Bacitracin is a peptide antibiotic nonribosomally produced by Bacillus licheniformis. The bcrABC genes which confer bacitracin resistance to the bacitracin producer encode ATP binding cassette (ABC) transporter proteins, which are hypothesized to pump out bacitracin from the cells. Bacillus subtilis 168, which has no bacitracin synthesizing operon, has several genes homologous to bcrABC. It was found that the disruption of Bacitracin is a dodecapeptide antibiotic produced by some strains of Bacillus licheniformis and Bacillus subtilis (2, 11). The synthesis is nonribosomally catalyzed by a multienzyme complex composed of three subunits, BacA, BacB, and BacC, whose genes have been cloned and sequenced (6,9,12,18,22). Bacitracin has potent antibiotic activity against gram-positive bacteria (30). The inhibition of peptidoglycan biosynthesis is the best-characterized bactericidal effect of bacitracin (27). It forms a complex mediated by a metal ion (Zn 2ϩ ) with the lipid C 55 -isoprenyl pyrophosphate (IPP) (24, 26), which is a carrier of a peptidoglycan unit or a disaccharide with pentapeptide across the membrane. Bacitracin, by binding to IPP, inhibits the conversion of IPP to C 55 -isoprenyl phosphate, which is catalyzed by a membrane associated pyrophosphatase (25).B. licheniformis, a bacitracin producer, has an ABC transporter system which is hypothesized to pump out bacitracin for self-protection (19). The transporter is composed of two membrane proteins, BcrB and BcrC, and two identical ATP-binding subunits, BcrA. Neumüller et al. recently reported that in B. licheniformis, bcrABC genes are localized about 3 kb downstream of the bacitracin biosynthetic operon bacABC (14). Between the bacABC operon and bcrABC genes, they also identified bacR and bacS genes which encode proteins with high homology to response regulator and sensory kinase of two-component regulatory systems and are involved in the regulation of bcrA expression.The B. subtilis genome project determined the entire DNA sequence of strain 168 and found that there are two operons which encode nonribosomal peptide antibiotic synthetase complexes (13). One is a surfactin synthetase operon (4), and the other is a plipastatin (fengycin) synthetase operon (29, 31). There is no bacitracin synthetase operon in B. subtilis 168. B. subtilis 168 is more sensitive to bacitracin than B. licheniformis, a bacitracin producer. Heterologous expression of bcrABC transporter in B. subtilis results in an increase of bacitracin resistance to a level similar to that observed in a bacitracin producer (5,19,20). A homology search reveals that in B. subtilis 168, there are several homologues of BcrA, -B, and -C of B. licheniformis. In this study we showed that the disruption of the ywoA gene, which encodes the BcrC homologue, resulted in a marked decrease of resistance to bacitracin. We also reported that the transcription of the ywoA gene was dependent on extracytoplasmic function (ECF) factors, M and X . MATERIALS AND METHODSBacterial strains and plasmids. B. subtilis 168...

Naturally occurring furan fatty acids were synthesized and their antioxidant activity has been studied during the oxidation of linoleic acid in the phosphate buffer, pH 6.9, in the dark. The extent of the oxidation was followed both by the accumulation of conj ugated diene and by the measurement of the residual amounts of linoleic acid. The tetra-alkylsubstituted furan fatty acids were found to suppress the oxidation. The trialkylsubstituted compound also showed antioxidant activity, being about 50% as effective as the tetraalkylsubstituted ones. The di-alkylsubstituted one revealed no significant activity. The antioxidant activity of furan fatty acids depended on the number of substituents on the furan ring. Therefore, a tetraalkylsubstituted furan ring may be necessary for the antioxidant action of furan fatty acids. The tetraalkylsubstituted furan fatty acids reduced 1,1diphenyl-2-picrylhydrazyl, reacted with the peroxyl radical generated from the thermal decomposition of a radical initiator, 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH), and also suppressed the AAPHinduced oxidation of linoleic acid, indicating that, by scavenging, the peroxyl radical furan fatty acids inhibit the oxidation. KEY WORDS: Antioxidant, furan fatty acid, linoleic acid, peroxyl radical.

