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A transformation protocol mediated by Agrobacterium tumefaciens is described for the sapstaining fungus Ophiostoma piceae. We compared transformants obtained from Agrobacterium with those obtained from yeast-like cells made into spheroplasts and treated with CaCl2. For all putative transformants analyzed, Southern hybridization confirmed that the hygromycin resistance gene had been integrated into the genomic DNA. While all transformants obtained from the treated spheroplasts had multiple copy vector insertion, 85% of the Agrobacterium-mediated transformants had single copy vector insertion.Key words: Ophiostoma piceae, genetic transformation, Ti plasmid.

The desertomycin action upon Saccharomyces uvarum wall synthesis has been studied. Spheroblast regeneration was carried out in a liquid medium containing labeled glucose to monitor the synthesis of different wall components. In the presence of desertomycin, wall synthesis was affected; this was expressed as a net reduction of insoluble alkali constituents content, more precisely the insoluble acido-alkali fraction that, in yeasts, is constituted by chains of beta(1,3)-glucans linked among themselves by beta(1,6) bonds. Mannan formation was not inhibited such polymers that cannot be fixed to the glucan matrix of the wall were liberated in the regeneration medium. Because of desertomycin action, the decrease in insoluble alkali content revealed an interference with the enzymatic systems catalyzing glucan synthesis. In vitro, however, this antifungal had little effect upon glucan synthetase activity: doses 5 times superior to the subinhibiting level used in vivo caused only 30% inhibition. This result can be explained by an indirect action of desertomycin. Parietal disorders were the result of membrane structure disturbance, notably the phospholipids and localized enzymatic systems. This antifungal presents an analogical structure with macrolides with recognized membrane action.

A metabolite with antifungal activity, of non polyenic macrolide structure, was extracted and purified from the culture supernatant of a soil-isolated Streptomyces spectabilis strain, BT 352. This product was found to be related to (or being) desertomycin. Six yeast and five filamentous fungus strains were used to determine minimum concentration of the metabolite that inhibits growth by 80% (IMC); it was established at 50 micrograms/mL for the fungi and at 100 micrograms/mL or more for the yeasts tested. Short-term genotoxicity tests showed no antifungal effect on the bacterial genome, and desertomycin at concentration levels of 100 micrograms/mL or more affected protein synthesis. The antifungal metabolite had no immediate inhibiting effect upon yeast respiration, even at high concentrations; however, the respiration activity of cells grown in the presence of subinhibiting doses and collected during their growth phase was reduced by as much as 40%. Saccharomyces uvarum spheroplast regeneration in a liquid medium containing desertomycin was inhibited at doses fivefold weaker than the IMC determined with intact cells. Contrary to amphotericin B, desertomycin subinhibiting doses do not modify, and if so lightly, the yeast latent phase or the spheroplast wall regeneration phase, thus indicating a fungicidal action. Moreover, following a 30-min contact with desertomycin subinhibiting and inhibiting doses, yeasts liberated potassium in large amounts, indicating that plasma membranes were affected.

The location of cellulase in C. fulvus depends on the carbon source for growth and the age of the culture. When cells were grown on glucose or cellobiose all CMC-hydrolyzing enzyme was cell-bound but only part of the activity was located on the cell surface. Treatment of cells with EDTA, lysozyme, and detergents and subsequent fractionation experiments showed that cellulase was also located in the periplasm and bound to a membrane fraction.Growth on cellulose gave cell-free cellulase active against CMC. The enzyme was repressed by glucose but formed at a constant differential rate on cellobiose and amylose. This rate was 8–10 times lower than on cellulose and possible reasons for this are discussed.

If cells of Spirillum serpens (strain VHL) are heat-killed at 56C for 90 min the cells retain their original morphology remarkably well. When actively motile cells of Bdellovibrio bacteriovorus (strain 6-5-S) are inoculated into a suspension of S. serpens heat-killed at this temperature, B. bacteriovorus attacks and penetrates the dead cells, and undergoes a cycle of growth and replication similar to that which occurs in living cells. Intracellular growth of B. bacteriovorus in nonviable cells has not previously been reported.

