99A-26 |
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T. DUONG1, R. Barrangou1, W. M. Russell2, E. Altermann1, R. J. Cano3, and T. R. Klaenhammer1. (1) Southeast Dairy Foods Research Ctr., North Carolina State Univ., Genomic Sciences Program, Box 7624, Raleigh, NC 27695-7624, (2) Rhodia, Inc., 2802 Walton Commons W., Madison, WI 53718-6785, (3) Environmental Biotechnology Institute, California Polytechnic State Univ., San Luis Obispo, 1 Grand Ave., San Luis Obispo, CA 93407 Lactobacillus acidophilus is a probiotic organism used as a health-promoting bacterium routinely delivered through dairy foods. Freezing and lyophilization are common methods used for preservation and storage of microorganisms during the production of concentrated starter cultures destined for industrial fermentations. The disaccharide trehalose has physical and chemical properties that have been reported to protect bacterial, yeast and animal cells against a variety of environmental stresses, including cryogenic stress. Trehalose has been previously shown to confer cryoprotection upon Lb. acidophilus. The objective of this study was to determine whether or not intracellular uptake of trehalose is essential to exert cryoprotective effects. Computational analysis of the Lb. acidophilus genome revealed a putative trehalose utilization and catabolism locus (tre) locus that encoded a transcriptional regulator of the lacI family, a trehalose specific Enzyme II typical of phosphotransferase systems (PTS), and a trehalose-6-phosphate hydrolase. Plasmid insertion via homologous recombination was used to knockout the trehalose PTS transporter. The resulting mutant was compared to the wild-type for its ability to survive cryogenic stress. The results showed that extracellular trehalose conferred cryoprotection in both the tre-mutant and parent. However, it was also demonstrated that intracellular uptake of trehalose significantly augments the cryoprotective effects of trehalose. We speculate that the mechanism of this enhanced cryoprotection is due to the intracellular accumulation of trehalose, which binds water molecules that might otherwise participate in ice crystal formation.
Session 99A, Biotechnology: General
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