<Emphasis Type="Italic">Methylobacillus caricis</Emphasis> sp. nov., an obligate methylotroph isolated from roots of sedge (<Emphasis Type="Italic">Carex</Emphasis> sp.)

A new obligately methylotrophic bacterium, strain OV^T, was isolated from roots of sedge (Carex sp.). The isolate was represented by aerobic gram-negative motile, non-spore-forming rods, which divided by binary fission. Optimal growth occurred at 22?29°C and pH 7.5?8.5 in the presence of 0.5?2% NaCl; growth was inhibited by 3.5% NaCl. Strain OV^T utilized methanol as the only carbon and energy source. The organism used the KDPG variant of the ribulose monophosphate pathway (RuMP) of С₁ metabolism. Ammonium was assimilated by reductive amination of α-ketoglutarate. The major cellular fatty acids were C_16:0 (45.5%), C_16:1ω7c (40.7%), and C_17cyc (8.0%). The major ubiquinone was Q₈. The dominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G + C content of strain OV^T was 51.4 mol % (T_m). While the 16S rRNA gene sequence of strain OV^T exhibited high similarity to those of Methylobacillus species: M. gramineus Lap^T (99.6%) and M. glycogenes TK 0113^T (98.7%), the DNA-DNA hybridization level between strain OV^T and M. gramineus Lap^T was only 52%. Based on the data obtained, strain OV^T was assigned to the new species Methylobacillus caricis sp. nov. (=VKM B-3158^T = JCM 32031^T).     

Enhanced protein production by sorbitol co-feeding with methanol in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis> strains

Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system.     

The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene <Emphasis Type="Italic">Mth1</Emphasis> in methylotrophic yeast <Emphasis Type="Italic">Pichia methanolica</Emphasis>

Two independent mutant strains of methylotrophic yeast Pichia methanolica (mth1 arg1 and mth2 arg4) from the initial line 616 (ade1 ade5) were investigated. The mutant strains possessed defects in genes MTH1 and MTH2 which resulted in the inability to assimilate methanol as a sole carbon source and the increased activity of alcohol oxidase (AO). The function of the AUG2 gene encoding one of the subunits of AO and CTA1, a probable homolog of peroxisomal catalase of Saccharomyces cereviseae, was investigated by analyses of the molecular forms of isoenzymes. It was shown that optimal conditions for the expression of the AUG2 gene on a medium supplemented with 3% of methanol leads to an increasing synthesis of peroxisomal catalase. The mutant mth1 possessed a dominant formation of AO isoform with electrophoretic mobility which is typical for isogenic form 9, the product of the AUG2 gene, and a decreased level of peroxisomal catalase. The restoration of growth of four spontaneous revertants of the mutant mth1 (Rmth1) on the methanol containing medium was accompanied by an increase in activity of AO isogenic form 9 and peroxisomal catalase. The obtained results confirmed the functional continuity of the structural gene AUG2 in mutant mth1. The correlation of activity of peroxisomal catalase and AO isogenic form 1 in different conditions evidenced the existence of common regulatory elements for genes AUG2 and CTA1 in methilotrophic yeast Pichia methanolica.     

<Emphasis Type="Italic">Nibribacter flagellatus</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Hibiscus syriacus</Emphasis> and emended description of the genus <Emphasis Type="Italic">Nibribacter</Emphasis>

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16^T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10–40 °C (optimum 28–30 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16^T were identified as Nibribacter koreensis KACC 16450^T (98.6%), Rufibacter roseus KCTC 42217^T (94.7%), Rufibacter immobilis CCTCC AB 2013351^T (94.5%) and Rufibacter tibetensis CCTCC AB 208084^T (94.4%). The DNA G+C content of strain THG-T16^T was determined to be 46.7 mol%. DNA–DNA hybridization values between strain THG-T16^T and N. koreensis KACC 16450^T, R. roseus KCTC 42217^T, R. immobilis CCTCC AB 2013351^T, R.tibetensis CCTCC AB 208084^T were 33.5?±?0.5% (31.7?±?0.7% reciprocal analysis), 28.1?±?0.2% (25.2?±?0.2%), 17.1?±?0.9% (10.2?±?0.6%) and 8.1?±?0.3% (5.2?±?0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C_16:1 ω5c, C_17:1 ω6c, iso-C_15:0, summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and summed feature 4 (iso-C_17:1 I and/or anteiso-C_17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-T16^T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16^T(=?KACC 19188^T?=?CCTCC AB 2016246^T).     