Pseudomonas aeruginosa (ATCC 9027) releases four periplasm-located enzymes, i.e., ribonuclease (EC 3.1.4.22; EC 3.1.4.23), alkaline phosphatase (EC 3.1.3.1), cyclic-2′, 3′-phosphodiesterase (EC 3.1.4.d), and 5′-nucleotidase (EC 3.1.3.5) into the medium during growth. Ribonuclease and alkaline phosphatase are classed as enzymes which are readily extracted by osmotic shock and spheroplast formation whereas cyclic-2′,3′-phosphodiesterase and 5′-nucleotidase are classed as enzymes which are not readily extracted by these procedures. In view of the relative ease of extraction of the former enzymes it is suggested that the latter enzymes, cyclic-2′,3′-phosphodiesterase and 5′-nucleotidase, are bound and located in the periplasm in a manner different to ribonuclease and alkaline phosphatase.

Six mutants of Salmonella typhimurium LT2 with defects in the heptose region of the lipopolysaccharide (LPS) ("rough" mutants) were more sensitive to the growth-inhibitory effects of erythromycin, bacitracin, vancomycin, novobiocin, kanamycin, and cloxacillin and of deoxycholate than smooth strains, but less sensitive to tetracycline and ampicillin. In general, growth of the three rough mutants of chemotype Rd2, which lack the distal but not the proximal heptose unit in the LPS, was less inhibited than the three mutants of chemotype Re, which are heptose-deficient. In addition, inhibition of uracil-1-14C incorporation in the presence of actinomycin D and spheroplast formation in the presence of lysozyme occurred in the rough mutants without ethylenediaminetetraacetate (EDTA) treatment of the cells, while actinomycin D and lysozyme were effective on smooth strains only after EDTA treatment. Since the major part of the LPS is in the outer membrane of the cell envelope, and since the target of the toxic agents used is located inside this layer, these data indicate that the carbohydrate part of the LPS component of the outer membrane is an essential part of a barrier layer preventing penetration of large molecules.

The site of the cell barrier to actinomycin-D uptake was studied using a wild-type Escherichia coli strain P and its cell envelope-defective filamentous mutants, strains 6γ and 12γ, both of which 'leak' β-galactosidase and alkaline phosphatase into the medium during growth indicating both membrane and cell-wall defects. Actinomycin-D entered the cells of these two mutant strains as evidenced by the inhibition of both 14C-uracil incorporation and synthesis of the induced β-galactosidase system. Under similar conditions, no inhibition occurred in the wild-type strain and its sucrose-lysozyme prepared spheroplasts. Actinomycin-D did, however, inhibit the above-mentioned systems in the wild-type sucrose-lysozyme spheroplasts prepared in the presence of 2 mM EDTA. The experimental data indicate that although the cell wall may act as a primary barrier or sieve to actinomycin-D, the cytoplasmic membrane should be considered the final and determinative barrier to this antibiotic.

The serological activity of glycine-induced spheroplasts and normal intact cells of Salmonella typhi was assessed. No major differences could be detected in the somatic O and in the Vi antigens of spheroplasts and intact cells. The only major change associated with transformation of normal typhoid organisms into spheroplasts seems to be associated with the flagellar antigen. Spheroplasts evoked a high H agglutinin response in immunized rabbits but were poorly agglutinated by standard H antiserum.The immunogenic ability of the spheroplasts to protect mice from the typhoid challenge was about five times higher than that of intact cells and the toxicity was reduced at least 10-fold.

The particulate material (intact and dissociated phycobilisomes), adjacent to the thylakoids of Plectonema boryanum and Calothrix parietina, was examined in thin sections and in freshly prepared C-phycocyanin extracts. For the latter a method was developed using spheroplasts of log-phase cells. When examined by sedimentation velocity centrifugation, the sedimentation coefficients of the rapidly extracted C-phycocyanin were found to be 18 S and 5 S. Also, a 10-S boundary was observed with P. boryanum. When C. parietina was grown under red light the 18-S aggregates increased to 20 S. The 18 S particles, in electron micrographs of negatively stained preparations, displayed eight subunits surrounding a central one. The possibility that some subunits fall out of the plane of view is contemplated in proposing a dodecamer arrangement for such particles. The 20-S particles were also examined by electron microscopy. However, the precise number of monomer units associated with either the 18-S or 20-S particles has not yet been ascertained. Thin sections of intact algal cells are presented to demonstrate the presence of phycobilisomes along the outer faces of the thylakoid membranes.

