Morphological Phenomena Associated with Penicillinase Induction and Secretion in Bacillus licheniformis

Cells of uninduced Bacillus licheniformis (strain 749) in mid-logarithmic phase have no extensive intracytoplasmic membrane. After induction with cephalosporin C, characteristic organelles that contain tubules and vesicles with single-layered membranes and no visible internal substructure can be seen in thin sections in the periplasm. A magnoconstitutive penicillinase producer (749/C) contains similar structures. It is suggested that they represent a penicillinase secretory apparatus. In the first 15 min after induction, negatively stained preparations of induced 749 show large intracellular vesicles without individual contact with the cell surface. Negatively stained 749/C and fully induced 749 contain invaginations comparable to the structures seen in thin sections. When protoplasts of induced 749 and of 749/C are prepared, vesicles and tubules similar to those seen in thin sections of whole cells are liberated from the cell. Growing protoplasts of induced 749 show massive convolutions of the peripheral membrane, multiple layers of membrane, and characteristic long, slender tubules extending from the protoplast surface. These phenomena are not observed in uninduced 749 except for the production of a relatively small number of tubules. In 749/C, there were fewer convolutions than in induced 749, although tubule production was similar. Multiple layers of membrane were not observed in 749/C. The relation of the penicillinase secretory structures to mesosomes and to secretory structures of other organisms is discussed.     

Further evidence for a partially folded intermediate in penicillinase secretion by Bacillus licheniformis.

Protoplasis of Bacillus licheniformis 749/C (a mutant constitutive for penicillinase production) continued to synthesize and release penicillinase in hypertonic growth medium in the presence of trypsin and chymotrypsin at 25 mug each per ml. When the protoplasts were stripped of about half of their membrane-bound penicillinase by pretreatment at pH 9.5 or with a higher level of trypsin, penicillinase activity no longer increased in the presence of the proteases. This effect was immediately eliminated after addition of soybean trypsin inhibitor. These proteases do not significantly inhibit general protein synthesis. Stripped protoplasts of strain 749/C and of uninduced strain 749 (unable to synthesize penicillinase) were incubated with 50 mug of chymotrypsin per ml, and the supernatent fluids were examined immunochemically for peptides derived from the penicillinase chain. Such fargments were found only with the protoplasts capable of synthesizing penicillinase (strain 749/C). The direct detection of the products of protease degradation of a susceptible form of penicillinase provides strong evidence that, in stripped protoplasts of B. licheniformis 749/C, penicillinase synthesis continues in the presence of trypsin or chymotrypsin and that, in these modified membranes, the protease is able to act on an early form of the enzyme that has not yet attained the protease-resistant conformation characteristic of the membrane-bound and exopenicillinases. This finding is discussed in terms of the current models of penicillinase secretion.     

Characteristics of penicillinase release by washed cells of Bacillus licheniformis

Saline-washed cells of Bacillus licheniformis strain 749/C (constitutive for penicillinase) were able to release exopenicillinase in the presence of concentrations of chloramphenicol that prevented protein synthesis completely. The release reaction was strongly pH-dependent, occurring at a faster rate at alkaline pH in anionic or cationic buffers than at neutral pH. A strongly pH-dependent release reaction was noted in growing cells also. The reaction in washed cells can be stopped completely by changing the pH to 6.0. Within 30 min at pH 9.0, about 55% of the cell-bound penicillinase was released; thereafter, release continued at a greatly reduced rate. Suspensions of washed cells retained their capacity to release penicillinase at pH 9.0 for 90 min. Penicillinase released at pH 9.0 from either cells or protoplasts was not readsorbed over a 60-min period after changing the pH to 6.0. The release reaction was strongly temperature-dependent. We examined the effect of a large number of metabolic inhibitors and other compounds on the pH-dependent release phenomenon. Quinacrine hydrochloride, chloroquine diphosphate, and chlorpromazine hydrochloride reduced secretion substantially at 10(-4)m. Deoxycholate and Triton X-100 were active at 10(-3)m, but tungstate, arsenate, and molybdate had small effects at 10(-1)m. The rate of exopenicillinase release at pH 9.0 from fully stabilized protoplasts was one-half that of intact cells. Protoplasts lysed in hypotonic media or detergents showed even greater reduction in releasing activity. Penicillinase released from washed cells at pH 7.5 or 9.0 appeared to be derived from the periplasmic tubule and vesicle fraction that was released by protoplast formation.     

