Effect of penicillin on the morphology and reproduction ofAzotobacter chroococcum

The effect of penicillin on the morphology and reproduction of some strains ofA. chroococcum was studied on a number of solid media. When the growth was not entirely suppressed by the penicillin, filamentous cells and spheroplasts were formed. The formation of spheroplasts was stimulated by peptone. Gonidia were sometimes formed inside the spheroplasts and also inside giant cells. They were released from the cell after disruption or after lysis of the cell wall. In some cases they produced dwarf cells. Under certain conditions groups of gonidia present in a cell fused and formed one or more normal-looking cells inside the mother cell. Sometimes one or moreAzotobacter cells developed inside a spheroplast or at the site of a spheroplast with a lysed cell wall. Microcolonies consisting of small cocci representing gonidia and dwarf cells were also observed occasionally at the sites of spheroplasts with lysed cell walls. Occasionally tiny groups of small elements with a less marked structure were found at such sites, probably representing debris of lysed cells. The production of normal-looking cells inside filamentous cells was greatly stimulated on a medium containing 10 percent horse serum, with a drop of sterile water containing 200 or 250 I.U. penicillin added in the centre of the plate. The growth ofA. chroococcum was greatly retarded when the medium contained 10 U/ml penicillin and seemed to be checked entirely at concentrations of 20 U/ml penicillin or higher. Occasionally, however, even at concentrations of 100 and 300 U/ml penicillin, a few filamentous cells were found and also a few microcolonies, visible only through the microscope, consisting of gonidia or regenerative rods. By repeated exposure ofAzotobacter to penicillin populations could be obtained that were adapted to high concentrations of this antibiotic.     

Freeze-Etch Study of Pseudomonas aeruginosa: Localization Within the Cell Wall of an Ethylenediaminetetraacetate-Extractable Component

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.     

Spores of microorganisms

The relationship of the synthesis of new cell wall in the postgerminative development ofBacillus cereus spores to protein and ribonucleic acid synthesis was studied through the incorporation of¹⁴C-diaminopimelic acid. The spores were not capable of synthesizing cell wall immediately after germination. A very short, period of protein synthesis was first needed, the messenger ribonucleic acid for these proteins being formed at the end of the depolymerization phase. On blocking cell wall synthesis with penicillin or cycloserine, swelling and the outset of elongation were normal. In the presence of penicillin, the cells afterwards disintegrated during the elongation phase, while with cycloserine, elongation of the cells was only arrested and later atypical division occurred. The findings are discussed from the aspect of the possibility of the participation of part of the preexisting diaminopimelic acid-containing spore material in the envelope system of the outgrowing cell.     

Modification of the envelope and the protein content of Escherichia coli surviving in sea water

A toxigenic strain of Escherichia coli displayed important structural modifications when placed in seawater which naturally lacked nutritive elements, as observed by electron microscopy. These include cell wall and cell body distortion, modification of the membranes, central segregation of the chromosome, and retraction of the cytoplasm. These modifications were accompanied by a decrease in cell protein content of approximately 40%. Certain cytoplasmic membrane proteins were lost, and new ones appeared. The development of these changes was considerably slower in cells that had previously been grown in a seawater medium. This suggests that osmotic regulation mechanisms, which enable E. coli to survive much longer in marine conditions, may have a protective influence.     

Penicillin and Cell Wall Synthesis: a Study of Bacillus licheniformis by Electron Microscopy

The changes in wall structure of two penicillinase-negative strains of Bacillus licheniformis on addition of penicillin were studied. After addition of penicillin to give a concentration of 1 unit/ml, exponentially growing cells of strain 749 c/72 doubled once and then stopped. Strain 749c/72/IIIg was more resistant and continued growing, but synthesis appeared to become uncontrolled over the surface, producing localized wall thickening at the expense of elongation, and leading to distorted cells and growth in twisted and coiled chains, with an accompanying drop in growth rate. The continued growth can be explained by the existence of a less sensitive transpeptidase, but there is no obvious explanation for the uncontrolled synthesis. The effect of penicillin could be reversed by addition of penicillinase in both strains, although there appeared to be a persistent effect of penicillin which also produced distorted cells for a few generations and inhibited cell separation. The changes in wall structure produced by penicillin and penicillinase appeared all over the cell surface, suggesting that wall synthesis occurred all over the cell. Also a separate process for cross-wall synthesis is suggested since this appeared less sensitive than wall synthesis.     

