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.

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.

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.

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.

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.

A novel three-TMH Na+/H+ antiporter and the functional role of its oligomerization.

Na+/H+antiportersare a category of ubiquitous transmembrane proteins with various important physiological roles in almost all living organisms ranging from bacteria to humans. However, the knowledge of novel Na+/H+antiporters remains to be broadened, and the functional roles ofoligomerization in theseantiportershave not yet been thoroughly understood. Here, we reported functional analysis of an unknown transmembrane protein composed of 103 amino acid residues. This protein was found to function as a Na+(Li+, K+)/H+ antiporter. To the best of our knowledge, this antiporter is the minimal one of known Na+/H+antiporters and thus designated as NhaM to represent the minimal Na+/H+antiporter. NhaM and its homologs have not yet been classified into any protein family. Based on phylogenetic analysis and protein alignment, we propose NhaM and its homologs to constitute a novel transporter family designated as NhaM family. More importantly, we found that NhaM is assembled with parallel protomers into a homo-oligomer and oligomerization is vital for the function of this antiporter. This implies that NhaM may adopt and require an oligomer structure for its normal function to create a similar X-shaped structure to that of the NhaA fold. Taken together, current findings not only present the proposal of a novel transporter family but also positively contribute to the functional roles of oligomerization in Na+/H+antiporters.

A Novel MFS-MDR Transporter, MdrP, Employs D223 as a Key Determinant in the Na+ Translocation Coupled to Norfloxacin Efflux.

Multidrug resistance (MDR) transporters of the major facilitator superfamily (MFS) were previously believed to drive the extrusion of multiple antimicrobial drugs through the coupling to proton translocation. Here, we present the identification of the first Na+-coupled MFS-MDR transporter, MdrP, which also can achieve H+-coupled drug efflux independently of Na+. Importantly, we propose that MdrP can extrude norfloxacin in a mode of drug/Na+ antiport, which has not yet been reported in any MFS member. On this basis, we further provide the insights into a novel Na+ and H+ coupling mechanism of MFS-MDR transporters, even for all secondary transporters. The most important finding lies in that D223 should mainly act as a key determinant in the Na+ translocation coupled to norfloxacin efflux. Furthermore, our results partially modify the knowledge of the conformational stability-related residues in the motif A of MFS transporters and imply the importance of a new positively charged residue, R361, for the stabilization of outward-facing conformation of MFS transporters. These novel findings positively contribute to the knowledge of MFS-MDR transporters, especially about Na+ and H+ coupling mechanism. This study is based mainly on measurements in intact cells or everted membranes, and a biochemical assay with a reconstituted MdrP protein should be necessary to come to conclusion to be assured.

Polar or Charged Residues Located in Four Highly Conserved Motifs Play a Vital Role in the Function or pH Response of a UPF0118 Family Na+(Li+)/H+ Antiporter.

Functionally uncharacterized UPF0118 family has been re-designated as autoinducer-2 exporter (AI-2E) family since one of its members, Escherichia coli YdgG, was identified to function as an AI-2E. However, it's very likely that AI-2E family members may exhibit significantly distinct functions due to low identities between them. Recently, we identified one member of this family designated as UPF0118 to represent a novel class of Na+(Li+)/H+ antiporters. In this study, we presented that UPF0118, together with its homologs, should represent an independent group of AI-2E family, designated as Na+/H+ Antiporter Group. Notably, this group shows five highly conserved motifs designated as Motifs A to E, which are not detected in the majority of AI-2E family members. Functional analysis established that polar or charged residues located in Motif A to D play a vital role in Na+(Li+)/H+ antiport activity or pH response of UPF0118. However, three basic residues located in Motif E are not involved in the function of UPF0118, although the truncation of C terminus resulted in the non-expression of this transporter. Therefore, we propose that E179-R182-K215-Q217-D251-R292-R293-E296-K298-S30 7 located in Motifs A to D can be used for signature functional motifs to recognize whether AI-2E family members function as Na+(Li+)/H+ antiporters. Current findings positively contribute to the knowledge of molecular mechanism of Na+, Li+ transporting and pH response of UPF0118, and the functional prediction of uncharacterized AI-2E family members.

Implications for Cation Selectivity and Evolution by a Novel Cation Diffusion Facilitator Family Member From the Moderate Halophile Planococcus dechangensis.

In the cation diffusion facilitator (CDF) family, the transported substrates are confined to divalent metal ions, such as Zn2+, Fe2+, and Mn2+. However, this study identifies a novel CDF member designated MceT from the moderate halophile Planococcus dechangensis. MceT functions as a Na+(Li+, K+)/H+ antiporter, together with its capability of facilitated Zn2+ diffusion into cells, which have not been reported in any identified CDF transporters as yet. MceT is proposed to represent a novel CDF group, Na-CDF, which shares significantly distant phylogenetic relationship with three known CDF groups including Mn-CDF, Fe/Zn-CDF, and Zn-CDF. Variation of key function-related residues to "Y44-S48-Q150" in two structural motifs explains a significant discrimination in cation selectivity between Na-CDF group and three major known CDF groups. Functional analysis via site-directed mutagenesis confirms that MceT employs Q150, S158, and D184 for the function of MceT as a Na+(Li+, K+)/H+ antiporter, and retains D41, D154, and D184 for its facilitated Zn2+ diffusion into cells. These presented findings imply that MceT has evolved from its native CDF family function to a Na+/H+ antiporter in an evolutionary strategy of the substitution of key conserved residues to "Q150-S158-D184" motif. More importantly, the discovery of MceT contributes to a typical transporter model of CDF family with the unique structural motifs, which will be utilized to explore the cation-selective mechanisms of secondary transporters.