Vesicle penicillinase of Bacillus licheniformis: existence of periplasmic-releasing factor(s).

In earlier studies of the membrane-bound penicillinase of Bacillus licheniformis 749/C, the enzyme present in the vesicles that were released during protoplast formation and the enzyme retained in the plasma membrane of protoplasts appeared to differ (i) in their behavior on gel permeation chromatography in the presence or absence of deoxycholate and (ii) in their tendency to convert to the hydrophilic exoenzyme (Sargent and Lampen, 1970). We have now shown that these vesicle preparations contain a soluble, heat-sensitive enzyme(s) that is released along with the vesicles during protoplast formation. The enzyme will convert the vesicle penicillinase to a form that resembles exopenicillinase, and this conversion can be inhibited by deoxycholate under certain circumstances. Sedimentation of such vesicle preparations at 100,000 X g produces vesicles which contain penicillinase that behaves as the plasma membrane enzyme obtained from protoplasts. Exopenicillinases released by growing cells at pH 6.5 and by washed cells or protoplasts at pH 9.0 have the same NH2-terminal residues (lysine and some glutamic acid); in addition, the various release systems show a parallel sensitivity to inhibition by deoxycholate, quinacrine, chloroquine, and o-phenanthroline. The formation of exopenicillinase (by cleavage of the membrane-bound enzyme) may well be dependent on the action of the releasing enzyme.     

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.     

Characteristics of Penicillinase Secretion by Growing Cells and Protoplasts of Bacillus licheniformis

Cultures of the inducible penicillinase-producing strain 749 of Bacillus licheniformis, induced with small amounts of benzylpenicillin, synthesized penicillinase at a high rate for a short period, after which the rate of synthesis slowly declined. During the period of active synthesis, the rate of secretion, as a fraction of the level of cell-bound penicillinase (which is originally high), gradually decreased to a constant level. Chloramphenicol, at a concentration (40 mug/ml) which completely inhibited synthesis of penicillinase, partially inhibited secretion if added during the period of active synthesis. During the phase of reduced synthesis, chloramphenicol was without effect on secretion. Penicillinase secretion, by actively growing cultures of the constitutive penicillinase-producing mutant 749/C, was inhibited by 75% immediately after addition of chloramphenicol. The secretion of part of the penicillinase released during active growth is probably dependent on synthesis of penicillinase, but part of the secreted penicillinase can be released in the absence of synthesis. Protoplasts were obtained from which periplasmic penicillinase has been removed, and these protoplasts were capable of substantial growth and penicillinase synthesis without lysis. At pH 7.5, there was no net incorporation of penicillinase into the cell membrane; the enzyme released was almost entirely of the exo form and was roughly equivalent to the amount of new enzyme formed. At pH 6.0, there was some incorporation of penicillinase into the plasma membrane, and approximately half of the extracellular penicillinase was in the exo form; the remainder perhaps represented membrane fragments. In the presence of chloramphenicol, a small amount of penicillinase was released at pH 7.5 as the exo form; at pH 6.0, practically none was released. We suggest that, with the removal from protoplasts of the periplasmic penicillinase-containing particles, a restriction on secretion has been lifted.     

Penicillinase-releasing protease of Bacillus licheniformis: purification and general properties.