Osmotic imbalance in inositol-starved spheroplasts of Saccharomyces cerevisiae.

Physiological states associated with inositol starvation of spheroplasts of Saccharomyces cerevisiae were investigated and compared with conditions preceding death of starved whole cells. In the absence of synthesis of inositol-containing lipids, cell surface expansion terminated after one doubling of whole cells. In spheroplasts, cessation of membrane expansion was apparently followed by rapid development of an osmotic imbalance, causing lysis. Continued synthesis and accumulation of cytoplasmic constituents within the limited cell volume were implicated as a cause of the osmotic imbalance. In whole cells, an increase in internal osmotic pressure also follows termination of membrane and cell wall expansion. The cell wall prevents lysis, allowing a state of increasing cytoplasmic osmotic pressure to persist in the period preceding onset of inositol-less death.     

Lipid localization in bacterial cells through curvature-mediated microphase separation

Although many proteins are known to localize in bacterial cells, for the most part our understanding of how such localization takes place is limited. Recent evidence that the phospholipid cardiolipin localizes to the poles of rod-shaped bacteria suggests that targeting of some proteins may rely on the heterogeneous distribution of membrane lipids. Membrane curvature has been proposed as a factor in the polar localization of high-intrinsic-curvature lipids, but the small size of lipids compared to the dimensions of the cell means that single molecules cannot stably localize. At the other extreme, phase separation of the membrane energetically favors a single domain of such lipids at one pole. We have proposed a physical mechanism in which osmotic pinning of the membrane to the cell wall naturally produces microphase separation, i.e., lipid domains of finite size, whose aggregate sensitivity to cell curvature can support spontaneous and stable localization to both poles. Here, we demonstrate that variations in the strength of pinning of the membrane to the cell wall can also act as a strong localization mechanism, in agreement with observations of cardiolipin relocalization from the poles to the septum during sporulation in the bacterium Bacillus subtilis. In addition, we rigorously determine the relationship between localization and the domain-size distribution including the effects of entropy, and quantify the strength of domain-domain interactions. Our model predicts a critical concentration of cardiolipin below which domains will not form and hence polar localization will not take place. This observation is consistent with recent experiments showing that in Escherichia coli cells with reduced cardiolipin concentrations, cardiolipin and the osmoregulatory protein ProP fail to localize to the poles.     

Nature of Penicillin-Induced Growth Inhibition of Mycoplasma neurolyticum

When penicillin was added to cultures of Mycoplasma neurolyticum in amounts up to 1,000 units per ml, lag time and generation time were increased and the total population was reduced in proportion to the antibiotic concentration. Although growth suppression by penicillin was complete, the death rate was slow and linear over periods up to 12 days. Growth after induced lag was due to a decay in penicillin activity and was not the result of mutant selection. However, repeated transfer in media which contained increasing concentrations of penicillin resulted in normal growth of M. neurolyticum at penicillin levels as high as 1,000 units per ml. Penicillinase did not play a role in recovery from penicillin-induced lag, and the inactive penicillin molecule did not prevent normal growth of M. neurolyticum. Removal of penicillin from the medium by washing or penicillinase during the induced lag was immediately followed by normal growth of the organism. These results suggest a reversible antibiotic function for penicillin which prevents multiplication of the organism by means unrelated to cell wall formation.     

Chemical, Biological, and Structural Properties of Stable Proteus L Forms and Their Parent Bacteria

Proteus L forms were disrupted by osmotic shock, and the sedimentable material present in the homogenate was further fragmented in a Sorvall pressure cell. The pressure cell was also used for disrupting normal Proteus cells. The homogenates obtained were fractionated by differential centrifugation. Purified endotoxins were isolated from the major fractions by phenol extraction. Material extracted with phenol from the membrane fraction of the L forms was about as toxic and pyrogenic on a weight basis as the typical enterobacterial endotoxins isolated from cell walls of normal bacteria. The yield of extract from L forms was about one-third of that from an equal weight of normal bacteria. No differences in the gross chemical composition of the phenol extracts from the L forms and the normal cells could be ascertained. A close serological relationship existed between extracts obtained from two L forms and their respective parent bacteria, but no such relationship was found in the case of the third L form studied and its parent bacterium. Diaminopimelic acid was not detected in the membranes of the L forms, but these membranes contained most of the succinic dehydrogenase of the organisms. Only small amounts of this enzyme were present in the wall fraction of normal bacteria. The data obtained suggest that precursors of the Proteus endotoxins are formed either in the soluble protoplasm of normal cells and L forms or at sites on the membrane from which they are readily liberated into the protoplasm, whereas the final steps of the synthesis of these toxins take place at the cytoplasmic membrane. In normal cells, much endotoxin is transported to and concentrated in the walls.     

