Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Biosynthesis of Macromolecules and Wall During Successive Steps

Protoplasts of Bacillus subtilis plated on SDG medium formed L colonies in quantative yield and propagated in the L-form indefinitely. Protoplasts or L bodies placed in 25% gelatin medium formed bacillary colonies. Details of the reversion of these naked bodies to the walled form are reported here. Protoplasts prepared in minimal medium reverted fairly synchronously 3 to 4 hr after inoculation into gelatin, but protoplasts preincubated in casein hydrolysate (CH)-enriched minimal medium were primed to revert within 1 hr in the gelatin. Preincubation for 1.5 hr in 0.44% CH was required for good priming. Cells must be subjected to this preincubation (step 1) in the naked state; it is effective for L bodies as well as protoplasts. Priming was blocked by chloramphenicol, puromycin, and actinomycin D but was not affected by penicillin, lysozyme, or inhibition of deoxyribonucleic acid (DNA) synthesis. It is concluded that protein and ribonucleic acid (RNA) synthesis are required during step 1, that DNA synthesis is not required, and that wall mucopeptide is not made. The reversion of well-primed protoplasts in the gelatin (step 2) proceeded undisturbed in thymine-starved cells with chromosomes arrested at the terminus. It was scarcely slowed by chloramphenicol in the gelatin but was delayed about 3 hr by both puromycin and actinomycin D. Escape from inhibition occurred while the inhibitors were still actively blocking growth. Penicillin and cycloserine inhibited and lysozyme reversed reversion. Momentary melting of the gelatin delayed reversion. It is concluded that mucopeptide synthesis occurs in step 2, that concomitant RNA, DNA, or protein synthesis is not essential, but that physical immobilization of excreted cell products at the protoplast surface is necessary early in step 2. Newly reverted cells were misshapen and osmotically sensitive. Processes which confer osmotic stability after reversion (step 3) did not occur in the presence of chloramphenicol or actinomycin D.     

The interrelationship between mucopeptide and ribitol teichoic acid formation as shown by the effect of inhibitors

1. The biosynthesis of teichoic acid in cell suspensions of two strains of Staphylococcus aureus is partially inhibited by the same low concentrations of penicillin that inhibit mucopeptide synthesis by 90–100%. Further increase in the concentration of the antibiotic by several hundred-fold still fails to cause any greater inhibition of teichoic acid synthesis. 2. Other conditions, such as amino acid deficiency or the presence of cycloserine or 5-fluorouracil, that inhibit mucopeptide synthesis also inhibit teichoic acid formation. 3. The degree of inhibition of teichoic acid synthesis caused by relatively high concentrations (10μg./ml.) of benzylpenicillin depends critically on the age of the culture from which the cell suspensions have been prepared. 4. No significant amounts of soluble teichoic acid have been found in the fluid from cells incubated in the presence of penicillin. 5. A high proportion of the teichoic acid formed in the presence of penicillin can be removed from wall preparations at room temperature by 0·1n-ammonia. This is not true of the teichoic acid formed in the absence of penicillin. 6. The teichoic acid extracted with ammonia from preparations of cell walls made from cells treated with penicillin is excluded from Sephadex G-25, has a low molar ratio of glucosamine to phosphorus and contains muramic acid, alanine, glutamic acid, glycine and lysine. 7. The implications of these results for the mechanism of action of penicillin are discussed.     

Iodinated vancomycin and mucopeptide biosynthesis by cell-free preparations from Micrococcus lysodeikticus

A particulate preparation from Micrococcus lysodeikticus was used to synthesize cell-wall mucopeptide. Radioactive iodinated vancomycin became attached to the preparation simultaneously with a complete inhibition of mucopeptide synthesis. After mucopeptide synthesis had occurred in the absence of antibiotic, the preparation took up more vancomycin, suggesting that new binding sites terminating in acyl-d-alanyl-d-alanine had been produced. The mucopeptide product was divided into a soluble and an insoluble portion, both sensitive to lysozyme. The soluble portion did not combine with vancomycin and hence had presumably lost its terminal d-alanine residues, either by transpeptidation or because of carboxy-peptidase action. The synthesis of both portions was unaffected by the presence of penicillin, but the insoluble part showed increased affinity for vancomycin, thus indicating that penicillin had caused conservation of d-alanyl-d-alanine termini.     

