Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4.

Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.

Lipid Compositions in Escherichia coli and Bacillus subtilis During Growth as Determined by MALDI-TOF and TOF/TOF Mass Spectrometry.

Lipids in Escherichia coli and Bacillus subtilis were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and TOF/TOF tandem mass spectrometry. Lipids were extracted from bacterial cells using an equal volume mixture of dichloromethane, ethanol, and water, which formed a biphasic system with the lipids in the organic layer. The resulting mass spectra of the extracts from both bacteria showed a series of peaks corresponding to sodiated phospholipids - primarily phosphatidylethanolamines (PE) and phosphatidylglycerols (PG). The relative amounts of the phospholipids and the fatty acid compositions inferred from the spectra were in good agreement with previously reported values from GC/MS and thin-layer chromatography studies. E. coli and B. subtilis were easily differentiated by dissimilarities in the composition and relative amounts of the phospholipids present as well as by the presence of lysyl-phosphatidylglycerol and diglucosyl diglycerides solely in the B. subtilis mass spectra. Changes in lipid content in the bacteria during their growth phases were also monitored. In E. coli, the spectra indicated an increase in the amount of the unique C(cy-17) fatty acid (in which the fatty acid chain contains a cyclopropane ring) formed during exponential growth. During stationary growth, the spectra indicated an increase in the amount of saturated fatty acids. In B. subtilis, the phospholipid composition remained relatively unchanged during exponential growth, but the amount of PG slightly decreased while the amount of PE slightly increased during stationary growth. No significant changes were observed for the lysyl-phosphatidylglycerols or glycolipids during the exponential or stationary growth phases.