The influence of glucose, ammonium and magnesium availability on the production of protease and bacitracin by Bacillus licheniformis

Bacillus licheniformis was cultivated in a range of defined media varying in both the nature of the growth-limiting component and the concentration of excess nutrients. The compositions of the media were such as to ensure that the final absorbance (A430) of the culture was the same in each case. Samples taken during the stationary phase were assayed for their content of extracellular serine protease and bacitracin. The nature of the growth-limiting nutrient had a profound effect on the amounts of these products formed while those components which were present in excess also exerted an influence in proportion to their concentration. Thus, for example, a four-fold increase in serine protease production occurred when ammonium replaced glucose as the growth-limiting nutrient. Serine protease and bacitracin production responded differently to these varying cultural conditions suggesting they are subject to separate control mechanisms. The results are discussed in relation to the need for rigorously controlled cultural conditions during physiological studies of this nature.     

Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain

The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC(50) ) ranged from 20 μm for bacitracin F to 1050 μm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI-TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI.     

Bacitracin and protease production in relation to sporulation during exponential growth of Bacillus licheniformis on poorly utilized carbon and nitrogen sources.

Growth of Bacillus licheniformis in a chemically defined medium containing glucose and ammonium chloride yielded a doubling time of 1.00 h. Examination of the culture during exponential growth revealed a lack of heat-resistant spores together with a complete absence of detectable concentrations of bacitracin or extracellular serine protease. Replacement of glucose as the sole carbon source by glycerol, pyruvic acid, citric acid, or lactic acid resulted in doubling times of 1.13, 2.00, 3.16, and 3.95 h, respectively. Bacitracin, protease, and heat-resistant spores were produced during exponential growth in amounts related to these doubling times. A qualitatively similar pattern was observed when ammonium chloride was replaced by sodium nitrate, alanine, or glutamic acid which gave doubling times of 1.65, 1.77, and 1.90 h, respectively. Protease, but not bacitracin, concentrations were substantially higher when the growth rate was restricted by use of poor nitrogen rather than poor carbon sources. The relationships between bacitracin production, protease production, and the sporulation process are discussed.     

Effect of protease inhibitors on androgen receptor analysis in rat prostate cytosol

Prostate androgen receptors are liable to proteolytic digestion during in vitro analysis; thus, various proteolytic enzyme inhibitors were tested for their ability to improve the androgen receptor assay. The serine (phenylmethylsulfonylflouride, aprotinin, p-aminobenzamidine) and thiol-senine (leupeptin, bacitracin) protease inhibitors individually present in the homogenization buffer significantly increased the measurable androgen binding sites by 30-35% in rat prostate cytosol as determined by saturation analysis with [3H]-17 beta-hydroxy-17-methyl- 4,9 11-estratrien-3-one (R-1881) for 20 hr at 4 degrees C. The apparent binding affinity was also increased by these compounds. Various combinations were tried and aprotinin/bacitracin was found to be additive in effect. This combination was also shown to prevent receptor degradation as determined by sucrose density gradient centrifugation. The carboxyl protease inhibitor, pepstatin A, was ineffective in improving the receptor assay. Rabbit bile, an inhibitor of seminin, interfered with receptor binding thus rendering it ineffective for use in saturation analysis. The results show that the use of serine-thiol protease inhibitors significantly improves the cytosol androgen receptor yield and assay sensitivity; therefore, we recommend routine inclusion of these compounds(s) in the homogenization buffer for androgen receptor assays.     

Bacitracin: Substantiation and Elimination of Contaminating Proteolytic Activity and Use as an Affinity Chromatography Ligand to Purify a Siderophore-Degrading Enzyme

Bacitracin is a commercial general peptidase inhibitor that may be used to purify proteases. Significant protease contamination of a commercial bacitracin preparation was noted and four procedures were developed to overcome the contamination. Dialysis, gel-filtration chromatography, molecular weight cutoff filters, and heat inactivation were effective, resulting in the diminution or elimination of proteolysis while maintaining the inhibitory effect of bacitracin. Attachment of bacitracin to an affinity chromatography resin did not immobilize a siderophore-degrading enzyme, as has been noted with peptidases. It did, however, result in its partial purification from some of the contaminating proteins originally present. Received: 1 June 2001 / Accepted: 30 July 2001     

In vitro production of bacitracin by proteolysis of vegetative Bacillus licheniformis cell protein.

