Complete Genome Sequence of Halalkalicoccus jeotgali B3T,an Extremely Halophilic Archaeon

Halalkalicoccus jeotgali B3^T, isolated from salt-fermented seafood from South Korea, is an extremely halophilic archaeon belonging to the family Halobacteriaceae. Here, we present the complete genome sequence of the type strain H. jeotgali B3^T (3,698,650 bp, with a G+C content of 62.5%), which consists of one chromosome and six plasmids. This is the first complete genome sequence of the Halalkalicoccus species.Extremely halophilic archaea (haloarchaea) are adapted to hypersaline environments and grow optimally in NaCl solutions of 2.6 M or higher (12). These haloarchaea are classified within the family Halobacteriaceae in the order Halobacteriales; currently, this family comprises 28 genera (3), and only 11 complete genome sequences in Halobacteriaceae have been reported. In a study of archaeal diversity in salt-fermented small shrimp or shellfish from South Korea, our laboratory isolated and characterized 5 novel, extremely halophilic archaeal strains of Halobacteriaceae. These strains included Natronococcus jeotgali (9), Halalkalicoccus jeotgali (11), Halorubrum cibi (7), Haloterrigena jeotgali (10) and Haladaptatus cibarius (8). We have now sequenced the genome of Halalkalicoccus jeotgali B3^T; genome sequencing had not been completed or initiated for any strain in this genus when our sequencing project was begun. The genus Halalkalicoccus currently contains only two species, Halalkalicoccus tibetensis (13) and H. jeotgali, and these species exhibit 98.6% gene sequence similarity in their 16S rRNA. The genome of H. jeotgali B3^T is the first of this genus to be sequenced.The complete genome sequence of H. jeotgali B3^T was determined by a whole-genome shotgun strategy using Roche 454 GS (FLX Titanium) pyrosequencing (898,168 reads totaling ∼348 Mb; ∼94-fold coverage of the genome) and a fosmid library (514 reads totaling ∼680 kb) at the Genome Resource Center, KRIBB (Korea Research Institute of Bioscience and Biotechnology). Genome sequences from pyrosequencing were processed by Roche''s software according to the manufacturer''s instructions, and sequences from the fosmid library were processed by PESTAS (6). A total of 898,196 reads were assembled using Newbler Assembler 2.3 (454 Life Science), which generated 54 large contigs (>100 bp in size) with bases having quality scores of 40 and above. The gaps between contigs were closed by primer walking and sequencing of PCR products across the gaps. The annotation was done by merging results obtained from the RAST (Rapid Annotation using Subsystem Technology) pipeline (1), Glimmer 3.02 (2), tRNAscan-SE 1.21 (5), and RNAmmer 1.2 (4).The H. jeotgali B3^T genome is 3,698,650 bases long with a 62.5% G+C content. The chromosome consists of a single circular chromosome (2,809,118 bp, with a G+C content of 65.0%) and six plasmids (406,285 bp, 55.3%; 363,534 bp, 54.2%; 44,576 bp, 58.9%; 44,459 bp, 54.9%; 23,727 bp, 47.6%; 6,951 bp, 60.6%). The genome contains 3,860 predicted coding sequences and 52 RNA genes (determined using RAST). The chromosome is predicted to contain 3,101 coding sequences with a coding intensity of 90.0%, including 47 tRNA genes, 1 5S rRNA gene, 1 16S rRNA gene, and 1 23S rRNA gene. The largest plasmid contains 466 coding sequences with a coding intensity of 81.2% and 2 tRNA genes, while the other five plasmids contain 425, 44, 48, 29, and 5 coding sequences with coding intensities of 80.2%, 84.2%, 83.0%, 69.6%, and 22.8%, respectively (determined using Glimmer3). More detailed analysis of this genome and comparative analysis with other haloarchaea will provide further insight into the genomic differences and metabolism of the extremely halophilic archaea.     

N‐glycan maturation is crucial for cytokinin‐mediated development and cellulose synthesis in Oryza sativa

To explore the physiological significance of N‐glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N‐acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N‐glycan maturation and accumulated high‐mannose N‐glycans. Phenotypic analyses revealed that gnt1 shows defective post‐seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark‐induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A‐type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N‐glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.     

