Osmoprotection of Escherichia coli by peptone is mediated by the uptake and accumulation of free proline but not of proline-containing peptides

Abstract

The effect of meat peptone type I (Sigma) on the growth of Escherichia coli cells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth of E. coli cells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast to Listeria monocytogenes (M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41-49, 1995), E. coli does not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures of E. coli growing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline by E. coli, and the inability of E. coli to utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism.

FIG. 1

Effect of peptone on the growth and the intracellular amino acid pools of E. coli NCIMB 10214 at high osmolarity. (a) Comparison of the growth stimulation by peptone and glycine betaine in the following: 0.5 M NaCl (○); 0.5 M NaCl plus 0.5% (wt/vol) peptone (▴); 0.5 M NaCl plus 1 mM glycine betaine (●); and 0.5 M NaCl plus 0.5% (wt/vol) peptone plus 1 mM glycine betaine (■). Cells were grown to exponential phase in McIlvaine’s medium exactly as described in Materials and Methods. Samples for amino acid analysis of high osmolarity cultures in the presence (A) and absence (A′) of peptone were taken in mid-log phase of growth. In addition, samples for amino acid analysis in the presence of peptone were taken at points B and C. (b) Main intracellular pools of amino acids at high osmolarity in the absence and presence of peptone (0.5%, wt/vol). Samples were taken at points indicated in panel a, and the intracellular pools of amino acids were determined exactly as described in Materials and Methods. Levels for pools of free glutamate (E), glutamine (Q), and proline (P) are indicated.

FIG. 2

Proline becomes limiting to the growth of E. coli NCIMB 10214 at high osmolarity in the presence of peptone. (a) Growth stimulation by peptone and proline in the following: 0.5 M NaCl (○); 0.5 M NaCl plus 0.5% (wt/vol) peptone (▴); 0.5 M NaCl plus 1 mM proline (●); and 0.5 M NaCl plus 0.5% peptone plus 1 mM proline (■). (b) Extracellular free proline measured in cultures growing in the presence of peptone. Samples were taken when the culture reached the OD₆₅₀s indicated, and amino acid analyses were performed exactly as described in Materials and Methods. The asterisk represents undetectable levels. (c) Composite figure of the intracellular (filled bars) and extracellular (hatched bars) concentrations of free proline in cultures growing in the presence of peptone, at the OD₆₅₀s indicated.

FIG. 3

Effect of addition of peptone (0.5%, wt/vol) at high osmolarity on the growth of an E. coli strain deficient in the three proline permeases (EF063). Cells were grown to exponential phase in McIlvaine’s medium exactly as described in Materials and Methods. (a) EF063, 0.5 M NaCl (○); 0.5 M NaCl plus 0.5% (wt/vol) peptone (▴); and 0.5 M NaCl plus 1 mM proline (●). (b) MK11 (isogenic strain with ProP activity). Symbols are the same as in panel a.