The action of a sporulation-specific seryl protease on antibiotic-free extracts of Bacillus licheniformis cells yields a peptide that is identified as bacitracin by its biological activity, its spectral properties, and its comigration with genuine bacitracin in both paper and thin-layer chromatography. During proteolysis, a chemical structure is generated with the spectral properties of a delta-2 thiazoline ring. The yield in vitro, 4 microgram of bacitracin per mg of protein, is less than the maximal yield from sporulating cells, 75 microgram of bacitracin per mg of cell protein, but is a linear function of the amount of protein in the reaction system. Approximately 30% of the protein yielding the antibiotic is ribosomal associated, and only 25% of that amount can be removed by washing with 1 M NH4Cl. The substrate protein is a constant fraction of the cell protein throughout exponential growth and very early sporulation stages of culture development.     

Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structure-activity relationship

Bacitracin is a widely used metal-dependent peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(II) for its biological activity, and has been reported to bind several other transition metal ions, including Co(II), Ni(II), and Cu(II). Despite the wide use of bacitracin, a structure-activity relationship for this drug has not been established, and the structure of its metal complexes has not been fully determined. We report here one- and two-dimensional nuclear magnetic resonance (NMR) studies of the structure of the metal complexes of several bacitracin analogues by the use of paramagnetic Co(II) as a probe. The Co(II) complex of this antibiotic exhibits many well-resolved isotropically shifted (1)H NMR signals in a large spectral window ( approximately 200 ppm) due to protons near the metal, resulting from both contact and dipolar shift mechanisms. The assignment of the isotropically shifted (1)H NMR features concludes that bacitracin A(1), the most potent component of the bacitracin mixture, binds to Co(II) via the His-10 imidazole ring N(epsilon), the thiazoline nitrogen, and the monodentate Glu-4 carboxylate to form a labile complex in aqueous solutions. The free amine of Ile-1 does not bind Co(II). Several different analogues of bacitracin have also been isolated or prepared, and the studies of their Co(II) binding properties further indicate that the antimicrobial activity of these derivatives correlates directly to their metal binding mode. For example, the isotropically shifted (1)H NMR spectral features of the high-potent bacitracin analogues, including bacitracins A(1), B(1), and B(2), are virtually identical. However, Glu-4 and/or the thiazoline ring does not bind Co(II) in the bacitracin analogues with low antibiotic activities, including bacitracins A(2) and F.     

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

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

Enzyme-bound intermediates in the biosynthesis of bacitracin.

1. Bacitracin synthetase, a three-component enzyme complex which catalyzes synthesis of the dodecapeptide bacitracin A, has been prepared from Bacillus licheniformis strains ATCC 10716, AL and SB 319. During synthesis of bacitracin, the amino acids (smaller amounts) and peptides are covalently bound to the enzyme complex. The nature of the bindings suggest that the amino acids and peptides are thioester linked. 2. The peptides, identified by thin-layer chromatography after performic acid liberation were Ile-Cys, Ile-Cys-Leu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu-Ile, Ile-Cys-Leu-Glu-Ile-Lys-Orn, Ile-Cys-Leu-Glu-Ile-Ile-Orn-Ile, Ile-Cys-L-EU-Glu-Ile-Lys-Orn-Ile-Phe, Ile-Cys-Leu-Glu-Ile-L-YS-Orn-Ile-Phe-His-Phe-His and Ile-Cys-Leu-Glu-Ile-Lys-Orn-Ile-Phe-His-Asp. 3. The labelled peptides covalently bound to bacitracin synthetase were intermediates in bacitracin synthesis. 4. Chain growth is initiated on one enzyme component (A) by the addition of isoleucine and cysteine. The sequential addition of the other amino acids proceeds in the C-terminal direction until the pentapeptide is formed. Further addition of amino acids and production of bacitracin are obtained by adding the other enzyme components (B and C) to the incubation mixture.     

Receptor-linked degradation of 125I-insulin is mediated by internalization in isolated rat hepatocytes

When hepatocytes were freshly isolated from rat liver and incubated for various periods of time at 37 degrees C, the media from the incubation, when completely separated from the cells, actively degraded 125I-insulin. THis soluble protease activity was strongly inhibited by bacitracin but was unaffected by the lysosomatropic agent ammonium chloride (NH4Cl). When hepatocytes were incubated with 125I-insulin at 37 degrees C in the presence or absence of 8 mM NH4Cl the ligand initially bound to the plasma membrane and was subsequently internalized as a function of time. When hepatocytes were incubated at 37 degrees C for 30 minutes with 125I-insulin in the presence of bacitracin and NH4Cl or bacitracin alone and the cells were washed, diluted, and the cell-bound radioactivity allowed to dissociate, the percent intact 125I-insulin in the cell pellet and in the incubation media was greater in the presence of NH4Cl at each time point of incubation. Under these same conditions a higher proportion of the cell-associated radioactivity was internalized and a higher proportion was associated with lysosomes. The data suggest that receptor-mediated internalization is required for insulin degradation by the cell, and that this process, at least in part, involves lysosomal enzymes. Furthermore, the data demonstrate that internalization is not blocked by the presence of bacitracin or NH4Cl in the incubation media, but that degradation is inhibited.     

