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.     

Comparison of purple membrane from Halobacterium cutirubrum and Halobacterium halabium.

Direct comparison of purple membrane preparations from Halobacterium cutirubrum and Halobacterium halobium was carried out. Both preparations were found to be essentially identical with respect to their molecular weight, retinal content, lipid composition, fingerprinting of peptides from peptide digestion, electron micrographs and X-ray diffraction patterns, and behaviour as a light-activated proton pump. Thus, there would appear to be no species differences in the purple membranes from these two bacteria.     

The glycolipid of Halobacterium saccharovorum

Abstract Of the five Bacteriodes species of the 'fragilis group' only Bacteroides fragilis was able to grow in human plasma. Therefore the capacity of several iron sources to stimulate to growth of Bacteroides species under iron restricted conditions in vitro was tested. The iron chelator bipyridyl was used for the restriction of iron in the media. Ferrous sulphate, ferric ammonium sulphate and ferric citrate stimulated the growth of all five Bacteroides species tested to the same extent. B. fragilis , and to a lesser extent B.thetaiotaomicron and B. distasonis were better able than B. vulgatus and B. ovatus to use haem-compounds as an iron source in the presence of the iron chelator bipyridyl. All five Bacteroides species tested could use 30% iron-saturated transferrin. There was no correlation between the ability of the strains to grow in human plasma and the ability to use either haem-compounds of transferrin as a source of iron.     

Ecophysiology of Bacteriophage S5100 Infecting Halobacterium cutirubrum

Increasing salinity reduces burst size and increases the latent period of infection of Halobacterium cutirubrum by lytic bacteriophage S5100. Cells become reversibly and persistently infected at saturation-level concentrations of NaCl. We propose that high salinity provides a natural refuge for sensitive host bacteria and that phage S5100 acts as a scavenger, proliferating when host viability is threatened by dilution of the environment.     

The fate of thymine-containing dimers in ultraviolet-irradiated Halobacterium cutirubrum

Extremely halophilic bacteria do not recover in the dark from the effects of ultraviolet radiation, although they can be completely photoreactivated, thus proving that pyrimidine dimers are the cytotoxic photoproducts. We have now shown, by studying the fate of thymine-containing dimers during dark liquid-holding of Halobacterium cutirubrum, that the lack of dark repair in these bacteria is due to their inability to excise such lesions, in contrast to Escherichia coli B. Nevertheless, extensive nonspecific degradation of DNA occurs in H. cutirubrum following ultraviolet irradiation, so the lack of dimer excision is not due to a generalised inability to degrade DNA after such treatment. These findings raise interesting questions concerning the basis of the resistance of halophiles to ultraviolet radiation.     

Evidence for two restriction-modification systems in Halobacterium cutirubrum.

Data from plating experiments indicated that Halobacterium cutirubrum NRC34001 has at least two separate restriction-modification systems. A spontaneous or induced loss of one or both systems resulted in four restriction-modification phenotypes. There was a positive correlation between changes in gas vacuolation phenotypes and either restriction-modification system.     

Partial purification and properties of Halobacterium cutirubrum L-alanine dehydrogenase.

1. Halobacterium cutirubrum L-alanine dehydrogenase was purified approx. 100-fold. 2. It has a mol. wt. of 72 500, about one-third that of two well-studied alanine dehydrogenases from non-halophiles. 3. The activity of the enzyme increases with temperature up to 70 degrees C, but the protein itself is not thermostable. 4. In the reductive amination reaction, the enzyme is fully active in the presence of high concentrations of K+, Na+ or NH4+ and partially active with Cs+ or Li+, but for oxidative deamination it has an absolute requirement for K+.     

Superoxide dismutase from the extremely halophilic archaebacterium Halobacterium cutirubrum.

Halobacterium cutirubrum, a member of the archaebacteria, contains one superoxide dismutase (EC 1.15.1.1). This enzyme functions in the high-ionic-strength intracellular environment and protects the organism against the toxic effects of the superoxide anion. The enzyme has been purified to about 90% homogeneity by a four-step procedure which never removes it from conditions of high ionic strength. The subunits of the purified enzyme have a molecular weight of 25,000 and are possibly in tetrameric association. The enzyme shows anomalously high resistance to azide inhibition and sensitivity to inactivation by hydrogen peroxide. Metal analysis indicates 0.2 atom of Mn, less than 0.03 atom of Cu, and less than 0.001 atom of Fe per subunit. The low content of Mn may explain the low specific activity found for this enzyme compared with that of eubacterial enzymes. Optimum activity occurs in 2 M KCl; KCl gives about twice as much activity as NaCl over the range of 2 to 4 M. The enzyme appears to be related to those isolated from other archaebacteria but also exhibits several novel features.