Production and purification of bacilysin

1. Bacilysin, a hydrophilic substance formed by certain aerobic spore-forming bacteria that causes lysis in cultures of growing staphylococci, has been produced in aerated cultures of a strain of Bacillus subtilis (A14). A chemically defined medium was used, which contained glucose, Czapek-Dox salts and ferric iron. Production of bacilysin occurred, after a lag, while the culture was still undergoing rapid growth. 2. Bacilysin was adsorbed from the culture medium on Zeo-Karb 225 (SR5) (H(+) form) and eluted with aqueous pyridine. The crude material was purified by chromatography in pyridine-acetate buffers on columns of Dowex 50 (X2) and Dowex 50 (X8) respectively and by chromatography in aq. 70% (v/v) propan-2-ol on Sephadex G-25. 3. Purified bacilysin behaved as a single ninhydrin-positive substance when subjected to chromatography on paper in butan-1-ol-acetic acid-water and to electrophoresis on paper at pH4.5 or pH1.8. At pH4.5 the substance behaved as though it had no net change and at pH1.8 it migrated towards the cathode.     

Isolation of bacilysin and a new amino acid from culture filtrates of Bacillus subtilis

1. Bacilysin, a labile dipeptide antibiotic that lyses growing staphylococci, was isolated from culture fluids of Bacillus subtilis by a process giving higher yields than those previously obtained. 2. The process involves adsorption on a cation-exchange resin and elution with aqueous trimethylamine, separation from neutral amino acids and glutamic acid by chromatography on DEAE-Sephadex at pH8.7 and separation from other neutral peptides by chromatography in aqueous propan-2-ol on Sephadex G-25. 3. A new amino acid, which is chemically related to bacilysin, was isolated from the fraction containing neutral amino acids. 4. Two substances that yield alanine on hydrolysis, in addition to bacilysin, were obtained from the neutral peptide fraction.     

Some properties of rat-liver glucose–adenosine triphosphate phosphotransferases

1. Bacilysin, a peptide which yields l-alanine and l-tyrosine on acid hydrolysis, was produced by a strain of Bacillus subtilis (A 14) in a chemically defined medium containing glucose, ammonium acetate or ammonium chloride, potassium phosphate and other inorganic salts, and ferric citrate. 2. Under the conditions used growth was diphasic. Bacilysin was formed during the second phase of slower growth, and there was little production during the stationary phase. Nevertheless, bacilysin production occurred when protein synthesis was inhibited by chloramphenicol. It thus appears that there is no obligatory coupling of protein synthesis and bacilysin synthesis. 3. When dl-[1-(14)C]alanine was added to a growing culture of B. subtilis, (14)C was incorporated into bacilysin, which contains an N-terminal alanine residue. 4. Under similar conditions virtually no (14)C was incorporated into bacilysin from dl-[2-(14)C]tyrosine, l-[U-(14)C]tyrosine or [1-(14)C]acetate, although these compounds were used by the cell for the biosynthesis of other substances. These results indicate that neither tyrosine nor acetate is a precursor of the fragment of bacilysin which yields tyrosine on hydrolysis with hot 6n-hydrochloric acid. 5. The tyrosine-yielding fragment of bacilysin was labelled with (14)C from [1,6-ring-(14)C(2)]shikimic acid. The biosynthesis of bacilysin thus appears to involve a diversion from the pathway leading to aromatic amino acids at the shikimic acid stage, or a subsequent one.     

Regulation of biosynthesis of bacilysin byBacillus subtilis

Summary Production of the dipeptide antibiotic bacilysin byBacillus subtilis 168 was growth associated and showed no evidence of repression by glucose or sucrose. Carbohydrates other than glucose and sucrose yielded lower specific titers of bacilysin. Bacilysin production in three such carbon sources (maltose, xylose, ribose) was delayed until growth slowed down. Ammonium salts were poor for bacilysin production when used as the sole nitrogen source. When added to the standard medium containing glutamate, they suppressed antibiotic production. Aspartate was slightly better than glutamate for antibiotic production as sole nitrogen source. No other nitrogen source tested, including inorganic, organic or complex, approached the activity of glutamate or aspartate. When added to glutamate, casamino acids, phenylalanine and alanine (a substrate of bacilysin synthetase) suppressed bacilysin production while stimulating growth. Phosphate provided for optimum growth and production at 7.5 mM and both processes were inhibited at higher concentrations. Ferric citrate stimulated growth and inhibited bacilysin production, the effects being due to both the iron and the citrate components. Elimination of ferric citrate stimulated production as did increasing the concentration of Mn to its optimum concentration of 6.6×10^–4M.     

