The osmoprotectant glycine betaine inhibits salt-induced cross-tolerance towards lethal treatment in Enterococcus faecalis

The response of Enterococcus faecalis ATCC 19433 to salt stress has been ch aracterized previously in complex media. In this report, it has been demons trated that this bacterium actively accumulates the osmoprotectant glycine betaine (CB) from salt-enriched complex medium BHI. To further understand t he specific effects of CB and other osmoprotective compounds in salt adapta tion and salt-induced cross-tolerance to lethal challenges, a chemically de fined medium lacking putative osmoprotectants was used. In this medium, bac terial growth was significantly reduced by increasing concentrations of NaC l. At 0.75 M NaCl, 90% inhibition of the growth rate was observed; GB and i ts structural analogues restored growth to the non-salt-stressed level. In contrast, proline, pipecolate and ectoine did not allow growth recovery of stressed cells. Kinetic studies showed that the uptake of betaines shows st rong structural specificity and occurs through a salt-stress-inducible high -affinity porter [K-m = 3.3 mu M; V-max = 130 nmol min(-1) (mg protein)(-1) ; the uptake activity increased 400-fold in the presence of 0.5 M NaCl]. Mo reover, CB and its analogues were accumulated as non-metabolizable cytosoli c osmolytes and reached intracellular levels ranging from 1.3 to 1.5 mu mol (mg protein)(-1). In contrast to the beneficial effect of CB on the growth of salt-stressed cultures of E. faecalis, its accumulation inhibits the sa lt-induced cross-tolerance to a heterologous lethal challenge. Indeed, pret reatment of bacterial cells with 0.5 M NaCl induced resistance to 0.3 % bil e salts (survival of adapted cells increased by a factor of 6800). The pres ence of GB in the adaptation medium reduced the acquisition of bile salts r esistance 680-fold. The synthesis of 11 of the 13 proteins induced during s alt adaptation was significantly reduced in the presence of GB, These resul ts raise questions about the actual beneficial effect of CB in natural envi ronments where bacteria are often subjected to various stresses.