Nematicidal activity of Lysobacter capsici YS1215 and the role of gelatinolytic proteins against root-knot nematodes

Lysobacter capsici YS1215 is a soil-borne strain that could inhibit the growth of phytopathogenic fungi, including Phytophthora capsici, Rhizoctonia solani and Fusarium oxysporum, as well as root-knot nematodes. The effect of different concentrations of bacterial culture filtrate (BCF) of L. capsici YS1215 on the mortality of second-stage juveniles (J2) of Meloidogyne incognita was studied using 24-well plates. The J2 mortality increased with increasing concentrations of BCF. YS1215 also produces gelatinases in the culture filtrate. To study its role in nematicidal activities, the partial purification and the characterisation of gelatinolytic proteins were done from the culture medium of the YS1215. The partially purified proteins showed three clear bands with molecular weights estimated using zymography to be 255.7, 232.1 and 146.4 kDa. The optimal pH and temperature for the proteins were 8.0 and 40°C, respectively. The activity of the proteins was inhibited by ethylenediaminetetraacetic acid, FeCl₃ and 1,10-phenanthroline, whereas it was activated by MnCl₂. The proteins may belong to the group of metalloproteases. Moreover, the proteins could hydrolyse skimmed milk, collagen, gelatin and bovine serum albumin (BSA) as substrates, but not casein. The proteins could induce 75% J2 mortality in five days and degrade the J2 bodies. The present study demonstrates the role of the gelatinolytic proteins in the nematicidal potential of L. capsici YS1215.     

Autophagy and its implication in Chinese hamster ovary cell culture

Chinese hamster ovary (CHO) cells, that are widely used for production of therapeutic proteins, are subjected to apoptosis and autophagy under the stresses induced by conditions such as nutrient deprivation, hyperosmolality and addition of sodium butyrate. To achieve a cost-effective level of production, it is important to extend the culture longevity. Until now, there have been numerous studies in which apoptosis of recombinant CHO (rCHO) cells was inhibited, resulting in enhanced production of therapeutic proteins. Recently, autophagy in rCHO cells has drawn attention because it can be genetically and chemically controlled to increase cell survival and productivity. Autophagy is a global catabolic process which involves multiple pathways and genes that regulate the lysosomal degradation of intracellular components. A simultaneous targeting of anti-apoptosis and pro-autophagy could lead to more efficient protection of cells from stressful culture conditions. In this regard, it is worthwhile to have a detailed understanding of the autophagic pathway, in order to select appropriate genes and chemical targets to manage autophagy in rCHO cells, and thus to enhance the production of therapeutic proteins.     

<Emphasis Type="Italic">Luteimonas aestuarii</Emphasis> sp. nov., isolated from tidal flat sediment

A novel bacterium B9^T was isolated from tidal flat sediment. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were characterized. Colonies of this strain are yellow and the cells are Gram-negative, rod-shaped, and do not require NaCl for growth. The 16S rRNA gene sequence similarity indicated that strain B9^T is associated with the genus Lysobacter (≤ 97.2%), Xanthomonas (≤ 96.8%), Pseudomonas (≤ 96.7%), and Luteimonas (≤ 96.0%). However, within the phylogenetic tree, this novel strain shares a branching point with the species Luteimonas composti CC-YY255^T (96.0%). The DNA-DNA hybridization experiments showed a DNA-DNA homology of 23.0% between strain B9^T and Luteimonas mephitis B1953/27.1^T. The G+C content of genomic DNA of the type strain is 64.7 mol% (SD, 1.1). The predominant fatty acids are iso-C_11:0, iso-C_15:0, iso-C_16:0, iso-C_17:0, iso-C_17:0 ω9c, and iso-C_11:0 3-OH. Combined analysis of the 16S rRNA gene sequences, fatty acid profile, and results from physiological and biochemical tests indicated that there is genotypic and phenotypic differentiation of the isolate from other Luteimonas species. For these reasons, strain B9^T was proposed as a novel species, named Luteimonas aestuarii. The type strain of the new species is B9^T (= KCTC 22048^T, DSM 19680^T).     