The structure of bacilysin and other products of Bacillus subtillis

1. Mass spectra of the trimethylsilyl derivative and the methyl ester of the N-trifluoroacetyl derivative of bacilysin indicated that the antibiotic had a molecular weight of 270. Several peaks in the spectrum of the methyl ester were consistent with the presence of an N-terminal alanine residue in the molecule. 2. The proton-magnetic-resonance spectrum of bacilysin confirmed that the antibiotic contained an epoxide group and the spin-spin splitting of the protons of the epoxide group indicated that the side chain of the epoxycyclohexanone ring was attached at C-4 and was alphabeta to the keto group. 3. The formation of an alphabeta-unsaturated ketone on reduction of bacilysin with chromous chloride also showed that the epoxide was alphabeta to the keto group. 4. The optical-rotatory-dispersion curve of bacilysin showed a positive Cotton effect. On the assumption that the reversed Octant rule for alphabeta-epoxyketones was applicable this revealed the absolute stereochemistry and enabled a definitive structure to be assigned to the molecule. 5. Similar measurements showed that substance AA1, isolated from culture supernatants, was the C-terminal amino acid of bacilysin. 6. Hydrolysis of substance P2 with leucine aminopeptidase and the mass spectrum of the methyl ester of its N-trifluoroacetyl derivative showed that this substance was l-analyl-l-alanine. 7. These results are discussed in relation to the biogenesis of bacilysin.     

Synthesis of bacilysin by Bacillus subtilis branches from prephenate of the aromatic amino acid pathway.

Previously characterized mutants of Bacillus subtilis 168 were analyzed for the accumulation of the antibiotic bacilysin in culture broths and for the presence of bacilysin synthetase in cell extracts. All aro mutants tested were deficient in bacilysin biosynthesis but had synthetase activity. Mutants with lesions in tyrA and pheA produced normal levels of bacilysin, suggesting that prephenate is the primary metabolic precursor of bacilysin.     

Molecular insights into the antifungal mechanism of bacilysin

Bacilysin is one of the simplest antimicrobial peptides and has drawn great attention for its excellent performance against Candida albicans. In this study, the antifungal mechanism of bacilysin was investigated. The target enzyme glucosamine-6-phosphate synthase (GFA) was expressed heterologously in Escherichia coli and its inhibition by bacilysin and derivatives was studied. It was concluded that bacilysin could be hydrolyzed by a proteinase of C. albicans, and that the released product, anticapsin, then inhibited the aminotransferase activity of GFA. This result was verified by molecular simulation, and the interaction mode of anticapsin with GFA was detailed, which provides data for the development of novel antifungal drugs. Transport of bacilysin into fungal cells was also simulated and it was shown that bacilysin is more readily transported into cells than anticapsin. Thus, our findings support a mechanism whereby bacilysin is transported into fungal pathogens, hydrolyzed to anticapsin, which then inhibits GFA.     

Observations on the structure of bacilysin.

1. Elementary analysis and other properties of a highly purified preparation of bacilysin indicated that a possible molecular formula for the substance is C(12)H(18)N(2)O(5). The results of electrometric titration were consistent with the hypothesis that the substance was a peptide containing one free alpha-amino group and one free carboxyl group. 2. Hydrolysis of bacilysin with 6n-hydrochloric acid at 105 degrees yielded l-alanine and l-tyrosine, but the ultraviolet spectrum of the substance showed that no tyrosine residue was present in the molecule and a nuclear-magnetic-resonance spectrum indicated that olefinic and aromatic protons were absent. The dinitrophenyl (DNP) derivative of bacilysin yielded DNP-alanine on acid hydrolysis. 3. Bacilysin was hydrolysed by leucine aminopeptidase (EC 3.4.1.1) and by Pronase to give alanine and an uncharacterized amino acid. Its infrared spectrum was consistent with the presence of a peptide grouping in the molecule. 4. The optical rotatory dispersion of bacilysin and its reaction with thiosemicarbazide indicated that the substance contained an aldehyde or ketone group. Its behaviour on catalytic reduction and its reaction with sodium thiosulphate and with certain thiols suggested that an epoxide group was present. 5. A possible type of structure for bacilysin is considered in the light of its known properties.     

Changes in patterns of alkaline serine protease and bacilysin formation caused by common effectors of sporulation inBacillus subtilis 168

Bacilysin biosynthesis and alkaline serine protease production inBacillus subtilis 168 were monitored and compared in batch cultures when various effectors of sporulation were added at different stages of growth in a medium containing sucrose and glutamate. Depending on the time of addition, glucose affected sporulation and serine protease formation to the same extent, but had no effect on bacilysin production. Ammonium andl-alanine additions suppressed all three processes. Casamino acids severely interfered with bacilysin formation and sporulation, but not with protease formation. Decoyinine, a well-known inducer of sporulation, induced protease formation as well, but did not affect bacilysin biosynthesis. The extent of the observed effects depended largely on the time of metabolite additions. The results are discussed with reference to a possible coregulation of sporulation and the formation of bacilysin and alkaline serine protease inB. subtilis.     

Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin

Transposon mutagenesis was employed to isolate the gene(s) related with the biosynthesis of dipeptide antibiotic in Bacillus subtilis PY79 (a prototrophic derivative of the standard 168 strain). The blocked mutants were phenotypically selected from the transposon library by bioassay and the complete loss of biosynthetic ability was verified through ESI-mass spectrometry analysis. Four different bacilysin nonproducer mutants (Bac(-)::Tn10(ori-spc)) were isolated from the transposon library. The genes involved in bacilysin biosynthesis were identified as thyA (thymidilate synthetase), ybgG (unknown; similar to homocysteine methyl transferase) and oppA (oligopeptide permease), respectively. The other blocked gene was yvgW (unknown; similar to heavy metal-transporting ATPase); however, backcross studies did not verify its involvement in bacilysin biosynthesis. This gene, on the other hand, appeared to be necessary for efficient sporulation and transformation. Opp involvement was significant as it suggested that bacilysin biosynthesis is under or a component of the quorum sensing pathway which has been shown to be responsible for the establishment of sporulation, competence development and onset of surfactin biosynthesis. For verification, it was necessary to check the involvement of peptide pheromones (PhrA or PhrC) internalized by the Opp system and response regulator ComA as the essential components of this global control. phrA, phrC and comA deleted mutants of PY79 were thus constructed and the latter two genes were shown to be essential for bacilysin biosynthesis.     

Cell-free synthesis of the dipeptide antibiotic bacilysin

Summary Bacilysin, a dipeptide antibiotic produced byBacillus subtilis A 14, was synthesized by a cell-free extract of the producing organism from its constitutent amino acids,l-alanine andl-anticapsin. The synthesis required ATP and Mg²⁺ and was optimal at pH 8.1. The same extract also synthesizedl-alanyl-l-alanine. The synthesis of bacilysin was not inhibited by chloramphenicol, DNase or RNase.     

Bacillus subtilis mutant deficient in the ability to produce the dipeptide antibiotic bacilysin: isolation and mapping of the mutation.

A Bacillus subtilis mutant which carries a lesion in a gene specific to the synthesis of the dipeptide antibiotic bacilysin was isolated. A derivative strain in which transposon Tn917 had inserted near the bacilysin lesion was isolated and used as the donor in PBS-1 transduction mapping experiments. The bac-1 locus was mapped between the ctrA and sacA loci, near 90% on the standard B. subtilis 168 chromosome map.     

Bacilysin from Bacillus amyloliquefaciens FZB42 Has Specific Bactericidal Activity against Harmful Algal Bloom Species

Harmful algal blooms, caused by massive and exceptional overgrowth of microalgae and cyanobacteria, are a serious environmental problem worldwide. In the present study, we looked for Bacillus strains with sufficiently strong anticyanobacterial activity to be used as biocontrol agents. Among 24 strains, Bacillus amyloliquefaciens FZB42 showed the strongest bactericidal activity against Microcystis aeruginosa, with a kill rate of 98.78%. The synthesis of the anticyanobacterial substance did not depend on Sfp, an enzyme that catalyzes a necessary processing step in the nonribosomal synthesis of lipopeptides and polyketides, but was associated with the aro gene cluster that is involved in the synthesis of the sfp-independent antibiotic bacilysin. Disruption of bacB, the gene in the cluster responsible for synthesizing bacilysin, or supplementation with the antagonist N-acetylglucosamine abolished the inhibitory effect, but this was restored when bacilysin synthesis was complemented. Bacilysin caused apparent changes in the algal cell wall and cell organelle membranes, and this resulted in cell lysis. Meanwhile, there was downregulated expression of glmS, psbA1, mcyB, and ftsZ—genes involved in peptidoglycan synthesis, photosynthesis, microcystin synthesis, and cell division, respectively. In addition, bacilysin suppressed the growth of other harmful algal species. In summary, bacilysin produced by B. amyloliquefaciens FZB42 has anticyanobacterial activity and thus could be developed as a biocontrol agent to mitigate the effects of harmful algal blooms.     

