title: Characterization of the tre locus and analysis of trehalose cryoprotection in Lactobacillus acidophilus NCFM.
abstract: 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.
authors.affiliation: Genomic Sciences Graduate Program, North Carolina State University, Raleigh, North Carolina 27695, USA.
authors.forename: Tri
authors.initials: T
authors.lastname: Duong
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Barrangou
authors.forename: W Michael
authors.initials: WM
authors.lastname: Russell
authors.forename: Todd R
authors.initials: TR
authors.lastname: Klaenhammer
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003451:Cryoprotective Agents / Q000378:metabolism / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D004186:Disaccharidases / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005798:Genes, Bacterial; D007779:Lactobacillus acidophilus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014199:Trehalose / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D003451:Cryoprotective Agents; D004269:DNA, Bacterial; D012097:Repressor Proteins; C094584:trehalose repressor protein, bacteria; D014199:Trehalose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C094583:phosphoenolpyruvate-trehalose phosphotransferase, bacteria; D004186:Disaccharidases; C040631:alpha, alpha-phosphotrehalase