A novel multifunctional peptide oligomer of bacitracin with possible bioindustrial and therapeutic applications from a Korean food-source Bacillus strain.

CONCLUSION: CSP32 has stable characteristics and may find bio-industrial and therapeutic applications.

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 sensing in Bacillus subtilis.

The extracellular presence of antibiotics is a common threat in microbial life. Their sensitive detection and subsequent induction of appropriate resistance mechanisms is therefore a prerequisite for survival. The bacitracin stress response network of Bacillus subtilis consists of four signal-transducing systems, the two-component systems (TCS) BceRS, YvcPQ and LiaRS, and the extracytoplasmic function (ECF) sigma factor sigma(M). Here, we investigated the mechanism of bacitracin perception and the response hierarchy within this network. The BceRS-BceAB TCS/ABC transporter module is the most sensitive and efficient bacitracin resistance determinant. The ABC transporter BceAB not only acts as a bacitracin detoxification pump, but is also crucial for bacitracin sensing, indicative of a novel mechanism of stimulus perception, conserved in Firmicutes bacteria. The Bce system seems to respond to bacitracin directly (drug sensing), whereas the LiaRS TCS and sigma(M) respond only at higher concentrations and indirectly to bacitracin action (damage sensing). The YvcPQ-YvcRS system is subject to cross-activation via the paralogous Bce system, and is therefore only indirectly induced by bacitracin. The bacitracin stress response network is optimized to respond to antibiotic gradients in a way that maximizes the gain and minimizes the costs of this stress response.

Metalloantibiotic Mn(II)-bacitracin complex mimicking manganese superoxide dismutase.

Superoxide dismutase (SOD) activities of various metallobacitracin complexes were evaluated using the riboflavin-methionine-nitro blue tetrazolium assay. The radical scavenging activity of various metallobacitracin complexes was shown to be higher than those of the negative controls, e.g., free transition metal ions and metal-free bacitracin. The SOD activity of the complex was found to be in the order of Mn(II)>Cu(II)>Co(II)>Ni(II). Furthermore, the effect of bacitracin and their complexation to metals on various microorganisms was assessed by antibiotic susceptibility testing. Moreover, molecular modeling and quantum chemical calculation of the metallobacitracin complex was performed to evaluate the correlation of electrostatic charge of transition metal ions on the SOD activity.

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.

1H NMR study on the conformation of bacitracin A in aqueous solution.

The conformation of bacitracin A, a widely used cyclic dodecapeptide antibiotic in aqueous solution, has been investigated using 500 MHz 1H NMR and molecular modeling. Findings revealed that a region (residues 1-6) is folded over the cyclic ring, resulting in metal coordination sites, a thiazoline ring, and Glu4 and His10 being proximate to each other.

DNA base damage by beta-lactam, tetracycline, bacitracin and rifamycin antibacterial antibiotics.

Several antibacterial antibiotics have been shown to participate with transition metal ions in chemical reactions leading to the formation of reactive oxygen species. An important host defence mechanism for dealing with invading bacteria involves the production of reactive oxygen species, such as superoxide, hydrogen peroxide and hypochlorous acid, by phagocytic cells. The production of reactive oxygens by redox cycling antibacterial antibiotics has led us to suggest that a 'phagomimetic' contribution may also be made in vivo. Here we show that four structurally different antibacterial antibiotics, in the presence of added copper salt, bring about oxidative modification to bases in DNA detected using gas chromatography-mass spectrometry. The drug most damaging to DNA was rifamycin SV which was more active than a reference mixture of hydrogen peroxide and ascorbic acid.

X-Ray structure of the antibiotic bacitracin A.

Bacitracins are a group of widely used peptide antibiotics. There has been interest in determining the three-dimensional structure of the bacitracins. However, solution studies indicate significant flexibility in their structure and to date native bacitracins have resisted attempts at crystallisation despite considerable efforts over a number of years by several groups. Here we report the first three-dimensional X-ray structure of a bacitracin, complexed to a subtilisin proteinase. X-Ray diffraction data were collected using synchrotron radiation in combination with the Image Plate Scanner system. The complex structure including two enzymes, two bacitracins, 220 water molecules and two Ca2+ ions was refined by restrained least-squares to a crystallographic R factor ( = sigma [[Fo-Fc]]/sigma [Fo]]) of 16.3% at 2.0 A.

Conformational analysis of bacitracin A, a naturally occurring lariat.

The proton nmr spectra of bacitracin A in H2O and DMSO-d6 have been assigned and the conformational behavior of the peptide in the two solvents has been compared. Although bacitracin A shows a conformational equilibrium between at least two conformations differing in the relative position of the cyclic and linear domains of the molecule, the spectra in water can be interpreted in terms of a preferred conformation in which the linear part is folded over the cyclic moiety and a turn is present around Ile(8)-DPhe(9).

Isolation of bacitracins A and F by high-speed counter-current chromatography.

The major components of bacitracin were separated and purified using high-speed counter-current chromatography (HSCCC). A systematic search for optimum two-phase solvent systems resulted in two systems: chloroform-ethanol-methanol-water (5:3:3:4) and chloroform-ethanol-water (5:4:3). These were selected based on the determination of the partition coefficients of all the components and the settling time of the phases. HSCCC with these solvent systems separated two components, bacitracins A and F. Improvements in the flow-cell arrangement eliminated noise in detection, making in-line monitoring possible. A tandem mass spectrometric technique was used to characterize the isolated components.