Experiments, using the scanning-beam electron microscope, were conducted to compare the morphologic lesions induced in a strain of Proteus mirabilis by two semisynthetic penicillins, ampicillin and carbenicillin, and two cephalosporins, cephalothin and cephaloridine. The observations demonstrated that the semisynthetic penicillins and cephalosporins did not induce the same sequence of structural alterations. In the presence of cephalothin or cephaloridine, mid-cell spherical defects were induced which rapidly progressed to large bulbous enlargements at low concentrations (2 and 20 μg/ml) but only spheroplast-like forms were observed at high concentrations (200 and 2000 μg/ml). Ampicillin and carbenicillin caused similar alterations in morphology except that marked cell elongation without division occurred with 2 and 20 μg/ml. These variations may reflect subtle differences in sites, rates, sensitivity, or mechanisms of action between these two groups of cell-wall inhibiting antibiotics.

Sphaerotilus natans developed sheathed filaments in stationary liquid cultures and motile swarm cells in shaken ones. Electron microscopy of negatively stained preparations and thin sections showed that the sheath consists of fibrils. When the filaments were grown in broth with glucose added, the sheath was much thicker and the cells were packed with granules of poly-β-hydroxybutyrate.Swarm cells possess a subpolar tuft of 10 to 30 flagella and a polar organelle which is usually inserted in a lateral position and believed to be ribbon-shaped. The polar organelle consists of an inner layer joined by spokes to an accentuated plasma membrane. The flagellar hook terminates in a basal disk, consisting of two rings, which is connected by a central rod to a second basal disk.

Growth of Rhodotorula aurantiaca in a medium supplemented with sulfur amino acids led to the synthesis and accumulation of S-adenosylmethionine, which was accompanied by a reduction in the total contents of carotenoid pigments and an increased sensitivity of the cell walls to snail gut enzymes (Helix pomatia) as judged by spheroplast formation. Walls isolated from supplemented cultures contained increased amounts of lipid and mannan but had a reduced content of protein and alkali-insoluble carbohydrate. The chemical modifications of the cell wall, which resulted as an indirect consequence of excessive S-adenosylmethionine synthesis, are interpreted to explain the ease with which sulfur amino acid-supplemented cultures of R. aurantiaca are converted to spheroplasts as well as their tendency to aggregate.

S. typhi spheroplasts were produced by growing typhoid organisms in a liquid semisynthetic medium containing 1.5 to 2.0% glycine. The addition of traces of magnesium and glucose concentrations of 0.5 to 2.0% was required for optimal growth conditions. These cells were considered to be of the transition L form since they reproduced as spheroplasts as long as they were grown in the glycine medium, but unlike a true L organism they reverted to the normal bacillary form when subcultured to glycine-free medium.The lytic susceptibility of S. typhi spheroplasts was studied. They were found to be resistant to a number of detergents, alcohols, and enzymes. They could, however, be broken up by osmotic shock and were lysed by trypsin, pancreatic lipase, and, best of all, by Dornavac(R), a commercial preparation of pancreatic desoxyribonuclease which, in addition, contains traces of other enzymes, such as trypsin and lipase. Studies towards the production of an enzyme-lysed sphero-plast vaccine for the prevention of typhoid are now in progress.

Spheroplasts of Mycobacterium smegmatis and Mycobacterium tuberculosis were subjected to a wide range of centrifugal forces ranging from 583 g to 15 000 g after which their viability was determined by plating them on two types of media; one containing stabilizing agents and the other without them. Though there were minor fluctuations in the viable counts, there was no consistent decrease in their viability even in the absence of osmotic stabilizers.

Sensitivity to the antibiotic myxin was significantly increased in Escherichia coli 15T− cells treated with ethylenediaminetetraacetic acid (2 mM) for 5 min. This treatment did not reverse the resistance to myxin in cells of a myxin-resistant strain. Cells of both the susceptible and the resistant strains accumulated myxin intracellularly to the same extent.

The spontaneous formation of spheroplasts was observed in stationary phase cultures and in aging cultures of Bacteroides ruminicola when grown in synthetic or a rumen fluid medium. Unlike most other gram-negative bacteria, spheroplasts of this organism also were formed when log-phase cells were treated with lysozyme in the presence of 0.01 M Mg2+ in 0.01 M Tris, pH 8.4.

An efficient method is described for preparing spheroplasts and protoplasts by treating bacillary cells of Mycobacterium smegmatis with precise concentrations of L-glycine (followed by lysozyme). This improved procedure was widely applicable to many rapidly growing mycobacteria by selecting the concentrations of glycine suitable for the individual strains used. The process of reversion of spheroplasts to original bacillary form on solid and in liquid media, as revealed by electron microscopy, appeared to involve the formation of an internal elementary or initial body with subsequent budding from the spheroplast. The internal membrane systems appeared to function in the induction of initial bodies and in the maturation of elementary bodies to become dividing forms. Possible mechanisms involved in the development of bacilli from spheroplasts are discussed.

