Factors influencing the frequency of mesosomes observed in fixed and unfixed cells of Streptococcus faecalis.

Mesosomes of Streptococcus faecalis (American Type Culture Collection 9790) were seen about 92% less frequently in freeze fractures of unfixed cells than in freeze fractures and sections of fixed cells. This difference in frequency was not related to any period of unbalanced macromolecular synthesis induced by chemical fixation. All measured synthetic processes (DNA, RNA, and protein synthesis, and glycerol incorporation) were halted with either osmium tetroxide (OS) or glutaraldehyde fixation. That fewer mesosomes were seen in freeze fractures of unfixed cells was probably due to the difficulty of observing cross-fractured mesosomes in this organism in the unfixed state. Unfortunately, mesosomes probably preferentially cross fracture in the unfixed state and therefore are usually only observed, infrequently, in those cases where the freeze fracture follows the surface layer of a mesosomal membrane. However, the addition of glycerol to unfixed cells, especially in the chilled state, greatly increased the frequency of observation of cytoplasmic mesosomes in freeze fractures. It is thought that glycerol, like chemical fixation, increases the number of surface-fractured mesosomes, which in turn increases the frequency of mesosome observation. It was also observed that cellular autolysis occurring during OS fixation seemingly reduced the number of mesosomes observed in thin sections and freeze fractures of OS-fixed cells.

Fungal Morphogenesis: Ring Formation and Closure by Arthrobotrys dactyloides.

The formation and the closure of constricting rings by a nematode-trapping fungus were recorded by means of time-lapse cinephotomicrography. Analysis of the film revealed that hyphal rings resulted from a sequence of morphological events not previouisly described. Cell inflation and ring constriction were induced by touch, increased temperature, and electrical stimutlation. The inflation process was not particularly sensitive to metabolic inhibitors and appears to operate without an expenditutre of energy on the part of the cell.

In vitro simulation of neural trauma by laser.

A serious lack of knowledge about central nervous system trauma is encountered on the cellular level where the inability to create precise experimental lesions of known magnitude has limited our understanding of the reactions of single cells to injury. We used a laser cell surgery technique developed in this laboratory to manipulate neurons in a controlled environment, in order to observe pathologic reactions during and immediately after the injury. This technique is especially suited for axonal and dendritic amputations close to the perikaryon. The laser provided three different physical modes of injury to neurites: direct vaporization of cytoplasm, pressure wave damage from external vaporization of substrate material, and photobiologically-induced localized cytoskeletal destruction leading to the slow pinching of processes followed by transection. Our data indicated a great similarity between laser impact damage and the cellular damage produced by physical trauma to the central nervous system.

Neuronal survival and dynamics of ultrastructural damage after dendrotomy in low calcium.

To determine the contributions of calcium to development of ultrastructural damage and neuronal death after mechanical injury, we amputated primary dendrites from over 300 cultured mammalian spinal neurons under normal (1.8 mM) or low (less than or equal to 30 microM) calcium conditions. Two general categories of early ultrastructural change were seen in both normal and low calcium: (1) a lesion-dependent gradient of damage that moved centripetally through the proximal segment and penetrated the soma within 15 min and (2) dilation of the somal Golgi/smooth endoplasmic reticulum (SER), which preceded the wave of deterioration from the lesion. Although the somal Golgi/SER changes were similar in both normal and low calcium, the damage gradient in low calcium differed from the damage gradient in normal calcium. (1) Microtubules and neurofilaments were preserved, (2) mitochondria became more electron dense but did not develop electronlucent foci or high amplitude swelling, and (3) an extensive vesicular gradient formed consisting of rows of swollen SER vesicles. Sodium ionophores have been reported to cause similar changes. Survival studies showed that calcium reduction significantly delayed neuronal death. Survival was 63 +/- 16% vs 35 +/- 8% (p less than 0.003) at 2 h and 30 +/- 7% vs 23 +/- 8% at 6 h in low and normal calcium, respectively. Dead neurons that had been lesioned in low calcium also showed greater ultrastructural preservation than neurons that died after dendrotomy in normal calcium. We hypothesize that under low calcium conditions, the large sodium injury current plays an important role in neuronal deterioration and death after mechanical trauma.

15-Azasteroid blockage of cell permeability and mitochondrial respiration.

