Triggering receptor expressed on myeloid cells-2 fine-tunes inflammatory responses in murine Gram-negative sepsis.

During infections, TLR-mediated responses require tight regulation to allow for pathogen removal, while preventing overwhelming inflammation and immunopathology. The triggering receptor expressed on myeloid cells (TREM)-2 negatively regulates inflammation by macrophages and impacts on phagocytosis, but the function of endogenous TREM-2 during infections is poorly understood. We investigated TREM-2's role in regulating TLR4-mediated inflammation by studying wild-type and TREM-2(-/-) mice challenged with LPS and found TREM-2 to dampen early inflammation. Augmented early inflammation in TREM-2(-/-) animals was followed by an accelerated resolution and ultimately improved survival, associated with the induction of the negative regulator A20. Upon infection with Escherichia coli, the otherwise beneficial effect of an exaggerated early immune response in TREM-2(-/-) animals was counteracted by a 50% reduction in bacterial phagocytosis. In line with this, TREM-2(-/-) peritoneal macrophages (PMs) exhibited augmented inflammation following TLR4 stimulation, demonstrating the presence and negative regulatory functionality of TREM-2 on primary PMs. Significantly, we identified a high turnover rate because TREM-2 RNA is 25-fold down-regulated and the protein proteasomally degraded upon LPS encounter, thus ensuring a tightly regulated and versatile system that modulates inflammation. Our results illustrate TREM-2's effects on infection-triggered inflammation and identify TREM-2 as a potential target to prevent overwhelming inflammation while preserving antibacterial-effector functions.

Alkalilactibacillus ikkensis, gen. nov., sp. nov., a novel enzyme-producing bacterium from a cold and alkaline environment in Greenland.

Three novel Gram-positive, endospore-forming bacteria were isolated from a cold and alkaline environment. Phylogenetic analysis showed that the strains were almost identical, and that they were related to Natronobacillus azotifigens 24KS-1(T) (95.8% identity), Paraliobacillus quinghaiensis YIM-C158(T) (95.1%), Paraliobacillus ryukyuensis O15-7(T) (94.5%), and Halolactibacillus miurensis M23-1(T) (93.9%). The isolates produced amylase, α-galactosidase, ß-galactosidase, and ß-glucuronidase, and showed optimal growth at pH 10, at 20°C, and at 2-8% (w/v) NaCl. Major fatty acids were C(14:0) (10.6-11.6%), anteiso-C(15:0) (25.7-32.7%), C(16:1) ω11c (12.2-16.0%), and C(16:0) (14.0-20.4%). The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol, and meso-diaminopimelic acid was found in the cell-wall peptidoglycan. The G+C content was 38.4%. DNA-DNA hybridization between strain GCM68(T) and H. miurensis M23-1(T) was 32.4%, while hybridization to N. azotifigens 24KS-1(T), Amphibacillus tropicus Z-7792(T), and Paraliobacillus ryukyuensis O15-7(T) was below 30%. The phylogenetic analysis and G+C content place strain GCM68(T) in relation to species belonging to Bacillus rRNA group 1, but phylogenetic and physiologic data combined with chemotaxonomic analyses support our proposal for a new genus, Alkalilactibacillus, gen. nov., with the novel species Alkalilactibacillus ikkensis, sp. nov. (type strain is GCM68(T) = DSM 19937 = LMG 24405).

Identification, cloning and expression of a cold-active beta-galactosidase from a novel Arctic bacterium, Alkalilactibacillus ikkense.

A novel, cold-active beta-galactosidase was isolated from an Arctic Gram-positive bacterium, Alkalilactibacillus ikkense. The corresponding gene was cloned and expressed as an active enzyme in Escherichia coli. Denaturing gel electrophoresis of both the native and the recombinant beta-galactosidase showed a monomeric molecular weight of 115-120 kDa. Analysis of the DNA sequence showed sequence similarity to known Glycosyl Hydrolase Family 2 beta-galactosidases from the genera Bacillus, Paenibacillus, Geobacillus, and Lactobacillus. The beta-galactosidase from this study was purified and shown to be highly active at low temperatures with more than 60% of its maximal activity maintained at 0 degrees C. The apparent optimal activity was observed at temperatures between 20 degrees C and 30 degrees C and at pH 8. The purified recombinant enzyme was stable without stabilizing agents for more than 100 hours at temperatures at and below 10 degrees C. At temperatures 40 degrees C and above, the beta-galactosidase was irreversibly inactivated within 10 minutes. When lactose was present in substantial amounts, the enzyme displayed transgalactosylation activity. Comparison of the beta-galactosidase with a commercially available enzyme showed that the conversion rate of the A. ikkense enzyme was approximately two-fold higher at temperatures between 0 degrees C and 20 degrees C.

A lipase with broad temperature range from an alkaliphilic gamma-proteobacterium isolated in Greenland.

