AIMS Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach. METHODS AND RESULTS The significant factors influencing the protease production as screened by Plackett-Burman method were identified as soybean flour and FeCl(3). Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l(-1)): NaCl, 250; KCl, 2; MgSO(4), 10; tri-Na-citrate, 1.5; soybean flour, 10 and FeCl(3), 0.16. This statistical optimization approach led to production of 69.44 +/- 0.811 U ml(-1) of protease. CONCLUSIONS Soybean flour and FeCl(3) were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3.9-fold increase in extracellular protease production. SIGNIFICANCE AND IMPACT OF THE STUDY The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.

The genome of the halophilic archaeon Halobacterium sp. NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation:(1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins. This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity.(2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome.(3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain. This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer.(4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes. These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution.(5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome. This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character.

An energy-transducing pathway in halobacteria is described. Arginine mediates substrate level phosphorylation and allows the cells to grow anaerobically. Bacteriorhodopsin plus light can function as an alternative energy source under these conditions, provided the cells contain the pigment when transferred to the anaerobic environment. Therefore the selection of mutants functionally defective in ATP synthase or bacteriorhodopsin becomes possible.

Work is described on the changes in cell ions during growth of cultures of a species of Halobacterium isolated from the Dead Sea. Cell K concentration fell from 5.5 to 3.8 moles per kg cell water during the logarithmic phase of growth and maintained the latter value during the stationary phase (initial medium concentration, 7 mM). Cell Na and Cl followed a complex series of roughly parallel changes. The logarithmic phase ion concentrations were: Na, 1.0-2.3 moles/kg cell water; Cl, 2.3-3.7 moles/kg cell water. The final stationary phase values were: Na, 0.5 moles/kg cell water; Cl, 2.3-2.9 moles/kg cell water (medium NaCl concentration, 3.9 Molal). It is suggested that most of the K(+) is bound within the cytoplasm.

We describe optimization of a transformation system for the halophilic archaebacterium Halobacterium volcanii. Transformation of spheroplasts in the presence of polyethylene glycol permits the uptake and expression of high-molecular-weight linear fragments of genomic DNA as well as plasmid or bacteriophage DNA. Transformations can be performed with either fresh or frozen cell preparations. Auxotrophic mutants were transformed to prototrophy with genomic DNA from wild-type cells with efficiencies of 5 x 10(4)/micrograms of DNA and frequencies of 8 x 10(-5) per regenerated spheroplast. The overall efficiency of transformation with genomic DNA implies that genetic recombination is an efficient process in H. volcanii.

Halobacterium sp. GRB (Ebert, K., Goebel, W., and Pfeifer, F.(1984) Mol.& Gen. Genet. 194, 91-97) was used to isolate bacteriorhodopsin (BR) mutants. A procedure is described which allows the enrichment of any type of mutant unable to grow under the selection conditions applied. Its use for the isolation of phototrophically negative, retinal-positive mutants of Halobacterium sp. GRB is demonstrated. Single-cell clones of this phenotype were further characterized. The expression of bacterioopsin was tested with a monoclonal antibody directed against the C terminus of the protein. The expressed bacteriorhodopsins were characterized by their specific activity for proton pumping, their spectral properties, and photocycle kinetics. About 15 independent mutants carrying bacteriorhodopsins of three distinct phenotypic classes could be isolated, including BR with a different absorption maximum, BR of lower specific activity, and BR characterized by a slower photocycle and a lack of proton pumping activity.

Phototrophic growth conditions for halobacteria in complex and synthetic media have been established, demonstrating the photosynthetic capacity of this class of archaebacteria. Mutagenesis, 5-bromo-2'-deoxyuridine selection and screening techniques are described which are useful tools in the elucidation of the structure-function relationship of retinal proteins in halobacteria.

The extremely halophilic bacterium (formerly designated as strain J.F. 54) isolated from salt pools of the Death Valley, California, is a motile, Gram-negative, extremely pleomorphic organism, aerobe, and facultative anaerobe. A variety of carbohydrates are assimilated with or without acid production; soluble starch is hydrolyzed. The organism is not proteolytic; catalase, oxidase, and DNase reactions are positive; Tween 20 is slightly hydrolyzed, but Tweens 40, 60, and 80 are not. Nitrates are reduced to nitrites with gas production; nitrites are not reduced. Optimum growth temperature is 40 degrees C. Growth is inhibited by bacitracin and by novobiocin. The type strain J.F. 54 differs from described species of the genus Halobacterium and is assigned to a new species, Halobacterium vallismortis sp. nov.