The 15-azasteroid, 1,10,11,11a-tetrahydro-11a-methyl-2H naphth (1,2-g)indol-7-o1, inhibits the growth of the cell culture lines KB and L-M as well as several strains of bacteria. The inhibition of growth is reversed following removal of the steroid from the growth medium. Using in vitro grown L-M cells, the compound inhibited the transport of amino acids and uracil. The action was non-detergent like and at least 100 times more effective in terminating metabolite transport than sodium azide. The azasteroid inhibited the oxidation of glutamate in isolated rat liver mitochondria. The oxidation of succinate was not effected by the azasteroid alone but in the presence of glutamate, the azasteroid uncoupled the oxidation of succinate from the ADP-ATP control. It is suggested that the azasteroid may be acting directly on the electron transport system and/or acting indirectly through membrane perturbations which disrupts the electron transport process.

The precision of determinations of zinc content of food.

Seven different types of foodstuff (containing 3-100 ppm Zn) were each analysed for zinc by colorimetry, polarography and atomic-absorption spectroscopy. All the techniques yielded similar values for the zinc content of a given sample, but atomic-absorption spectroscopy had a smaller average coefficient of variation.

Release of sporangiospores by a strain of Actinoplanes.

Dehiscence of Actinoplanes sp. 7-10 sporangia is triggered by wetting of the spores. This process requires time because of the hydrophobic nature of the sporangial envelope; it can be speeded up and enhanced by a wetting agent. Once wetted, the spores swell, usually ripping the sporangial wall, and escape as motile elements when functional flagella are synthesized. Flagellation and motility are separate phenomena, both of which lose intensity with age. Spores from old sporangia can regain motility when supplied with an exogenous carbon source, but, when provided only with water, phosphate buffer, or amino acids, flagellation takes place without motility. Deflagellation-reflagellation experiments indicated that functional flagella can be reformed only in presence of both amino acids and glucose which must be added within 180 min of deflagellation. Inoperative flagella were formed in the presence of inhibitors of nucleic acid synthesis, such as 6-azauracil, but inhibitors of protein synthesis, such as chloramphenicol, did not interfere with reflagellation. Flagellated spores remained so after germination.

Three-dimensional reconstruction of whole cells of Streptococcus faecalis from thin sections of cells.

A new ultrastructural technique has been developed to study the geometry of cell wall assembly in Streptococcus faecalis, which is believed to occur between pairs of raised bands located on the organism's surface. Three-dimensional reconstructions of these new regions of envelope growth are produced from the mathematical rotation (around a central axis) of various measurements taken from central, longitudinal thin sections of cells. These reconstructions can be used to calculate the surface area and volume of the septal and peripheral walls that were supposedly present in any given cell before sectioning. In an accompanying paper, it is shown how such surface and volume estimations, coupled with other measurements of length, thickness, and curvature, can be used to characterize a cycle of envelope growth in this organism. The validity of the assumptions used to reconstruct cells by rotation and the possible sources of error in using this technique are discussed.

Cell cycle changes in the buoyant density of exponential-phase cells of Streptococcus faecium.

Cell buoyant densities were determined by centrifugation in Percoll gradients containing exponential-phase cells of Streptococcus faecium ATCC 9790 grown at a mass doubling time of about 33 min. This bacterium showed the highest average density values (1.13 g/ml) measured to date for any eucaryotic or procaryotic organism. Fractions having the highest densities were enriched with cells that were in the process of dividing or had just divided. These high-density fractions were also enriched with cells that had newly initiated sites of cell wall growth. It appears that S. faecium shows minimum cell densities in the midportion of its cycle.

Cytoplasmic damage gradients in dendrites after transection lesions.

Ultrastructural changes in the cytoplasm of transected dendrites have been studied with serial section electron microscopy 10 to 12 min after the lesion. Dendrites from large multipolar neurons in mouse spinal monolayer cultures were selected 4 weeks after seeding and cut with a pulsed UV laser microbeam. The data reveal cytoplasmic damage gradients which decrease in severity with distance from the lesion. The damage is expressed primarily in cytoskeletal disruption, the swelling and vacuolation of mitochondria (MC), and the swelling and vesiculation of the endoplasmic reticulum (ER). For a 3-4 microns diameter dendrite lesioned 150 microns from the soma and fixed 10 min after transection, the following results can be reported. The number of microtubules (MT) decreases almost linearly with a slope of 40 MT/10 microns until MT completely disappear 20 microns from the lesion. Neurofilaments (NF) decrease at approximately 50 NF/10 microns, with 20% of these structures still intact at or near the lesion. MC show both gradual and explosive swelling in the damaged process. The onset of these two phases of MC swelling are correlated with the initial and total loss of MT respectively. MT and NF densities in cut dendrites are drastically different from those in control processes and suggest a possible retrograde redistribution of these organelles. Damage from external shockwaves that cause process distortion but not transection results in a less severe but nevertheless similar loss of MT and NF.

