Glutamine, glutamate, and alpha-glucosylglycerate are the major osmotic solutes accumulated by Erwinia chrysanthemi strain 3937

Abstract

Erwinia chrysanthemi is a phytopathogenic soil enterobacterium closely related to Escherichia coli. Both species respond to hyperosmotic pressure and to external added osmoprotectants in a similar way. Unexpectedly, the pools of endogenous osmolytes show different compositions. Instead of the commonly accumulated glutamate and trehalose, E. chrysanthemi strain 3937 promotes the accumulation of glutamine and alpha-glucosylglycerate, which is a new osmolyte for enterobacteria, together with glutamine. The amounts of the three osmolytes increased with medium osmolarity and were reduced when betaine was provided in the growth medium. Both glutamine and glutamate showed a high rate of turnover, whereas glucosylglycerate stayed stable. In addition, the balance between the osmolytes depended on the osmolality of the medium. Glucosylglycerate and glutamate were the major intracellular compounds in low salt concentrations, whereas glutamine predominated at higher concentrations. Interestingly, the ammonium content of the medium also influenced the pool of osmolytes. During bacterial growth with 1 mM ammonium in stressing conditions, more glucosylglycerate accumulated by far than the other organic solutes. Glucosylglycerate synthesis has been described in some halophilic archaea and bacteria but not as a dominant osmolyte, and its role as an osmolyte in Erwinia chrysanthemi 3937 shows that nonhalophilic bacteria can also use ionic osmolytes.

FIG. 1.

¹³C nuclear magnetic resonance spectra of an ethanol extract of E. chrysanthemi 3937 grown in M63 medium with 0.5 M NaCl in the mid-exponential (A) and stationary (B) phases and ¹³C nuclear magnetic resonance spectrum control of chemically synthesized α-glucosylglycerate (C). The main peaks correspond to carbons of glutamate (e), glutamine (q), and glucosylglycerate (gg).

FIG. 2.

Infrared spectrum of the isolated glucosylglycerate along with its inverted second derivative (29) in the 1,400 to 900 cm⁻¹ frequency domain.

FIG. 3.

Evolution of osmolyte content during growth. Intracellular levels of glucosylglycerate (diamonds), glutamate (squares), and glutamine (triangles) during growth of E. chrysanthemi 3937 in 0.5 M NaCl-M63 medium with 15 mM (NH₄)₂SO₄. Growth (open circles) was monitored by optical density measurements at 570 nm.

FIG. 4.

Influence of medium osmolarity on the ratio of internal solutes. The effect of the NaCl concentration on the intracellular glucosylglycerate (GG, grey), glutamine (Gln, black), and glutamate (Glu, white) content of E. chrysanthemi 3937 grown to exponential phase in M63 medium was determined.

FIG. 5.

Glucosylglycerate accumulation is favored under NH₄ limitation. Intracellular levels of glucosylglycerate (diamonds), glutamate (squares), and glutamine (triangles) were determined during growth of E. chrysanthemi 3937 in M63 medium with 0.5 M NaCl and containing only 1 mM (NH₄)₂SO₄. Growth (open circles) was monitored by optical density measurements at 570 nm.

FIG. 6.

Suppression of endogenous solute accumulation. Intracellular levels of glucosylglycerate (diamonds), glutamate (squares), glutamine (triangles), and glycine betaine (closed circles) during growth of E. chrysanthemi 3937 in M63 medium with 0.5 M NaCl and 15 mM (NH₄)₂SO₄ was determined; 500 μM [methyl-¹⁴C]glycine betaine was added after 18 h of growth. Growth (open circles) was monitored by optical density measurements at 570 nm.