Enhancing the efficiency of the <Emphasis Type="Italic">Pichia pastoris AOX1</Emphasis> promoter via the synthetic positive feedback circuit of transcription factor Mxr1

Background

The methanol-regulated AOX1 promoter (P_AOX1) is the most widely used promoter in the production of recombinant proteins in the methylotrophic yeast Pichia pastoris. However, as the tight regulation and methanol dependence of P_AOX1 restricts its application, it is necessary to develop a flexible induction system to avoid the problems of methanol without losing the advantages of P_AOX1. The availability of synthetic biology tools enables researchers to reprogram the cellular behaviour of P. pastoris to achieve this goal.

Results

The characteristics of P_AOX1 are highly related to the expression profile of methanol expression regulator 1 (Mxr1). In this study, we applied a biologically inspired strategy to reprogram regulatory networks in P. pastoris. A reprogrammed P. pastoris was constructed by inserting a synthetic positive feedback circuit of Mxr1 driven by a weak AOX2 promoter (P_AOX2). This novel approach enhanced P_AOX1 efficiency by providing extra Mxr1 and generated switchable Mxr1 expression to allow P_AOX1 to be induced under glycerol starvation or carbon-free conditions. Additionally, the inhibitory effect of glycerol on P_AOX1 was retained because the synthetic circuit was not activated in response to glycerol. Using green fluorescent protein as a demonstration, this reprogrammed P. pastoris strain displayed stronger fluorescence intensity than non-reprogrammed cells under both methanol induction and glycerol starvation. Moreover, with single-chain variable fragment (scFv) as the model protein, increases in extracellular scFv productivity of 98 and 269% were observed in Mxr1-reprogrammed cells under methanol induction and glycerol starvation, respectively, compared to productivity in non-reprogrammed cells under methanol induction.

Conclusions

We successfully demonstrate that the synthetic positive feedback circuit of Mxr1 enhances recombinant protein production efficiency in P. pastoris and create a methanol-free induction system to eliminate the potential risks of methanol.

     

<Emphasis Type="Italic">Phenylobacterium terrae</Emphasis> sp. nov., isolated from a soil sample of Khyber-Pakhtun-Khwa,Pakistan

A Gram-stain negative, aerobic, motile, non-spore-forming and rod-shaped bacterial strain, designated YIM 730227^T, was isolated from a soil sample, collected from Karak district, Khyber-Pakhtun-Khwa, Pakistan. The bacterium was characterized using a polyphasic taxonomic approach. Pairwise comparison of the 16S rRNA gene sequences showed that strain YIM 730227^T is closely related to Phenylobacterium lituiforme FaiI3^T (97.5% sequence similarity), Phenylobacterium muchangponense A8^T (97.4%), Phenylobacterium panacis DCY109^T (97.1%), Phenylobacterium immobile E^T (97.1%) and Phenylobacterium composti 4T-6^T (97.0%), while also sharing 98.0% sequence similarity with Phenylobacterium hankyongense HKS-05^T after NCBI blast, showing it represents a member of the family Caulobacteraceae. The major respiratory quinone was Q-10 and the major fatty acids were C_16:0, summed feature 8 (comprising C_18:1ω7c and/or C_18:1ω6c), C_18:1ω7c 11-methyl and C_17:0. The polar lipids were phosphatidylglycerol, unidentified glycolipids, phospholipid and unidentified lipid. The G?+?C content of the genomic DNA was 68.2 mol%. The DNA–DNA relatedness values of strain YIM 730227^T with P. hankyongense HKS-05^T, P. lituiforme FaiI3^T, P. muchangponense A8^T, P. panacis DCY109^T, P. immobile E^T and P. composti 4T-6^T were 31.3?±?0.6, 26.1?±?0.2, 24.3?±?0.1, 21.8?±?0.9, 19.8?±?0.6 and 18.2?±?1.1%, respectively, values lower than 70%. Besides the morphological and chemotaxonomic characteristics, phylogenetic analyses of 16S rRNA gene sequences and the biochemical characteristics indicated that the strain YIM 730227^T represents a novel member of the genus Phenylobacterium, for which the name Phenylobacterium terrae sp. nov. (type strain YIM 730227^T =?KCTC62324^T?=?CGMCC 1.16326^T) is proposed.     