We have undertaken a detailed study of the antioxidant activity of allicin, one of the main thiosulfinates in garlic, in order to obtain quantitative information on it as a chain-breaking antioxidant. The antioxidant actions of allicin against the oxidation of cumene and methyl linoleate (ML) in chlorobenzene were studied in detail using HPLC. The hydroperoxides formed during the course of the inhibited oxidation of ML were analyzed as their corresponding alcohols by HPLC, and it is apparent that an allylic hydrogen atom of the allicin is responsible for the antioxidant activity. Furthermore, it is clear that the radical-scavenging reactions of allicin proceed via a one-step hydrogen atom transfer based on the results of the reaction with 2,2-diphenyl-1-picrylhydrazyl (DPPH) in the presence of Mg2+ and calculation of the ionization potential value. In addition, we determined the stoichiometric factor (n), the number of peroxyl radicals trapped by one antioxidant molecule, of allicin by measuring the reactivity toward DPPH in chlorobenzene, and the value of n for allicin was about 1.0. Therefore, we measured the rate constants, k(inh), for the reaction of allicin with peroxyl radicals during the induction period of the cumene and the ML oxidation. As a result, we found that allicin reacts with peroxyl radicals derived from cumene and ML with the rate constants k(inh) = 2.6 x 10(3) M(-1)s(-1) and 1.6 x 10(5) M(-1)s(-1) in chlorobenzene, respectively. Our results demonstrate for the first time reliable quantitative kinetic data and the antioxidative mechanism of allicin as an antioxidant.

Garlic extract significantly inhibited the oxidation of methyl linoleate in homogeneous acetonitrile solution, whereas the antioxidant effect of allicin-free garlic extract, prepared by removing allicin by prepared by removing allicin by preparative HPLC, was much lower than that of the garlic extract. These results suggest that the antioxidant properties are mostly attributed to the presence of allicin in the garlic extract. Allicin a major component of the thiosulfinates in garlic extract, was found to be effective for inhibiting methyl linoleate oxidation, but its efficiency was less than that of alpha-tocopherol. Next, the reactivity of allicin toward the peroxyl radical, which is a chain-propagating species, was investigated by direct ESR detection. The addition allicin to 2,2'-azobis(2,4-dimethylvaleronitrile)-peroxyl radical solution caused the signal intensity of the peroxyl radical to dose-dependently decrease, indicating that allicin is capable of scavenging the the peroxyl radical and acting as an antioxidant. Finally, we studied the structure-anioxidant activity relationship for thiosulfinates and suggested that the combination of the allyl group (-CH2CH=CH2) and the -S(O)S- group is necessary for the antioxidant action of thiosulfinates in the garlic extract. In addition, one of the two possible combinations, -S(O)S-CH2CH=CH2, was found to make a much larger contribution to the antioxidant activity of the thiosulfinates than the other, CH2=CH-CH2-S(O)S-.

As a part of our work on the antioxidant properties of naturally occurring furan fatty acids (F acids), we evaluated their hydroxyl radical (HO.) scavenging activity by an electron spin resonance (ESR) spin trapping technique with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The additions of F acids to the incubation mixture of Fe2(+)-diethylenetriaminepentaacetic acid complex, H2O2 and DMPO decreased the intensity of the DMPO-OH adduct signal in a dose-dependent way. This decrease was not attributed to the destruction of DMPO-OH adduct by F acids. Kinetic competition studies indicated that the decrease in DMPO-OH signal intensity was mainly due to the competition of F acids with DMPO for HO., and not to the inhibition of the HO. generation system itself. F acids were found to react rapidly with HO. at approximately a diffusion-controlled rate (1.7 x 10(10)M-1 s-1). Comparison with the common HO. scavengers indicated that the rate constant of F acids is higher than those of mannitol and ethanol, and is compatible with those of histidine and dimethylsulfoxide, demonstrating that F acids are a potent HO. scavenger. It is suggested that F acids may serve as antioxidants in biological systems through their ability to scavenge HO..