The membrane penicillinase of Bacillus licheniformis 749/C is a phospholipoprotein which differs from the exoenzyme in that it has an additional sequence of 24 amino acid residues and a phosphatidylserine at the NH2 terminus. In exponential-phase cultures, the conversion of membrane penicillinase to exoenzyme occurs at neutral and alkaline pH. An enzyme that will cleave the membrane penicillinase to yield the exoenzyme is present (in small amounts) in exponential-phase cells and is released during their conversion to protoplasts. The enzyme is found in the filtrate of a stationary-phase culture of the uninduced penicillinase-inducible strain 749 and has been purified to apparent homogeneity from this source. The protease has an approximate molecular weight of 21,500 and requires Ca2+ ions for stabilization. It has a pH optimum of 7.0 to 9.5 for hydrolysis of casein and for the cleavage of membrane penicillinase. Both activities are inhibited by diisopropylfluorophosphate; hence, the enzyme is a serine protease. This enzyme may be entirely responsible for the formation of exopenicillinase by this organism, since the other neutral and alkaline proteases of strain 749 have little, if any, activity in releasing exopenicillinase. The enzyme has been termed penicillinase-releasing protease.     

Variant of Penicillinase Mediated by an R Factor in Escherichia coli

The penicillinase from an Escherichia coli strain harboring an R factor R(GN823) was purified and its properties were compared with those of a known type I penicillinase mediated by R factors. The molecular weight and S(20,w) of the enzyme were 22,600 and 2.42S, respectively. The isoelectric point of the enzyme was 6.9. These values are clearly different from those of type I penicillinase. The specific activity of the enzyme was 84,700 units per mg of the purified enzyme protein, which is about 20 times higher than that of the type I penicillinase. However, similarities were observed between the enzyme and the type I-penicillinase at optimal pH (6.5 to 7.0), optimal temperature (40 to 45C), substrate specificity, Michaelis constants for penicillins and cephaloridine, and effect of inhibitors. Furthermore, antiserum against type I penicillinase showed cross-reaction against this enzyme. The enzyme was named type Ib penicillinase, and the original type I penicillinase was renamed type Ia-penicillinase.     

Characteristics of Secretion of Penicillinase, Alkaline Phosphatase, and Nuclease by Bacillus Species

The distribution of alkaline phosphatase and nuclease activity between cells and medium was examined in one strain of Bacillus licheniformis and four strains of B. subtilis. Over 95% of both activities was found in the medium of the B. licheniformis culture, but in the B. subtilis cultures the amount of enzyme activity found in the medium varied with the strain and the enzyme considered. B. licheniformis 749 and its penicillinase magnoconstitutive mutant 749/C were grown in continuous culture with phosphorous as the growth-limiting factor, and the kinetics of penicillinase formation and secretion were examined. Nutrient arrest halted secretion (usually after a lag of about 30 min) in both the inducible and constitutive strains. Chloramphenicol did not eliminate secretion, but under certain circumstances reduced its rate. In the inducible strain treated with a low level of inducer, the rate of secretion was more affected by the rate of synthesis than by the level of cell-bound enzyme. During induction, the onset of accretion of cell-bound penicillinase and secretion of the exoenzyme were nearly simultaneous. It seems unlikely that a long-lived, membrane- or cell-bound intermediate is mandatory in the secretion of the three enzymes by Bacillus species. In the case of penicillinase secretion, there are at least two different phases. When penicillinase synthesis is proceeding rapidly, the rate of secretion is five to six times greater at equivalent concentrations of membrane-bound penicillinase than it is when penicillinase synthesis is reduced. The data require that any membrane-bound intermediate in the formation of exoenzyme be much shorter-lived in cells with a high rate of synthesis than in cells with a low rate. Either there are two separate routes for the secretion of penicillinase or the characteristics of the process vary substantially between the early stages and the declining phase of induction.     

The isolation of protoplasts from the placental cells ofSolanum nigrum L.