<Emphasis Type="Italic">Gallaecimonas mangrovi</Emphasis> sp. nov., a novel bacterium isolated from mangrove sediment

A Gram-stain negative, rod-shaped, non-motile, strictly aerobic bacterium HK-28^T was isolated from a mangrove sediment sample in Haikou city, Hainan Province, China. Strain HK-28^T was able to grow at 10–45 °C (optimum 25–30 °C), pH 5.0–8.5 (optimum 6.0–7.0) and 0.5–12.0% (w/v) NaCl (optimum 1.0–3.0%, w/v). The major cellular fatty acids were C_16:0, Summed Feature 8 (C_18:1 ω7c and/or C_18:1 ω6c), Summed Feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), C_17:0, C_12:0 3-OH and C_17:1ω8c. Ubiquinone-8 (Q-8) was the predominant respiratory quinone. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, four unidentified phospholipids, two unidentified glycolipid, an unidentified glycophospholipid, an unidentified aminolipid and an unidentified lipid. The DNA G+C content was 50.2 mol%. Accoroding to 16S rRNA gene sequence similarities, strain HK-28^T shared 97.1 and 96.7% sequence similarities to the validly named species Gallaecimonas xiamenensis MCCC 1A01354^T and Gallaecimonas pentaromativorans MCCC 1A06435^T, respectively, and shared lower sequence similarities (<?92.0%) to all other genera. Phylogenetic analysis showed strain HK-28^T was clustered with G. pentaromativorans MCCC 1A06435^T and G. xiamenensis MCCC 1A01354^T. Strain HK-28^T showed low DNA–DNA relatedness with G. xiamenensis MCCC 1A01354^T (28.3?±?1.5%) and G. pentaromativorans MCCC 1A06435^T (25.2?±?2.4%). On the basis of phenotypic, chemotaxonomic and genotypic characteristics, strain HK-28^T is considered to represent a novel species in the genus Gallaecimonas, for which the name Gallaecimonas mangrovi sp. nov. is proposed. The type strain is HK-28^T (=?KCTC 62177^T?=?MCCC 1K03441).     

Assessment of active methanogenic archaea in a methanol-fed upflow anaerobic sludge blanket reactor

Methanogenic archaea enrichment of a granular sludge was undertaken in an upflow anaerobic sludge blanket (UASB) reactor fed with methanol in order to enrich methylotrophic and hydrogenotrophic methanogenic populations. A microbial community assessment, in terms of microbial composition and activity—throughout the different stages of the feeding process with methanol and acetate—was performed using specific methanogenic activity (SMA) assays, quantitative real-time polymerase chain reaction (qPCR), and high-throughput sequencing of 16S ribosomal RNA (rRNA) genes from DNA and complementary DNA (cDNA). Distinct methanogenic enrichment was revealed by qPCR of mcrA gene in the methanol-fed community, being two orders of magnitude higher with respect to the initial inoculum, achieving a final mcrA/16S rRNA ratio of 0.25. High-throughput sequencing analysis revealed that the resulting methanogenic population was mainly composed by methylotrophic archaea (Methanomethylovorans and Methanolobus genus), being also highly active according to the RNA-based assessment. SMA confirmed that the methylotrophic pathway, with a direct conversion of methanol to CH₄, was the main step of methanol degradation in the UASB. The biomass from the UASB, enriched in methanogenic archaea, may bear great potential as additional inoculum for bioreactors to carry out biogas production and other related processes.     

<Emphasis Type="Italic">Flavobacterium zaozhuangense</Emphasis> sp. nov., a new member of the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from metolachlor-contaminated soil

