Ultrastructure of the obligate halophilic bacterium Halobacterium halobium

The fine structure of Halobacterium halobium was studied by means of a modified double-fixation technique. The cell envelope is shown to consist of both a “wall” and a plasma membrane. Some electron-dense strands were seen inside the cytoplasm running parallel to the cell envelope. An unusual organelle (or organelles) appeared inside the cytoplasm in the form of parallel striated strands.     

High-pressure adaptation by salt stress in a moderately halophilic bacterium obtained from open seawater

High-pressure adaptation was examined using a moderately halophilic bacterium (Micrococcus roseus), which was isolated from open seawater and capable of growing in 15% w/v NaCl (optimum NaCl concentration: 3% w/v). After treatment at 207 MPa, colony-forming units (CFUs) significantly decreased; however, the loss of integral cells after pressure was only 30% when direct cell count was performed microscopically. In order to investigate the piezotolerance of M. roseus under high pressure without morphological change, the survival of cells was examined under pressure at 138 MPa for 2 h. M. roseus in 3% NaCl was still sensitive to pressure at 138 MPa. However, the cells in the third generations showed remarkably increased pressure resistance, and no significant loss of viability was confirmed. Furthermore, when M. roseus was cultured in 1, 3, 5, 10 and 15% NaCl, the survival ratio proportionally increased at increased NaCl concentration. M. roseus cultured in 15% NaCl was remarkably resistant (94.7% viability) to pressure at 138 MPa, even when suspended in lower concentration of NaCl. This suggests that NaCl concentrations in growth culture affect the piezotolerance of M. roseus and that this species has an ability to adapt to high pressure.     

Halobellus rufus sp. nov., an extremely halophilic archaeon isolated from non-purified solar salt

A halophilic archaeon, designed strain CBA1103^T, was isolated from non-purified solar salt. The cells of strain CBA1103^T were observed to be Gram-stain negative and pleomorphic, and the colonies appear red. Strain CBA1103^T was observed to grow between 20 and 55 °C (optimum 37 °C), and in NaCl concentrations of 10–30 % (w/v) (optimum 15 %) with 0–0.5 M MgSO₄·7H₂O (optimum 0.1 M) and at pH 6.0–9.0 (optimum pH 7.0). Additionally, the cells lyse in distilled water. The major polar lipids of strain CBA1103^T are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and two glycolipids chromatographically identical to sulfated mannosyl glucosyl diether and manosyl glucosyl diether. Strain CBA1103^T is shown to belong to the Halobellus genus and exhibits similarity to related taxa; the 16S rRNA gene sequence similarity between strain CBA1103^T and Halobellus rarus 18362^T, Hbs. limi 16811^T, Hbs. litoreus JCM 17118^T, Hbs. inordinatus YC20^T, Hbs. clavatus TNN18^T and Hbs. salinus CSW2.24.4^T is 97.3, 96.5, 96.5, 94.5, 94.5 and 93.7 %, respectively. The RNA polymerase subunit B gene sequence of strain CBA1103^T shows 93.7 % similarity with the sequence of Hbs. litoreus JCM 17118^T; the similarity is lower with sequences from the type strains of other species of Halobellus. The genomic DNA G+C content of strain CBA1103^T was determined to be 67.0 mol% a value which is in the range of the genomic DNA G+C content of members of the genus Halobellus (61.5–69.2 mol%). These results suggest that strain CBA1103^T should be considered to represent a new taxon for which the name Halobellus rufus sp. nov. is proposed, with the type strain CBA1103^T (=CECT 8423^T =JCM 19434^T).     

A novel moderately halophilic bacterium for decolorizing azo dye under high salt condition

Halomonas sp strain GTW was newly isolated from coastal sediments contaminated by chemical wastewater and was identified to be a member of the genus Halomonas by 16S rDNA sequence analysis and physical and biochemical tests. The optimal decolorization conditions were as follows: temperature 30°C, pH 6.5.0–8.5, NaCl 10–20% (w/v) and the optimal carbon source was yeast exact. The results of experiments demonstrated that the bacteria could decolorize different azo dyes under high salt concentration conditions, and the decolorization rate of five tested azo dyes could be above 90% in 24 h. The exploitation of the salt-tolerant bacteria in the bio-treatment system would be a great improvement of conventional biological treatment systems and the bio-treatment concept.     

Kocuria marina BS-15 a biosurfactant producing halophilic bacteria isolated from solar salt works in India

Biosurfactant screening was made among the eight halophilic bacterial genera isolated from Kovalam solar salt works in Kanyakumari of India. After initial screening, Kocuria sp. (Km), Kurthia sp. (Ku) and Halococcus sp. (Hc) were found to have positive biosurfactant activity. Biosurfactant derived from Kocuria sp. emulsified more than 50% of the crude oil, coconut oil, sunflower oil, olive oil and kerosene when compared to the other strains. Further, Kocuria marina BS-15 derived biosurfactant was purified and characterized by TLC, FTIR and GC–MS analysis. The TLC analysis revealed that, the purified biosurfactants belong to the lipopeptide group. The IR spectrum results revealed that functional groups are R₂CNN, alkenes and N–H. The GC–MS analysis confirmed the compound as Nonanoic acid and Cyclopropane with the retention time of 12.78 and 24.65, respectively.     

