Halophilic Archaeon Halobacterium Salinarum Research Articles

Kinetic regulation of an alkaline p-nitrophenylphosphate phosphatase from Halobacterium salinarum in low water system by Mn2+ and monovalent cations.

Reversed micelles were used as a cytoplasmic model to study the effect of the multi-ionic equilibria on kinetics of extreme halophilic enzymes. The enzymatic system used was an alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium). This enzyme was solubilised in reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The p-nitrophenylphosphate phosphatase is a good system to study the regulation of the enzymatic activity, because it utilises manganese, water and potassium or sodium as cofactors and reacts with p-nitrophenylphosphate. Kinetic behaviour was determined by the ratio between [Mn2+] and [Na+] or [K+]. When the [Mn2+] increased and [Na+] or [K+] decreased, the kinetics showed cooperative behaviour. Rabin's model describes the kinetic behaviour of the p-nitrophenylphosphate phosphatase in reversed micelles.

Stability of an extreme halophilic alkaline phosphatase from Halobacterium salinarium in non-conventional medium

Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w 0 ([H 2O]/[surfactant]), salt concentration, with and without Mn +2. At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25°C). The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn +2-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.

Expression and fast-flow purification of a polyhistidine-tagged myoglobin-like aerotaxis transducer

A Co 2+-affinity, fast-flow perfusion chromatography method to purify a polyhistidine-tagged myoglobin-like aerotaxis transducer HemAT- Hs has been developed. The method relies upon a six-histidine affinity tag fused to the C-terminus and N-terminus of HemAT- Hs for expression in the native host, an extremely halophilic Archaeon Halobacterium salinarum, and in the heterologous host Escherichia coli, respectively. The His-tagged HemAT- Hs can be purified rapidly using either low or high ionic strength buffers. Purified His-tagged HemAT- Hs in high or low salt buffers demonstrated no difference in spectral characteristics and retained reversible oxygen binding capacity. This fast-flow Co 2+-affinity perfusion chromatography provides a simple method for preparation of halophilic heme containing soluble proteins for biophysical and structural studies.

Enzymatic activity of an extremely halophilic phosphatase from the Archaea Halobacterium salinarum in reversed micelles

Alkaline p-nitrophenylphosphate phosphatase ( pNPPase) from the halophilic archaeon Halobacterium salinarum (previously halobium) was solubilized in reversed micelles of cetyltrimethylammonium bromide (CTAB) in cyclohexane with 1-butanol as cosurfactant. The hydrolysis reaction appears to follow Michaelis–Menten kinetics. The dependency of the maximum reaction rate ( V max) on the water content θ (% v/v) (or ω 0 value: molar ratio of water to surfactant concentrations) showed a bell-shaped curve for 0.3 M CTAB, but not for 0.2 M CTAB. The enzyme activity increased with the surfactant concentration at a constant ω 0 value (10.27). When the surfactant concentration was increased at a constant θ, the enzyme activity decreased. The enzyme was more stable in reversed micelles than in aqueous media.

Evidence for Post-translational Membrane Insertion of the Integral Membrane Protein Bacterioopsin Expressed in the Heterologous Halophilic Archaeon Haloferax volcanii

The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome. In each case, bacterioopsin was present both in the membrane and in the cytoplasmic fractions. Pulse-chase labeling experiments showed that the fusion protein in the cytoplasmic fraction is the precursor of the membrane-bound species. Bacterioopsin mutants that lack the seventh helix (BopDelta7) were found to accumulate only in the cytoplasmic fraction, whereas bacterioopsin mutants that lack either helices four and five (BopDelta4-5), or helices one and two (BopDelta1-2), were found in the cytoplasmic as well as in the membrane fractions. The seventh helix, when expressed alone, could target in trans the insertion of a separately expressed bacterioopsin mutant protein that has only the first six helices. These results support a model in which bacterioopsin is produced in H. volcanii as a soluble protein and in which its insertion into the membrane occurs post-translationally. According to this model, membrane insertion is directed by the seventh helix.