The inhibition of mucopeptide synthesis by benzylpenicillin in relation to irreversible fixation of the antibiotic by staphylococci

1. Benzylpenicillin is irreversibly fixed to staphyloccoci by a reaction that obeys second-order kinetics, whereas the progress of inhibition of mucopeptide synthesis obeys first-order kinetics after a short lag during which the antibiotic has no effect. 2. When the micro-organisms are saturated with benzylpenicillin they can still make mucopeptide in solutions containing chloramphenicol at a normal rate after a lag period. 3. About 90% of the benzylpenicillin stays fixed to the cells after mucopeptide synthesis has reached its maximum and constant rate. 4. During the phase when mucopeptide synthesis by cells saturated with benzylpenicillin is accelerating, a small number of additional sites that fix benzylpenicillin is revealed. The number of these sites reaches a maximum and constant value at about the same time as mucopeptide biosynthesis reaches a maximum and constant rate. 5. Staphylococci saturated with benzylpenicillin are exceedingly sensitive to fresh additions of the antibiotic. 6. The degree of inhibition of mucopeptide synthesis caused by these small amounts of antibiotic agrees with the degree of substitution by benzylpenicillin of the newly revealed or `sensitive' sites. 7. Since these sensitive sites are revealed during incubation of the bacteria with chloramphenicol it is unlikely that they are due to newly formed protein. 8. On the basis of these results, a hypothesis for the inhibition by penicillin of the cross-linking reaction in the terminal stages of mucopeptide synthesis is suggested.     

Activity of membrane ATPase during inhibition and reversion ofEscherichia coli cell division

Activity of Mg²⁺-dependent ATPase from the fraction of cell-free homogenate sedimenting at 35 000×g was studied during the growth and division ofEscherichia coli B. It decreased with the transition to stationary growth phase and after a specific inhibition of cell division. During the reversion of the division of filamentous forms the activity sharply increased; with the end of the reversion it dropped again to the level prior to the inhibition. The possible connection of the activity of Mg²⁺-dependent ATPase with the cell division ofEscherichia coli B is discussed.     

Antibiotic- and hormonally induced alterations inStaphylococcus aureus

The ability of penicillin to induce permeability changes inStaphylococcus aureus was markedly enhanced by selected gonadal steroids. Subinhibitory concentrations of penicillin and subinhibitory physiological concentrations of progesterone also acted in concert to reduce the incorporation of¹⁴C-alanine into staphylococcal mucopeptides by 18 to 21%. The minimal concentration of the antibiotic which significantly interfered with the incorporation of alanine into the staphylococcal mucopeptide was 3.30 units/ml. When progesterone was added to the system, the minimal concentration was lowered to 0.50 units/ml. The 17α-hydroxy-progesterone interfered with mucopeptide synthesis only when used in conjunction with penicillin. On the contrary, progesterone, dehydroepiandrosterone and β-estradiol exerted an additive effect in decreasing the incorporation of alanine into the staphylococcal mucopeptide. These results extend our previous studies and suggest an extracellular site of hormonal action located on the cell envelope.     

Inhibitory effect of penicillin on spore-specific functions inBacillus polymyxa 2459

Penicillin at concentrations non-inhibitory to the vegetative growth was found to inhibit sporulation inBacillus polymyxa 2459. The effect of penicillin was shown to be at the level of spore-specific mucopeptide synthesis. Penicillin had no effect on the early events such as DNA and protein synthesis in sporogenesis The sensitive period of inhibition was between T₀ to T₂ hours of sporulation.     

