ALBERT

Notes: Abstract The cellulolytic enzyme complex from Clostridium thermocellum JW 20 was purified from the cellulose to which the enzyme was bound during growth. After centrifugation and gel filtration the enzyme complex was analyzed by SDS-PAGE. Three subunits with apparent molecular weights of 195 000 Da, 97 000 Da and 72 000 Da were purified by preparative SDS-PAGE and electroelution. Polyclonal antibodies directed against these three subunits were raised in rabbits. The specificity of the antisera was tested with immunochemical methods. Cross reactions with other subunits of the cellulase complex were observed. Immunoelectron microscopy of protein-A gold labeled, resin embedded cells indicated that the three types of subunits were located in the outer region of the cytoplasm and on structures at the outside of the cell wall.

Notes: Abstract Cells of Pseudomonas carboxydovorans from the exponential growth phase revealed the major portion (87%) of CO dehydrogenase attached to the inner aspect of the cytoplasmic membrane. In stationary cells only about half of the total amount of the enzyme remained membrane-bound, and a drop of the CO-oxidizing activity with O2 was observed. The CO-oxidizing activity with the unphysiological electron acceptor methylene blue, which does not need any contact of the enzyme with the membrane, always exceeded that with O2. Measurements of respiration rates of extracts with different electron donors in addition to CO suggested that the electron transport chain is not rate-limiting. It is concluded that the electron flow from CO to O2 in intact cells of P. carboxydovorans is controlled by the amount of CO dehydrogenase attached to a membrane-bound electron acceptor.

Notes: Abstract The membrane-bound hydrogenase was localized in cells of Alcaligenes eutrophus by electron microscopic immunocytochemistry. Post-embedding labeling performed on ultrathin sections revealed that the enzyme was located predominantly (80%) at the cell periphery in autotrophically and heterotrophically grown bacteria harvested from the exponential phase of growth. In the stationary growth phase, however, only 50% of the enzyme was found at the cell periphery; the remaining 50% was distributed over the cytoplasm. The relative amount of electron microscopic label per cell as seen by application of the protein A—gold technique was higher in cells grown autotrophically as compared to cells grown heterotrophically on fructose. Derepression of the enzyme was followed electron microscopically in a substrate-shift experiment (growth on fructose, followed by a shift to glycerol). Major amounts of the enzyme appeared to undergo a reattachment to the cytoplasmic membrane under these conditions, starting with a reduced location of the enzyme in the cytoplasm and an accumulation in cell areas close to the cytoplasmic membrane. These findings indicate that the ‘membrane-bound’ hydrogenase (i.e., that material enriched as membrane-bound enzyme according to the appropriate activity test) is not, in fact, membrane bound or membrane integrated but membrane associated. It may or may not interact with the cytoplasmic face of the cytoplasmic membrane, depending on the growth phase and conditions.

Notes: Marked changes in cell envelope structure were observed when Clostridium sp. strain EM1 cells grown in continuous culture under glucose limitation were compared with cells grown under starch limitation. The increase in the level of extracellular α-amylase and pullulanase during starch-limited growth was parallelled by degradation of the cell-envelope layer and the formation of blebs and a high number of vesicles, which apparently originated from the cytoplasmic membrane. These vesicles were covered by a delicate layer of small particles and were shown to be released into the culture fluid. It is assumed that the overproduction of enzyme destined to remain associated with the cells led to these changes.

Notes: Abstract Most bacteria lack obvious compartmentation, i.e., structural partition of the cell into functional entities (organelles) formed by a closed biological membrane. Nevertheless, these organisms exhibit sophisticated regulation and interactions of their catabolic and anabolic pathways; they are able to exploit a great variety of carbon and energy sources, and they conserve and transform energy in an efficient manner. In a less stringent sense, ‘compartments’ are also present in bacteria if one accepts that bacterial ‘compartments’ are not necessarily surrounded by a membrane, but are rather defined as mere functional entities characterized by their structural components, their enzymes and other functional proteins such as binding proteins. This view would mean that the bacterial cell can be described as a highly organized structured system comprised of these functional entities. Regulated transport processes within ‘compartments’ and across boundaries involving low and high molecular mass compounds, solutes, and ions take place within the ‘framework’ constituted by this structured system. Special emphasis is given to the fact that many of the transport processes take place involving the functional entity ‘energized membrane’. This ‘framework’, the structural basis for the functional potential of a bacterial cell, can be studied by electron microscopy. Advanced sample preparation techniques and imaging modes are available which keep the danger of artefact formation low; they can be applied at cellular and macromolecular levels. Recent developments in immunoelectron microscopy and affinity labelling techniques provide tools which allow to unequivocally locate enzymes and other antigens in the cell and to identify polypeptide chains in enzyme complexes. Application of these approaches in studies on cellular and macromolecular organization of bacteria and their enzyme systems confirmed some old views but also extended our knowledge. This is exemplified by a description of selected enzyme complexes located in the bacterial cytoplasm, in the cytoplasmic membrane or attached to it, in the periplasmic space, and attached to the cell wall or set free into the surrounding medium.