Effect of glycerol on carotenogenesis in the extreme halophile, Halobacterium cutirubrum

Growth of cells of Halobacterium cutirubrum in complex medium in the presence of 0.1 to 0.5% glycerol was slightly stimulated (about 10%), but the content of the major red pigment, bacterioruberin, was reduced fourfold and that of the minor red pigments, mono- and bisanhydrobacterioruberins, was also reduced but to a lesser extent. The content of the C40 carotenes, beta-carotene and lycopene, however, was greatly increased in 0.1 to 0.5% glycerol but was then reduced to negligible amounts at higher glycerol concentrations (1 to 5%). Similar effects of glycerol on carotenogenesis were observed when cells were grown in a chemically defined medium (BSMK), but growth of cells was considerably slower.     

Mechanism of dissolution of envelopes of the extreme halophile Halobacterium cutirubrum

Onishi, H. (National Research Council, Ottawa, Ontario, Canada), and D. J. Kushner. Mechanism of dissolution of envelopes of the extreme halophile Halobacterium cutirubrum. J. Bacteriol. 91:646-652. 1966.-Envelopes of Halobacterium cutirubrum dissolved rapidly in media of low ionic strength. Heating partially inhibited breakdown, probably because of nonspecific protein coagulation rather than inactivation of a lytic enzyme(s). Dissolution of envelopes in water did not involve splitting of peptide bonds or protein-lipid bonds, or any extensive breakdown of carbohydrate polymers. Dissolution was increased by alcohols and urea, even at high salt concentrations, but was not affected by metabolic inhibitors. Thus, no evidence was found for a dilution-activated lytic enzyme that contributes to envelope breakdown. Cells of H. cutirubrum were stable in 2 m NaCl, but lysis occurred in 2 m KCl or NH(4)Cl. This lysis did not involve an extensive breakdown of the envelope. No evidence for different sites of Na(+), K(+), and NH(4) (+) action was obtained from the pattern of release of envelope constituents in different concentrations of these salts. Ultracentrifugation studies showed that adding salts to envelopes that had been dissolved in water led to a nonspecific reaggregation of envelope material. No difference was seen between the effects of KCl and NaCl, except at 3 to 4 m concentrations where KCl caused more aggregation. The preferential effect of Na(+) on intact cells is probably due to its ability specifically to prevent leakage rather than to an overall effect on envelope integrity.     

8-Hydroxy-5-deazaflavin-dependent electron transfer in the extreme halophile Halobacterium cutirubrum

Abstract Cell extracts of the extreme halophile Halobacterium cutirubrum were found to contain 8-hydroxy-5-deazaflavin as well as 8-OH-5-deazaflavin: NADPH oxidoreductase activity. The oxidoreductase was partially purified and showed maximum activity at pH 5.4, which is unusually low for halobacteria, and 5.3 M NaCl, close to the intracellular salt concentration. The results indicate the presence of an 8-OH-5-deazaflavin-dependent electron transfer system in a nonmethanogenic organism.     

Structure determination of the glycolipid sulfate from the extreme halophile Halobacterium cutirubrum

A sulfur-containing glycolipid, accounting for ca. 25% of the total polar lipids, has been isolated from the extreme halophile Halobacterium cutirubrum. The ammonium salt of the lipid was found to have the molecular formula C(61)H(117)O(21)S.NH(4), and on strong acid hydrolysis it yielded 2,3-di-O-phytanyl-sn-glycerol, glucose, mannose, galactose, and sulfate in equimolar proportions. Infrared and NMR spectra indicated the presence of a secondary sulfate group. Solvolysis of the lipid in 0.004 m HCl in tetrahydrofuran resulted in rapid release of inorganic sulfate and formation of galactosyl-mannosyl-glucosyl diphytanyl glycerol ether. With higher acid concentration (0.25 m methanolic HCl), stepwise hydrolysis of monosaccharide units occurred, giving mannosyl-glucosyl glycerol diphytanyl ether and monoglucosyl glycerol diphytanyl ether. The position of attachment of the sugars and of the sulfate group was determined by methylation of the free acid form of the glycolipid sulfate, followed by acid hydrolysis and gas-liquid chromatographic analysis of the partially methylated sugars as the alditol acetates. The configuration of the glycosidic linkages was established both by optical rotation measurements and by specific enzymatic hydrolysis. The results obtained established the structure as 2,3-di-O-phytanyl-1-O-[beta-d-galactopyranosyl-3'-sulfate-(1' -->6')-O-alpha-d-mannopyranosyl-(1' --> 2')-O-alpha-d-glucopyranosyl]-sn-glycerol.     

Some properties of the citrate synthase from the extreme halophile, Halobacterium cutirubrum.

1. Citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7] was purified about 400-fold from the extreme halophile, Halobacterium cutirubrum, by a method involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite and gel filtration on Sephadex G-200. 2. The purified enzyme was best activated by high concentrations of KCl (3M); the chlorides of other cations and K+ salts of other anions (Br-, NO3-, SCN-) were less effective than KCl as activators. The enzyme was best stabilized by high concentrations of NaCl or KCl. Cold-lability was found in the presence of 3M-KCl, but not in the presence of NaCl at concentrations up to 5M. The results suggest that both the shielding of negative charges on the enzyme molecule and the stabilization of hydrophobic bonds by high KCl concentrations were required for maximum activity of the enzyme. 3. The double-reciprocal plots for acetyl-CoA or oxaloacetate at several concentrations of the co-substrate intersected at the abscissa in the presence of either KCl or NaCl, at either 1 or 3M. The Km for oxaloacetate increased about fivefold with the salt concentration, from 1 to 3M.     