The two-component regulatory system BacRS is associated with bacitracin 'self-resistance' of Bacillus licheniformis ATCC 10716.

Bacitracin is a peptide antibiotic produced by several Bacillus licheniformis strains that is most active against other Gram-positive microorganisms, but not against the producer strain itself. Recently, heterologous expression of the bacitracin resistance mediating BcrABC transporter in Bacillus subtilis and Escherichia coli was described. In this study we could determine that the transporter encoding bcrABC genes are localized about 3 kb downstream of the 44-kb bacitracin biosynthetic operon bacABC. Between the bac operon and the bcrABC genes two orfs, designated bacR and bacS, were identified. They code for proteins with high homology to regulator and sensor proteins of two-component systems. A disruption mutant of the bacRS genes was constructed. While the mutant displayed no effects on the bacitracin production it exhibited highly increased bacitracin sensitivity compared to the wild-type strain. Western blot analysis of the expression of BcrA, the ATP-binding cassette of the transporter, showed in the wild-type a moderate BcrA induction in late stationary cells that accumulate bacitracin, whereas in the bacRS mutant cells the BcrA expression was constitutive. A comparison of bacitracin stressed and nonstressed wild-type cells in Western blot analysis revealed increasing amounts of BcrA and a decrease in BacR in the stressed cells. From these findings we infer that BacR acts as a negative regulator for controlling the expression of the bcrABC transporter genes.     

Requirement for peptidoglycan synthesis during sporulation of Bacillus subtilis.

Cultures of Bacillus subtilis were treated during sporulation with antibiotics (bacitracin and vancomycin) that affect peptidoglycan synthesis. The cells were resistant to the effects of the antibiotics only when the drugs were added about 2 h after the beginning of sporulation. This was about 1 h later than the escape time of a temperature-sensitive sporulation mutant that is unable to complete prespore septation. Similar experiments were done with a mutant temperature sensitive for peptidoglycan synthesis. This showed an escape curve similar to that shown by the antibiotics. When sporulating cells were treated with antibiotics, they produced alkaline phosphatase earlier than normal. Enzyme production was unaffected by inhibition of deoxyribonucleic acid synthesis but was inhibited by chloramphenicol. Sporulation mutants that are unable to make alkaline phosphatase under normal conditions were able to make it in the presence of bacitracin. The alkaline phosphatase made under these conditions was under "sporulation-type" control since its synthesis was repressible by casein hydrolysate and unaffected by inorganic phosphate. When cells were treated with bacitracin in the growth medium as well as in the sporulation medium, alkaline phosphatase synthesis was at the same level as in an untreated control. A number of other antibiotics and surfactants were tested for the ability to cause premature production of the phosphatase of those tested, only taurodeoxycholate whowed this behavior. Moreover, incubation of cells with taurodeoxycholate in the growth medium as well as in the sporulation medium prevented premature enzyme production.     

Characterization of insulin degradation by rat-liver low-density vesicles

When incubated in vitro, isolated rat liver low-density vesicles degrade endocytosed insulin intraluminally. The rate of intravesicular degradation suggests that this pathway contributes significantly to insulin degradation in vivo. The vesicles can be selectively disrupted with digitonin at concentrations that abolish the latency of NADH pyrophosphatase, with minimal effect on the cisternal Golgi marker, galactosyl transferase. The results suggest that latent NADH pyrophosphatase may act as a marker enzyme for the vesicles within which insulin is degraded. The possible role of insulin-glucagon protease, a candidate enzyme for insulin degradation by the liver, was investigated. The activity of latent insulin-glucagon protease associated with low-density vesicles is sufficient to account for the rate of intravesicular proteolysis. However, the rate of intravesicular proteolysis is insensitive to membrane-permeant thiol reagents under conditions which strongly inhibit insulin-glucagon protease. This shows that insulin-glucagon protease is not rate-limiting for insulin degradation by these vesicles, and is unlikely to be involved in the regulation of degradation. After disruption with Brij, internalized insulin remains associated with the membrane. Degradation is not inhibited by addition of excess unlabelled insulin to the medium, and occurs more rapidly than the degradation of an equal activity of iodo-insulin added to the disrupted membranes. This implies that degradation of endocytosed insulin occurs while it is still bound to the inner surface of the vesicles. When bacitracin is coinjected with iodo-insulin, it inhibits degradation of internalized insulin both by intact and Brij-disrupted vesicles, but not the degradation of added exogenous insulin, confirming that degradation is membrane-associated, and that it does not require the release of insulin into free solution.     

