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.

Application of protoplast transformation and single-and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na ؉ /H ؉ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na ؉ -sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na ؉ /H ؉ -deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na ؉ concentrations equal to or greater than 10 mM. Even at lower Na ؉ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na ؉ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na ؉ /H ؉ antiport activity available to extrude the Na ؉ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na ؉ /H ؉ antiporter, N13 exhibited a defect in diffusion potentialenergized efflux of 22 Na ؉ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na ؉ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na ؉ , comparable efflux was observed in preparations from all the strains.

Membrane vesicles of Clostridium thermoautotrophicum prepared by osmotic lysis after lysozyme treatment contained carbon monoxide dehydrogenase and methylenetetrahydrofolate dehydrogenase with specific activities three-to fourfold higher than the specific activity of the cytoplasm. The membrane-associated carbon monoxide dehydrogenase mediated the reduction with CO or the oxidation with CO2 of b-type cytochromes and other electron carriers in the membrane.Acetogenic bacteria, such as Clostridium thermoautotrophicum, grow heterotrophically on several sugars and autotrophically on H2-CO2, CO, and methanol-CO2 (3,23 C. thermoautotrophicum 701/5 was grown in anaerobic medium on glucose (14) under CO2 atmosphere at 59°C at pH 6.5 and harvested during exponential growth (A660, 1.5).Membrane vesicles were prepared by lysozyme treatment by the method of Kaback (11) modified by Otto et al. (16). A lysozyme concentration of 0.8 mg/ml and a temperature of 47°C were used during lysis of the cells. The membranes were washed with 50 mM potassium phosphate buffer (pH 7.0) containing 3 mM dithiothreitol and all metals present in the growth medium (14). The membranes were then stored at 40C in 50 mM potassium phosphate buffer containing 3 mM dithiothreitol and 20% glycerol. Membrane vesicles were made permeable with the addition of 1% toluene. Cell extracts and membranes were also prepared with the French pressure cell as described previously (9). Anaerobic conditions were applied throughout all procedures by using a type B anaerobic chamber (Coy Laboratory Products).H+-ATPase (9), carbon monoxide dehydrogenase (17), hydrogenase (18), formate dehydrogenase (13), methyleneH4folate reductase (2), and formyl-H4folate synthetase (19) were assayed as described previously. Protein was measured by the rose bengal dye-binding assay (5) in the presence of 0.2% Triton X-100 to solubilize the membrane proteins.

Membrane vesicles and the Fl-ATPase from Clostridium thermoaceticum were examined by electron microscopy. Fl-ATPase particles projecting from the vesicles have a diameter of 10 to 12 nm. The Fl-ATPase has an 3,3Y8b structure. The a and , subunits are most likely arranged in an alternating sequence around a central protein mass consisting of the -y and 8 subunits.Clostridium thermoaceticum is an obligate anaerobic thermophile that carries out homoacetogenic fermentation of sugars (10). It also grows autotrophically (8) by using a novel pathway for the incorporation of CO or CO2 into acetate (10,15). The ability of this bacterium to grow autotrophically, along with other findings (2, 5) has led to the suggestion that C. thermoaceticum can couple electron transport with ATP synthesis via an H+-translocating ATPase. In a previous report (6), we described the purification of the F1-ATPase from C. thermoaceticum and showed that it is an oligomer with an Mr of about 370,000 consisting of four different subunits in the apparent molar ratio of a33_Y8. The quaternary structure of this enzyme appears to be similar to that of the Fl-ATPases isolated from several respiratory organisms, (4,13,14), but different from that of the enzymes isolated from two nonrespiratory bacteria (3,17). In this report, we describe an electron microscopic study of the three-dimensional structure of the Fl-ATPase from C. thermoaceticum.The preparation of membrane vesicles and purified F1-ATPase from C. thermoaceticum was as described earlier (6), except that ATP (1 mM) was included in all buffers used for purification. Also, the final DEAE Figure 2 is an overview of a negatively stained sample, whereas Fig. 3 and 4 depict results of tilting experiments. Figure 2 shows that the F1-ATPase sample used for the electron microscopic analysis was homogeneous. The diameter of the particle types 1 to 4, assumed to be face views of F1-ATPase, is 11 + 1.5 nm. Type 6 particle projections are approximately 11 nm in the direction parallel to the long axis and 8 to 9 nm perpendicular to that axis and are interpreted to be projections of F1-ATPase particles attached to the support film in a tilted fashion. Type 5 projections are also interpreted to be views of attached F1-ATPase particles slightly tilted with respect to the most commonly seen face-view.Type 1 projections in Fig. 2 reveal six intensity maxima arranged in a circle at average intervals of approximately 60°. The center of these projections appears to be white, indicating the presence of a proteinaceous mass therein. The difference between type 1 and type 2 projections is that type 2 projections exhibit negative stain trapped in the center of the particles, causing a dark central dot. Type 3 is very similar to types 1 and 2; however, the six intensity maxima arranged in a circle in types 1 and 2 appear to be arranged into two groups, each group being made up of three intensity maxima arranged in a plane at 1200 intervals. One plane appears to be rotated 600 with respect to the other, thus giving r...