The 5-S RNA . protein complex from an extreme halophile, Halobacterium cutirubrum. Purification and characterization.

A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Halobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamide gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.     

The effects of ultraviolet radiation on the moderate halophile Halomonas elongata and the extreme halophile Halobacterium salinarum

Both the moderately halophilic bacterium, Halomonas elongata, and the extremely halophilic archaea, Halobacterium salinarum, can be found in hypersaline environments (e.g., salterns). On complex media, H. elongata grows over a salt range of 0.05-5.2 M, whereas, H. salinarum multiplies over a salt range of 2.5-5.2 M. The purpose of this study was to illustrate the effect that solar (UV-A and UV-B) and germicidal radiation (UV-C) had on the growth patterns of these bacteria at varied salt concentrations. Halomonas elongata grown on a complex medium at 0.05, 1.37, and 4.3 M NaCl was found to be more sensitive to UV-A and UV-B radiation, as the salt concentration of the medium increased. Halobacterium salinarum grown on a complex medium at 3.0 and 4.3 M NaCl did not show a significant drop in viability after 39.3 kJ.m-2 of UV-A and UV-B exposure. When exposed to UV-C, H. elongata exhibited substantially more sensitivity than H. salinarum. In H. elongata, differential sensitivity to UV-C was observed. At 0.05 M NaCl, H. elongata was less sensitive to UV-C than at 1.37 and 4.3 M NaCl. Both bacteria showed some photoreactivation when incubated under visible light following both UV-A, UV-B, and UV-C exposure. Mutagenesis following UV-C exposure was demonstrated by both organisms.     

The membrane lipids of Halobacterium halobium

The lipid content of the cell membrane of Halobacterium halobium increased from about 15% to 21% during exponential growth of the organism. Total lipid phosphorus more than doubled during the growth cycle. The mixture of membrane lipids from stationary-phase organisms was similar to lipid mixtures from whole cells of other halobacteria inasmuch as 80% of the lipid phosphorus occurred in a diether analogue of phosphatidylglycerophosphate and an additional 7.5% occurred in the ether analogue of phosphatidylglycerol. The lipid mixture was more complex than those reported for other halophils, however, 12 components being recognized in the acetone-insoluble fraction and 17 in the acetone-soluble fraction. There were major changes in the proportions of some minor components of the acetone-insoluble fraction during a growth cycle. Three nitrogenous lipids were recognized in the acetone-insoluble fraction, but all were present in relatively low proportion. One, which was not a phospholipid, contained a bound peptide. Of the 17 acetonesoluble compounds, 15 were pigments. The major carotenoids were alpha- and beta-bacteriorubrin. The carotenoid pigments occurred at maximal concentration after 6-7 days' growth.     

Surface characteristics of phosphatidylglycerol phosphate from the extreme halophile Halobacterium cutirubrum compared with those of its deoxy analogue, at the air/water interface.

The relationship between area per molecule and surface pressure of monolayers of phosphatidylglycerol phosphate from extreme halophile Halobacterium cutrirubrum and its deoxy analogue, deoxyphosphatidylglycerol phosphate, spread at an air/water interface was examined. The effect of ionization of the primary and secondary acidic functions of the phosphate groups of the two lipids on surface characteristics of compression isotherms was determined by spreading monolayers on subphases with pH values ranging from below the apparent pKa of the primary ionization (pH 0) to greater than that of secondary ionization (pH 10.9). The limiting molecular area increases with decreasing pH below 2. Ionization of the primary phosphate functions of both phospholipids (with bulk pK1 values close to 4) is associated with a marked expansion of the films, as judged by values of limiting molecular area. Ionization of the secondary phosphate functions causes further expansion of the films, with the apparent pK2 of deoxyphosphatidylglycerol phosphate slightly less than that indicated for phosphatidylglycerol phosphate. Values of surface-compressibility modulus calculated from the surface characteristics of the phosphatidylglcerol phosphate monolayers showed that films spread on subphases with a pH of about the apparent pK1 of the primary phosphate functions were the least compressible. Increasing or decreasing subphase pH caused an increase in compressibility; this effect on compressibility was much less with monolayers of deoxyphosphatidylglycerol phosphate at high pH. The effect of inorganic counter-ions on monolayer characteristics of phosphatidylglycerol phosphate was examined by using subphases of NaCl concentrations varying from 0.01 to 1 M. The limiting molecular area was found to increase exponentially with respect to the subphase NaCl concentration.     

Solution studies of elongation factor Tu from the extreme halophile Halobacterium marismortui.

The activity, stability and structure in solution of polypeptide elongation factor hEF-Tu from Halobacterium marismortui have been investigated. The protein is stable in aqueous solutions only at high concentrations of NaCl, KCl or ammonium sulphate, whereas it is more active in exchanging GDP at lower salt concentrations. It is more active and stable at lower pH values than is non-halophilic EF-Tu. The structure in solution of the protein was determined by complementary density, ultracentrifugation, dynamic light-scattering and neutron-scattering measurements. The protein has large hydration interactions, similar to those of other halophilic proteins: 0.4 (+/- 0.1) g of water and 0.20 (+/- 0.05) g of KCl associated with 1 g of protein, with a water/KCl mass ratio always remaining close to 2. The kinetics of inactivation at low salt concentrations showed a stabilizing effect of NaCl when compared to KCl. At low salt concentration, inactivation, protein unfolding and aggregation were strongly correlated. The results suggest that the stabilization model proposed for halophilic malate dehydrogenase by Zaccai et al., involving extensive protein interactions with hydrated salt ions, is also valid for hEF-Tu.