Tissue distribution of avian pancreatic polypeptide-degrading activity

Degradation of avian pancreatic polypeptide (APP) by subcellular fractions from homogenates of chicken kidney, liver, and brain was characterized in this study. Chicken kidney cytosol exhibited the highest degrading activity of all kidney subcellular fractions studied including nuclear, mitochondrial, and microsomal. The cytosolic kidney APP-degrading activity was inhibited in a dose-dependent manner by bacitracin, serine protease inhibitors, and dithiothreitol, and eluted in the void volume of a Sephadex G-100 column, indicating that it is a soluble, serine protease-like activity with a Mr greater than 100,000 kDa and with some dependence on disulfide bonds. Soluble cytosol fractions from chicken liver, kidney, and brain all exhibited greater APP-degrading activity than that of corresponding membrane fractions and, furthermore, were similar in activity between one another. It is concluded that APP degradation by tissue homogenates occurs via a soluble, cytosolic protease which is inhibited by selected serine protease inhibitors; the activity does not differ among liver, kidney, and brain, three tissues which show different receptivity for APP.     

Differential influence of bacitracin on plant proteolytic enzyme activities

The effect of bacitracin on the activity of proteases extracted from pollen and sprouts of various plant species and compared to five commercially available proteases was studied. Bacitracin stimulates some pollen proteolytic enzyme activities, contrary to its inhibitory influence on proteases from the other sources. Proteases from maize pollen, inhibited by pepstatin and phenylmethylsulfonyl fluoride, immediately accelerate their activities after addition of bacitracin to the reaction mixture. The stimulating influence of peptide antibiotic on pollen proteases of some plants is unexpected and molecular mechanism of this phenomenon requires a further elucidation. The augmentation of allergenic response caused by pollen enzymes and drugs containing bacitracin is discussed.     

Induction of penicillinase by bacitracin.

Bacitracin preparations are shown to induce penicillinase (β-lactamase I) formation in strain 569 of $\text{Bacillus cereus}$. At high bacitracin concentrations (2–4 mg/ml) the level of induced enzyme obtained reaches a maximum which is comparable to that induced by optimal concentrations (1–2 mcg/ml) of β-lactam antibiotics. Penicillinase formation induced by short exposure to bacitracin, continues at a normal rate after all free bacitracin has been removed. The inducing activity of bacitracin is highly, but not completely, resistant to β-lactamase and can be entirely eliminated by prolonged treatment with penicillinase of $\text{B. cereus}$. The site of induction by bacitracin is, however, different from that mediating induction by β-lactam antibiotics. The inducing component has been isolated by thin layer chromatography; it seems to be closely related to, but not identical with, bacitracin A,B or F.     

Sporulation of a bacitracin-sensitive mutant of Bacillus licheniformis is self-inhibited by bacitracin

A mutant of Bacillus licheniformis (BLU166) sensitive to its own antibiotic bacitracin was isolated and the mutation bcr-l was mapped close to the bacitracin synthetase genes. The sensitivity was shown to be specific for bacitracin. Two further bacitracin-sensitive strains were constructed, one (BLU171) with normal ability to synthesize bacitracin, and one (BLU170) a bacitracin non-producer. In addition to an increased sensitivity of growing cells to bacitracin, sporulation of the mutant strain BLU171 was self-inhibited by bacitracin. It is concluded that (1) there might exist at least two levels of resistance to bacitracin; (2) mutation bcr-1 affects a 'structural' component, which may protect the sensitive reaction of cell-wall biosynthesis; (3) sporulation is affected to a greater extent by bacitracin than vegetative growth; and (4) synthesis of bacitracin is independent of the presence of this resistance mechanism since the sensitive mutant produces similar amounts of the antibiotic to the wild-type strain.     

Biosynthesis of bacitracin on a protein thiotemplate.

The dodecapeptide bacitracin A is the major constitutent of a family of antibacterial peptides produced by Bacillus licheniformis. The non-ribosomal biosynthesis of bacitracin has been studied in cell-free extracts. Bacitracin synthetase has been fractionated on Sephadex G 200 column into two fractions; both fractions were required for bacitracin biosynthesis. On the other hand, on a Sepharose affinity chromatography column, using L-leucine as ligand, three fractions were obtained; all three were required for bacitracin biosynthesis. During bacitracin synthesis, the enzyme components contain a number of thioester bound peptides. The nature of the peptides suggested that the synthesis proceeds towards the C-terminal end of the molecule. It is assumed that by sequential addition of thioester-bound amino acids, bacitracin A could be synthesized on the surface of the enzyme containing phosphopantetheine.