小麦赤霉病原菌拮抗菌Bacillus amyloliquefaciens 7M1产抗菌素的研究

【目的】从小麦根际土壤分离鉴定一株赤霉病拮抗菌,对该菌产的抗菌素进行生物学性质研究、种类鉴定和抑菌实验。【方法】利用牛津杯法和光照培养箱实验对其抑菌活性进行测定,通过16S r RNA基因序列分析对目标菌株的种属进行初步鉴定,根据抗菌素相关基因进行PCR扩增和测序,利用在线软件Pro Param tool和TMHMM对编码蛋白进行生物信息学分析。【结果】7M1菌体和抗菌素对禾谷镰刀菌的抑菌圈直径分别为16.33±0.13 mm和15.43±0.21 mm,16S r RNA基因序列分析结果显示其为芽孢杆菌,并与解淀粉芽孢杆菌具有较近的亲缘关系,菌株7M1抗菌素对小麦赤霉病的温室防治效果为76.41%,而且热稳定性好,可被蛋白酶K、胰蛋白酶、胃蛋白酶降解,在p H 5.0-10.0有较好的抑菌活性,但是紫外线辐射会降低其抑菌活性。菌株7M1含有bac AB、itu C、bam D 3种基因,通过与Gen Bank中相关的抗菌素基因进行比对,发现其编码的氨基酸序列与Gen Bank库中的芽孢杆菌溶素、伊枯草菌素和杆菌抗霉素D等抗菌素的相似性达到99%。bac AB编码蛋白和itu C编码蛋白是稳定蛋白,bam D编码蛋白是不稳定蛋白,此外,3种基因的编码产物不具有明显的跨膜结构。【结论】从该菌发酵液提取的抗菌素有很好的抗禾谷镰刀菌活性而且性质稳定,因而在小麦赤霉病的生物防治中具有潜在的应用价值。     

Inhibition of glucosamine synthase by bacilysin and anticapsin

L-Glutamine:D-fructose-6-phosphate amidotransferase ('glucosamine synthase', EC 5.3.1.19) from Escherichia coli MRE 600 was purified at least 75-fold. It catalysed the formation of 21.1 mumol glucosamine 6-phosphate (mg protein)-1 in 30 min at 37 degrees C. Its molecular weight, estimated by gel filtration, was about 90000 and it was inhibited by thiol group reagents. Anticapsin, the C-terminal amino acid of the dipeptide antibiotic bacilysin, and to a lesser extent bacilysin itself, inhibited glucosamine synthase activity. Kinetic studies indicated that the inhibition was non-competitive with respect to fructose 6-phosphate as substrate but partly competitive with respect to L-glutamine. Incubation of the enzyme with anticapsin brought about a time-dependent and irreversible inhibition. It is suggested that anticapsin behaves as a glutamine analogue and that a reaction of its epoxide group with a thiol group of glucosamine synthase results in its linkage to the enzyme by a covalent bond.     

ScoC Regulates Bacilysin Production at the Transcription Level in Bacillus subtilis

Bacillus subtilis mutants with high expression of the bacilysin operon ywfBCDEFG were isolated. Comparative genome sequencing analysis revealed that all of these mutants have a mutation in the scoC gene. The disruption of scoC by genetic engineering also resulted in increased expression of ywfBCDEFG. Primer extension and gel mobility shift analyses showed that the ScoC protein binds directly to the promoter region of ywfBCDEFG. Our results indicate that the transition state regulator ScoC, together with CodY and AbrB, negatively regulates bacilysin production in B. subtilis.Gram-positive model bacterium Bacillus subtilis produces the dipeptide antibiotic bacilysin, which consists of an l-alanine and an unusual amino acid, l-anticapsin (15). We previously reported that a polycistronic operon, ywfBCDEFG, and a monocistronic gene, ywfH, are required for bacilysin production (7). The gene products of ywfB and ywfG are thought to participate in the l-anticapsin biosynthesis pathway, while the ywfE gene product has been assigned as an amino acid ligase involved in alanine-anticapsin ligation (14). The protein encoded by the ywfF gene is necessary for self-protection against bacilysin (13). Thus, the ywfBCDEFG operon has an obligate role in bacilysin production.We previously showed that a certain rifampin (rifampicin) resistance mutation can activate the B. subtilis dormant secondary metabolism, neotrehalosadiamine (3,3′-diamino-3,3′-dideoxy-α,β-trehalose) synthesis (8). Subsequently, we attempted to activate bacilysin production in the same way. Unexpectedly, we found that the expression of the bacilysin operon ywfBCDEFG was induced by a mechanism independent of the rifampin resistance mutation. Although the expression of the bacilysin operon ywfBCDEFG was previously reported to be negatively regulated by transition state regulators CodY (7) and AbrB (11), the mechanism we found was apparently different from these known mechanisms. Here, we report a novel regulatory mechanism involved in bacilysin production.