Strain ZZ-8^T, a Gram-negative, aerobic, non-spore-forming, non-motile, yellow-pigmented, rod-shaped bacterium, was isolated from metolachlor-contaminated soil in China. The taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain ZZ-8^T is a member of the genus Flavobacterium and shows high sequence similarity to Flavobacterium humicola UCM-46^T (97.2%) and Flavobacterium pedocola UCM-R36^T (97.1%), and lower (<?97%) sequence similarity to other known Flavobacterium species. Chemotaxonomic analysis revealed that strain ZZ-8^T possessed MK-6 as the major respiratory quinone; and iso-C_15:0 (28.5%), summed feature 9 (iso-C_17:1 w9c/C_16:0 10-methyl, 22.9%), iso-C_17:0 3-OH (17.0%), iso-C_15:0 3-OH (8.9%), iso-C_15:1 G (8.6%) and summed feature 3 (C_16:1 w7c/C_16:1 w6c, 5.7%) as the predominant fatty acids. The polar lipids of strain ZZ-8^T were determined to be lipids, a glycolipid, aminolipids and phosphatidylethanolamine. Strain ZZ-8^T showed low DNA–DNA relatedness with F. pedocola UCM-R36^T (43.23?±?4.1%) and F. humicola UCM-46^T (29.17?±?3.8%). The DNA G+C content was 43.3 mol%. Based on the phylogenetic and phenotypic characteristics, chemotaxonomic data and DNA–DNA hybridization, strain ZZ-8^T is considered a novel species of the genus Flavobacterium, for which the name Flavobacterium zaozhuangense sp. nov. (type strain ZZ-8^T?=?KCTC 62315 ^T?=?CCTCC AB 2017243^T) is proposed.     

<Emphasis Type="Italic">Limnobacter humi</Emphasis> sp. nov., a thiosulfate-oxidizing,heterotrophic bacterium isolated from humus soil,and emended description of the genus <Emphasis Type="Italic">Limnobacter</Emphasis> Spring <Emphasis Type="Italic">et al.</Emphasis> 2001

Three Gram-negative, strictly aerobic, chemolithoheterotrophic bacterial strains, designated UCM-30, UCM-33, and UCM-39^T, were isolated in South Korea. Based on their 16S rRNA gene sequences, the three isolated strains were found to be similar to Limnobacter thiooxidans CS-K2^T (97.41–97.68%), Limnobacter litoralis KP1-19^T (95.55–95.76%), and various genera belonging to the class Betaproteobacteria (90.34–93.34%). DNA-DNA hybridization showed 79.3–83.9% similarity between the genomic DNA of UCM-39^T, UCM-30, and UCM-33, while the sequence similarity between UCM-39^T and L. thiooxidans KACC 13837T or L. litoralis LMG 24869T was 23.7% and 18.6%, respectively. The DNA G+C content of UCM 39T was 59.7 mol%, the major ubiquinone was Q-8, and the optimal oxidation rate was observed at 10 mM thiosulfate. The major fatty acids (≥ 10%) were summed features 3 (C_16:1 ω7c and/or C_16:1 ω6c) and 8 (C_18:1 ω7c and/or C_18:1 ω6c), and C_16:0. The major polar lipids (diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol) were found in all members of genus Limnobacter. Based on phenotypic, physiological, and phylogenetic analyses, the UCM-39T strain was found to be significantly distinct to represent a novel species affiliated to the genus Limnobacter. We propose to name it Limnobacter humi sp. nov. with the type strain UCM-39^T (=KACC 18574^T =NBRC 111650^T).     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

<Emphasis Type="Italic">Paradevosia shaoguanensis</Emphasis> gen. nov., sp. nov., Isolated from a Coking Wastewater

A Gram staining negative, rod-shaped, aerobic bacterial strain J5-3^T with a single polar flagellum was isolated from coking wastewater collected from Shaoguan, Guangdong, China. It was motile and capable of optimal growth at pH 6–8, 30 °C, and 0–2 % (w/v) NaCl. Its predominant fatty acids were 11-methyl C_18:1 ω7c (29.2 %), C_16:0 (20.6 %), C_19:0 cyclo ω8c (18.2 %), C_18:0 (11.0 %), and C_18:1 ω7c/C_18:1 ω6c (10.9 %) when grown on trypticase soy agar. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids (GL1, GL2), and two unknown phospholipid (PL1, PL2). The predominant ubiquinone was Q-10, and the genome DNA G+C content was 61.7 mol %. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain J5-3^T belonged to the family Hyphomicrobiaceae in Alphaproteobacteria. It shared the 16S rRNA gene sequence similarities of 93.8–96.1 % with the genus Devosia, 94.5–94.8 % with the genus Pelagibacterium, and <92.0 % with all the other type strains in family Hyphomicrobiaceae. It can be distinguished from the closest phylogenetic neighbors based on several phenotypic and genotypic features, including α-galactosidase activity, tetracycline susceptibility, major fatty acid composition, polar lipid profile, DNA gyrase B subunit (gyrB) gene sequence, and random-amplified polymorphic DNA profile. Therefore, we consider strain J5-3^T to represent a novel species of a novel genus within the family Hyphomicrobiaceae, for which the name Paradevosia shaoguanensis gen. nov., sp. nov. is proposed. The type strain of Paradevosia shaoguanensis is J5-3^T (=CGMCC 1.12430^T =LMG 27409^T).     

