Macromolecular organization of F1-ATPase isolated from Clostridium thermoaceticum as revealed by electron microscopy

Abstract

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...

Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

“…With cellulolytic organisms such as Tichoderma reesei, Neocallismastix frontalis, Pseudomonas fluorescens, Cellulomonas fimi, Fibrobacter succinogenes, and Ruminococcus flavefaciens, it is not certain whether the enzymes essential for the degradation of crystalline cellulose function individually or whether they are present on the cell or substrate surface as a complex (1,15,20). There is evidence that in Clostridium thernocellum as well as in Clostridium strain C7, the enzymes are present in a complex, termed the "cellulosome" (3, 18,19,22,23). There is evidence that the enzymes present in Clostndium cellulovorans are also present in a complex.…”

Characterization of the extracellular cellulase from a mesophilic clostridium (strain C7)

“…The strain C7 cylindrical particles seem to be composed of two rings of subunits with a cleft or groove visible at the juncture of the two rings. Similar flattened cellulosomes, consisting of subunits arranged so that they formed two halves of the cellulosome separated by a groove, were observed by Mayer and co-workers (18) in cultures of C. thermocellum JW20. The existence of similar ultrastructural features in multiprotein complexes from different cellulolytic bacteria suggests that a particular arrangement of the proteins within the complexes is required for crystalline cellulose hydrolysis.…”

“…The particles observed by electron microscopy in negatively stained preparations of the 700,000-Mr enzyme complex of strain C7 are similar in ultrastructure to cellulosomes of C. thermocellum (6,13,18). Both the strain C7 particles and the cellulosomes are composed of subunits.…”

Paenibacillus curdlanolyticus Strain B-6 Multienzyme Complex: A Novel System for Biomass Utilization

“…The observation of cell surfaces at the late exponential growth phase by scanning electron microscopy (SEM) revealed that the cells adhered to xylan (Fig. 5A), similar to the cells of the cellulosome producing anaerobic bacterium, C. thermocellum, which is a cell associated entity that mediates the adhesion of the bacterium to cellulose (Lamed et al, 1987;Mayer et al, 1987), whereas the surface of the cells of strain B-6 at the late stationary growth phase lacked such structures because the multienzyme complex was released into the medium from the cell surfaces (Fig. 5B).…”