Following tyrothricin peptide production by Brevibacillus parabrevis with electrospray mass spectrometry.

The tyrocidines and analogues are cationic cyclodecapeptides [cyclo (D-Phe1-L-Pro2-L-(Phe3/Trp3)-D-(Phe4/Trp4)-L-Asn5-L-Gln6-L-(Tyr7/Phe7/Trp7)-L-Val8-L-(Orn9/Lys9)-L-Leu10], produced together with the neutral linear pentadecapeptide gramicidins, in the antibiotic tyrothricin complex by Brevibacillus parabrevis. Despite discovery 80 years ago, it was still uncertain whether these peptides are secreted or sequestered intracellularly. We resolved this by utilising high resolution electrospray mass spectrometry to confirm the predominantly intracellular sequestration of the peptides in the tyrothricin complex. A "peptidomics" approach allowed us to map the intracellular production of 16 cyclodecapeptides and 6 gramicidins over 16 days of culturing. Gramicidin production remained relatively constant, with Val-gramicidin A the predominant analogue produced throughout the 16 day fermentation period. The tyrothricin cyclodecapeptides have four variable positions and there was a culturing time related shift from the Phe-rich A analogues, containing a L-Phe3-D-Phe4 aromatic dipeptide unit, to the Trp-rich C analogues with L-Trp3-D-Trp4. For the other variable aromatic residue position, Tyr7 was preferentially incorporated above Trp7, with a minor incorporation of Phe7 over the whole culturing period. For the variable basic amino acid residue, there was time-sensitive shift from Orn9 to Lys9 incorporation. Modulation of the cyclodecapeptide profile over time does not correlate with the reported non-ribosomal peptide synthetase affinity, specifically for Trp in the variable aromatic residue positions, indicating additional supply-demand control in the cyclodecapeptides production by B. parabrevis. These novel observations are not only of importance for production and purification of selected peptide analogues from the tyrothricin complex, but also for insight into microbial control of non-ribosomal peptide production that extends beyond the peptide synthetase machinery.

Suppression of tyrocidine production by purine nucleotides and related substances in Bacillus brevis.

Bacillus brevis (ATCC 8185) produces an antibiotic peptide, tyrocidine. We found that adenosine or 5'-AMP suppressed the production of tyrocidine with half-maximum inhibition at 100-300 microM. This inhibition was specific to the production of tyrocidine since neither adenosine nor 5'-AMP showed any effect on bacterial growth. Cyclic nucleotides had no effect. These results suggest that adenosine, 5'-AMP or its metabolite was specifically involved in the regulation of tyrocidine production.

Peptide antibiotics and sporulation: induction of sporulation in asporogenous and peptide-negative mutants of Bacillus brevis.

Mutants of Bacillus brevis ATCC 8185 were isolated which were unable to produce detectable amounts of either tyrocidine or linear gramicidin, or both peptide antibiotics. Tyrocidine-negative mutants (BM5, BM21, BM44) sporulated normally. Gramicidin-negative mutants (BM2, BM24) were oligosporogenous, and mutants unable to produce both peptides (S18, S19) were asporogenous. Addition of tyrocidine and/or gramicidin to asporogenous mutants in rich medium did not stimulate sporulation. However, these mutants formed normal spores after being transferred to nitrogen-free medium and upon the addition of tyrocidine. It was demonstrated that nutrient broth has a suppressive effect on tyrocidine-induced sporulation of S18. The tyrocidine-negative mutant BM44, sporogenous in rich medium, could sporulate under nitrogen deprivation only if supplemented with tyrocidine. The significance of the peptide antibiotics for a regulatory role in sporogenesis of B. brevis is discussed.

Isolation and properties of Bacillus brevis mutants unable to produce tyrocidine.

Mutants of Bacillus brevis ATCC 10068 were isolated which produced less than 1/100 of the amount of tyrocidine produced by the parent strain. These mutants produced spores at the same frequency and which were as resistant to heating at 80 degrees C for up to 3 h as were those produced by the parent strain. A partially purified tyrocidine synthetase from strain ATCC 10068 catalyzed [32P]PPi-ATP exchange reactions dependent on added tyrocidine-constituent amino acids. These activities were separated into three groups (I, II, and III) by fractionation on an Ultrogel AcA34 column. Each group was similar to one of the three components (heavy, intermediate, and light, respectively) found previously for strain ATCC 8185 except that glutamate-dependent activity was not detected in the group I activities and some amino acyl-tRNA synthetase activities were associated with the group III activities. Some of the mutants were shown to have defective tyrocidine synthetase enzymes. Mutant BH30 was defective in two of the group II amino acid-dependent [32P]PPi-ATP exchange reactions, mutant BH16 was defective in one of the group I and one of the group II reactions, and mutant BH34 had alterations to activities in all of the groups. It is unlikely that any of these mutants could synthesise tyrocidine. We conclude that tyrocidine is not involved in either the sporulation process or the resistance of spores of B. brevis ATCC 10068 to heating at 80 degrees C for up to 3 h.

Isolation of amino acid activating subunit--pantetheine protein complexes: their role in chain elongation in tyrocidine synthesis.