A gamma-proteobacterium related to the genera Alteromonadales and Pseudomonadales, isolated from a cold and alkaline environment in Greenland, has been shown to produce a lipase active between 5 degrees C and 80 degrees C, with optimal activity at 55 degrees C and pH 8. PCR-based screening of genomic DNA from the isolated bacterium, followed by genome walking, resulted in two complete open reading frames, which were predicted to encode a lipase and its helper protein, a lipase foldase. The amino acid sequence derived for the lipase showed resemblance to lipases from Pseudomonas, Rhodoferax, Aeromonas and Vibrio. The two genes were cloned into different expression systems in E. coli with or without a putative secretion sequence, but despite the fact that both recombinant lipase and lipase foldase were observed on SDS-PAGE, no recombinant lipase activity was detected. Attempts to refold the recombinant lipase in vitro using a purified lipase foldase remained unsuccessful.

Arsukibacterium ikkense gen. nov., sp. nov, a novel alkaliphilic, enzyme-producing gamma-Proteobacterium isolated from a cold and alkaline environment in Greenland.

A novel aerobic, Gram-negative, non-pigmented bacterium, GCM72(T), was isolated from the alkaline, low-saline ikaite columns in the Ikka Fjord, SW Greenland. Strain GCM72(T) is a motile, non-pigmented, amylase- and protease-producing, oxidase-positive, and catalase-negative bacterium, showing optimal growth at pH 9.2-10.0, at 15 degrees C, and at 3% (w/v) NaCl. Major fatty acids were C(12:0) 3-OH (12.2+/-0.1%), C(16:00) (18.0+/-0.1%), C(18:1)omega7c (10.7+/-0.5%), and summed feature 3 comprising C(16:1)omega7c and/or iso-C(15:0) 2-OH (36.3+/-0.7%). Phylogenetic analysis based on 16S rRNA gene sequences showed that isolate GCM72(T) was most closely related to Rheinheimera baltica and Alishewanella fetalis of the gamma-Proteobacteria with a 93% sequence similarity to both. The G+C content of DNA isolated from GCM72(T) was 49.9mol% and DNA-DNA hybridization between GCM72T and R. baltica was 9.5%. Fatty acid analysis and G+C content supports a relationship primarily to R. baltica, but several different features, such as a negative catalase-response and optimal growth at low temperature and high pH, together with the large phylogenetic distance and low DNA similarity to its closest relatives, lead us to propose a new genus, Arsukibacterium, gen. nov., with the new species Arsukibacterium ikkense sp. nov. (type strain is GCM72(T)).

Rhodonellum psychrophilum gen. nov., sp. nov., a novel psychrophilic and alkaliphilic bacterium of the phylum Bacteroidetes isolated from Greenland.

A novel alkaliphilic and psychrophilic bacterium was isolated from the cold and alkaline ikaite tufa columns of the Ikka Fjord in south-west Greenland. According to 16S rRNA gene sequence analysis, strain GCM71(T) belonged to the family 'Flexibacteraceae' in the phylum Bacteroidetes. Strain GCM71(T), together with five related isolates from ikaite columns, formed a separate cluster with 86-93 % gene sequence similarity to their closest relative, Belliella baltica. The G+C content of the DNA from strain GCM71(T) was 43.1 mol%, whereas that of B. baltica was reported to be 35 mol%. DNA-DNA hybridization between strain GCM71(T) and B. baltica was 9.5 %. The strain was red pigmented, Gram-negative, strictly aerobic with non-motile, rod-shaped cells. The optimal growth conditions for strain GCM71(T) were pH 9.2-10.0, 5 degrees C and 0.6 % NaCl. The fatty acid profile of the novel strain was dominated by branched and unsaturated fatty acids (90-97 %), with a high abundance of iso-C(17 : 1)omega9c (17.5 %), iso-C(17 : 0) 3-OH (17.5 %) and summed feature 3, comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c (12.6 %). Phylogenetic, chemotaxonomic and physiological characteristics showed that the novel strain could not be affiliated to any known genus. A new genus, Rhodonellum gen. nov., is proposed to accommodate the novel strain. Strain GCM71(T) (=DSM 17998(T)=LMG 23454(T)) is proposed as the type strain of the type species, Rhodonellum psychrophilum sp. nov.

Bacterial diversity in permanently cold and alkaline ikaite columns from Greenland.

Bacterial diversity in alkaline (pH 10.4) and permanently cold (4 degrees C) ikaite tufa columns from the Ikka Fjord, SW Greenland, was investigated using growth characterization of cultured bacterial isolates with Terminal-restriction fragment length polymorphism (T-RFLP) and sequence analysis of bacterial 16S rRNA gene fragments. More than 200 bacterial isolates were characterized with respect to pH and temperature tolerance, and it was shown that the majority were cold-active alkaliphiles. T-RFLP analysis revealed distinct bacterial communities in different fractions of three ikaite columns, and, along with sequence analysis, it showed the presence of rich and diverse bacterial communities. Rarefaction analysis showed that the 109 sequenced clones in the 16S rRNA gene library represented between 25 and 65% of the predicted species richness in the three ikaite columns investigated. Phylogenetic analysis of the 16S rRNA gene sequences revealed many sequences with similarity to alkaliphilic or psychrophilic bacteria, and showed that 33% of the cloned sequences and 33% of the cultured bacteria showed less than 97% sequence identity to known sequences in databases, and may therefore represent yet unknown species.