Photolithographic fabrication of poly(ethylene glycol) microstructures for hydrogel-based microreactors and spatially addressed microarrays

We describe the fabrication of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures with a high aspect ratio and the use of hydrogel microstructures containing the enzyme beta-galactosidase (beta-Gal) or glucose oxidase (GOx)/horseradish peroxidase (HRP) as biosensing components for the simultaneous detection of multiple analytes. The diameters of the hydrogel microstructures were almost the same at the top and at the bottom, indicating that no differential curing occurred through the thickness of the hydrogel microstructure. Using the hydrogel microstructures as microreactors, beta-Gal or GOx/HRP was trapped in the hydrogel array, and the time-dependent fluorescence intensities of the hydrogel array were investigated to determine the dynamic uptake of substrates into the PEG-DA hydrogel. The time required to reach steady-state fluorescence by glucose diffusing into the hydrogel and its enzymatic reactions with GOx and HRP was half the time required for resorufin beta-D-galactopyranoside (RGB) when used as the substrate for beta-Gal. Spatially addressed hydrogel microarrays containing different enzymes were micropatterned for the simultaneous detection of multiple analytes, and glucose and RGB solutions were incubated as substrates. These results indicate that there was no cross-talk between the beta-Gal-immobilizing hydrogel micropatches and the GOx/HRP-immobilizing micropatches.     

NM23-H2 involves in negative regulation of Diva and Bcl2L10 in apoptosis signaling

The Bcl-2 family members are evolutionally conserved and crucial regulators of apoptosis. Diva (Boo), an ortholog of Bcl2L10 or Bcl-B, is a member of the Bcl-2 family that has contradictory functions in apoptosis. To understand the signaling mechanisms of Diva, we searched for proteins that interact with Diva using the yeast two-hybrid system. We identified a nucleoside diphosphate kinase isoform, NM23-H2. Here, we show that Diva bound to NM23-H2 in cells in which the transmembrane domain of Diva was required, and both proteins were colocalized in cytoplasm. Of interest, Diva protein level was significantly down-regulated by NM23-H2 as knock down of NM23-H2 restored Diva expression. Overexpression of NM23-H2 induced apoptosis, and the depletion of NM23-H2 led to the increase of Diva's apoptotic activity. Thus, these results indicate the existence of a previously undiscovered mechanism by which NM23-H2 involves in the regulation of Diva-mediated apoptosis.     

Airway exposure levels of lipopolysaccharide determine type 1 versus type 2 experimental asthma

Allergic asthma is characterized by airway inflammation initiated by adaptive immune responses to aeroallergens. Recent data suggest that severe asthma may be a different form of asthma rather than an increase in asthma symptoms and that innate immune responses to LPS can modulate adaptive immune responses to allergens. In this study, we evaluated the hypothesis that airway exposure to different doses of LPS induces different form of asthma. Our study showed that neutrophilic inflammation and IFN-gamma expression were higher in induced sputum from severe asthma patients than from mild to moderate asthmatics. Animal experiments indicated that allergen sensitization with low-dose LPS (0.1 microg) induced type 2 asthma phenotypes, i.e., airway hyperresponsiveness, eosinophilic inflammation, and allergen-specific IgE up-regulation. In contrast, allergen sensitization with high-dose LPS (10 microg) induced asthma phenotypes, i.e., airway hyperresponsiveness and noneosinophilic inflammation that were not developed in IFN-gamma-deficient mice, but unaffected in the absence of IL-4. During the allergen sensitization period, TNF-alpha expression was found to be enhanced by both low- and high-dose LPS, whereas IL-12 expression was only enhanced by high-dose LPS. Interestingly, the asthma phenotypes induced by low-dose LPS, but not by high-dose LPS, were completely inhibited in TNF-alpha receptor-deficient mice, whereas the asthma phenotypes induced by high-dose LPS were abolished in the homozygous null mutation of the STAT4 gene. These findings suggest that airway exposure levels of LPS induces different forms of asthma that are type 1 and type 2 asthma phenotypes by high and low LPS levels, respectively.