Summary Living protoplasts were isolated from the interplacental regions ofSolanum nigrum berries by the removal of the walls from cells in tissue slices treated for 1–2 hours with 12% pectinase in 0.33 M to 0.38 M sucrose solution. Protoplasts thus isolated, then washed and transferred to microculture chambers for observation, invariably tended to be spherical. Comparative measurements of cell and protoplast volumes revealed that 10% of the isolated structures were subunits of protoplasts. From diameter changes in protoplasts studied in a hypotonic (0.20 M) sucrose solution, the maximum expansion of the plasma membrane was determined. Slightly hypertonic solutions (0.33 M to 0.38 M sucrose) promote stability of isolated protoplasts for several days. The importance to stability of osmotic concentration and ion balance in the medium is here established. Probably of equal importance is the optimal combination of several common constituents of culture media. Further studies on some aspects of specific medium requirements are in progress.This work was supported by a special grant from the Office of Advanced Studies and Research, University of South Carolina.     

Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis.

By using plasmid pMB9, penicillinase genes (penP and penI) from both the wild-type and constitutive strains of Bacillus licheniformis 9945A were cloned in EScherichia coli. When a low-copy-number plasmid was used, both wild-type and constitutive penicillinase genes could be transferred into Bacillus subtilis. However, when a high-copy-number plasmid was used, only the genes of the wild type could be transferred. These recombinant plasmids in B. subtilis could all be transferred by the protoplast transformation procedure into B. licheniformis. Transformants of E. coli were resistant to ampicillin (20 micrograms/ml) in spite of the low penicillinase activities (7 U/mg of cells). However, transformants of B. subtilis and B. licheniformis were sensitive to ampicillin (20 micrograms/ml) even in high penicillinase activities (more than 10,000 U/mg of cells). The secretion of penicillinase was rarely observed in E. coli. In contrast, penicillinases secreted from transformants of B. subtilis and B. licheniformis were around 30 and 60% of the total activities, respectively. We took advantage of the plasmids to permit the construction of hetero- and mero-polyploid structures in host cells, and we discuss a regulatory mechanism of penicillinase synthesis in B. licheniformis.     

Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from ~75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from ~50% of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.     

Resistance of Escherichia coli to penicillins. V. Physiological comparison of two isogenic strains, one with chromosomally and one with episomally mediated ampicillin resistance

Two essentially isogenic strains of Escherichia coli K-12 were compared: D31 had chromosomally and D1-R1 episomally mediated resistance to ampicillin. The two strains had the same ability to form colonies on ampicillin plates, but in other tests they were quite different. In serial dilution tests as well as in exponentially growing cultures, D1-R1 was far more resistant to ampicillin than was D31. The inoculum effect with D1-R1 was large and with D31 was rather small. On plates, D31 was more resistant to penicillin G than was D1-R1. The penicillinase activity of buffer suspended cells against dl-ampicillin was 15 times higher for D1-R1 than for D31, but the two strains showed about the same rate of hydrolysis of penicillin G. With dl-ampicillin as substrate, for D1-R1 the apparent K(m) was 1.7 x 10(-4)m, whereas D31 gave a slightly sigmoid curve with a half-saturation concentration of about 5 x 10(-3)m. No induction of penicillinase activity was found. When the growth rate was varied by a factor of four, the amount of penicillinase per cell mass was constant in both D1-R1 and D31, whereas in two wild-type strains the amounts of penicillinase increased with increasing growth rates. With exponentially growing D1-R1, ampicillin disappearance started within 3 min, but at low ampicillin concentrations the rate was less than 10% of the rate of hydrolysis by buffer-suspended cells. Before D31 started hydrolysis, there was a lag period that lasted at least one generation and depended on the concentration of ampicillin. After this lag period, the rate of hydrolysis was 10 times higher than that observed with buffer-suspended cells. These differences between growing and nongrowing cells indicate that both the chromosomally and the episomally mediated penicillinases are controlled by some products present in growing cells.     

