutamate and maintain its low concentrations in the synaptic cleft for avoiding neuronal cytotoxicity. Dysfunction of EAATs can lead to many psychiatric diseases. Here we report cryo-EM structures of human EAAT2 in an inward-facing conformation, in the presence of substrate glutamate or selective inhibitor WAY-213613. The glutamate is coordinated by extensive hydrogen bonds and further stabilized by HP2. The inhibitor WAY-213613 occupies a similar binding pocket to that of the substrate glutamate. Upon association with the WAY-213613, the HP2 undergoes a substantial conformational change, and in turn stabilizes the inhibitor binding by forming hydrophobic interactions. Electrophysiological experiments elucidate that the unique S441 plays pivotal roles in the binding of hEAAT2 with glutamate or WAY-213613, and the I464-L467-V468 cluster acts as a key structural determinant for the selective inhibition of this transporter by WAY-213613.

Impact of dietary calcium and oxalate, and Oxalobacter formigenes colonization on urinary oxalate excretion.

PURPOSE: Enteric colonization with Oxalobacter formigenes, a bacterium whose main energy source is oxalate, has been demonstrated to decrease the risk of recurrent calcium oxalate kidney stone formation. We assessed the impact of diets controlled in calcium and oxalate contents on urinary and fecal analytes in healthy subjects who were naturally colonized with O. formigenes or not colonized with O. formigenes. MATERIALS AND METHODS: A total of 11 O. formigenes colonized and 11 noncolonized subjects were administered diets controlled in calcium and oxalate contents. We assayed 24-hour urine collections and stool samples obtained on the last 4 days of each 1-week diet for stone risk parameters and O. formigenes levels. Mixed model analysis was used to determine the effects of colonization status on these variables. RESULTS: Urinary calcium and oxalate excretion were significantly altered by the dietary changes in O. formigenes colonized and noncolonized individuals. Mixed model analysis showed significant interaction between colonization status and oxalate excretion on a low calcium (400 mg daily)/moderate oxalate (250 mg daily) diet (p = 0.026). Urinary oxalate excretion was 19.5% lower in O. formigenes colonized subjects than in noncolonized subjects on the low calcium/moderate oxalate diet (mean ± SE 34.9 ± 2.6 vs 43.6 ± 2.6 mg, p = 0.031). CONCLUSIONS: Results suggest that O. formigenes colonization decreases oxalate excretion during periods of low calcium and moderate oxalate intake.