The crystal structure of biotin synthase from Escherichia coli in complex with S-adenosyl-Lmethionine and dethiobiotin has been determined to 3.4 angstrom resolution. This structure addresses how "AdoMet radical" or "radical SAM" enzymes use Fe 4 S 4 clusters and S-adenosyl-L-methionine to generate organic radicals. Biotin synthase catalyzes the radical-mediated insertion of sulfur into dethiobiotin to form biotin. The structure places the substrates between the Fe 4 S 4 cluster, essential for radical generation, and the Fe 2 S 2 cluster, postulated to be the source of sulfur, with both clusters in unprecedented coordination environments.Biotin synthase (BioB) catalyzes the final step in the biotin biosynthetic pathway, the conversion of dethiobiotin (DTB) to biotin. This remarkable reaction uses organic radical chemistry for the insertion of a sulfur atom between nonactivated carbons C6 and C9 of DTB (Scheme 1). BioB is a member of the "AdoMet radical" or "radical SAM" superfamily, which is characterized by the presence of a conserved CxxxCxxC sequence motif (C, Cys; x, any amino acid) that coordinates an essential Fe 4 S 4 cluster, as well as by the use of S-adenosyl-Lmethionine (AdoMet or SAM) for radical generation (1-3). AdoMet radical enzymes act on a wide variety of biomolecules. For example, BioB and lipoyl-acyl carrier protein synthase (LipA) are involved in vitamin biosynthesis; lysine 2,3-aminomutase (LAM) facilitates the fermentation of lysine; class III ribonucleotide reductase (RNR) activase and pyruvate formatelyase (PFL) activase catalyze the formation of glycyl radicals in their respective target proteins; and spore photoproduct lyase repairs ultraviolet light-induced DNA damage.AdoMet has been referred to as the "poor man's adenosylcobalamin" (4) because of the ability of both cofactors to generate a highly reactive 5′-deoxyadenosyl radical (5′-dA·), formed through homolytic cleavage of a C-Co bond in the case of adenosylcobalamin (AdoCbl) and through reductive cleavage of a C-S bond in the case of AdoMet (5). In AdoMet radical enzymes, the formation of 5′-dA· requires the addition of one electron, provided in E. coli by reduced flavodoxin and transferred first into an Fe 4 S 4 cluster and then into AdoMet (3). In the reaction catalyzed by BioB, there is general agreement that 5′-dA· generated from AdoMet oxidizes DTB (6), but the number and types of FeS clusters and of other cofactors involved in the reaction have been a subject of controversy (7)(8)(9)(10)(11)(12)(13)(14). Protein preparation-dependent cofactor differences have led to two mechanistic proposals for the method of S insertion in BioB. One proposal involves the use of an Fe 2 S 2 cluster as the sulfur source for biotin, and is consistent with 34 S isotopic labeling studies (15) and with the observed destruction of an Fe 2 S 2 cluster

Basil essential oils, including basil sweet linalool (BSL) and basil methyl chavicol (BMC), were screened for antimicrobial activity against a range of Gram‐positive and Gram‐negative bacteria, yeasts and moulds using an agar well diffusion method. Both essential oils showed antimicrobial activity against most of the micro‐organisms examined except Clostridium sporogenes, Flavimonas oryzihabitans, and three species of Pseudomonas. The minimum inhibitory concentration (MIC) of BMC against Aeromonas hydrophila and Pseudomonas fluorescens in TSYE broth (as determined using an indirect impedance method) was 0·125 and 2% (v/v), respectively; the former was not greatly affected by the increase of challenge inoculum from 103 to 106 cfu ml−1. Results with resting cells demonstrated that BMC was bactericidal to both Aer. hydrophila and Ps. fluorescens. The growth of Aer. hydrophila in filter‐sterilized lettuce extract was completely inhibited by 0·1% (v/v) BMC whereas that of Ps. fluorescens was not significantly affected by 1% (v/v) BMC. In addition, the effectiveness of washing fresh lettuce with 0·1 or 1% (v/v) BMC on survival of natural microbial flora was comparable with that effected by 125 ppm chlorine.

Essential oils extracted by hydrodistillation from five different varieties of Ocimum basilicum L. plants (Anise, Bush, Cinnamon, Dark Opal and a commercial sample of dried basil) were examined for antimicrobial activity against a wide range of foodborne Gram-positive and -negative bacteria, yeasts and moulds by an agar well diffusion method. All five essential oils of basil showed antimicrobial activity against most of the organisms tested with the exception of Flavimonas oryzihabitans and Pseudomonas species. The inhibitory effect of Anise oil, in comparison with mixtures of the predominant components of pure linalool and methyl chavicol, against the acid-tolerant organisms, Lactobacillus curvatus and Saccharomyces cerevisiae, was examined in broth by an indirect impedance method. Synergistic effects between Anise oil, low pH (pH 4.2) and salt (5% NaCl) were determined. The antimicrobial effect of Anise oil was also assessed in a tomato juice medium by direct viable count, showing that the growth of Lact. curvatus and S. cerevisiae was completely inhibited by 0.1% and 1% Anise oil, respectively. The results of the current study indicate the need for further investigations to understand the antimicrobial effects of basil oils in the presence of other food ingredients and preservation parameters.

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Aims:To characterize the effect of bovine lactoferrin and lactoferricin B against feline calicivirus (FCV), a norovirus surrogate and poliovirus (PV), as models for enteric viruses. Methods and Results: Crandell-Reese feline kidney (CRFK) cells were used for the propagation of FCV and monkey embryo kidney (MEK) cells for PV. The assays included visual assessment of cell lines for cytopathic effects and determination of the percentage cell death using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] dye reduction assay. Incubation of bovine lactoferrin with CRFK cells either prior to or together with FCV inoculation substantially reduced FCV infection. In contrast, the interference of lactoferrin with the infection of cells with PV was demonstrated only when lactoferrin was present with cell lines and virus for the entire assay period. Using indirect immunofluorescence, lactoferrin was detected on the surface of both CRFK and MEK cells, suggesting that the interference of viral infection may be attributed to lactoferrin binding to the surfaces of susceptible cells, thereby preventing the attachment of the virus particles. Lactoferricin B, a cationic antimicrobial peptide derived from the N-terminal domain of bovine lactoferrin, reduced FCV but not PV infection. Conclusion: Lactoferrin was shown to interfere with the infection of cells for both FCV and PV. However, lactoferricin B showed no interference of infection with PV and interference with infection for FCV required the presence of lactoferricin B together with the cell line and virus. Significance and Impact of the Study: An in vitro basis is provided for the effects of bovine lactoferrin and lactoferricin B in moderating food-borne infections of enteric viruses.

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