An Uncharacterized Major Facilitator Superfamily Transporter From Planococcus maritimus Exhibits Dual Functions as a Na+(Li+, K+)/H+ Antiporter and a Multidrug Efflux Pump.

Within major facilitator superfamily (MFS), up to 27 unknown major facilitator families and many members of 60 well-characterized families have been functionally unknown as yet, due to their sharing no or significantly low sequence identity with characterized MFS members. Here we present the first report on the characterization of one functionally unknown MFS transporter designated MdrP with the accession version No. ANU18183.1 from the slight halophile Planococcus maritimus DS 17275T. During the screening of Na+/H+ antiporter genes, we found at first that MdrP exhibits Na+(Li+, K+)/H+ antiport activity, and propose that it should represent a novel class of Na+(Li+, K+)/H+ antiporters. However, we speculate that MdrP may possess an additional protein function. The existence of the signature Motif A of drug/H+antiporter (DHA) family members and phylogenetic analysis suggest that MdrP may also function as a drug efflux pump, which was established by minimum inhibitory concentration tests and drug efflux activity assays. Taken together, this novel MFS transporter exhibits dual functions as a Na+(Li+, K+)/H+ antiporter and a multidrug efflux pump, which will be very helpful to not only positively contribute to the function prediction of uncharacterized MFS members especially DHA1 family ones, but also broaden the knowledge of Na+/H+ antiporters.

Characterization of a Functionally Unknown Arginine-Aspartate-Aspartate Family Protein From Halobacillus andaensis and Functional Analysis of Its Conserved Arginine/Aspartate Residues.

Arginine-aspartate-aspartate (RDD) family, representing a category of transmembrane proteins containing one highly conserved arginine and two highly conserved aspartates, has been functionally uncharacterized as yet. Here we present the characterization of a member of this family designated RDD from the moderate halophile Halobacillus andaensis NEAU-ST10-40T and report for the first time that RDD should function as a novel Na+(Li+, K+)/H+ antiporter. It's more interesting whether the highly conserved arginine/aspartate residues among the whole family or between RDD and its selected homologs are related to the protein function. Therefore, we analyzed their roles in the cation-transporting activity through site-directed mutagenesis and found that D154, R124, R129, and D158 are indispensable for Na+(Li+, K+)/H+ antiport activity whereas neither R35 nor D42 is involved in Na+(Li+, K+)/H+ antiport activity. As a dual representative of Na+(Li+, K+)/H+ antiporters and RDD family proteins, the characterization of RDD and the analysis of its important residues will positively contribute to the knowledge of the cation-transporting mechanisms of this novel antiporter and the roles of highly conserved arginine/aspartate residues in the functions of RDD family proteins.

Characterization of a novel two-component Na+(Li+, K+)/H+ antiporter from Halomonas zhaodongensis.

In this study, genomic DNA was screened for novel Na+/H+ antiporter genes from Halomonas zhaodongensis by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. Co-expression of two genes designated umpAB, encoding paired homologous unknown membrane proteins belonging to DUF1538 (domain of unknown function with No. 1538) family, were found to confer E. coli KNabc the tolerance to 0.4 M NaCl and 30 mM LiCl, and an alkaline pH resistance at 8.0. Western blot and co-immunoprecipitation establish that UmpAB localize as a hetero-dimer in the cytoplasmic membranes. Functional analysis reveals that UmpAB exhibit pH-dependent Na+(Li+, K+)/H+ antiport activity at a wide pH range of 6.5 to 9.5 with an optimal pH at 9.0. Neither UmpA nor UmpB showed homology with known single-gene or multi-gene Na+/H+ antiporters, or such proteins as ChaA, MdfA, TetA(L), Nap and PsmrAB with Na+/H+ antiport activity. Phylogenetic analysis confirms that UmpAB should belong to DUF1538 family, which are significantly distant with the above-mentioned proteins with Na+/H+ antiport activity. Taken together, we propose that UmpAB represent a novel two-component Na+(Li+, K+)/H+ antiporter. To the best of our knowledge, this is the first report on the functional analysis of unknown membrane proteins belonging to DUF1538 family.

A UPF0118 family protein with uncharacterized function from the moderate halophile Halobacillus andaensis represents a novel class of Na+(Li+)/H+ antiporter.

In this study, genomic DNA was screened from Halobacillus andaensis NEAU-ST10-40T by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. One gene designated upf0118 exhibiting Na+(Li+)/H+ antiport activity was finally cloned. Protein alignment showed that UPF0118 shares the highest identity of 81.5% with an unannotated gene encoding a protein with uncharacterized protein function belonging to UPF0118 family from H. kuroshimensis, but shares no identity with all known specific Na+(Li+)/H+ antiporter genes or genes with Na+(Li+)/H+ antiport activity. Growth test, western blot and Na+(Li+)/H+ antiport assay revealed that UPF0118 as a transmembrane protein exhibits pH-dependent Na+(Li+)/H+ antiport activity. Phylogenetic analysis indicated that UPF0118 clustered with all its homologs belonging to UPF0118 family at a wide range of 22-82% identities with the bootstrap value of 92%, which was significantly distant with all known specific single-gene Na+(Li+)/H+ antiporters and single-gene proteins with the Na+(Li+)/H+ antiport activity. Taken together, we propose that UPF0118 should represent a novel class of Na+(Li+)/H+ antiporter. To the best of our knowledge, this is the first report on the functional analysis of a protein with uncharacterized protein function as a representative of UPF0118 family containing the domain of unknown function, DUF20.

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).

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)).

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).

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)).

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.

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)).