Dissociation of the multienzymes of tyrocidine synthesis by prolonged incubation of crude extracts of Bacillus brevis (Dubos strain, ATCC 8185) has yielded, on Sephadex G-100 chromatography, two fractions of amino acid activating subunits, a larger one of 70,000 daltons and a smaller one of 90,000 daltons; the latter was a complex consisting of the 70,000 dalton subunit and the pantetheine-carrying protein of about 20,000 daltons. When it dissociated, the intermediate enzyme, which activates three amino acids, contained two-thirds of the subunits in the 70,000 dalton and one-third in the 90,000 dalton fraction; the heavy enzyme, which activates six amino acids, contained five-sixths of the subunits in the former fraction and one-sixth in the latter. Both fractions showed ATP-PP(i) exchange with all amino acids that are activated by the respective polyenzymes. With proline as an example, the 70,000 dalton subunit exhibited a single low-affinity binding site, which should correspond to the peripheral thiol acceptor site, whereas the 90,000 dalton subunit showed both a low-affinity binding site and an additional high-affinity site for proline; the high-affinity site is attributed to the pantetheine present on the pantetheine-carrying protein, and suggests that amino acids are translocated from the peripheral SH to the pantetheine-carrying moiety during chain elongation. This was confirmed by the observation that the 90,000 dalton complex, when incubated with the light enzyme in the presence of phenylalanine and proline, produced DPhe-Pro dipeptide that cyclized into DPhe-Pro diketopiperazine, but the 70,000 dalton activating subunit, when similarly incubated, did not. After subunit dissociation, however, no further elongation occurred after the transfer from phenylalanine to proline.

Adenine nucleotide pool and energy charge during growth of a tyrothricin-producing strain of Bacillus brevis.

The adenine nucleotide levels and derived energy charge value of a tyrothricin-producing strain of Bacillus brevis under aerobic conditions were in good agreement with published values for other bacteria. When growing cultures of B. brevis underwent a transition from aerobic to anaerobic conditions, cyclic variations in the level of adenine nucleotides were observed and the energy charge value oxcillated between 0.87 and 0.70. The significance of these changes is considered in relation to antibiotic production as a possible regulatory mechanism in energy metabolism. It is concluded that tyrothricin is not directly involved in the observed changes in energy charge value.

The relation between sporulation and the induction of antibiotic synthesis and of amino acid uptake in Bacillus brevis.

The induction and localization of tyrocidine-synthesizing enzymes is shown to be parallel, during growth of Bacillus brevis (ATCC 8185, American Type Culture Collection, Rockville, Md.), with the induction of uptake of constitutive amino acids and of components of pantetheine, a coenzyme of tyrocidine synthesis. Antibiotic synthesis appears at the end of logarithmic growth when the first soluble enzymes may be obtained from homogenates. During this period, binding proteins for metabolite uptake were isolated by intensive sonication which, when studied by chromatography, were identified by the appearance of low molecular weight fractions binding the radioactively marked metabolites; their induction was prevented by addition of rifampicin. The major purpose of this study was a comparison of antibiotic production and sporulation, the progress of which was followed by electron microscopy. The onset of tyrocidine synthesis and metabolite uptake coincided with the appearance of septum formation indicating that sporulation had progressed to stage II. With the progress of spore encapsulation, the tyrocidine production migrated from the soluble fraction into the forespore, terminating with the separation of forespores from the sporangium membrane. The resulting concentration of antibiotic in the forespore may indicate its function in sporulation, the nature of which, however, was not explored.

Isolation of a peptidyl-pantetheine-protein from tyrocidine-synthesizing polyenzymes.

The polyenzyme complex responsible for the synthesis of tyrocidine in Bacillus brevis (ATCC 8185) was found to contain 4'-phosphopantetheine, which appeared to be connected with the production of growing peptide chains. Confirmation of this assumption has now been obtained by purifying from bacterial lysates a polyenzyme-dissociation product; this was labeled with [(14)C]pantothenic acid and peptide chains containing tritiated amino acids, and had a molecular weight of 17,000. To obtain these results, organisms were grown udner conditions favorable for incorporation of radioactive pantothenic acid into tyrocidine-synthesizing enzymes. A crude lysate of the [(14)C]pantothenic acid-labeled organisms was preincubated with the tritiated amino acids to form enzyme-bound growing peptide chains. The doubly labeled fragments were purified from the polyenzyme-dissociation products produced by prolonged lysis. In a second set of experiments, the three enzymes responsible for tyrocidine synthesis, including the two polyenzymes containing pantetheine, were purified and incubated with radioactive amino acids and ATP to form polyenzyme-bound peptide chains. Thereupon, a Triton X-100 extract of the 20,000 x g fraction of crude homogenate was added to dissociate the purified polyenzymes. The dissociation products were purified and yielded, on dodecyl sulfate gel electrophoresis, peptidyl-marked products ranging in molecular weight from 90,000 to 17,000, the latter being most abundant. Electrophoresis of analogous preparations after preincubation with higher concentrations of dodecyl sulfate and dithiothreitol at 100 degrees yielded a single product of 17,000 molecular weight, indicating that the larger molecular weight fractions were aggregates thereof.