Comparative Analysis of Mono- and Bifunctional Chorismate Synthases in <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells Capable and Incapable of Phenylalanine Production

The activity of chorismate synthase, the terminal enzyme of the common aromatic pathway, is absolutely dependent on reduced flavin mononucleotide. The bifunctional chorismate synthase of Saccharomyces cerevisiae (product of the ARO2 gene) can reduce flavin in a reaction that involves NADPH, in contrast to the monofunctional chorismate synthase of Escherichia coli (product of the _aro C gene). The latter enzyme does not have the capacity for flavin reduction, and its activity therefore depends on the flavin reductase function of the cell. Chemical synthesis of the structural part of the ARO2 gene that involved the substitution of rare E. coli codons was performed for an in vivo comparison of the two types of chorismate synthase. ARO2 expression was tested in the T7 system, and isogenic E. coli strains TG1Δ _aro CP_tac-ARO2 and TG1Δ _aro CP_tac- _aro C were obtained. Comparative analysis of proteins from the cell extracts of these strains and in silico assessment of hybrid RBS efficiency showed that the level of AroC protein synthesis in TG1Δ _aro CP_tac- _aro C was higher than the level of ARO2 synthesis in the TG1Δ _aro CP_tac-ARO2 cells. The introduction of Ptac-ARO2 and Ptac- _aro C modifications led to complete recovery of the growth of the aromatic auxotroph TG1Δ _aro C on minimal mineral medium supplemented with glucose and restored phenylalanine production in the E. coli strain DV1017Δ _aro C, which lacked chorismate synthase activity. The similar positive effects of P_tac- _aro C and P_tac-ARO2 on phenylalanine biosynthesis in the DV1017ΔtyrR strain, in which chorismate synthase played a “bottleneck” role, indicated the absence of a limiting effect of reduced flavin on monofunctional chorismate synthase overexpressed in E. coli cells.     

Description of <Emphasis Type="Italic">Hymenobacter daejeonensis</Emphasis> sp. nov., isolated from grass soil,based on multilocus sequence analysis of the 16S rRNA gene, <Emphasis Type="Italic">gyr</Emphasis>B and <Emphasis Type="Italic">tuf</Emphasis> genes

A polyphasic taxonomic study was carried out on strains PB105^T and PB108 isolated from a grass soil in Korea. The cells of the strains were Gram-stain negative, non-spore-forming, non-motile, and rod-shaped. Comparative 16S rRNA gene sequence studies showed a clear affiliation of these strains with Bacteroidetes, which showed high pairwise sequence similarities with Hymenobacter algoricola VUG-A23a^T (99.2%), Hymenobacter fastidiosus VUG-A124a^T (97.4%), and Hymenobacter daecheongensis Dae14^T (96.9%). The phylogenetic analysis based on 16S rRNA gene sequences showed that the strains formed a clear phylogenetic lineage with the genus Hymenobacter. The major fatty acids were identified as C_15:0 iso, C_15:0 anteiso, C_16:1 ω5c, C_15:0 iso 3-OH, C_17:0 iso 3-OH, summed feature 3 (C_16:1 ω6c and/or C_16:1 ω7c/t), and summed feature 4 (C_17:1 anteiso B and/or C_17:1 iso I). The major cellular polar lipids were identified as phosphatidylethanolamine, an unidentified aminolipid, and two unidentified lipids. The respiratory quinone was identified as MK-7 and the genomic DNA G+C content was determined to be 64.5 mol% for strain PB105^T and 64.1 mol% for strain PB108. DNA–DNA hybridization value of type strain PB105^T with H. algoricola VUG-A23a^T was 32.3% (reciprocal 39.2). Based on the combined genotypic and phenotypic data, we propose that strains PB105^T and PB108 represent a novel species of the genus Hymenobacter, for which the name Hymenobacter daejeonensis sp. nov. is proposed. The type strain is PB105^T (=?KCTC 52579^T?=?JCM 31885^T).     