Growth,cell wall formation and differentiation in the protonema of the moss,Funaria hygrometrica: Effects of plasmolysis on the developmental program and its expression

Summary Cultivation ofFunaria protonemata under plasmolytic or slightly subplasmolytic conditions initially causes a cessation of growth which is accompanied by a transient disappearance (or strong reduction in frequency, respectively) of putative cellulose synthesizing particle rosettes in the plasma membrane. Simultaneously, the formation and exocytosis of cell wall materialsecreting Golgi vesicles is slowed down. The latter process does not become apparent for several hours, though the reduction in activity can be proved indirectly. As a consequence of the imbalance between exocytosis, cell wall material accumulates in the plasmolytic space, generally at the cell tip. This indicates that the pattern of local, polar deposition of cell wall formation and cell elongation, membrane debris as well as wall material is maintained for some time. Later, however, the whole protoplast may become covered by new wall layers. Potentially growing filament tips and the distal region of nontip cells increase in diameter after longer cultivation in subplasmolytic conditions. It is suggested that normal wall growth results from a softening of the existing wall, its stretching and simultaneous stabilization by the apposition of new wall layers. We believe that the swelling is caused by a change in the equilibrium between the obviously less affected softening process and the imperfect stabilization by new wall layers because the wall layers which are formed at reduced turgor pressure are looser than normal and may have a changed composition.Kinetin-induced buds do not develop under plasmolytic conditions. Instead, spiral filaments are formed which readily give rise to buds when the osmotic value of the (kinetin-containing) medium is normalized. The results show that plasmolysis affects the expression of the developmental program rather than its initiation or maintenance.     

EXOCYTOSIS OF LATEX BEADS DURING THE ENCYSTMENT OF ACANTHAMOEBA

Cells of Acanthamoeba castellanii (Neff) are known to form mature cysts characterized by a cellulose-containing cell wall when transferred to a nonnutrient medium. Amebas which engulfed latex beads before encystment formed mature cysts essentially devoid of bead material. The encystment of bead-containing cells appeared to be similar to that of control cells since no important differences between the two were observed with respect to cellular levels of glycogen or protein, cellulose synthetase activity, the amount of cyst wall polysaccharide formed, or the percentage of cysts formed. Actinomycin D and cycloheximide inhibited encystment as well as bead expulsion. Ultrastructural analysis revealed that the beads, which initially were contained in phagocytic vesicles, were released from the cell by fusion of vesicular membranes with the plasma membrane. Exocytosis was observed in cells after 3 hr of encystment, with most of the beads being lost before cyst wall formation. Each bead-containing vesicle involved in expulsion was conspicuously demarcated by an area of concentrated cytoplasm, which was more homogeneously granular than the surrounding cytoplasm. Beads were not observed in the cytoplasm of mature cysts but were occasionally found in the cyst wall.     

Cell wall and cytoskeleton reorganization as the response to hyperosmotic shock in Saccharomyces cerevisiae

Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7-1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate. During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed. The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose. The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls. Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface. The rate of incorporation of D-[U-14C]glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in cell wall composition between osmotically stressed yeast and control cells were found. Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments. After 2-3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds. The response of S. cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain. We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis.     

Effect of osmotic stress on the ultrastructure and viability of the yeast Saccharomyces cerevisiae

Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell shrinkage, without plasmolysis. The relationship between cell volume and osmolality was non-linear; between 1 and 4 osM there was a plateau in cell volume, with apparently a resistance to further shrinkage; beyond 4 osM cell volume was reduced further. The loss of viability of S. cerevisiae after hypertonic stress was directly related to the reduction in cell volume in the shrunken state. The plasma membrane is often considered to be the primary site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted in a major loss of viability, all cells were osmotically responsive. The effects of osmotic stress on mitochondrial activity and structure were investigated using the fluorescent probe rhodamine 123. The patterns of rhodamine staining were altered only after extreme stress and are assumed to be a pathological feature rather than a primary cause of injury. Changes in the ultrastructure of the cell envelope were examined by freeze-fracture and scanning electron microscopy. In shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the cytoplasmic side buckled with irregular projections into the cytoplasm. On return to isotonic solutions these structural alterations were reversible, suggesting a considerable degree of plasticity of the wall. However, the rate of enzyme digestion of the wall may have been modified, indicating that changes in wall structure persist.     

