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 characterized previously in complex media. In this report, it has been demonstrated that this bacterium actively accumulates the osmoprotectant glycine betaine (CB) from salt-enriched complex medium BHI. To further understand the specific effects of CB and other osmoprotective compounds in salt adaptation and salt-induced cross-tolerance to lethal challenges, a chemically defined medium lacking putative osmoprotectants was used. In this medium, bacterial growth was significantly reduced by increasing concentrations of NaCl. At 0.75 M NaCl, 90% inhibition of the growth rate was observed; GB and its 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 strong 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]. Moreover, CB and its analogues were accumulated as non-metabolizable cytosolic 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 salt-induced cross-tolerance to a heterologous lethal challenge. Indeed, pretreatment of bacterial cells with 0.5 M NaCl induced resistance to 0.3 % bile salts (survival of adapted cells increased by a factor of 6800). The presence of GB in the adaptation medium reduced the acquisition of bile salts resistance 680-fold. The synthesis of 11 of the 13 proteins induced during salt adaptation was significantly reduced in the presence of GB, These results raise questions about the actual beneficial effect of CB in natural environments where bacteria are often subjected to various stresses.