Studies on the behavior of meiotic protoplasts II. Induction of a high fusion frequency in protoplasts from liliaceous plants

A method for the induction of high fusion frequencies in meioticprotoplasts of a lily and Trillium is described. Fusion wasinduced during the rapid isolation of protoplasts by quicklybringing about physical contact between protoplasts. Fused protoplastswere obtained in 50 to 90% yields from prophase cells and in30 to 60% yields from cells in later phases. Large protoplastswith more than 10 nuclei were frequently produced. During culture,meiotic development was not synchronous in these protoplasts;each multinudeate maintaining a high degree of synchrony. (Received February 28, 1973; )     

A frameshift mutation that elongates the penicillinase protein of Bacillus licheniformis

A penicillinase mutant penP102, isolated after ICR (acridine mustard) mutagenesis of Bacillus licheniformis strain 749/C, retains about 50% of the wild-type penicillinase specific activity. The penicillinase produced by this mutant differs from the wild-type protein in its sensitivity to pH and its electrophoretic behaviour. The penP102 mutation appears to have several other phenotypic effects, including an increase in the efficiency of release of the extracellular form of the enzyme.The penP102 penicillinase has been purified and its amino acid sequence compared to that of the wild-type enzyme. The mutation has resulted in the replacement of the last three amino acids of the wild-type enzyme and the addition of 17 residues at the carboxy-terminus. Comparison of the wild-type and mutant amino acid sequences shows that the mutational event is a single nucleotide deletion from the codon for asparagine²⁶⁵. Consideration of the possible nucleotide sequence for the region beyond the carboxy-terminus of the wild-type protein shows that there are no possible termination codons until four and six triplets beyond the codon for the carboxy-terminal lysine, indicating that the carboxy-terminus of the wild-type extracellular penicillinase is generated by proteolytic cleavage of a larger precursor protein.     

Protein synthesis by long-lived messenger ribonucleic acid in bacteria

1. Experiments were performed to investigate two hypotheses about the function of long-lived messenger RNA in bacteria. After RNA synthesis had been stopped by the addition of actinomycin, continuing protein synthesis was used as a measure of persistent messenger RNA. 2. The hypothesis that messenger RNA responsible for the synthesis of membrane protein is exceptionally long-lived was tested in experiments with protoplasts of Bacillus megaterium. However, this messenger RNA proved to be of approximately average stability. 3. The hypothesis that long-lived messenger RNA is responsible for the synthesis of constitutive proteins was tested by comparing the synthesis of penicillinase in an inducible and a constitutive strain of Bacillus licheniformis. After the addition of actinomycin, penicillinase synthesis continued for far longer in the constitutive than in the inducible strain. This difference is attributed to a difference in stability of the penicillinase-messenger RNA in the two strains, which does not extend to all messenger RNA indiscriminately. 4. A model is tentatively proposed to account for the altered stability of messenger RNA in the constitutive mutant.     

Regulation of Staphylococcal Penicillinase Synthesis

5-Methyl tryptophan was found to be an efficient inducer of penicillinase synthesis in Staphylococcus aureus. Addition of actinomycin D or tryptophan to the culture medium shuts off the 5-methyl tryptophan-induced synthesis of penicillinase with an apparent half-life of approximately 1 to 2 min, respectively. Hence, in the induction of penicillinase synthesis, 5-methyl tryptophan seems to function as a structural analogue of penicillin rather than by becoming incorporated in proteins and thereby creating faulty penicillinase repressor or antirepressor. This conclusion is supported by similarities in the structures of the two compounds as revealed by solid atomic models. The fact that S. aureus exposed to (14)C-penicillin in the absence of protein synthesis failed to synthesize penicillinase at an increased level when cell growth was resumed strongly suggests that a protein involved in the regulation of penicillinase synthesis must be synthesized in the presence of the penicillinase inducer. In turn, this observation suggests that the penicillinase inducer promotes penicillinase synthesis by directing the penicillinase regulatory protein (i.e., the penicillinase antirepressor) to acquire a different conformation when it is synthesized in the presence of the penicillinase inducer. A working model for the regulation of penicillinase synthesis based on these and other data has been constructed and is presented.     

