Bacillus licheniformis 749-C plasma membrane penicillinase, a hydrophobic polar protein

Plasma membrane penicillinase from Bacillus licheniformis 749/C is hydrophobic in nature, although it is virtually identical to its riydrophilic exoenzyme counterpart in amino acid composition and sequence. Unlike the exoenzyme, however, the purified membrane enzyme retains [³³P]phosphate and [³H]glycerol. By isoelectricfocusing the membrane enzyme is more acidic than the exoenzyme; it has a lower mobility in SDS gel electrophoresis, consistent with the presence of a very hydrophobic moiety. Unlike the exoenzyme, which binds no taurodeoxycholate, the membrane enzyme binds 10 molecules tightly and approximately 37 molecules in the presence of excess taurodeoxycholate (0.1% solution). The membrane enzyme is identical to the exoenzyme in its reaction with antibodies to exopenicillinase as determined by a radioimmune inhibition assay and immunodiffusion in agar. Heat stability studies indicate a slightly less stable conformation for the membrane enzyme, but this difference largely disappears in the presence of antibody to the exoenzyme. Conversion of membrane enzyme to exoenzyme has been achieved by brief treatment with trypsin, or by incubation of impure preparations at pH 9.0 in 25% potassium phosphate.Since the two forms of penicillinase are very similar in conformation, the hydrophobicity of the membrane form of the enzyme would seem to derive from combination with a hydrophobic moiety, probably phospholipid.     

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.     

Purification of plasma membrane penicillinase from Bacillus licheniformis 749/C and comparison with exoenzyme.

The membrane penicillinase of Bacillus licheniformis 749/C has been demonstrated to be a phospholipoprotein. The homogeneous enzyme gives a positive reaction for phosphorous and for unsaturated fatty acids, has a molecular weight of 33,000 in contrast to 29,000 for the exoenzyme, and contains 8 to 9 additional residues of aspartate or asparagine, 4 to 5 of serine, 7 of glutamate or glutamine, and 4 to 5 of glycine per mole. The COOH-terminal sequence of both membrane and exoenzymes is -Met-Asn-Gln-Lys-COOH; hence the extra peptide portion present in the membrane enzyme is not attached to the COOH-terminus of the exoenzyme. Procedures which readily detected the lysine residue at the NH2 terminus of the exoenzyme did not yield a positive test with the membrane form. The NH2 terminus of the membrane enzyme may be blocked by or linked to the phospholipid. A procedure for the preparation of membrane penicillinase on a large scale and an improved method for purification of the exoenzyme have been developed.     

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.     

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 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.     

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.     

Evidence for phospholipid in plasma membrane penicillinase of Bacilluslicheniformis749C

The plasma membrane-bound penicillinase of $\text{Bacillus}$$\text{licheniformis}$$\text{749}\text{C}$ has been purified. Amino acid analysis showed no significant differences in composition between the enzyme and exopenicillinase. Enzyme purified from cultures containing H₃³³PO₄ or [³H]-glycerol contained ³³P or [³H]-glycerol activity and treatment with 8 M urea, 0.2% sodium dodecyl sulfate at 80° C did not remove the ³H-activity from the enzyme protein. Trypsin readily cleaved the glycerol-containing moiety from the enzyme protein, forming enzyme with molecular weight and heat stability like that of the exoenzyme. Phospholipase D and C also produced enzyme resembling the exo-form.     

In vitro synthesis of hydrophobic penicillinase in extracts of Bacillus licheniforms 749/C.

Extracts of $\text{Bacillus licheniformis}$ 749/C in an $\text{in vitro}$ protein synthesis system produced the hydrophobic penicillinase containing covalently-bound phospholipid. The hydrophilic penicillinase (exoenzyme) and the hydrophobic enzyme without the phospholipid were scarcely detectable.     

Membrane associated phospholipoproteins of Bacillus licheniformis 749

The membrane-bound penicillinase of Bacillus licheniformis 749/C is a phospholipoprotein that differs from the hydrophilic exoenzyme in that its polypeptide chain carries an additional 25 residues (mostly hydrophilic) with phosphatidylserine as the NH₂-terminus. To determine if other phospholipoproteins are present in the plasma membrane, the penicillinase-inducible strain 749 was grown without inducer in the presence of [2-³H] glycerol. Electrophoretic separation of the membrane proteins (after removel of free lipids) showed an association of ³H-activity with certain of the proteins which could not be broken by lipid solvents and strongly denaturing conditions. Pronase digestion of the membrane proteins (after solvent extraction) released phosphatidylserine, thus indicating the covalent linkage of protein and phospholipid. Treatment of the isolated membranes with trypsin solubilized the protein portion of some of the phospholipoproteins (as with penicillinase), but not the ³H-labelled fragment. Penicillinase should be considered as the first observed example of a group of phosphatidylserine-containing proteins present in the plasma membrane of B. licheniformis 749 and 749/C.     

