A special morphogenetic wall defect and the subsequent activity of “murosomes” as the very reason for penicillin-induced bacteriolysis in staphylococci

The actual reason for the penicillin-induced bacteriolysis of staphylococci was shown to be the punching of one or a few minute holes into the peripheral cell wall at predictable sites. These perforations were the result of the lytic activity of novel, extraplasmatic vesicular structures, located exclusively within the bacterial wall material, which we have named murosomes.In untreated staphylococci the punching of holes into the peripheral wall is a normal process which follows cross wall completion and represents the first visible step of cell separation. Under penicillin, however, analogous holes are punched by the murosomes at sites of presumptive cell separation even if no sufficient cross wall material had been assembled before at this site (but had rather been deposited at other sites). Consequently, because of the internal pressure of the protoplast, lytic death is the inevitable result of this perforation of the protective peripheral wall.Hence, the real mechanism of penicillin-induced bacteriolysis in staphylococci is considered to be mainly the result of a special morphogenetic wall defect: bacteriolysis is taking place regularly when a cell separation process is no longer preceeded by sufficient cross wall assembly at the correct place. However, hypotheses which are based purely on some variations of overall biochemical processes like total wall enzyme activities or total wall synthesis are not regarded to be sufficient to explain this type of lytic death.Dedicated to Prof. Dr. Gerhart Drews on the occasion of his 60th birthday     

A novel, “hidden” penicillin-induced death of staphylococci at high drug concentration,occurring earlier than murosome-mediated killing processes

In log-phase cells of staphylococci, cultivated under high, non-lytic concentrations of penicillin G, there occurred a novel killing process hitherto hidden behind seemingly bacteriostatic effects. Two events are essential for the apprearance of this hidden death: (i) the failure of the dividing cell to deposit enough fibrillar cross-wall material to be welded together, and (ii) a premature ripping up of incomplete cross walls along their splitting system. Hidden death started as early as 10–15 min after drug addition, already during the first division cycle. It was the consequence of a loss of cytoplasmic constituents which erupted through peripheral slit-like openings in the incomplete cross walls. The loss resulted either in more or less empty cells or in cell shrinkage. These destructions could be prevented by raising the external osmotic pressure. In contrast, the conventional non-hidden death occurred only much later and exclusively during the second division cycle and mainly in those dividing cells, whose nascent cross walls of the first division plane had been welded together. These welding processes at nascent cross walls, resulting in tough connecting bridges between presumptive individual cells, were considered as a morphogenetic tool which protects the cells, so that they can resist the otherwise fatal penicillin-induced damages for at least an additional generation time (morphogenetic resistance system). Such welded cells, in the virtual absence of underlying cross-wall material, lost cytoplasm and were killed via ejection through pore-like wall openings or via explosions in the second division plane and after liberation of their murosomes, as it was the case in the presence of low, lytic concentrations of penicillin. Bacteriolysis did not cause any of the hitherto known penicillin-induced killing processes.Dedicated to Prof. Dr. Georg Henneberg on the occasion of his 85th birthday     

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.     

Two alternative mechanisms of cell separation in staphylococci: one lytic and one mechanical

Electron microscopy studies revealed two different mechanisms of cell separation in Staphylococcus aureus. Both mechanisms were initiated by the centrifugal lytic action (directed outward from the center) of murosomes, which perforated the peripheral cell wall. Thereafter, during the first type of cell separation, murosomes also lysed large parts of the cross wall proper in the opposite, i.e., centripetal direction, forming spokelike lytic lesions (“separation scars”) next to the most prominent structure of the cross wall, the splitting system. This bidirectional lytic action of murosomes revealed that the staphylococcal cross wall is composed of permanent and transitory parts; transitory parts constituted about one-third of the volume of the total cross wall and seemed to be digested during cell separation. The second mechanism of cell separation was encountered within the splitting system, which has been regarded as the main control unit for lytic cell separation for more than 25 years. The splitting system, however, represents mainly a mechanical aid for cell separation and becomes effective when cell-wall autolytic activities are insufficient. Received: 5 August 1996 / Accepted: 4 December 1996     

Penicillin-induced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis

To analyze if chemical cell wall alterations contribute to penicillin-induced bacteriolysis, changes in the amount, stability, and chemical composition of staphylococcal cell walls were investigated. All analyses were performed before onset of bacteriolysis i.e. during the first 60 min following addition of different penicillin G doses. Only a slight reduction of the amount of cell wall material incorporated after penicillin addition at the optimal lytic concentration was observed as compared to control cells. However, the presence of higher penicillin G concentrations reduced the incorporation of wall material progressively without bacteriolysis. Losses of wall material during isolation of dodecylsulfate insoluble cell walls were monitored to assess the stability of the wall material following penicillin addition. Wall material grown at the lytic penicillin concentration was least stable but about 30% of the newly incorporated wall material withstood even the harsh conditions of mechanical breakage and dodecylsulfate treatment. Dodecylsulfate insoluble cell walls were used for chemical analyses. While peptidoglycan chain length was unaffected in the presence of penicillin, other wall parameters were considerably altered: peptide cross-linking was reduced in the wall material synthesized after addition of penicillin; reductions from approx. 85% in controls to about 60% were similar for lytic and also for very high penicillin concentrations leading to nonlytic death. O-acetylation was also reduced after treatment with penicillin; this effect paralleled the occurence of subsequent bacteriolysis at different drug concentrations. The results are not consistent with hypotheses explaining penicillin-induced lysis as a result of an overall weakened cell wall structure or an overall activation of autolytic wall enzymes but not conflicting with the model that ascribes penicillin-induced bacteriolysis as the result of a very restricted, local perforation of the peripheral cell wall (murosome-induced bacteriolysis).Abbreviations CL Cross-linking - DNFB 2,4-dinitro-1-fluorobenzole - MIC Minimal inhibitory concentration - OD Optical density at 578 nm - PEN Penicillin G     

Division and death rates of Salmonella typhimurium inside macrophages: use of penicillin as a probe.

In mouse peritoneal macrophages infected in vitro with Salmonella typhimurium the number of viable bacteria and the number of stainable bacteria detected by light microscopy both increased at similar rates with a doubling time of more than 1 h. Antibiotics were not present; instead extracellular bacteria were removed by frequently rinsing the cells. The bacterial doubling time in the same medium in the absence of macrophages was about 20 min. Penicillin added to macrophage monolayers rapidly entered the macrophages, reaching a diffusion equilibrium. The penicillin-induced bacterial death rate appeared to depend on the bacterial division rate as well as on the penicillin concentration. These properties of penicillin were used to monitor intracellular bacterial division and death rates. The results indicated that intracellular killing, with the disappearance of stainable bacteria, did not contribute to the extended doubling time in macrophages. It was concluded that the intracellular environment of the bacteria was probably growth inhibitory but not bactericidal.     

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.     

The arrangement of microfibrils in sporangial walls of Allomyces

Summary The microfibrillar component of the walls of zoosporangia and resistant sporangia of the phycomycete Allomyces arbusculus has been studied in the electron microscope, after chemical removal of the amorphous wall materials. In the zoosporangium wall the microfibrils are randomly arranged, as in the outer layer of the hyphal walls, and the sporangial wall is of even thickness. In the resistant sporangia the microfibrillar layer of the wall is perforated by numerous pores 0.25 in diameter. The microfibrils are randomly arranged over much of the wall but tend to be concentrically arranged in the vicinity of the pores. On the inside of the wall the microfibrils form a thickened rim around the pore.     

Differential Recovery of Auxotrophs After Penicillin Enrichment in Escherichia coli

Various auxotrophs are recovered from a penicillin enrichment cycle with differing efficiencies. Reconstruction experiments indicate that, under starvation conditions in the presence of penicillin, most auxotrophs undergo some death, whereas prolineless mutants are virtually immune to penicillin-induced killing.     

Mode of cell separation and arrangement of Staphylococcus

The process of cell separation and arrangement of Staphylococcus was investigated using a scanning electron microscope. After two cycles of cell division, the Staphylococcal cells cultured on an agar medium were generally observed to be arranged in three morphological types: linear, square, and crooked arrangements. Results of the examination of cell surface structure revealed that separations had occurred in these clustered cells following two patterns. One type of second separation occurred parallel to the transversal axis of the preceding pair of the parental cells (X-type) and the other occurred tangential to it (Y-type). In the former type, the four daughter cells were usually arranged tetragonally after the separations, and in the latter type they were arranged either linearly or crookedly depending on the direction of the second separation. The final pattern of the cell arrangement was thus determined by the type of septal wall formation and the direction of cell separation. After several cycles of cell divisions, the cells were finally arranged in an irregular grape-like cluster, even though the cross walls were formed regularly at the rectangular face of the preceding cross walls.     

