Ureases of extreme halophiles of the genus Haloarcula with a unique structure of gene cluster

We searched for urease activities in 71 strains of extreme halophiles by a urea-phenol red-agar plate method. Positive strains were further investigated by measuring the ammonia released from urea in cell-free extracts. Only 4 strains of the genus Haloarcula, Har. aidinensis, Har. hispanica, Har. japonica, and Har. marismortui were finally shown as the urease producers. A partially purified urease from Har. hispanica was a typical halophilic enzyme in that it showed maximum activity at 18-23% NaCl and lost the activity irreversibly in the absence of NaCl. Partial genes (1596 bp) of the urease encoding from upstream of the beta subunit down to the N-terminal 139 amino acids of the alpha subunit, were PCR amplified from the four strains, as well as from five urease-negative Haloarcula strains. Strains of other genera, which were urease-negative, did not yield PCR products. The deduced amino acid sequences of the beta subunit and partial alpha subunit were similar to each other (92-100% similarities) and to those from other organisms. Analysis of the draft genome sequence of Har. marismortui, however, suggested that the order of the genes encoding the three subunits (with the total number of amino acids of 834) and four accessory proteins was beta-alpha-gamma-UreG-UreD-UreE-UreF. This order is quite unique, since in other microorganisms the order is gamma-beta-alpha-UreE-UreF-UreG-UreD in most cases. No open reading frames were detected in the PCR-amplified upstream of the beta subunit, suggesting that all Haloarcula species have the same unique structure of the urease gene cluster.     

Geometry Regulates Traction Stresses in Adherent Cells

Cells generate mechanical stresses via the action of myosin motors on the actin cytoskeleton. Although the molecular origin of force generation is well understood, we currently lack an understanding of the regulation of force transmission at cellular length scales. Here, using 3T3 fibroblasts, we experimentally decouple the effects of substrate stiffness, focal adhesion density, and cell morphology to show that the total amount of work a cell does against the substrate to which it is adhered is regulated by the cell spread area alone. Surprisingly, the number of focal adhesions and the substrate stiffness have little effect on regulating the work done on the substrate by the cell. For a given spread area, the local curvature along the cell edge regulates the distribution and magnitude of traction stresses to maintain a constant strain energy. A physical model of the adherent cell as a contractile gel under a uniform boundary tension and mechanically coupled to an elastic substrate quantitatively captures the spatial distribution and magnitude of traction stresses. With a single choice of parameters, this model accurately predicts the cell’s mechanical output over a wide range of cell geometries.     

Genetic analysis of the gas vesicle gene cluster in haloarchaea

Gas vesicles are buoyant intracellular organelles composed of a rigid proteinaceous membrane surrounding a gas-filled space. Many prokaryotic microorganisms including photosynthetic and heterotrophic bacteria and halophilic and methanogenic archaea produce gas vesicles. In the majority of cases gas vesicles function in providing vertical motility to cells in aquatic environments. Recent genetic analysis of several halophilic archaeal (haloarchaeal) species has shown that a large cluster of genes [gvpMLKJIHGFEDACN(O)] is necessary for gas vesicle formation.     

Insulin-like growth factors promote vasculogenesis in embryonic stem cells

The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1α and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.     

Antigen presentation using novel particulate organelles from halophilic archaea

A presentation vehicle was developed based on particulate gas vesicles produced by halophilic archaea. Gas vesicle epitope displays were prepared using standard coupling methods or recombinant DNA technology. When presented in the context of gas vesicle preparations, either the hapten, TNP, or a model six amino acid recombinant insert in the outer gas vesicle protein, GvpC was rendered immunogenic. Assays to quantify humoral responses indicated that each preparation elicited strong antibody responses in the absence of exogenous adjuvant. Thus, each preparation elicited a humoral response when injected into mice and this response was long lived and exhibited immunologic memory. Recombinant gas vesicle preparations therefore constitute a new, self-adjuvanting carrier/display vehicle for presentation of an array of peptidyl epitopes.     

Function and biotechnology of extremophilic enzymes in low water activity

ABSTRACT: Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology.