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Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite.

The cell wall of Sulfolobus acidocaldarius has been isolated. Cells were mechanically disrupted with a French press, and the cytoplasmic membrane was removed by extracting cell-envelope fragments with Triton X-100. The Triton-insoluble cell wall material retained the characteristic subunit structure when examined in the electron microscope. Isolated cell wall fragments formed in open sheets that were easily separated from cytoplasmic contamination. Chemical studies showed that the Triton-insoluble cell wall fragments consisted of lipoprotein with small amounts of carbohydrate and hexosamine. The amino acid composition indicated a highly charged hydrophobic cell surface. The presence of diaminopimelic acid with only traces of muramic acid indicates that the cell envelope does not have a rigid peptidoglycan layer. The results of chemical analyses and electron microscopy suggest a wall-membrane interaction stabilizing the cell envelope. The chemical and physical properties of this type of cell envelope would appear to form the basis for a new major division of bacteria with the definitive characteristics of a morphologically distinct subunit cell wall devoid of peptidoglycan.

A variety of temperature-sensitive mutants of host-dependent Bdellovibrio bacteriovorus 109D were selected after ethyl methane sulfonate mutagenesis. Mutants that demonstrated plaque-forming ability reversion frequencies of 10(-8) to 10(-9) were chosen for further study. Representatives of these mutants were then characterized by phase-contrast and electron microscopy, temperature-shifted one-step growth experiments, attachment kinetics, and macromolecular capabilities. Representative mutants demonstrate various types of blockage corresponding to the previously described morphological stages of Bdellovibrio predatious life cycle, i.e., attachment blockage (109D153), penetration blockage (109D3 and 109D48), and blockage of intracellular growth (109D4 and 109D152). The time of release from temperature repression for the mutant classes was found to correspond to the apparent morphological stage of blockage via temperature-shifted, one-step growth experiments. Mutants characterized as exhibiting blockage in the penetration or intracellular stages of the infection cycle exhibited, at the permissive and nonpermissive temperatures, kinetics of attachment to Escherichia coli WP2 similar to those of the wild type. One mutant, 109D153, exhibited depressed attachment at the restrictive temperature even though the Bdellovibrio cells were motile. The extent of 38.5 C attachment of 109D153 to E. coli is at the same level as that of wild-type 109D to Bacillus subtilis, a gram-positive, non-host organism. Subsequent detachments were revealed in the wild-type 109D-B. subtilis or mutant 109D153-Escherichia coli (38.5 C) cultures. These studies reveal a biphasic attachment phenomenon in the early interaction of Bdellovibrio with its host. It appears that, at the restrictive temperature, 109D153 is capable only of the initial, nonspecific type of attachment.

The nuclear apparatus of Hyphomicrobium sp. strain B-522 is examined by various microscopy and radiolabeling techniques to determine its behavior during the reproductive cycle of these bacteria. The young, swarmer cell contains a single nucleoid comprised of a deoxyribonucleic acid (DNA) molecule with a molecular weight of 3.1 x 10(9). After development of the swarmer into a mature mother cell with a hypha and bud, the nucleoid replicates and separates into two daughter nucleoids during the initial stages of bud formation. After further development of the bud, one of the daughter nucleoids in the mother cell is rapidly transferred through the hypha to the bud. Half of the old DNA strands pass to each consecutive generation of daughter cells, but only 43% of the stable ribonucleic acid is transferred. The role which the hypha plays in the developmental cycle of these bacteria is discussed, and a mechanism for nuclear transfer is proposed.

The behavior of a number of motile flagellated bacteria toward viscosity characteristics of their fluid environments was observed. All showed an increase in velocity (micrometers per second) in more viscous solutions. Velocity reached a maximum at a characteristic value, however, and thereafter decreased with higher viscosities. Peritrichously flagellated bacteria had maximum velocities at higher viscosities than polarly flagellated bacteria. Effects of temperature, and possible utilization of chemical constituents in the viscous solutions, were studied and found to be negligible factors under the experimental conditions used. Different agents produced the same phenomenon, thus indicating that there probably were no chemically induced metabolic effects. Loss of available water and the possibility of a variable energy supply to the flagellar propulsive system were considered but are believed minimal. Theoretically derived thermodynamic equations were utilized and suggest that the conformation of the flagellar helix affects efficiency of propulsion. Such a relationship between helix waveform and velocity was experimentally observed with Thiospirillum jenese.