Ultrastructure of the Cell Wall and Cell Division of Unicellular Blue-green Algae

The fine structure of the cell wall and the process of cell division were examined in thin sections of two unicellular blue-green algae grown under defined conditions. Unilateral invagination of the photosynthetic lamellae is the first sign of cell division in the rod-shaped organism, Anacystis nidulans. Symmetrical invagination of the cytoplasmic membrane and inner wall layers follows. One wall layer, which appears to be the mucopolymer layer, is then differentially synthesized to form the septum; the outer wall layers are not involved in septum formation. Centripetal splitting of the inner layer separates the two daughter cells. A second division, in a plane parallel to the first, usually occurs before the first daughter cells are separated. In the coccoid organism, Gleocapsa alpicola, the features of cell division are broadly similar; however, unilateral invagination of the lamellae is not observed and the second division takes place in a plane perpendicular to the plane of the previous division.     

Onset of penicillin-induced bacteriolysis in staphylococci is cell cycle dependent.

Synchronously growing staphylococci were treated with "lytic" concentrations of penicillin at different stages of their division cycle. Coulter Counter measurements and light microscopy were used to determine the onset of bacteriolysis. Independent of the stage of the division cycle at which penicillin was added, (i) the cells were always able to perform the next cell division; (ii) the following division, however, did not take place; and (iii) instead, at this time, when the onset of the subsequent cell separation was observed in control cultures, lysis of the penicillin-treated cells occurred. These results support a recent model (P. Giesbrecht, H. Labischinski, and J. Wecke, Arch. Microbiol. 141:315-324, 1985) explaining penicillin-induced bacteriolysis of staphylococci as the result of a special morphogenetic mistake during cross wall formation.     

The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance

Previous studies in our laboratory have shown that the Staphylococcus aureus LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a lrgAB mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the lrgAB mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the lrgAB defect by providing the lrgAB genes in trans restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the lrgAB mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the lrgAB mutation enhanced penicillin-induced killing of cells approaching the stationary phase of growth, the time at which the lrgAB operon was shown to be maximally expressed. This effect of the lrgAB mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the lrgAB mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which lrgAB expression was shown to be minimal. However, expression of the lrgAB operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of S. aureus involves a novel regulator of murein hydrolase activity.     

Fine Structure of Selected Marine Pseudomonads and Achromobacters

The fine structure of more than 20 marine pseudomonads and more than 15 achromobacters was examined. Under the conditions extant, clear differences between members of these two groups were seen. The pseudomonads displayed the characteristic gram-negative morphology: the cell wall was irregularly undulant and the cytoplasmic membrane more nearly planar, ribonucleoprotein (RNP) particles were loosely packed throughout the periphery of the cytoplasm, and the deoxyribonucleic acid (DNA) was axially disposed. Cell division appeared to be by constriction. Some strains characteristically produced evaginations or blebs of the cell wall. Occasionally, thick, densely stained ring structures were seen which are possibly analogous to mesosomes. In contrast, the achromobacters demonstrated a regularly undulant outer cell wall element and a planar inner wall. The cytoplasmic membrane was thin and not readily observed. RNP particles were densely stained and tightly packed in the cytoplasm; the DNA was most often lobate in disposition. Cellular division was mediated by the formation of a septum which consisted of the cytoplasmic membrane and the inner element of the cell wall. Mesosomes were observed in all of the strains examined. Dense inclusion bodies were also seen in many strains.     

Ultrastructural observation of paracrystalline aggregates of microtubules in anterior pituitary cells of the chinchilla (Chinchilla laniger)

Summary Unusual paracrystalline aggregates of microtubules which have not been described in any other mammalian species were observed in cultured anterior pituitary cells of normal chinchillas as well as in situ in the pituitary glands of these animals. These aggregates appeared as regularly arranged tubular structures in the longitudinal plane, and as a checkerboard pattern of closely and regularly packed microtubules when examined in transverse section. Supplementation with vinblastine, colcemide or colchicine in the culture medium did not change these structures morphologically. Each unit of tubules consisted of an outer wall or parellelogram profile and an inner wall composed of a single hexagonal doublet or in a figure 8 form. The outer wall of the parallelogram was 35×28 nm in length for both sides, while the diagonal of the inner wall was 18×28 nm. These paracrystalline aggregates of microtubules in the chinchilla pituitary cells are morphologically distinct from the paracrystalline assembly of cytoplasmic microtubules induced by vinblastine or other alkaloids.The function and significance of these paracrystalline aggregates in anterior pituitary cells of the chinchilla are uncertain.Supported by USPHS Grant HD 11826     

