Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60°C. Glycinecin A is a heterodimer consisting of 39-and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes, glyA and glyB, encoding the 39-and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences of glyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with either glyA or glyB separately did not show the bacteriocin activity.Bacteriocins are bactericidal compounds, usually proteinaceous, whose activities are often restricted to bacterial strains that are closely related to the producing bacterium (6, 23). Bacteriocins are produced in all major groups of Eubacteria and Archaebacteria (22). Some bacteriocins, the halocins from Halobacteria, have no protein sequence homology to any known bacteriocins (20), whereas others, such as the S-type pyocins of Pseudomonas aeruginosa, some of the colicins of Escherichia coli, and a cloacin of Enterobacter cloacae, reveal protein sequence homologies (16,17,19).Despite their diversity, the bacteriocins share several characteristics (6, 23). They are generally high-molecular-weight proteins that gain entry into target cells by binding to cell surface receptors. Their bactericidal mechanisms vary and include pore formation, degradation of cellular DNA, disruption of specific cleavage of 16S rRNA, and inhibition of peptidoglycan synthesis.Many phytopathogenic bacteria, including members of the corynebacteria, erwinias, pseudomonads, and xanthomonads, produce proteinaceous bacteriocins (1, 2, 4, 5, 21, 24, 25). Since these bacteriocins are highly specific, can be produced at low cost, and are likely to be safe for both users and the environment, they appear to be excellent candidates for agricultural use in controlling plant pathogens. However, little is known about the chemical compositions, structures, modes of action, and genetics of most bacteriocins. Only a few isolated and/or purified bacteriocins have been reported from Agrobacterium radiobacter (18), Corynebacterium ulcerans (1), Erwinia carotovora subsp. carotovora (4), and Pseudomonas syringae pv. syringae (21).Thus far, bacteriocin production by xanthomonads has received little attention. Bacteriocin production by X...
Polymerization by microbial transglutaminase (mTGase) of disulfide bond-cleaved bovine serum albumin treated with 2-mercaptoethanol was investigated in an oil-in-water model emulsion system. Fluorescence and circular dichroism spectroscopy revealed that conformational changes in tertiary structure were more distinct as compared with the secondary structure. Reduced protein was polymerized more effectively by mTGase. Highest storage modulus was observed in the emulsion gel formed by both 2-ME and mTGase treatments. The phase angle was less than 45° in all samples with 2-ME and enzyme treatments, an indication that the conformational changes due to the reduction and polymerization contribute significantly to the formation of emulsion gels.
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