We present a practical description of polyethylene glycol mediated spheroplast transformation of Halobacterium halobium and Halobacterium volcanii. This method has been applied to phage DNA transfection, plasmid DNA transformation, and transformation with linear fragments of high molecular weight genomic DNA. Efficient spheroplast regeneration allows uncomplicated recovery of transformed progeny. Transformations can be performed equally well using fresh or frozen cell preparations. These methods should find application in molecular cloning, genetic fine mapping, and strain construction.Key words: archaebacteria, Halobacterium, transformation methods, spheroplast.

Strains of Methanospirillum hungatii, when treated with dithiothreitol at alkaline pH, formed spheroplasts which lysed in the absence of an osmotic stabilizer. Other methanogenic bacteria showed no response to this treatment, including Methanobacterium thermoautotrophicum. Methanobacterium strain M.o.H., Methanobacterium strain G2R, and Methanosarcina barkeri. The reaction with M. hungatii proceeded slowly at pH 8.0, but increased dramatically up to a pH of 10. Spheroplasts did not form in the presence of MgCl2 or if large amounts of FeS precipitate had been deposited on the cells during growth. Ultra-thin sections of M. hungatii GPI confirmed that individual cells, surrounded by a membrane and an inner wall, were contained within an outer wall or sheath. Adhesive material, which stained in the region of the cell spacer, was observed to bind together the inner and outer wall layers. After treatment with dithiothreitol, the spheroplasts, which retained the flexible inner wall and membrane, were released from the rigid outer wall. Examination of the isolated outer cell wall revealed that dithiothreitol (and dodecyl sodium sulfate) dissolved the adhesive material and damaged the cell spacer at the attachment site to the outer wall. It is proposed that dithiothreitol at alkaline pH loosens both the cell spacer and inner wall attachments to the outer wall, thereby allowing the osmotic pressure of the cytoplasm to eject the spheroplast. Resistance of the cells to lysis by Triton X-100 is lost upon spheroplast formation, indicating that protection to the cell is conferred by the outer cell wall.

The subcellular distribution of invertase was examined after synthesis and secretion by sphaeroplasts had been uncoupled by the addition of 30 μg mL−1 trypsin. Sphaeroplasts secreted only the high molecular weight invertase during uncoupling by trypsin. The level of low molecular weight, 'small' invertase in the soluble internal pool was found to be elevated by over fivefold, and the membrane-associated pool was found to contain low molecular weight invertase in addition to intermediate molecular weight invertase, after 1.5 h of trypsin treatment. Purified plasma membranes from trypsin-treated sphaeroplasts had no detectable mannan synthetase activity. On the basis of these and previous findings, a working hypothesis wherein invertase is synthesized on the internal surface of the plasma membrane and glycosylated during its transit to the external surface is presented.

The optimal conditions for activation of Dictyostelium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation.Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 °C in 8 M urea or incubating spores in 4–8 M urea for 10 h at 23.5 °C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and promise causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.

A significantly modified procedure for investigating enzyme secretion from yeast sphaeroplasts, and results from its application are described. Sphaeroplasts were derepressed for invertase biosynthesis in the presence of helicase and fractionated to reveal the distribution of high and low molecular weight forms of invertase. Secreted enzyme was found to be of high molecular weight, exclusively. Less than 10% of the total invertase activity was present in washed sphaeroplasts and of this, 43% was soluble, consisting of both high and low molecular weight forms of invertase. Washed membranes retained 32% of the internal invertase activity, and on solubilization with Triton X-100 the enzyme was found to be of an intermediate molecular weight. These results are consistent with the hypothesis that invertase is glycosylated at the plasma membrane.

A p-nitrophenyl α-galactoside hydrolase is partially released when whole cells of Pseudomonas atlantica are converted to spheroplasts. The p-nitrophenyl α-glactoside hydrolase is completely inactivated by treatment of whole cells with diazonaphthalene – disulfonic acid (NDS), a reagent which does not penetrate the cytoplasmic membrane. Under the conditions used no inactivation of lactic acid dehydrogenase was observed. A specific staining procedure for this enzyme for use in electron microscopy was developed. The results with this technique in conjunction with the results of spheroplasting and NDS localization suggest that p-nitrophenyl α-galactoside hydrolase is located in or on the double-track membranes, primarily on the outer double track.