Formation of branched plasmodesmata in regenerating Solanum nigrum-protoplasts

During the early stages of culture, discontinuous branched half-plasmodesmata were found randomly scattered in the newly formed outer cell walls of regenerating Solanum nigrum L. protoplasts. During later culture stages, most of these outer-wall plasmodesmata, which had been exposed to the culture medium, disappeared, except for those near the periphery of division walls between daughter cells and those near non-division walls between secondarily associated unrelated cells. Moreover, in the peripheral parts of older division walls, there were continuous branched plasmodesmata which showed the typical morphological characteristics of secondary cell connections: several cytoplasmic strands joined in the median plane of the cell wall and were often linked by so-called median cavities. Evidence is presented that this type of continuous plasmodesma originates from the fusion of the half-plasmodesmata which persisted in the outer walls adjacent to the division wall. Due to growth of the cells after division, opposite parts of the outer walls of the daughter cells come into close contact and fuse, elongating the original division wall peripherally. Opposite half-plasmodesmata remaining in these parts of the outer wall may thereby also be brought into contact and fuse to form a continuous secondary cell connection in the secondarily coalesced wall part. Our assumption is supported by further experiments: (i) longterm video observations of living cells showed differences in the development of the shapes of regenerating cells and (ii) electron-microscopical investigations showed differences in the frequency of the, presumably secondary, cell connections in the peripheral parts of the division walls — both related to the firmness of the embedding medium. In the central parts of division walls, unbranched primary cell connections were found as well as a second type of continuous branched plasmodesma showing an entirely different branching pattern: the region of the middle lamella was always traversed by straight, unbranched parts of these plasmodesmata and the branches occurred exclusively within the younger wall layers. Evidence is given that these branches are modifications of originally unbranched primary plasmodesmata, developing during subsequent thickening of the division wall.The authors are indebted to Prof. H. Binding, Botanisches Institut, Universität Kiel, for making his cell-culture laboratory available to us and to Dr. F. Grundler, Institut für Phytopathologie, Universität Kiel, for placing the video equipment at our disposal. The work was supported by the Deutsche Forschungsgemeinschaft.     

The interaction of naturally elaborated blebs from serum-susceptible and serum-resistant strains of Neisseria gonorrhoeae with normal human serum

We studied the interaction of normal human serum immunoglobulins with outer-membrane bleb antigens of Neisseria gonorrhoeae. Gonococcal 68,000 Dalton and Lip (H.8 antigen) outer-membrane proteins were recognized by normal human serum immunoglobulins in blebs from serum-resistant strains, but not in blebs from serum-susceptible strains. The addition of blebs from a serum-resistant strain to bactericidal assays resulted in significantly greater inhibition of serum killing than the addition of blebs from a serum-susceptible strain. Our results indicate that blebs from two serum-resistant gonococcal strains have an enhanced ability to bind and remove cell-targeted bactericidal factors, and that outer-membrane blebbing may contribute to serum resistance.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Lymphoid structures in the wall of the human esophagus during postnatal ontogenesis

Lymphoid aggregates in the wall of the human oesophagus have been studied in corpses of 107 persons died a sudden death and who had not any disease in the digestive organs and in the immune system (total plane preparations, slices stained with hematoxylin--eosin, azure-2-eosin and after van Gieson). The lymphoid aggregates are arranged as longitudinal chains. Their number increases from birth towards the second childhood, and then decreases towards the old age. In the upper oesophageal parts these aggregates are more numerous than in the lower ones. The lymphoid aggregates look like convexoconvex lenses. Their size is maximal during the second childhood, the longitudinal dimensions are nearly two times as great as their thickness. With time, the sizes of the formations become small. The arrangement density of the lymphoid cells in the subepithelial layer of the mucous membrane is maximal in newborns, and then it decreases up to the old age. The lymphoid aggregates are situated in the oesophageal wall, mainly, near the ducts of the oesophageal glands.     

Atomic force microscopy of cell growth and division in Staphylococcus aureus

The growth and division of Staphylococcus aureus was monitored by atomic force microscopy (AFM) and thin-section transmission electron microscopy (TEM). A good correlation of the structural events of division was found using the two microscopies, and AFM was able to provide new additional information. AFM was performed under water, ensuring that all structures were in the hydrated condition. Sequential images on the same structure revealed progressive changes to surfaces, suggesting the cells were growing while images were being taken. Using AFM small depressions were seen around the septal annulus at the onset of division that could be attributed to so-called murosomes (Giesbrecht et al., Arch. Microbiol. 141:315-324, 1985). The new cell wall formed from the cross wall (i.e., completed septum) after cell separation and possessed concentric surface rings and a central depression; these structures could be correlated to a midline of reactive material in the developing septum that was seen by TEM. The older wall, that which was not derived from a newly formed cross wall, was partitioned into two different surface zones, smooth and gel-like zones, with different adhesive properties that could be attributed to cell wall turnover. The new and old wall topographies are equated to possible peptidoglycan arrangements, but no conclusion can be made regarding the planar or scaffolding models.