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.     

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.     

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.     

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.     

Phosphatidylseryl-transfer RNA in Bacillus licheniformis 749/C.

Phosphatidylserine has been found in extracts of Bacillus licheniformis made under alkaline conditions but not under neutral or acidic ones and was derived from the tRNA fraction. In tRNA preparations kept below neutrality during purification, phosphatidylserine was the only phospholipid released when the pH was raised to 9.0. The amount of bound phosphatidylserine could be increased by incubating tRNA from B. licheniformis or Escherichia coli with CTP and phosphatidic acid in the presence of an S-30 extract from either organism. The tRNA carrying phosphatidylserine has been separated from the bulk of the tRNA by DEAE-Sephadex chromatography in the presence of a detergent. On deaminoacylation of this material and rechromatography on DEAE-Sephadex, a number of peaks were found, indicating that this behavior is not confined to a single isoaccepting species.     

Nutrient-stimulated methylation of a membrane protein in Bacillus licheniformis.

When nitrogen-starved vegetative cells of Bacillus licheniformis A5 were presented with a good nitrogen source in the presence of chloramphenicol and methyl-labeled methionine, a 40-kilodalton (kDa) protein was found to be reversibly methylated, with a half-life of approximately 10 to 15 min. The 40-kDa protein was strongly methylated in response to the addition of ammonia, glutamine, or sodium glutamate nitrogen sources that produce generation times of less than or equal to 90 min) but was very poorly methylated in the absence of a nitrogen source or in the presence of potassium glutamate or histidine (generation times of greater than 150 min). The methylated protein was found to be membrane associated, but the methylation reaction did not appear to be related to chemotaxis, because the spectrum of nutrients that promoted methylation was different from that which prompted a chemotactic response. In addition, the methyl residue on the 40-kDa protein was found to be alkali stable. Approximately 180 to 640 molecules of the methylated protein were found per cell. The characteristics of this methylated protein were consistent with the hypothesis that the reversible methylation of the protein functions in nutrient sensing to regulate growth, cell division, and the initiation of sporulation.     

Immunoelectron microscopic localization of penicillinase in Bacillus licheniformis.

Penicillinase was localized in log-phase cells of Bacillus licheniformis 749/C by labeling with ferritin-anti-penicillinase immunoglobulin G conjugate. Mildly fixed homogenized cells, isolated subcellular fractions, and frozen thin sections were labeled. The label was distributed in discrete patches in the cell envelope. The patches extended from the inside part of the membrane to the outside part of the wall. The inside part of the membrane was labeled more extensively than the outside part. The cytoplasm also bound some ferritin-immunoglobulin G conjugate. Immunoelectrophoresis and biochemical assay of cytosol material suggest that the cytoplasmic antigenic sites are a protease-sensitive form of penicillinase.     

Membrane-bound and secreted forms of penicillinase from Bacillus licheniformis

Three forms of penicillinase from Bacillus licheniformis have been isolated. Two are secreted into the extracellular medium and one is membrane-bound. The secreted proteins are water-soluble; one has been previously described and sequenced, the other contains an amino-terminal extension of eight amino acid residues. The membrane-bound form behaves in all respects as a typical amphiphilic membrane protein. It binds one micelle of Triton XI00 and reassociates with egg lecithin to lipid vesicles into which the protein is incorporated. No lipids are covalently associated with the purified protein. Membrane penicillinase contains an amino-terminal peptide extension as compared to the exo forms. This tail is the most likely explanation to its amphiphilic properties.     

Beta-lactamase of Bacillus licheniformis 749/C at 2 A resolution

Two crystal forms (A and B) of the 29,500 Da Class A beta-lactamase (penicillinase) from Bacillus licheniformis 749/C have been examined crystallographically. The structure of B-form crystals has been solved to 2 A resolution, the starting model for which was a 3.5 A structure obtained from A-form crystals. The beta-lactamase has an alpha + beta structure with 11 helices and 5 beta-strands seen also in a penicillin target DD-peptidase of Streptomyces R61. Atomic parameters of the two molecules in the asymmetric unit were refined by simulated annealing at 2.0 A resolution. The R factor is 0.208 for the 27,330 data greater than 3 sigma (F), with water molecules excluded from the model. The catalytic Ser-70 is at the N-terminus of a helix and is within hydrogen bonding distance of conserved Lys-73. Also interacting with the Lys-73 are Asn-132 and the conserved Glu-166, which is on a potentially flexible helix-containing loop. The structure suggests the binding of beta-lactam substrates is facilitated by interactions with Lys-234, Thr-235, and Ala-237 in a conserved beta-strand peptide, which is antiparallel to the beta-lactam's acylamido linkage; an exposed cavity near Asn-170 exists for acylamido substituents. The reactive double bond of clavulanate-type inhibitors may interact with Arg-244 on the fourth beta-strand. A very similar binding site architecture is seen in the DD-peptidase.     

Functional domains of the penicillinase repressor of Bacillus licheniformis.

The penicillinase repressor (PENI) negatively regulates expression of the penicillinase gene (penP) in Bacillus licheniformis by binding to its operators located within the promoter region of penP.penI codes for a protein with 128 amino acids. Filter-binding analyses suggest that the active form of the repressor is a dimer. Genetic analyses of PENI derivatives showed that the repressor carrying either a 6-amino-acid deletion near the N terminus or a 14-amino-acid deletion at the C terminus was functionally inactive in vivo. A repressor derivative carrying a 6-amino-acid deletion within its N-terminal region was extensively purified and used in DNA footprinting and subunit cross-linking analyses. The results of these studies showed that the repressor derivative had lost its ability to bind operator specifically even though it could dimerize effectively. In similar studies, we demonstrated that an N-terminal portion of PENI with a molecular mass of 10 kDa derived by digestion with papain was able to bind operator specifically but with reduced affinity and had completely lost its ability to dimerize. These data suggest that the repressor has two functional and separable domains. The amino-terminal domain of the repressor is responsible for operator recognition, and the carboxyl-terminal domain is involved in subunit dimerization.     

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.     

Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C.

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.