<Emphasis Type="Italic">Zunongwangia flava</Emphasis> sp. nov., belonging to the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from <Emphasis Type="Italic">Salicornia europaea</Emphasis>

A yellow pigmented bacterium designated strain MBLN094^T within the family Flavobacteriaceae was isolated from a halophyte Salicornia europaea on the coast of the Yellow Sea. This strain was a Gram-stain negative, aerobic, non-spore forming, rod-shaped bacterium. Phylogenetic analysis of the 16S rRNA gene sequence of strain MBLN094^T was found to be related to the genus Zunongwangia, exhibiting 16S rRNA gene sequence similarity values of 97.0, 96.8, 96.4, and 96.3% to Zunongwangia mangrovi P2E16^T, Z. profunda SM-A87^T, Z. atlantica 22II14-10F7^T, and Z. endophytica CPA58^T, respectively. Strain MBLN094^T grew at 20?37°C (optimum, 25?30°C), at pH 6.0?10.0 (optimum, 7.0?8.0), and with 0.5?15.0% (w/v) NaCl (optimum, 2.0?5.0%). Menaquinone MK-6 was the sole respiratory quinone. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, and four unidentified lipids. Major fatty acids were iso-C_17:0 3-OH, summed feature 3 (C_16:1ω6c and/or C16:1 ω7c), and iso-C_15:0. The genomic DNA G + C content was 37.4 mol%. Based on these polyphasic taxonomic data, strain MBLN094^T is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia flava sp. nov. is proposed. The type strain is MBLN094^T (= KCTC 62279^T = JCM 32262^T).     

<Emphasis Type="Italic">Nocardia zhihengii</Emphasis> sp. nov., an actinobacterium isolated from rhizosphere soil of <Emphasis Type="Italic">Psammosilene tunicoides</Emphasis>

A Nocardia-like actinobacterial strain, designated YIM TG2190^T, was isolated from rhizosphere soil of Psammosilene tunicoides collected from Gejiu, Yunnan province, China. The cells of strain YIM TG2190^T were observed to be Gram-stain positive and non-motile. The strain forms extensively branched substrate mycelia that fragments into rod-shaped elements. The 16S rRNA gene sequence analysis showed that strain YIM TG2190^T is closely related to Nocardia nova (97.5%), Nocardia jiangxiensis (97.1%) and Nocardia miyunensis (96.8%). Growth occurs at 4–30?°C (optimum 28?°C), pH 6.0–8.0 (optimum pH 7.0) and the strain can tolerate NaCl (w/v) up to 3% (optimum 0–1%). The cell walls were found to contain meso-diaminopimelic acid. The whole-cell sugars were identified as glucose, mannose, ribose, galactose, arabinose and fucose. The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides, phosphatidylglycerol and an unidentified phospholipid. The menaquinones detected were MK-9 (H₂) and MK-8 (H₄). The major fatty acids (>?5%) were found to be C_16:0 (33.9%), summed feature 3 (21.7%), C_18:0 10-methyl TBSA (13.7%) and C_18:1ω9c (7.0%). The DNA G+C content was determined to be 61.1 mol%. DNA-DNA relatedness between the strain YIM TG2190^T and N. nova CGMCC 4.1705^T, N. jiangxiensis CGMCC 4.1905^T and N. miyunensis CGMCC 4.1904^T were 46.9?±?2.6, 36.8?±?1.3, and 35.7?±?2.6%, respectively, values which are less than the threshold value (70%) for the delineation of prokaryotic genomic species. The phenotypic, chemotaxonomic and phylogenetic data indicates that strain YIM TG2190^T represents a novel species of the genus Nocardia, for which the name Nocardia zhihengii sp. nov. is proposed. The type strain is YIM TG2190^T (=KCTC 39596^T?=?DSM 100515^T).     

The crystal structure of methanol dehydrogenase,a quinoprotein from the marine methylotrophic bacterium <Emphasis Type="Italic">Methylophaga aminisulfidivorans</Emphasis> MP<Superscript>T</Superscript>