Synthesis of heat-shock proteins in Saccharomyces cerevisiae protoplasts

The effect of cellular capsule elimination in Saccharomyces cerevisiae yeasts (protoplast formation) on the heat-shock protein synthesis and the synthesis of the proteins in protoplasts were studied. The methods of mono- and dimeric electrophoresis have demonstrated that (1) about 18 heat-shock proteins with the molecular masses 26-98 Kd are synthesized in cells at 41 degrees C; (2) protoplast formation per se does not induce the synthesis of heat-shock proteins, but the induction of these proteins in protoplasts at 41 degrees C is similar to the one in intact cells. The protoplast formation induces the synthesis of specific proteins different from heat-shock proteins and the synthesis is inhibited by the heat-shock. The heat-shock induces modification of 88 and 86 Kd heat-shock proteins. It inhibits the synthesis of a number of peptides (15-50 Kd) in cells and protoplasts.     

Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants

The rate of respiratory CO2 evolution from the leaves of Zea mays, Panicum miliaceum, and Panicum maximum, representing NADP-ME, NAD-ME, and PEP-CK types of C4 plants, respectively, was increased by approximately two to four times after a period of photosynthesis. This light-enhanced dark respiration (LEDR) was a function of net photosynthetic rate specific to plant species, and was depressed by 1% O2. When malate, aspartate, oxaloacetate or glycine solution at 50 mM concentration was introduced into the leaves instead of water, the rate of LEDR was enhanced, far less in Z. mays (by 10-25%) than in P. miliaceum (by 25-35%) or P. maximum (by 40-75%). The enhancement of LEDR under glycine was relatively stable over a period of 1 h, whereas the remaining metabolites caused its decrease following a transient increase. The metabolites reduced the net photosynthesis rate in the two Panicum species, but not in Z. mays, where this process was stimulated by glycine. The bundle sheath cells from P. miliaceum exhibited a higher rate of LEDR than those of Z. mays and P. maximum. Glycine had no effect on the respiration rate of the cells, but malate increased in cells of Z. mays and P. miliaceum by about 50% and 30%, respectively. With the exception of aspartate, which stimulated both the O2 evolution and O2 uptake in P. maximum, the remaining metabolites reduced photosynthetic O2 evolution from bundle sheath cells in Panicun species. The net O2 exchange in illuminated cells of Z. mays did not respond to CO2 or metabolites. Leaf mesophyll protoplasts of Z. mays and P. miliaceum, and bundle sheath protoplasts of Z. mays, which are unable to fix CO2 photosynthetically, also produced LEDR, but the mesophyll protoplasts, compared with bundle sheath protoplasts, required twice the time of illumination to obtain the maximal rate. The results suggest that the substrates for LEDR in C4 plants are generated during a period of illumination not only via the Calvin cycle reactions, but also by the conversion of endogenous compounds present in leaf cells. The stimulation of LEDR under glycine is discussed in relation to its direct or indirect effect on mitochondrial respiration.     

Chemical and electron microscopic studies of factors associated with the release of penicillinase from Staphylococcus aureus

The effect of various factors such as sodium chloride, sodium citrate, pH, buffers, and enzymatic and physical disruption of cells on the release of penicillinase by Staphylococcus aureus ATCC 14458 was investigated. Penicillinase was measured at selected time intervals from supernates of cultures grown in Antibiotic Medium 3 broth containing various concentrations of salts or buffers or from supernates of cultures treated with lysostaphin and subsequently disrupted by French press treatment.Incubation of cells with media containing either sodium chloride (5, 10, and 15%), sodium citrate (5 and 10%), or organic buffers (Tris-HC1, 2.5, 5.0, and 7.5%; BES, 10 and 20%) resulted in a significant stimulation of the release of penicillinase when compared to control cells. It was also observed that pH 7.0–7.5 was optimal for penicillinase activity and release. From studies of enzymatic and mechanical disruption of cells, it was observed that an increase in ionic strength of the suspending medium to certain optimal levels appeared to stimulate the conversion of penicillinase to an extracellular form.Electron microscopic studies revealed that a large number of mesosomal vesicles seemed to be present in cells incubated for 4 hours in media containing various concentrations of sodium chloride. It is proposed that either appearance of vesicles or convolution of cell membrane, which may be caused by further synthesis of new membrane, is involved in stimulation of the synthesis and release of membrane-bound penicillinase.