Partially purified cellulase and a cellulase-containing polygalacturonase but not lysozyme extensively degraded the walls of Chlamydomonas reinhardtii and Ulothrix fimbrata and converted intact cells of the algae to spheroplasts. A streptomycete cellulase cochromatographed with the enzyme system releasing glucose from walls of these organisms, and this preparation also converted the algal cells to spheroplasts. The dominant constituent in the walls was carbohydrate, and glucose and small quantities of galacturonic acid but no amino sugars were present in acid hydrolysates of the walls. Glucose accounted for essentially all of the material solubilized by the cellulase preparation. Lysozyme acted on Cylindrospermum sp. walls, and it, but not the other enzymes, converted some of the Cylindrospermum sp. cells to spheroplasts. Streptomycete enzymes lysing Micrococcus lysodeikticus cochromatographed with the proteins releasing reducing sugars from Cylindrospermum sp. walls, and components in the active fraction converted cells of this alga into spheroplasts. X-ray diffraction revealed that the walls of C. reinhardtii and U. fimbrata but not those of Cylindrospermum sp. contained cellulose. The data suggest that the susceptibility of the first two species to microbial degradation in natural ecosystems results from an attack on the cellulose in their walls, and the susceptibility of the third is linked with the microbial production of a lysozyme.

Most of the cell-bound protease activity of Bacteroides amylophilus was attached to lysozyme spheroplasts (79%) and to the ruptured spheroplast envelope (48%). Protease activity also remained bound to mechanically ruptured cell envelopes from which it could be liberated by n-butanol treatment.Butanol-liberated protease activity was purified 80-fold, contained 23% RNA, and was excluded from G-200 Sephadex. Small molecular weight protease activity could not be liberated from this complex by treatment with acetone, EDTA, or urea. RNA could not be separated from the protease activity by treatment with streptomycin sulfate, MgCl2, or sodium dodecyl sulfate but was separated from the protein by phenol extraction. Protamine sulfate precipitation gave variable results. It is concluded that the cell-bound protease remains firmly bound to cellular components from which it cannot be liberated easily.

Exogenously added trypsin arrested invertase secretion by sphaeroplasts of Saccharomyces strain 1016. The mechanism of inhibition is presumed due to attack on plasma membrane protein(s). Gross membrane damage by trypsin was not apparent, as evidenced by the absence of leakage of intracellular alkaline phosphatase, after trypsin treatment. Trypsin treatment did induce an increased sensitivity to lysis, observed only when changes in osmotic pressure were made and fresh glucose added. While synthesis of invertase was eventually inhibited by trypsin, a greater than twofold increase in internal invertase was observed, due to complete inhibition of secretion. This is the first report of the uncoupling of synthesis and secretion in yeast.

Protoplasts–spheroplasts of a human isolate of Cryptococcus neoformans were prepared using the gut enzyme of Helix pomatia. The induction process, as studied by transmission electron microscopy, occurred in two stages. Early in the induction process, protoplasts–spheroplasts emerged from whole cells through a break in the cell wall – capsule envelope. Later, a gradual dissolution of the entire cell wall occurred releasing the intact protoplast–spheroplast. A comparison of protoplasts–spheroplasts with normal untreated cells revealed that the degree of cellular vacuolation as well as the resolution of cellular organelles was similar.

A method is described whereby Clostridium botulinum 33A and 51B spore spheroplasts are produced in two sequential steps: (1) by reducing spore coats for 3 h with 50% mercaptoacetic acid – 8 M urea – 0.01 M EDTA, and (2) by lysis of the cortex for 2 h with 500 μg/ml lysozyme in 0.01 M Tris-HCl (pH 8.0) – 0.01 M EDTA – 1 mg/ml dithiothreitol and 25% lactose added as osmotic stabilizing agent.Optical density measurements (absorption spectra) of ultraviolet (UV)-absorbing material released after submitting spheroplasts to osmotic lysis in distilled water showed absorption maxima around 270 nm (dipicolinic acid) and 260 nm (nucleic acids). Radioactivity profiles of 3H-labeled TCA-insoluble material from centrifugation of alkaline sucrose gradients showed that the above schedule for obtaining spore spheroplasts resulted in optimal DNA extraction and did not cause extensive breakage of DNA.Phase-contrast microscopy indicated that spore appearance is not visibly affected by chemical coat reduction; however, spores become phase dark after treatment with lysozyme. Electron microscopy showed that after chemical reduction coat layers are less compact, of less electron density, and dissolved in some areas. Furthermore, lysozyme treatment disintegrates and to a large degree removes the cortex and structural details become visible in the core.

