Biohydrogenation of C18 unsaturated fatty acids to stearic acid by a strain of Butyrivibrio hungatei from the bovine rumen.

AIMS: To identify a ruminal isolate which transforms oleic, linoleic and linolenic acids to stearic acid and to identify transient intermediates formed during biohydrogenation. METHODS AND RESULTS: The stearic acid-forming bacterium, isolated from the rumen of a grazing cow, was a Gram-negative motile rod which utilized a range of growth substrates including starch and pectin but not cellulose or xylan. From its 16S rRNA gene sequence, the isolate was identified as a strain of Butyrivibrio hungatei. During conversion of linoleic acid, 9,11-conjugated linoleic acid formed as a transient intermediate before trans-vaccenic acid accumulated together with stearic acid. Unlike previously studied ruminal biohydrogenating bacteria, B. hungatei Su6 was able to convert alpha-linolenic acid to stearic acid. Linolenic acid was converted to stearic via conjugated linolenic acid, linoleic acid and trans-vaccenic acid as intermediates. Oleic acid and cis-vaccenic acid were converted to a series of trans monounsaturated isomers as well as stearic acid. An investigation of these isomers indicated that mixed trans positional isomers are intermediate in the biohydrogenation of cis monounsaturated fatty acids to stearic acid. CONCLUSION: This, the first rigorous identification and characterization of a ruminal bacterium which forms stearic acid, shows that B. hungatei plays an important role in unsaturated fatty acid transformations in the rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: Biohydrogenating bacteria which convert C18 unsaturated fatty acids to stearic acid have not been available for study for many years. Access to B. hungatei Su6 now provides a fresh opportunity for understanding biohydrogenation mechanisms and rumen processes which lead to saturated fat in ruminant products.

Bioenergetics and solute uptake under extreme conditions.

The ion and particularly the proton and sodium ion permeabilities of cytoplasmic membranes play crucial roles in the bioenergetics of microorganisms. The proton and sodium permeabilities of membranes increase with temperature. Psychrophilic and mesophilic bacteria and mesophilic, (hyper)thermophilic, and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains constant (homeoproton permeability). Thermophilic bacteria are an exception. They rely on the less permeable sodium ions to generate a sodium motive force, which is subsequently used to drive energy-requiring membrane-bound processes. Transport of solutes across bacterial and archaeal membranes is mainly catalyzed by primary ATP-driven transport systems or by proton- or sodium-motive-force-driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary uptake systems. Several high-affinity ATP-binding cassette (ABC) transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.

Adaptations of the archaeal cell membrane to heat stress.

In extreme environments varying from hot to cold, acidic to alkaline, and highly saline, mainly Archaea are found. Thermophilic and extremely acidophilic Archaea have a membrane that contains membrane spanning tetraether lipids. These tetra-ether membranes have a limited permeability for protons even at the high temperatures of growth and this property makes it possible for thermophilic archaea to maintain a viable proton motive force under the extreme conditions. -Ether lipids cannot be degraded easily and are highly stable which is also a requirement for life under extreme conditions. Psychrophilic and mesophilic Bacteria, and all Archaea adjust the lipid composition of their membranes so that the proton permeability of their membranes remains within a narrow range. This phenomenon is termed 'homeoproton permeability adaptation'. Thermophilic Bacteria are the only prokaryotes that are unable to control the proton permeability of their membranes. These organisms have to rely on the less permeable sodium ions in energy transducing processes in their membrane.

Lipid membranes from halophilic and alkali-halophilic Archaea have a low H+ and Na+ permeability at high salt concentration.

The influence of pH and the salt concentration on the proton and sodium ion permeability of liposomes formed from lipids of the halophile Halobacterium salinarum and the haloalkaliphile Halorubrum vacuolatum were studied. In contrast with liposomes formed from Escherichia coli lipids, liposomes formed from halophilic lipids remained stable up to 4M of NaCl and KCl. The proton permeability of the liposomes from lipids of halophiles was independent of the salt concentration and was essentially constant between pH 7 and pH 9. The sodium ion permeability increased with the salt concentration but was 10- to 100 fold lower than the proton permeability. It is concluded that the membranes of halophiles are stable over a wide range of salt concentrations and at elevated pH values and are well adapted to the halophilic conditions.

Homeostasis of the membrane proton permeability in Bacillus subtilis grown at different temperatures.

Bacillus subtilis was grown at its growth temperature limits and at various temperatures in between the lower and upper growth temperature boundary. Liposomes were made of the extracted membrane lipids derived from these cells. The headgroup composition of the cytoplasmic membrane lipids did not differ significantly at the lower (13 degrees C) and upper (50 degrees C) temperature boundary. The averaged lipid acyl chain length, degree of saturation, and ratio of iso- and anteiso-branched fatty acids increased with the temperature. At the temperature of growth, the membranes were in a liquid-crystalline phase, but liposomes derived from cells grown at 13 degrees C were almost threefold more viscous than those derived from 50 degrees C grown cells. The temperature dependence of the proton permeability of the liposomes was determined using the acid-pulse method with monitoring of the outside pH with the fluorescent probe pyranine. The proton permeability of each liposome preparation increased with the temperature. However, the proton permeability of the liposomes at the growth temperature of the cells from which the lipids were derived was almost constant. These data indicate that the growth temperature dependent variation in lipid acyl chain composition permits maintenance of the proton permeability of the cytoplasmic membrane. This 'homeo-proton permeability adaptation' precludes futile cycling of protons at higher growth temperatures and allows cells to sustain the proton motive force as a driving force for essential energy transducing processes.

The essence of being extremophilic: the role of the unique archaeal membrane lipids.

In extreme environments, mainly Archaea are encountered. The archaeal cytoplasmic membrane contains unique ether lipids that cannot easily be degraded, are temperature- and mechanically resistant, and highly salt tolerant. Moreover, thermophilic and extreme acidophilic Archaea possess membrane-spanning tetraether lipids that form a rigid monolayer membrane which is nearly impermeable to ions and protons. These properties make the archaeal lipid membranes more suitable for life and survival in extreme environments than the ester-type bilayer lipids of Bacteria or Eukarya.

Sodium-coupled energy transduction in the newly isolated thermoalkaliphilic strain LBS3.

Strain LBS3 is a novel anaerobic thermoalkaliphilic bacterium that grows optimally at pH 9.5 and 50 degrees C. Since a high concentration of Na+ ions is required for growth, we have analyzed the primary bioenergetic mechanism of energy transduction in this organism. For this purpose, a method was devised for the isolation of right-side-out membrane vesicles that are functional for the energy-dependent uptake of solutes. A strict requirement for Na+ was observed for the uptake of several amino acids, and in the case of L-leucine, it was concluded that amino acid uptake occurs in symport with Na+ ions. Further characterization of the leucine transport system revealed that its pH and temperature optima closely match the conditions that support the growth of strain LBS3. The ATPase activity associated with inside-out membrane vesicles was found to be stimulated by both Na+ and Li+ ions. These data suggest that the primary mechanism of energy transduction in the anaerobic thermoalkaliphilic strain LBS3 is dependent on sodium cycling. The implications of this finding for the mechanism of intracellular pH regulation are discussed.

Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea.

Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is the sole energy-coupling ion. To test whether sodium is the preferred coupling ion at high temperatures, the proton- and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature. The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures. Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature. In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature. The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.