Permeabilization of Fungal Hyphae by the Plant Defensin NaD1 Occurs through a Cell Wall-dependent Process

The antifungal activity of the plant defensin NaD1 involves specific interaction with the fungal cell wall, followed by permeabilization of the plasma membrane and entry of NaD1 into the cytoplasm. Prior to this study, the role of membrane permeabilization in the activity of NaD1, as well as the relevance of cell wall binding, had not been investigated. To address this, the permeabilization of Fusarium oxysporum f. sp. vasinfectum hyphae by NaD1 was investigated and compared with that by other antimicrobial peptides, including the cecropin-melittin hybrid peptide CP-29, the bovine peptide BMAP-28, and the human peptide LL-37, which are believed to act largely through membrane disruption. NaD1 appeared to permeabilize cells via a novel mechanism that required the presence of the fungal cell wall. NaD1 and Bac2A, a linear variant of the bovine peptide bactenecin, were able to enter the cytoplasm of treated hyphae, indicating that cell death is accelerated by interaction with intracellular targets.     

Locus of the Action of Serum and the Role of Lysozyme in the Serum Bactericidal Reaction

The mechanism of the lethal action of human serum on a rough strain of Escherichia coli was investigated by use of serum with and without lysozyme, in medium of low and high osmotic pressure, with cells radioactively labeled in the peptidoglycan polymer, and by electron microscopy. The results suggested that there are two separate components in the bacterial cell wall that afford structural support for the cell. Lysozyme attacked one of these, the peptidoglycan polymer. Serum damaged the other, which is probably the peripherally located lipopolysaccharide-phospholipid complex. The cell wall damage caused by lysozyme-free serum promptly resulted in cell death under usual conditions. In plasmolyzed cells, however, the wall damage was not lethal, presumably because the membrane of the plasmolyzed cell was protected from secondary lethal changes which otherwise occur.     

Intracellular localization of integrin-like protein and its roles in osmotic stress-induced abscisic acid biosynthesis in Zea mays

Summary. Plants have evolved many mechanisms to cope with adverse environmental stresses. Abscisic acid (ABA) accumulates significantly in plant cells in response to drought conditions, and this is believed to be a major mechanism through which plants enhance drought tolerance. In this study, we explore the possible mechanisms of osmotic stress perception by plant cells and the consequent induction of ABA biosynthesis. Immunoblotting and immunofluorescence localization experiments, using a polyclonal antibody against human integrin β1, revealed the presence of a protein in Zea mays roots that is similar to the integrin proteins of animals and mainly localized in the plasma membrane. Treatment with GRGDS, a synthetic pentapeptide containing an RGD domain, which interacted specifically with the integrin protein and thus blocked the cell wall–plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, and the GRGDS analog which does not contain the RGD domain had no effect. Our results show that a strong interaction exists between the cell wall and plasma membrane and that this interaction is largely mediated by integrin-like proteins. They also imply that the cell wall and/or cell wall–plasma membrane interaction plays important roles in the perception of osmotic stress. Accordingly, we conclude that the cell wall and/or cell wall–plasma membrane interaction mediated by the integrin-like protein plays important roles in osmotic stress-induced ABA biosynthesis in Zea mays. Correspondence: J. S. Liang, College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, People’s Republic of China.     

Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans

The plasma membrane of the fungal pathogen Candida albicans forms a protective barrier that also mediates many processes needed for virulence, including cell wall synthesis, invasive hyphal morphogenesis, and nutrient uptake. Because compartmentalization of the plasma membrane is believed to coordinate these diverse activities, we examined plasma membrane microdomains termed eisosomes or membrane compartment of Can1 (MCC), which correspond to ∼200-nm-long furrows in the plasma membrane. A pil1∆ lsp1∆ mutant failed to form eisosomes and displayed strong defects in plasma membrane organization and morphogenesis, including extensive cell wall invaginations. Mutation of eisosome proteins Slm2, Pkh2, and Pkh3 did not cause similar cell wall defects, although pkh2∆ cells formed chains of furrows and pkh3∆ cells formed wider furrows, identifying novel roles for the Pkh protein kinases in regulating furrows. In contrast, the sur7∆ mutant formed cell wall invaginations similar to those for the pil1∆ lsp1∆ mutant even though it could form eisosomes and furrows. A PH-domain probe revealed that the regulatory lipid phosphatidylinositol 4,5-bisphosphate was enriched at sites of cell wall invaginations in both the sur7∆ and pil1∆ lsp1∆ cells, indicating that this contributes to the defects. The sur7∆ and pil1∆ lsp1∆ mutants displayed differential susceptibility to various types of stress, indicating that they affect overlapping but distinct functions. In support of this, many mutant phenotypes of the pil1∆ lsp1∆ cells were rescued by overexpressing SUR7. These results demonstrate that C. albicans eisosomes promote the ability of Sur7 to regulate plasma membrane organization.     

Staphylococcal Cell Wall: Morphogenesis and Fatal Variations in the Presence of Penicillin

The primary goal of this review is to provide a compilation of the complex architectural features of staphylococcal cell walls and of some of their unusual morphogenetic traits including the utilization of murosomes and two different mechanisms of cell separation. Knowledge of these electron microscopic findings may serve as a prerequisite for a better understanding of the sophisticated events which lead to penicillin-induced death. For more than 50 years there have been controversial disputes about the mechanisms by which penicillin kills bacteria. Many hypotheses have tried to explain this fatal event biochemically and mainly via bacteriolysis. However, indications that penicillin-induced death of staphylococci results from overall biochemical defects or from a fatal attack of bacterial cell walls by bacteriolytic murein hydrolases were not been found. Rather, penicillin, claimed to trigger the activity of murein hydrolases, impaired autolytic wall enzymes of staphylococci. Electron microscopic investigations have meanwhile shown that penicillin-mediated induction of seemingly minute cross wall mistakes is the very reason for this killing. Such “morphogenetic death” taking place at predictable cross wall sites and at a predictable time is based on the initiation of normal cell separations in those staphylococci in which the completion of cross walls had been prevented by local penicillin-mediated impairment of the distribution of newly synthesized peptidoglycan; this death occurs because the high internal pressure of the protoplast abruptly kills such cells via ejection of some cytoplasm during attempted cell separation. An analogous fatal onset of cell partition is considered to take place without involvement of a detectable quantity of autolytic wall enzymes (“mechanical cell separation”). The most prominent feature of penicillin, the disintegration of bacterial cells via bacteriolysis, is shown to represent only a postmortem process resulting from shrinkage of dead cells and perturbation of the cytoplasmic membrane. Several schematic drawings have been included in this review to facilitate an understanding of the complex morphogenetic events.     

Integrin-like Protein Is Involved in the Osmotic Stress-induced Abscisic Acid Biosynthesis in Arabidopsis thaliana

We studied the perception of plant cells to osmotic stress that leads to the accumulation of abscisic acid （ABA） in stressed Arabidopsis thaliana L. cells. A significant difference was found between protoplasts and cells in terms of their responses to osmotic stress and ABA biosynthesis, implying that cell wall and/or cell wall-plasma membrane interaction are essential in identifying osmotic stress. Western blotting and immunofluorescence localization experiments, using polyclonal antibody against human integrin β1, revealed the existence of a protein similar to the integrin protein of animals in the suspension-cultured cells located in the plasma membrane fraction. Treatment with a synthetic pentapeptide, Gly-Arg-Gly-Asp-Ser （GRGDS）, which contains an RGD domain and interacts specifically with integrin protein and thus blocks the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, but not in protoplasts. These results demonstrate that cell wall and/or cell wall-plasma membrane interaction mediated by integrin-Iike proteins played important roles in osmotic stress-induced ABA biosynthesis in Arabidopsis thaliana.     

Procedure to Isolate Viable Sperm Cells from Corn (Zea mays L.) Pollen Grains

Sperm cells were isolated from corn (Zea mays L.) tricellular pollen grains. They were released using a light osmotic chock, and separated from pollen contaminants (especially starch grains) by a Percoll gradient centrifugation. Isolated sperm cells (3 × 10⁶ per milliliter) show a high viability score (90%) as demonstrated with the fluorochromatic reaction. They appeared as spherical cells which lack cell wall and plastids, and can be considered as haploid protoplasts.