Photoactive yellow protein from the halophilic bacterium Salinibacter ruber

A gene for photoactive yellow protein (PYP) was identified from the genome sequence of the extremely halophilic aerobic bacterium Salinibacter ruber (Sr). The sequence is distantly related to the prototypic PYP from Halorhodospira halophila (Hh) (37% identity) and contains most of the amino acid residues identified as necessary for function. However, the Sr pyp gene is not flanked by its two biosynthetic genes as in other species. To determine as to whether the Sr pyp gene encodes a functional protein, we cloned and expressed it in Escherichia coli, along with the genes for chromophore biosynthesis from Rhodobacter capsulatus. The Sr PYP has a 31-residue N-terminal extension as compared to other PYPs that appears to be important for dimerization; however, truncation of these extra residues did not change the spectral and photokinetic properties. Sr PYP has an absorption maximum at 431 nm, which is at shorter wavelengths than the prototypical Hh PYP (at 446 nm). It is also photoactive, being reversibly bleached by either blue or white light. The kinetics of dark recovery is slower than any of the PYPs reported to date (4.27 x 10(-4) s(-1) at pH 7.5). Sr PYP appears to have a normal photocycle with the I1 and I2 intermediates. The presence of the I2' intermediate is also inferred on the basis of the effects of temperature and alchohol on recovery. Sr PYP has an intermediate spectral form in equilibrium with the 431 nm form, similar to R. capsulatus PYP and the Y42F mutant of Hh PYP. Increasing ionic strength stabilizes the 431 nm form at the expense of the intermediate spectral form, and the kinetics of recovery is accelerated 6.4-fold between 0 and 3.5 M salt. This is observed with ions from both the chaotropic and the kosmotropic series. Ionic strength also stabilizes PYP against thermal denaturation, as the melting temperature is increased from 74 degrees C in buffer alone to 92 degrees C in 2 M KCl. Sr accumulates KCl in the cytoplasm, like Halobacterium, to balance osmotic pressure and has very acidic proteins. We thus believe that Sr PYP is an example of a halophilic protein that requires KCl to electrostatically screen the excess negative charge and stabilize the tertiary structure.     

Identification, phylogenetic analysis and characterization of obligate halophilic fungi isolated from a man-made solar saltern in Phetchaburi province, Thailand

A total of 17 species from 43 isolates were obtained through serial dilutions of soil samples isolated from one of the man-made solar salterns located in Ban Laem district of Phetchaburi province, Thailand. Soil analysis of the sample revealed high salinity and moisture content, slight alkalinity and low amounts of nitrogen, total organic carbon and organic matter in the habitat. Morphological analysis was performed on all isolates, and molecular identification and phylogenetic analysis were carried out only on the halophilic fungi isolated. Six halophilic fungi, belonging to four species, were identified among the isolates, including five strains of Aspergillus genus [Aspergillus flavus, A. gracilis, A. penicillioides (2 strains) and A. restrictus]. One species was found to be a yeast, namely, Sterigmatomyces halophilus, which was the most frequent isolate found among the halophilic fungi. All other isolates were halotolerant fungi. Characterization of the halophilic fungal isolates showed that they were best adapted to conditions of 10–15 % NaCl (w/v), slight alkalinity (pH 7.0–7.5) and a temperature range of 30–35 °C.     

Halomonas aidingensis sp. nov., a moderately halophilic bacterium isolated from Aiding salt lake in Xinjiang, China

Two Gram-negative moderately halophilic bacterial strains, designated Ad-1(T) and C-12, were isolated from Aiding salt lake of Xinjiang in China. The novel isolates were subjected to a polyphasic taxonomic study. Cells of these strains were cocci or short rods and motile with polar flagella. Colonies produced brown-red pigment. The isolates grew in the range of 0.5-25% (w/v) NaCl, pH 5.5-10.5 and 4-45°C. Analysis of their 16S rRNA gene sequences indicated that strains Ad-1(T) and C-12 belonged to the genus Halomonas showing 92.7-98.4% similarity with the type species. The isoprenoid quinones of the isolates were Q-9 and Q-8. The major cellular fatty acids were C18:1ω7c, C16:1ω7c/6c, C16:0, C12:0-3OH and C10:0. The DNA G + C contents of strains Ad-1(T) and C-12 were 64.6 and 63.9 mol%, respectively. The DNA relatedness between the two isolates was 89.2%. The similarities of these newly isolated strains with closely related type strains were lower than 35% at the genetic level. Based on phenotypic, chemotaxonomic and genetic characteristics, the representative strain Ad-1(T) is considered to be a novel species of the genus Halomonas, for which the name Halomonas aidingensis sp. nov. is proposed, with Ad-1(T) (= CGMCC 1.10191(T) = NBRC 106173(T)) as the type strain.     