Effects of selected inhibitors on growth and cell division inAgmenellum

Summary Concentrations of chloramphenicol and penicillin G which permit growth induce the formation of temporary filaments, morphologically and ultrastructurally identical to stable, chemically-induced filamentous mutants ofAgmenellum quadruplicatum strainBG-1. These induced filaments were propagated by serial transfers in the presence of inhibitor and underwent an immediate, synchronous reversion upon its removal. The reversion of penicillin-induced filaments was insensitive to inhibitors of DNA synthesis but sensitive to inhibitors of protein synthesis until the completion of the mucopolymer septum. Penicillin G blocked the early stages or initiation of cell division. Chloramphenicol blocked the terminal stages of cell division, the cleavage of the mucopolymer septum by the outer wall layers.     

Role of the cell surface in bacterial mating: requirement for intact mucopeptide in donors for the expression of surface exclusion in R+ strains of Escherichia coli.

Two derivatives of the F-like R factor R1drd19 carrying mutually exclusive resistance determinants were used to study the role of the mucopeptide in the expression of conjugal functions. The use of metabolically active penicillin spheroplasts in R+ times R- matings had no effect on the ability of the cells to donate or accept a plasmid. However, in R+ times R+ matings it was found that surface exclusion was totally abolished if the donor, but not the recipient, was a spheroplast. This result implies that the traS gene, expressed by the excluding plasmid, is dependent for its action on an intact mucopeptide layer in the donor cell, and that this interaction is independent of the transfer ability of the excluding plasmid.     

Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus megaterium

Hancock, R. (Harvard Medical School, Boston, Mass.), and P. C. Fitz-James. Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus megaterium. J. Bacteriol. 87:1044-1050. 1964.-Penicillin and cycloserine do not inhibit the growth of protoplasts of Bacillus megaterium, indicating that inhibition of cell-wall synthesis is the only significant process by which they inhibit growth of bacteria. In contrast, bacitracin and vancomycin inhibit growth of protoplasts and bacteria at similar concentrations, indicating that they have important sites of action other than their known inhibition of cell-wall synthesis. At concentrations which inhibit mucopeptide synthesis, penicillin, bacitracin, and vancomycin each cause an increased rate of efflux of K ions from growing bacteria. This effect of penicillin is prevented by chloramphenicol or hypertonic sucrose, whereas the effects of bacitracin and vancomycin are unchanged under these conditions. It is concluded that bacitracin and vancomycin have direct effects on the cytoplasmic membrane, and it is proposed that their inhibition of cell-wall synthesis could be a consequence of these effects. Bacitracin and vancomycin do not compete with penicillin for binding to cells of B. megaterium, a further indication that they have a different primary site of action.     

Inhibition of bacterial cell wall mucopeptide synthesis: A new function of RNA bacteriophage Qβ

Studies of the morphology of Escherichia coli showed that these bacilli when infected with RNA phage Qβ in broth containing hypertonic sucrose and Mg²⁺ formed osmotically labile spherical cells or spheroplasts. Phage-induced spheroplasts readily released their burst of phage when diluted into ordinary culture broth without sucrose. Investigation of the mechanism of host cell lysis revealed that incorporation of [³H]diaminopimelic acid (DAP) into the mucopeptide layer of the cell wall was markedly inhibited starting at about the midpoint of the phage replication cycle. The major site of inhibition is the DAP-containing mucopeptide layer since the synthesis of the lipoprotein-lipopolysaccharide layer, making up the bulk of the cell wall of E. coli, was not affected. A model for Qβ-mediated cell lysis is discussed which is analogous to penicillin-induced cell rupture.     