The cell walls of three species of rumen bacteria (Bacteroides ruminicola, Bacteroides succinogenes, and Megasphaera elsdenii) were studied by a variety of morphological methods. Although all the cells studied were gram-negative and had typical cytoplasmic membranes and outer membranes, great variation was observed in the thickness of their peptidoglycan layers. Megasphaera elsdenii evidenced a phenomenally thick peptidoglycan layer whose participation in septum formation was very clearly seen. All species studied have cell wall "coats" external to the outer membrane. The coat of Bacteroides ruminicola is composed of large (approximately 20 nm) globules that resemble the protein coats of other organisms, whereas the coat of Bacteroides succinogenes is a thin and irregular carbohydrate coat structure. Megasphaera elsdenii displays a very thick fibrillar carbohydrate coat that varies in thickness with the age of the cells. Because of the universality of extracellular coats among rumen bacteria we conclude that the production of these structures is a protective adaptation to life in this particular, highly competitive, environment.

Chemotaxis toward yeast extract is demonstrated in obligately and facultatively parasitic strains of Bdellovibrio bacteriovorus.

Treatment of the spirochete-like Sex Ratio Organism with sodium deoxycholate results in the release of fibrils having a diameter of 3.6 nm and a period of 9 nm.

The relationship between chromosome replication and morphogenesis in the budding bacterium Hyphomicrobium neptunium has been investigated. Nalidixic acid was found to completely inhibit deoxyribonucleic acid synthesis, but not ribonucleic acid synthesis. The antibiotic was bacteriostatic to the organism for the initial 5 h of exposure; thereafter it was bacteriocidal. Observation of inhibited cultures revealed cells that had produced abnormally long stalks, but no buds. These results indicate that bud formation is coupled to chromosome replication in H. neptunium. They do not exclude the possibilities that cross wall formation and bud separation may also be coupled to chromosome replication.

Scanning electron microscopy has been used to visualize the residents of microbial communities populating habitats on epithelial surfaces in the gastrointestinal tracts of mice. In the stomach, bacteria form a dense layer on the stratified squamous epithelium of the nonsecreting area. Microbes of at least three morphological types can be seen in this layer, including short rods with round ends, rods in chains, and tapering filaments composed of repeating units of rod- or coccal-shaped elements varying in size from large at one end of the filament to small at the other end. These three forms all attach by one end to the epithelium. The latter two forms can be found only so attached; in both cases, the end is inserted into a hole or depression in the keratinized epithelium. In the small intestine, a microbe of morphology similar to that of the tapering filaments found in the stomach can be seen attached end-on to the epithelium. Again each filament has one end inserted into a hole in the epithelium. In this case, however, the repeating elements of each filament are all about the same size. In the cecum and colon, predominantly fusiform- and spiral-shaped microbes can be seen mixed together in layers on the epithelium. At least three types of fusiform-shaped microbes can be distinguished on the basis of surface texture, and one type of spiral-shaped microbe can be found. These microorganisms appear to be attached to each other and to the epithelium by weblike filaments. The numerous microbial types present in the various epithelial habitats associate intimately surface-to-surface with each other and with the epithelium. Such surface-surface association may be an important autogenic factor contributing to the stability of the murine gastrointestinal ecosystem.

A method is described for obtaining synchronously growing swarmer cell populations of Hyphomicrobium sp. strain B-522. This was accomplished by isolating young swarmers from random cultures by centrifugation and filtration. Cell multiplication occurred during 38% of the growth cycle in populations synchronized in this manner. Observations were made of the changes in cellular morphology which occurred during the growth cycle. Of the 14.25 h required for the doubling in cell numbers, an average of 5 h passed before the swarmer cells began to develop their hyphae. This time varied over a range of 10 h. The time interval between the beginning of hyphal development and the beginning of bud formation was 3.5 to 4.5 h. The maturation of the first buds and their separation from the mother cells were completed in 5.5 h. The duration of these steps is compared to those measured previously in agar slide cultures.