Exopenicillinase Synthesis in Staphylococcus aureus

In Staphylococcus aureus, penicillinase remaining cell-bound (60 to 75% of original total) after treatment with citrate does not become exopenicillinase. Exopenicillinase in these cells appears only under conditions permitting de novo penicillinase synthesis. By use of (14)C-labeled cells, it was shown that exopenicillinase consists of newly synthesized molecules which have not equilibrated with preformed membrane-bound enzyme.     

Periplasmic Structure of Frozen-Etched and Negatively Stained Cells of Bacillus licheniformis as Correlated with Penicillinase Formation

Bacillus licheniformis strain 749/C (constitutive for penicillinase formation) and uninduced cells of strain 749 (penicillinase-inducible) were examined after freezeetching. In the early stationary phase, strain 749/C organisms had clusters of vesicles (30 to 40 nm in diameter) on the outer surface of the plasma membrane. These are randomly distributed on the membrane, including the region of septum formation. The vesicles are not intimately associated with the plasma membrane, and their inner and outer surfaces are devoid of particles. Periplasmic vesicles were not detected by freeze-etching in strain 749 (uninduced) or in young cells of 749/C; however, the membrane of mid-logarithmic phase 749/C cells had a corrugated appearance. Negatively stained 749/C cells (logarithmic phase) also showed many vesicular and tubular bodies in the periplasm as well as septal and cytoplasmic mesosomes of typical morphology. The periplasmic structures appear to be formed either by evagination of plasma membrane or by migration of vesicular bodies from the membranous pockets of the cytoplasm. Stationary phase cells of 749/C still have many periplasmic vesicular bodies; however, the mesosomes are greatly reduced both in number and size. In sharp contrast, strain 749 organisms have very few structures similar to the periplasmic bodies of strain 749/C. These findings support our previous view that penicillinase-producing cells of 749/C have periplasmic membranous structures that are rare in the uninduced strain 749, though there is some lack of correspondence between freeze-etching, negative staining, and thin section data. These structures may be important for the retention or storage of penicillinase in the cell.     

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.     

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.     

Lifetime of messenger ribonucleic acid for penicillinase synthesis in several strains of bacilli

1. Previous studies of penicillinase synthesis in Bacillus licheniformis showed that enzyme synthesis after the addition of actinomycin continues for far longer in the constitutive mutant 749/C than in the parental inducible strain (Yudkin, 1966). This result was interpreted as indicating a difference in the lifetime of specific messenger RNA in the two strains. Other bacilli have now been examined in an attempt to see whether this difference is general. 2. There was no difference in the lifetime of messenger RNA for penicillinase synthesis between an inducible and a constitutive strain of Bacillus cereus. 3. Three freshly isolated constitutive mutants of B. licheniformis also had short-lived messenger RNA, like their inducible parent. 4. A reinvestigation of mutant 749/C confirmed the original finding that, on treatment with actinomycin, it continued to synthesize penicillinase far longer than did its parent. 5. An inducible revertant of mutant 749/C was indistinguishable from the original inducible strain, and appeared to have lost both constitutivity and long-lived messenger RNA in the back mutation.     

Kinetics of Penicillinase Induction and Variation of Penicillinase Translation in Staphylococcus aureus

At neutral pH, the rate of penicillinase synthesis by staphylococci declines gradually after removal of free inducer, while at pH 5.4 enzyme formation is generally linear for an extended period. Linear synthesis of penicillinase was observed at neutral pH in nonsaturating concentrations (1 μg/ml) of actinomycin D. The rate of enzyme synthesis, corrected for inhibition of growth caused by the antibiotic, was relatively independent of the time of actinomycin addition. The lag preceding linear enzyme formation increased with the interval between induction and the addition of actinomycin. The findings are consistent with the concept that, at neutral pH, “operons” activated by induction are rapidly repressed, while at pH 5.4, this process is delayed.

