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.Freezing and freeze-drying are methods commonly used for preservation and storage of microorganisms and for production of concentrated starter cultures used in industrial fermentations. The sensitivity of microorganisms to cryogenic stress results in significant cell injury and death, ultimately reducing productivity or activity. The mechanisms by which freezing causes cell damage include efflux of water from the cell, resulting in precipitation of cytoplasmic solutes and components (25), mechanical stress to cellular components, and rupture of cell membranes due to ice crystal formation (26,36,37

Global analysis of carbohydrate utilization by Lactobacillus acidophilus using cDNA microarrays

“…In contrast, FOS and raffinose are transported by ABC transporters of the MsmEFGK family, La502-La505 and La1437-La1442, respectively. In the case of trehalose and FOS, microarray results correlate well with functional studies in which knock out of transporters and hydrolases modified the saccharolytic potential of L. acidophilus (9,21). Differential expression of the EIIABC Tre is consistent with recent work in L. acidophilus, indicating that La1012 is involved in trehalose uptake, because knock out of the trehalose PTS transporter resulted in a loss of the ability to grow on trehalose (21).…”

“…In the case of trehalose and FOS, microarray results correlate well with functional studies in which knock out of transporters and hydrolases modified the saccharolytic potential of L. acidophilus (9,21). Differential expression of the EIIABC Tre is consistent with recent work in L. acidophilus, indicating that La1012 is involved in trehalose uptake, because knock out of the trehalose PTS transporter resulted in a loss of the ability to grow on trehalose (21). Similarly, differential expression of the fos operon is consistent with previous work in L. acidophilus indicating that those genes are involved in uptake and catabolism of FOS, induced in the presence of FOS, and repressed in the presence of glucose (9).…”

Technology and potential applications of probiotic encapsulation in fermented milk products

“…Analysis of the L. acidophilus NCFM genome revealed that a putative trehalose utilization locus consisting of a transcriptional regulator (treR), a trehalose phosphoenolpyruvate transferase system transporter (tre-B) and a trehalose-6-phosphate hydrolase (treC). It was demonstrated that disruption of both the hydrolase genes and trehalose transporter abolished the ability of L. acidophilus NCFM to grow on trehalose and reduced the survival of this microorganism when subjected to repeated cycles of freezing and thawing in the presence of trehalose, demonstrating that not only is the internalization of trehalose important, but also its subsequent hydrolysis is an important contributing factor (Duong et al, 2006). It has been demonstrated that different bacterial species vary with respect to spray-drying tolerance, indicating the importance of strain selection.…”

Transcriptional and functional analysis of galactooligosaccharide uptake bylacSinLactobacillus acidophilus

“…The L. acidophilus NCFM genome was recently sequenced to reveal that the molecular machinery responsible for carbohydrate uptake and catabolism in NCFM accounts for 17% of the genes present in the genome (23). Broad carbohydrate utilization of L. acidophilus NCFM was demonstrated and included transporters for trehalose (24), fructooligosaccharides (25), and several other mono-, di-, and trisaccharides (26).…”