Bacillus subtilis, which grows under aerobic conditions, employs fatty acid desaturase (Des) to fluidize its membrane when subjected to temperature downshift. Des requires molecular oxygen for its activity, and its expression is regulated by DesK-DesR, a two-component system. Transcription of des is induced by the temperature downshift and is decreased when membrane fluidity is restored. B. subtilis is also capable of anaerobic growth by nitrate or nitrite respiration. We studied the mechanism of cold adaptation in B. subtilis under anaerobic conditions that were predicted to inhibit Des activity. We found that in anaerobiosis, in contrast to aerobic growth, the induction of des expression after temperature downshift (from 37 degrees C to 25 degrees C) was not downregulated. However, the transfer from anaerobic to aerobic conditions rapidly restored the downregulation. Under both aerobic and anaerobic conditions, the induction of des expression was substantially reduced by the addition of external fluidizing oleic acid and was fully dependent on the DesK-DesR two-component regulatory system. Fatty acid analysis proved that there was no desaturation after des induction under anaerobic conditions despite the presence of high levels of the des protein product, which was shown by immunoblot analysis. The cold adaptation of B. subtilis in anaerobiosis is therefore mediated exclusively by the increased anteiso/iso ratio of branched-chain fatty acids and not by the temporarily increased level of unsaturated fatty acids that is typical under aerobic conditions. The degrees of membrane fluidization, as measured by diphenylhexatriene fluorescence anisotropy, were found to be similar under both aerobic and anaerobic conditions.

• MeSH
• Aerobiosis MeSH
• Anaerobiosis MeSH
• Bacillus subtilis metabolism   physiology   MeSH
• Bacterial Proteins biosynthesis   MeSH
• Cell Membrane chemistry   MeSH
• Fatty Acid Desaturases biosynthesis   MeSH
• Membrane Fluidity MeSH
• Adaptation, Physiological * MeSH
• Fatty Acids metabolism   MeSH
• Cold Temperature * MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Fatty Acid Desaturases MeSH
• Fatty Acids MeSH

The temperature dependence of fluorescence anisotropy, lifetime and differential tangent of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its polar trimethylammonium derivative (TMA-DPH) were investigated in cytoplasmic membranes of Bacillus subtilis. The fluorescence parameters were compared in the two types of membranes prepared from bacteria cultivated at 20 and 40 degrees C. Steady-state anisotropy measurements showed that within a broad range of temperatures, membranes cultivated at 20 degrees C exhibit significantly lower values than those prepared from cells cultivated at 40 degrees C. The temperature dependence of lifetime and differential tangent measurements (differential polarized phase fluorimetry) were fully consistent with steady-state anisotropy data of both DPH and TMA-DPH. The low anisotropy values in the case of TMA-DPH could be explained by a shorter lifetime and higher temperature-induced decrease as compared with DPH. Surprisingly, the temperature dependence of rotational rate R calculated according to the model of hindered rotations (Lakowicz 1983) gave misleading results. When increasing the temperature from 5 to 25 degrees C, a marked drop of rotational relaxation rate was observed. The minimum R values were measured between 25 and 30 degrees C and further increase of temperature (up to 60 degrees C) was reflected as increase of the R values. Therefore, a new model of "heterogeneous rotations" was developed. This model assumes that even at low temperatures (approaching 0 degrees C) where the differential tangent reaches zero, a fraction of fast rotating molecules exists. The ratio between fast and slowly rotating molecules may be expressed by this model, the newly calculated rotational rates are fully consistent with anisotropy, lifetime and differential tangent measurements and represent the monotonically increasing function of temperature.

• MeSH
• Bacillus subtilis metabolism   ultrastructure   MeSH
• Cell Membrane metabolism   MeSH
• Diphenylhexatriene analogs & derivatives   metabolism   MeSH
• Fluorescence Polarization MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Diphenylhexatriene MeSH

alpha-Amylase was found to be the main protein secreted by Bacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45 degrees C, respectively. A change in temperature can affect the pattern of proteins secreted as detected by gel electrophoresis. 14C-Leucine incorporation into extracellular proteins and their proportion at the end of the growth phase was higher at 30 degrees C than that at 40 or 45 degrees C. The effect of temperature on alpha-amylase synthesis as determined by its enzymic activity and on the extracellular protein synthesis followed a similar pattern.

• MeSH
• alpha-Amylases biosynthesis   metabolism   MeSH
• Bacillus subtilis enzymology   metabolism   MeSH
• Bacterial Proteins biosynthesis   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Leucine metabolism   MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• alpha-Amylases MeSH
• Bacterial Proteins MeSH
• Leucine MeSH

The validity of the principle of homeoviscous adaptation for Bacillus subtilis was tested by comparing fluorescence anisotropy (1,6-diphenyl-1,3,5-hexatriene) and electron-spin resonance (16-doxylstearate) measurements carried out in isolated plasma membranes and in phospholipid fractions. The physical measurements were supplemented by fatty-acid analysis. The results support our previous findings on intact cells. The thermoadaptive mechanism of B. subtilis manifested as an increase in relative proportion of branched anteiso-C15 and anteiso-C17 fatty acids, are not strong enough to compensate for the marked physical change of membrane fluidity induced by temperature decrease.

• MeSH
• Bacillus subtilis analysis   physiology   MeSH
• Electron Spin Resonance Spectroscopy MeSH
• Membrane Fluidity * MeSH
• Fluorescence Polarization MeSH
• Phospholipids analysis   MeSH
• Adaptation, Physiological * MeSH
• Fatty Acids analysis   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phospholipids MeSH
• Fatty Acids MeSH