Degradation and turnover of bacterial cell wall mucopeptides in growing bacteria

The mucopeptide layer of the cell wall ofBacillus megaterium is broken down into separate components during growth of the cells. The released diaminopimelic acid is partly decarboxylated to lysine, which is incorporated in the proteins and partly used for cell wall resynthesis. The smaller portion of the degraded mucopeptide is released into the medium in the form of non-utilized fragments. The rate of the mucopeptide turnover is a function of the rate of growth of the culture. About 15–20% of the rigid layer of the cell wall is degraded during on cell division. The sensitivity ofBacillus megaterium to lysozyme and the rate of its conversion to protoplasts is also proportionate to the rate of growth of the culture. There is no measurable mucopeptide turnover in non-growing cells, either in the stationary phase of the culture or in starvation in nitrogen-free medium. The resistance of the cell wall to lysozyme also increases during the stationary phase. The rigid component of the cell wall is probably also broken down during growth ofBacillus cereus andEscherichia coli cultures.     

Production of thiol-penicillin-binding protein 3 of Escherichia coli using a two primer method of site-directed mutagenesis.

The active site serine residue of penicillin-binding protein 3 of Escherichia coli that is acylated by penicillin (Ser-307) has been converted to a cysteine residue using a simple and efficient two primer method of site-directed mutagenesis. The resulting thiol-penicillin-binding protein 3 was expressed under the control of the lacUV5 promoter in a high copy number plasmid. Constitutive expression of the thiol-enzyme (but not of the wild-type enzyme) was lethal, and the plasmid could only be maintained in E. coli strains that carried the lacIq mutation. Induction of the expression of the thiol-enzyme resulted in inhibition of cell division and the growth of the bacteria into very long filamentous cells. The inhibition of septation was probably due to interference of the function of the wild-type penicillin-binding protein 3 in cell division by the enzymatically inactive thiol-enzyme, and this implies that penicillin-binding protein 3 acts as part of a complex in vivo. We were unable to detect any acylation of the thiol-enzyme by penicillin, but it is not yet clear if this was because the thioester was not formed at an appreciable rate, or if it was formed but was too unstable to be detected by a modified penicillin-binding protein assay.     

Synthesis of macromolecules in the yeastCandida utilis as influenced by a specific factor inducing cell division

From the yeastCandida utilis a compound was isolated which uncouples the process of cell growth and division by accelerating the cell division without influencing outgrowth of cell mass when applied on another population of identical yeasts. This compound accelerated the initiation of DNA synthesis and had no influence on the synthesis of RNA and protein. Moreover, in the presence of division inducing factor the yeasts started multiplication before the content per cell reached the control level. The stimulating effect of division inducing factor was not obvious when the proteosynthesis of yeast cells was inhibited. We concluded that the division inducing factor regulates the formation of a protein which is synthesized in a very small amount and has a role in the initiation of DNA replication.     

Latent root primordia in poplar stems

Root primordia initiate in poplar stems in the secondary growing parts, that is in the parts where the elongation growth is terminated and the leaves are mature. Their initiation is connected with the occurrence of unusual biseriate, rarely multiseriate rays. A small cell group in the secondary phloem is initiated by cell division of the ray. It gradually enlarges by continuing cell division, by the addition of cells adjacent to the cell group and by cambial activity. Thus, a hemispherical root primordium is formed, for which a permanent occurrence of reserve lipids is characteristic. In stems several years old the intraprimordial mitotic activity is rhythmically renewed together with the cambium function renewal. Latent root primordia slightly enlarge with the passing years, whereas mainly the cells localized in their centre divide. Further organization and root histogenesis was not observed either in older root primordia. Adjacent to root primordia, cambial initials produce the secondary xylem elements increasingly. Xylem protuberances are thus formed under root primordia. Primordia initiation is most frequent within the first year of stem development, though they can also initiate in later years.     

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.     