Lysozyme-free antiserum and complement treatment of strain 1885 of Pseudomonas aeruginosa was observed to destroy the penetration barrier of the outer cell wall to lysozyme but not to induce leakage of acid-soluble nucleotides through the cell membrane. The same treatment did not produce a significant increase in uptake of 3H-tetracycline or 3H-streptomycin by the resistant strain 1885 in spite of the destruction of the penetration barrier to lysozyme. A significant increase in both streptomycin and tetracycline uptake occurred in carbenicillin-treated strains but the increase was similar for both susceptible and resistant (to tetracycline and streptomycin) strains of P. aeruginosa. These data suggest (1) the outer cell wall is not a significant penetration barrier to these drugs; (2) the peptidoglycan layer does function as a penetration barrier of similar magnitude in resistant and susceptible cells; (3) the resistance of the strains is a property of the cell membrane or materials intimately associated with the cell membrane. The latter conclusion was further supported by the differential uptake of streptomycin in NaCl-lysozyme-induced spheroplasts of strains 1885 and 2379.

The spheroplast-inducing activity of glycine in solid and sen~isolid media was studied, particularly with reference to groivth, reversion to the bacillary state, and properties of the spheroplasts. The combination of 3% NaCI, 1.75% glycine, 10% horse serum, 2% dextrose, and 0.01 Ai MgS04.7HzO was optimal for L colony formation. Resistance to glycine induction was apparent by the third generation in glycine-containing media, while the absence of the inducer invariably resulted in Inass reversion. Glycine-induced spheroplasts were essentially similar to the parent bacilli in virulence, antigenicity, phage type, antibiotic sensitivity, and biochemical fermentations. These forms are not filtrable through membrane filters. It is suggested that the term "L" form should be applied to these spheroplasts, even though stability was not one of their characteristics. In earlier reports we described the development of a typhoid somatic antigen vaccine which was prepared from spheroplasts grown in a semisynthetic liquid medium containing 1.5% glycine (3, 4). During these studies a number of interesting biological characteristics were noted in both intact and lysed spheroplasts. These preliminary investigations were extended to study, in greater detail, the inducing activity of glycine in both solid and semisolid media, the reversion to the original bacillary form, and the biological properties of the spheroplasts. We also considered the possibility of producing stabilized "L" forms which could be useful as an oral vaccine. Glycine was used throughout as the inducer because of the reproducibility of its activity under widely different experimental conditions.

Stationary phase cells of Streptococcus lactis resisted lysis by lysozyme under a variety of test conditions, although they lost 20% of their hexosamine. Lysozyme was bound by the cells and caused the optical density of the suspensions to rise. Incubation of the cells (0.5 mg dry weight/ml) in buffer with lysozyme (1 mg/ml), NaCl (0.5 M), or sodium dodecylsulfate (0.1%) caused prompt lysis. The cells were stabilized with 10% polyethylene glycol but not by 10–30% sucrose. Lysozyme and NaCl-treated cells in polyethylene glycol were spheroplasts because they were osmotically fragile, lost their shape but retained a rhamnose wall polymer. The spheroplasts retained all the DNA of the original cells, and 10% of the original numbers could be cultured on agar. However, they lost all their muramic acid and 60% of their hexosamine. In contrast to NaCl, sodium dodecylsulfate-treated cells were killed.

Preliminary evidence has been obtained which suggests that the intracellular invertase of Saccharomyces cerevisiae may not be localized in the vacuole per se. Alkaline phosphatase, an intracellular enzyme, and acid protease, a typically lysosomal enzyme, both showed high specific activity in the vacuole fraction prepared by equilibrium centrifugation of lysed sphaeroplasts in Ficoll gradients. Invertase activity has been found to be associated with vacuoles only when glucose-repressed cells are derepressed. Cells derepressed for invertase biosynthesis contained a population of vesicles which were virtually absent from the repressed cells. Evidence is presented which strongly suggests that these vesicles rather than the vacuoles are the vehicle by which invertase is secreted from the cell.