Control of wall band splitting in Streptococcus faecalis.

Computer reconstructions of 659 and 1325 whole mounted, shadowed cells, randomly chosen from cultures of Streptococcus faecalis undergoing balanced growth and doubling in mass every 83 min and 30 min, respectively, were used to analyse the cell cycle. The size limits and duration of phases of the cell cycle were estimated by applying a method previously described by the authors, details of which are given here to allow others to use the method. Deeply constricted cells whose primary septal radius, Rs, was less than or equal to 0.18 micron were considered as belonging to an E-phase ending the cell cycle. The statistical parameters of these E-phase cells were used to calculate the mean and coefficient of variation of dividing cells. These latter values, in turn, predicted the moments of the total population well enough so that the method's assumptions were judged adequately satisfied. Therefore, the method was considered applicable to other phases and sub-phases of the cell cycle of these two cultures. The E-phase cells were further classified as having either 0, 1 or 2 secondary growth zones, allowing us to calculate the percentage of newborn cells without growth zones. In the slow-growing cells, 69% of the cells arose with no growth zone. On the other hand, in more rapidly growing cells 16% of the cells or less arose with no growth zone. Our calculations showed that they could exist without a growth zone for only 2 and 0.1 min, respectively. We also classified cells as possessing a 'birth site' if the volume between the two daughter bands was greater than 0, but less than 0.06 micron3. From the statistical properties of such cells with new growth zones, the mean pole time, W, was estimated. We also estimated W from the size of cells in E-phase. The major conclusion is that the pole time is only slightly greater than the mass doubling time at both growth rates. Since DNA synthesis in S. faecalis takes longer (C = 50 to 52 min) than the mass doubling time in rich medium (30 min), a new round of chromosome replication must be initiated before the old round of synthesis is completed (dichotomous replication). Consequently, wall band splitting and initiation of chromosome replication do not occur simultaneously. It was also concluded that the cell initiates wall band splitting, resulting in pole formation and cell division, when the growth zones cannot function rapidly enough to allow the increase of surface area required to accommodate continuing production of cytoplasm.

Septal membrane fusion--a pivotal event in bacterial spore formation?

Formation of the asymmetrically located septum divides sporulating bacilli into two distinct cells: the mother cell and the prespore. The rigidifying wall material in the septum is subsequently removed by autolysis. Examination of published electron micrographs indicates that the two septal membranes then fuse to form a single membrane. Membrane fusion would be expected to have profound consequences for subsequent development. For example, it is suggested that fusion activates processing of pro-sigma E to sigma E in the cytoplasm by exposing it to a membrane-bound processing enzyme. Asymmetry of the fused membrane could restrict processing to one face of the membrane and hence explain why sigma E is associated with transcription in the mother cell but not in the prespore. Asymmetry of the fused membrane might also provide a mechanism for restricting the activity of another factor, sigma F, to the prespore. Attachment of the flexible fused septal membrane to the condensing prespore nucleoid could help drive the engulfment of the prespore by the mother cell.

Study of pole assembly in Bacillus subtilis by computer reconstruction of septal growth zones seen in central, longitudinal thin sections of cells.

