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Applied and Environmental Microbiology, February 2006, p. 1218-1225, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1218-1225.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of the tre Locus and Analysis of Trehalose Cryoprotection in Lactobacillus acidophilus NCFM

Tri Duong,1,2 Rodolphe Barrangou,1,2 W. Michael Russell,3 and Todd R. Klaenhammer2*

Genomic Sciences Graduate Program, North Carolina State University, Raleigh, North Carolina 27695,1 Department of Food Science, North Carolina State University, Raleigh, North Carolina 27695,2 Danisco, Inc., Madison, Wisconsin 537183

Received 14 November 2005/ Accepted 22 November 2005

Freezing and lyophilization are common methods used for preservation and storage of microorganisms during the production of concentrated starter cultures destined for industrial fermentations or product formulations. The compatible solute trehalose has been widely reported to protect bacterial, yeast and animal cells against a variety of environmental stresses, particularly freezing and dehydration. Analysis of the Lactobacillus acidophilus NCFM genome revealed a putative trehalose utilization locus consisting of a transcriptional regulator, treR; a trehalose phosphoenolpyruvate transferase system (PTS) transporter, treB; and a trehalose-6-phosphate hydrolase, treC. The objective of this study was to characterize the tre locus in L. acidophilus and determine whether or not intracellular uptake of trehalose contributes to cryoprotection. Cells subjected to repeated freezing and thawing cycles were monitored for survival in the presence of various concentrations of trehalose. At 20% trehalose a 2-log increase in survival was observed. The trehalose PTS transporter and trehalose hydrolase were disrupted by targeted plasmid insertions. The resulting mutants were unable to grow on trehalose, indicating that both trehalose transport into the cell via a PTS and hydrolysis via a trehalose-6-phosphate hydrolase were necessary for trehalose fermentation. Trehalose uptake was found to be significantly reduced in the transporter mutant but unaffected in the hydrolase mutant. Additionally, the cryoprotective effect of trehalose was reduced in these mutants, suggesting that intracellular transport and hydrolysis contribute significantly to cryoprotection.

* Corresponding author. Mailing address: Department of Food Science, Box 7624, North Carolina State University, Raleigh, NC 27695. Phone: (919) 515-2972. Fax: (919) 513-0014. E-mail: klaenhammer{at}ncsu.edu.

Applied and Environmental Microbiology, February 2006, p. 1218-1225, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1218-1225.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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