At a concentration of 4 μg/ml, actinomycin D blocked penicillinase messenger synthesis and also elicited a short-lived acceleration of the increase of penicillinase activity in uninduced and, late after induction, in induced cultures. This effect did not require a functional genomic repressor mechanism since it occurred also in a penicillinase-constitutive strain. It required protein synthesis and could not be attributed to a greater enzyme stability in the presence of actinomycin. The results suggest enhanced penicillinase translation after addition of actinomycin D.

     

The unique stability of Vibrio proteolyticus neutral protease under alkaline conditions affords a selective step for purification and use in amino acid-coupling reactions.

A procedure is described for the purification of a neutral protease from fermentation broths of Vibrio proteolyticus. The key feature of the purification scheme is the selective, irreversible inactivation of a contaminating exoenzyme, aminopeptidase, by alkali treatment, rather than removal of this enzyme by conventional chromatographic methods. Fermentation broths or concentrates were brought to pH 11.5 to 11.7 by Na2CO3-NaOH addition and incubated at 25 degrees C until aminopeptidase activity was diminished. The alkali treatment resulted in greater than 99% reduction of aminopeptidase activity with minimal loss of neutral protease activity. The neutral protease could be further purified to apparent homogeneity by QA-52 cellulose chromatography. The alkali treatment of fermentation concentrates was also useful for preparation of V. proteolyticus neutral protease to effect the coupling of N-protected aspartic acid and phenylalanine methyl ester for the production of N-aspartylphenylalanine methyl ester, a precursor for the sweetener aspartame.     

The unique stability of Vibrio proteolyticus neutral protease under alkaline conditions affords a selective step for purification and use in amino acid-coupling reactions

A procedure is described for the purification of a neutral protease from fermentation broths of Vibrio proteolyticus. The key feature of the purification scheme is the selective, irreversible inactivation of a contaminating exoenzyme, aminopeptidase, by alkali treatment, rather than removal of this enzyme by conventional chromatographic methods. Fermentation broths or concentrates were brought to pH 11.5 to 11.7 by Na2CO3-NaOH addition and incubated at 25 degrees C until aminopeptidase activity was diminished. The alkali treatment resulted in greater than 99% reduction of aminopeptidase activity with minimal loss of neutral protease activity. The neutral protease could be further purified to apparent homogeneity by QA-52 cellulose chromatography. The alkali treatment of fermentation concentrates was also useful for preparation of V. proteolyticus neutral protease to effect the coupling of N-protected aspartic acid and phenylalanine methyl ester for the production of N-aspartylphenylalanine methyl ester, a precursor for the sweetener aspartame.     

Immunoelectron microscopic double labeling of alkaline phosphatase and penicillinase with colloidal gold in frozen thin sections of Bacillus licheniformis 749/C.

The subcellular distribution of alkaline phosphatase and penicillinase was determined by double labeling frozen thin sections of Bacillus licheniformis 749/C with colloidal gold-immunoglobulin G (IgG). Antipenicillinase and anti-alkaline phosphatase antibodies were used to prepare complexes with 5- and 15-nm colloidal gold particles, respectively. The character of the labeling of membrane-bound alkaline phosphatase and penicillinase was different: the immunolabels for alkaline phosphatase (15-nm particles) were bound to a few sites at the inner surface of the plasma membrane, and the gold particles formed clusters of various sizes at the binding sites; the immunolabels for penicillinase (5-nm particles), on the other hand, were bound to the plasma membrane in a dispersed and random fashion. In the cytoplasm, immunolabels for both proteins were distributed randomly, and the character of their binding was similar. The labeling was specific: pretreating the frozen thin sections with different concentrations of anti-alkaline phosphatase or penicillinase blocked the binding of the immunolabel prepared with the same antibody. Binding could be fully blocked by pretreatment with 800 micrograms of either antibody per ml.     

Affinity chromatography purification of penicillinase of Bacillus licheniformis 749-C and its use to measure tuurnover of the cell bound enzyme

Affinity chromatography purification of small amounts of penicillinase using cephalosporin C covalently linked to Sepharose 4B has been used in examining the turnover of cell-bound penicillinase by B. licheniformis 749/C. Under conditions in which most of the nascent penicillinase is retained by the cells, turnover could be demonstrated (15% of the cell-bound enzyme in 2.5 hours), although most of the secreted enzyme is derived from newly-formed molecules.