The septal growth of Bacillus subtilis 168/s has been studied by making a number of observations from thin sections of cells from exponentially growing cultures. The process was initiated by the formation of a new cross wall under a preexisting layer of cylindrical wall. An annular notch appeared to cut through the overlying wall and presumably allowed the cross wall to split into two layers of peripheral wall. During this initial notching process, two raised bands of wall material were produced which resembled those previously observed in morphological studies of Streptococcus faecalis. Through an improved fixation technique, it was possible to preserve the bands seen in B. subtilis to the extent that they were used as markers to study the subsequent stages of septal growth. These stages included (i) the continued displacement of the two bands from the cross wall (as the two nascent polar surfaces enlarged and as the diameter of the cross wall decreased), (ii) the closure of the cross wall, and (iii) the final severance of the common cross wall connection between two completed poles. To study this process in a more quantitative manner, three-dimensional reconstructions of the envelope observed between pairs of the raised bands were made from axial thin sections of cells. The process of reconstruction was based on a technique by which x, y coordinates were taken from thin sections and were rotated around the cell's central axis. These reconstructions were used to estimate the surface area or volume of the reconstructed zones or their parts. A round of septal growth was then simulated by arranging 118 reconstructions in order of increasing surface area or volume. The topology of the process was studied by noting how various measurements of septal thickness, length, surface area, and volume varied as a function of increasing septal zone size. This analysis was based on several assumptions, of which three of the most important are: (i) the bands produced by the initial notching process are markers which separate septal from cylindrical wall growth; (ii) a septal zone observed between pairs of bands is made up of two nascent poles and a single cross wall; and (iii) as septal zones develop in terms of relative age they increase in size (volume or surface area) or amount of wall. The data suggested that the S. faecalis model of surface growth (in which polar growth occurs through a regulated constrictive separation and expansion of a cross wall) also seems applicable to the pattern of septal growth observed here for B. subtilis. This was indicated from measurements which showed that increases in the size of nascent polar surfaces were correlated with decreases in cross wall diameter. An explanation of these observations may be that decreases in cross wall diameter were due to a progressive splitting of the cross wall that removed surface from the outer circumference of the cross wall and converted it into new polar surface. Calculations further suggested that if the poles of B. subtilis were made by this model a sizeable and variable increase in surface area of the cross wall would also be required to convert these separating cross wall layers into two curved polar structures. Measurements of wall thickness taken from various locations within septal zones indicated that while the thickness of the polar wall of B. subtilis was constant over its surface, the width of the cross wall varied considerably during a round of synthesis. Again, one of the simplest explanations compatible with these observations and those previously made in S. faecalis is that the B. subtilis cross wall is brought to a constant thickness (possibly by remodeling or precursor addition) before or during separation. Although most observations made from the reconstruction of the septal zones of B. subtilis may fit the S. faecalis model of surface growth, differences in the pattern of septal growth were seen when the two organisms were compared. These have been discussed in terms of differences in the regulation of their respective septal growth sites and basic mechanisms of wall assembly and modification.

Effect of temperature on the distribution of membrane particles in Streptococcus faecalis as seen by the freeze-fracture technique.

When cells of Streptococcus faecalis ATCC 9790 were incubated at temperatures above 10 C before being frozen for freeze-fracture, a random distribution of particles was observed on the outer fracture face of the freeze-cleaved cell membrane. However, when cells were incubated below 10 C before freezing, particleless patches were seen on this membrane surface. The size of the patches produced on chilling could be increased by centrifugation or by storing the chilled cells overnight at about 3 C. Patch formation appeared readily reversible, since the medium and large patches that formed on chilling could not be observed in cells warmed for 10 s at 25 C. However, during the transition from the patch to patchless state, smaller patches not seen in the chilled cells were observed. This suggested that the smaller patches might have been intermediate forms produced by the fragmentation of larger patches on warming.

Poorly lytic bacteriophage from Dactylosporangium thailandensis (Actinomycetales).

Dactylosporangiophage A1 has a polygonal head (75 nm) with spherical capsomeres (3 nm) and a noncontractile tail (200 by 10 nm) with cross-striations which is terminated with at least three prongs which are used for attachment. It contains double-stranded deoxyribonucleic acid and produces very little lysis. Intracellular phage multiplication leads to the formation of crystalline aggregates of apparently complete virions. Plaques are formed only on certain substrains of Dactylosporangium thailandensis L1 and are always small (0.5 mm or less). They are clear on some substrains and turbid on others. Formation of plaques occurs only on one medium, Czapek agar with 0.2 to 0.4% yeast extract, 0.2% peptone, or a defined mixture of amino acids. Over 100 strains of bacteria, mainly actinomycetes, were screened in a futile attempt to find an indicator strain which is not a substrain of L1. The Dactylosporangium-phage system studied is considered to be a semiresistant carrier state.