Reduced bacterial adherence to silicone plastic neurosurgical prosthesis.

Bacteria have been shown to adhere to smooth surfaces, such as shunts, by secreting a complex polysaccharide coat called the glycocalyx. We assume that if bacterial adherence could be reduced to zero, foreign-body-related infections would be essentially eliminated. This study describes a new technique for quantitating bacterial adherence to plastic using radioactive chromium, and demonstrates that presoaking the silicone plastic surgical tubing used for ventriculoperitoneal and ventriculoatrial shunts in bacitracin A solution (50,000 units in 250 ml) reduces the adherence of Staphylococcus epidermidis by 54%. We conclude that pretreatment of a hydrocephalic shunt tubing with an aqueous bacitracin solution before its implantation may help to reduce the postoperative shunt infections due to direct contamination of the shunt at the time it is inserted.

[Sporulation in Bacillus licheniformis during altered bacitracin synthesis]. / Sporoobrazovanie u Bacillus licheniformis pri izmenenii sinteza batsitratsina.

Sporulation in different strains of Bacillus licheniformis, 10716 and 1001 in connection with changes in synthesis of bacitracin was studied. It was shown that the sporulation efficiency did not depend on the synthesis of the antibiotic: in some strains with low potency for the antibiotic production, the sporulation level was lowered, while in the others, it was not lowered. Moreover, normal sporulation was also observed, when the synthesis of bacitracin was inhibited. Therefore, it is suggested that there is no correlation between the sporulation and antibiotic production.

Studies on the complex formed between bacitracin A and divalent cations.

Bacitracin A is a peptide antibiotic which forms stoichiometric complexes with divalent cations, including Ni2+ and Zn2+. In this paper it is shown that the metal-bacitracin complex contains a group which has a pKa near pH 5.5. Deprotonation of the group is concomitant with the aggregation and precipitation of the metal-bacitracin complex. Bacitracin A, in the absence of metals, does not contain any group which has a pKa in this range. It is postulated that this group is the N-terminal amino of isoleucine, which was previously postulated not to be directly involved in metal coordination based on proton release measurements. An attempt was made to demonstrate directly that the N-terminal amino group is not coordinated to the metal by examining the reactivity of this group with 2,4,6-trinitrobenzene sulfonate. It was clearly shown that bound metals protect the N-terminal amino group from reacting with this reagent. It is speculated that this metal-protection results from a combination of factors, including steric hindrance.

[Physico-chemical properties of the complexes of bacitracin with DNA and its effect on the process of transcription in vitro]. / Fiziko-khimicheskie svoistva kompleksov batsitratsina s DNK i vliianie ego na protsess transkriptsii in vitro.

The aim of the present paper was to study the action of one of the peptide antibiotics, bacitracin, as the regulator of gene activity at the transcription level. Therefore the commercial bacitracin has been fractionated into two main parts by paper chromotography. These two fractions have been identified as bacitracin A (biologically active) and bacitracin F (biologically inactive). The binding of both fractions to DNA has been studied. It has been shown that bacitracin A stabilizes DNA to a lesser degree than bacitracin F does. DNA-bacitracin complexes are formed in the major groove of the DNA helix by hydrogen bonds. The analysis of the the obtained experimental data allows us to suppose that bacitracin binding to DNA has a very specific character and that this antibiotic may act as the regulator of gene activity.

Binding of nickel and zinc ions to bacitracin A.

Bacitracin A is a cyclic dodecapeptide antibiotic produced by Bacillus licheniformis. Bacteriocidal activity requires the presence of divalent cations such as Zn2+. The metal-bacitracin A complex binds to bactoprenyl pyrophosphate, a lipid intermediate required for cell wall biosynsthesis which is found within the bacterial membrane. In this paper, the pH dependence of the metal binding to bacitracin A is investigated in an effort to define the sites of metal coordination. Most of the studies described in this report were performed with Ni2+ and Zn2+. Metal binding was monitored by observing changes in the ultraviolet absorption spectrum of bacitracin A and by monitoring the proton release which is concomitant with metal binding to the peptide. It was determined that both Ni2+ and Zn2+ form 1:1 complexes with bacitracin A in solution. These complexes are soluble in acidic solutions, but above approximately pH 5.5 they become insoluble. On the basis of the data reported as well as results previously reported from other laboratories, a model for divalent metal ion binding to bacitracin is suggested. It is proposed that the metal coordinates directly to the glutamate carboxyl, the histidine imidazole, and the thiazoline ring. The aspartate carboxyl and N-terminal amino group are not directly involved in metal binding. It is further proposed that due to the proximity of the metab, the pK of the N-terminal amino is shifted from 7.7 to 5.7 upon metal binding. Deprotonation of this group is suggested to cause precipitation of the bacitracin A-metal complex. This model is consistent with all the metal binding data and, furthermore, is consistent with the 1H NMR data presented in the accompanying paper [Mosberg, H. I., Scogin, D. A., Storm, D. R., & Gennis, R. B. (1980) Biochemistry (following paper in this issue)].