The first crystal structure of a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) from a marine methylotrophic bacterium, Methylophaga aminisulfidivorans MP^T (MDH_Mas), was determined at 1.7 Å resolution. The active form of MDH_Mas (or MDHI_Mas) is a heterotetrameric α₂β₂, where each β-subunit assembles on one side of each of the α-subunits, in a symmetrical fashion, so that two β-subunits surround the two PQQ-binding pockets on the α-subunits. The active site consists of a PQQ molecule surrounded by a β-propeller fold for each α-subunit. Interestingly, the PQQ molecules are coordinated by a Mg²⁺ ion, instead of the Ca²⁺ ion that is commonly found in the terrestrial MDHI, indicating the efficiency of osmotic balance regulation in the high salt environment. The overall interaction of the β-subunits with the α-subunits appears tighter than that of terrestrial homologues, suggesting the efficient maintenance of MDHI_Mas integrity in the sea water environment to provide a firm basis for complex formation with MxaJ_Mas or Cyt c_L. With the help of the features mentioned above, our research may enable the elucidation of the full molecular mechanism of methanol oxidation by taking advantage of marine bacterium-originated proteins in the methanol oxidizing system (mox), including MxaJ, as the attainment of these proteins from terrestrial bacteria for structural studies has not been successful.     

<Emphasis Type="Italic">Roseovarius tibetensis</Emphasis> sp. nov., a halophilic bacterium isolated from Lake LongmuCo on Tibetan Plateau

Two Gram-stain negative halophilic strains, designated as LM2^T and LM4, were isolated from Lake LongmuCo on Tibetan Plateau. These two strains were aerobic, catalaseand oxidase-positive, nonmotile and rod-shaped organisms. Phylogenetic analysis based on 16S rRNA gene sequences indicated that LM2^T and LM4 belong to the genus Roseovarius, with Roseovarius tolerans EL-172^T (97.3% and 97.4% 16S rRNA gene sequence similarity, respectively) and Roseovarius azorensis SSW084^T (95.5% and 95.6% 16S rRNA gene sequence similarity, respectively) as their closest neighbors. Q-10 was the sole respiratory quinone of these two strains. The major fatty acids were C_18:1ω7c/C_18:1ω6c, C_16:0, C_19:0 cyclo ω8c, and 11-methyl C_18:1ω7c. The polar lipids included phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phospholipid of unknown structure containing glucosamine, and unidentified aminolipid. The DNA G + C content was between 64.2 and 64.5 mol%. DNA-DNA hybridization showed 96.7% relatedness between LM2^T and LM4, 24.9% relatedness between LM2^T and R. tolerans EL-172^T, and 36.3% relatedness between LM4 and R. tolerans EL-172^T. Based on phylogenetic analysis, DNA-DNA hybridization, a range of physiological and biochemical characteristics, LM2^T and LM4 belong to the same species and were clearly distinguished from the type strains of the genus Roseovarius. It was evident that LM2^T and LM4 could be classified as a novel species of the genus Roseovarius, for which the name Roseovarius tibetensis sp. nov. is proposed. The type strain is LM2^T (= CGMCC 1.16230^T = KCTC 62028^T).     

<Emphasis Type="Italic">Halomonas tibetensis</Emphasis> sp. nov., isolated from saline lakes on Tibetan Plateau

Strains pyc13^T and ZGT13 were isolated from Lake Pengyan and Lake Zigetang on Tibetan Plateau, respectively. Both strains were Gram-negative, catalase- and oxidase-positive, aerobic, rod-shaped, nonmotile, and nonflagellated bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains pyc13^T and ZGT13 belong to the genus Halomonas, with Halomonas alkalicola 56-L4-10aEn^T as their closest neighbor, showing 97.4% 16S rRNA gene sequence similarity. The predominant respiratory quinone of both strains was Q-9, with Q-8 as a minor component. The major fatty acids of both strains were C_18:1ω6c/C_18:1ω7c, C_16:1ω6c/C_16:1ω7c, C_16:0, and C_12:0 3OH. The polar lipids of both strains consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, glycolipid, phospholipids of unknown structure containing glucosamine, and unidentified phospholipids. The DNA G + C content of pyc13^T and ZGT13 were 62.6 and 63.4 mol%, respectively. The DNA-DNA hybridization values of strain pyc13^T were 34, 41, 61, 35, and 35% with the reference strains H. alkalicola 56-L4-10aEn^T, H. sediminicola CPS11^T, H. mongoliensis Z-7009^T, H. ventosae Al12^T, and H. fontilapidosi 5CR^T, respectively. Phenotypic, biochemical, genotypic, and DNA-DNA hybridization data showed that strains pyc13^T and ZGT13 represent a new species within the genus Halomonas, for which the name H. tibetensis sp. nov. is proposed. The type strain is pyc13^T (= CGMCC 1.15949^T = KCTC 52660^T).