Characterization of halophilic alkaline phosphatase from Halomonas sp. 593, a moderately halophilic bacterium

A halophilic alkaline phosphatase was highly purified (about 510-fold with about 21% yield) from a moderate halophile, Halomonas sp. 593. The N-terminal 35 amino acid sequence of this enzyme was found to be more acidic than those previously isolated from Vibrio spp., and this enzyme was partially resistant to SDS. Several enzymatic properties demonstrated that it showed higher halophilicity than those enzymes from Vibrio spp.     

A new dehydrogenase specific towards aromatic aldehydes from a halophilic bacterium

A new enzyme showing a dehydrogenase activity towards aromatic aldehydes was isolated, purified and characterized from a halophilic strain isolated from saline environment. The enzyme is a monomer of 54 kDa; it is rather thermostable (optimal temperature: 50 degrees C) showing a broad spectrum of activity in a large pH range with the maximum at pH 9.5. The substrate specificity and the effect of ions were evaluated and compared with analogous described proteins.     

A bacteriophage of a moderately halophilic bacterium

A bacteriophage of an aerobic, gram-negative, rod-shaped halophilic bacterium, provisionally named Pseudomonas sp. G3, is described. The phage has a head and a tail and is similar in appearance to Salmonella phage Beccles. It infects its bacterial host at all salt concentrations in which the bacteirum is able to grow. In contrast to phages of halophilic archaebacteria, the newly-described phage is relatively stable in the absence of salt. It also infects Vibrio costicola and two unidentified halophilic eubacteria.Abbreviations PPT proteose peptone-tryptone medium - pfu plaque-forming unit - G+C guanine + cytidine content, mol %     

Glycine betaine transport in the obligate halophilic archaeon Methanohalophilus portucalensis

Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed K(t) of 23 microM and a V(max) of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [(14)C]betaine uptake. The proton-conducting uncoupler 2, 4-dinitrophenol and the ATPase inhibitor N, N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 microM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, alpha-glutamate, beta-glutamine, and N(epsilon)-acetyl-beta-lysine. These investigations demonstrate that this model halophilic methanogen, M. portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.     

Halomonas sinaiensis sp. nov., a novel halophilic bacterium isolated from a salt lake inside Ras Muhammad Park, Egypt

An alkalitolerant and halotolerant bacterium, designated strain Sharm was isolated from a salt lake inside Ras Muhammad. The morphological, physiological and genetic characteristics were compared with those of related species of the genus Halomonas. The isolate grew optimally at pH 7.0, 5–15% NaCl at 35°C. The cells were Gram-negative rods, facultative anaerobes. They accumulated glycine-betaine, as a major osmolyte, and ectoine and glutamate as minor components. The strain Sharm^T biosynthetised α-glucosidase. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and a novel phosphoglycolipid as major components. Ubiquinone with nine repetitive unities (Q9) was the only quinone found and, nC16:0 and C19:0 with cyclopropane were the main cellular fatty acids, accounting for 87.3% of total fatty acids. The G + C content of the genomic DNA was 64.7 mol %. The 16S rRNA sequence analysis indicated that strain Sharm was a member of the genus Halomonas. The closest relatives of the strain Sharm were Halomonas elongata and Halomonas eurihalina. However, DNA–DNA hybridisation results clearly indicated that strain Sham was a distinct species of Halomonas. On the basis of the evidence, we propose to assign strain Sharm as a new species of the genus Halomonas, H. sinaiensis sp. nov, with strain Sharm^T as the type strain (DSM 18067^T; ATCC BAA-1308^T). The EMBL accession number for the 16S rRNA sequence of Halomonas sinaiensis strain Sharm^T is AM238662.     

An extreme halophilic enzyme active at low salt in reversed micelles.

Possible biotechnological applications of extreme halophilic enzymes are strongly determined by their high salt requirement of around 4 M NaCl. Consequently, the use of these in organic media seemed to be unlikely. However, we have succeeded in dissolving a halophilic enzyme, p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum, in an organic medium by creating a reverse micellar system with very low salt concentration. The enzyme retained its catalytic properties in reversed micelles made with an anionic surfactant (dioctyl sodium sulphosuccinate) or with a cationic surfactant (hexadecyltrimethylammonium bromide) in cyclohexane plus 1-butanol as co-surfactant. The dependence of the rate of hydrolysis of p-nitrophenylphosphate phosphate on the molar water/surfactant ratio (w(0) value) showed a bell-shaped curve for each surfactant system. Kinetic parameters were determined in each system. The enzymatic reaction appeared to follow Michaelis-Menten kinetics with the anionic surfactant only. The kinetic behaviour was determined at different concentrations of Mn(2+) in reversed micelles of dioctyl sodium sulphosuccinate as surfactant.