Structure of the cell wall of lactobacilli. Role of muramic acid phosphate in Lactobacillus fermenti

1. The polysaccharide and mucopeptide components of the cell wall of Lactobacillus fermenti, serological group F, were separated by mild conditions of acid hydrolysis; the polysaccharide was composed of glucose and galactose. 2. Soluble cell-wall products were isolated from cell wall lysed by lysozyme and a Streptomyces enzyme preparation. The lysozyme-dissolved fraction contained a greater proportion of mucopeptide. 3. The soluble preparations were heated in dilute acid to hydrolyse the linkage between the polysaccharide and mucopeptide components and then incubated with acid phosphatase. 4. Inorganic phosphate was released from products of Streptomyces enzyme action but not from products of lysozyme action. 5. The phosphate was shown to be present in the mucopeptide as muramic acid phosphate. It is concluded that in the intact wall polysaccharide is joined to muramic acid by a phosphodiester linkage.     

THE INITIAL STRUCTURAL LESION OF PENICILLIN ACTION IN BACILLUS MEGATERIUM

The effect of penicillin on the structure of Bacillus megaterium cells was followed in media with and without osmotic stabilization. In peptone without osmotic support the cells showed a distortion of the normal membrane-wall relationship by 20 minutes. This appeared to be a combination of both membrane distortion and cytoplasmic leakage. Lytic changes quickly followed. With osmotic support a clean-cut lesion at the transverse-septal site developed by 10 minutes' growth in penicillin. The membrane lost its normal relationship to the cell wall and formed a pocket which was filled with a fibrous material which appeared to be unorganized wall mucopeptide. The pocket of fibers enlarged until the cell either lysed or formed a protoplast.     

Conserved relationship between FtsZ and peptidoglycan in the cyanelles of Cyanophora paradoxa similar to that in bacterial cell division

Cyanelles of the biflagellate protist Cyanophora paradoxa have retained the peptidoglycan layer, which is critical for division, as indicated by the inhibitory effects of β-lactam antibiotics. An FtsZ ring is formed at the division site during cyanelle division. We used immunofluorescence microscopy to observe the process of FtsZ ring formation, which is expected to lead cyanelle division, and demonstrated that an FtsZ arc and a split FtsZ ring emerge during the early and late stages of cyanelle division, respectively. We used an anti-FtsZ antibody to observe cyanelle FtsZ rings. We observed bright, ring-shaped fluorescence of FtsZ in cyanelles. Cyanelles were kidney-shaped shortly after division. Fluorescence indicated that FtsZ did not surround the division plane at an early stage of division, but rather formed an FtsZ arc localized at the constriction site. The constriction spread around the cyanelle, which gradually became dumbbell shaped. After the envelope’s invagination, the ring split parallel to the cyanelle division plane without disappearing. Treatment of C. paradoxa cells with ampicillin, a β-lactam antibiotic, resulted in spherical cyanelles with an FtsZ arc or ring on the division plane. Transmission electron microscopy of the ampicillin-treated cyanelle envelope membrane revealed that the surface was not smooth. Thus, the inhibition of peptidoglycan synthesis by ampicillin causes the inhibition of septum formation and a marked delay in constriction development. The formation of the FtsZ arc and FtsZ ring is the earliest sign of cyanelle division, followed by constriction and septum formation.     

Properties of rod cells reversed from penicillin spheroplasts in Escherichia coli

Hirokawa, Hideo (The University of Tokyo, Tokyo, Japan). Properties of rod cells reversed from penicillin spheroplasts in Escherichia coli. J. Bacteriol. 91:125-128. 1966.-Spheroplasts of Escherichia coli B-054 were formed by penicillin, and the properties of rod cells reversed from the spheroplasts were examined. It was found that the rod cells were not capable of forming colonies when placed in a minimal medium immediately after the reversion, the postaddition of the nutrient to this medium being without effect. In this respect, the so-called residual cells which did not convert to spheroplasts by penicillin treatment behaved just as the reversed rod cells. These two types of cells, the reversed and residual rod cells, however, could grow up to form colonies when they were cultivated in a complex medium immediately after the reversion. The nutrient requirement and penicillin sensitivity of the progeny of these cells were found to be quite similar to those of the original untreated cells. Accordingly, it was concluded that the nutritional defect induced by penicillin treatment was not a hereditary change.