Abstract
Brevibacterium linens can grow in media of relatively high osmotic strength (up to 3 M-NaCl). Optimal growth in minimal medium occurred with either added NaCl (0.5-1 M) or other osmolytes developing an equivalent osmotic pressure. Above 1 M-NaCl the growth rate slowed, but was enhanced by adding the osmoprotectant glycine betaine or its precursor choline (1 mM) to the media. Neither soluble carbohydrates nor ninhydrin-reacting compounds accumulated during osmotic treatment. However, a Dragendorff-positive compound, together with K+ ions, accumulated (7-fold) as a consequence of elevating the medium osmolality. Spectral characteristics of the purified compound were similar to those of synthesized 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine). In the absence of exogenously supplied osmoprotectants, a strict dependence between the intracellular ectoine content and the external salt concentration (up to 1 M-NaCl) was observed. From 1 to 2 M-NaCl, the ectoine content decreased, and this signalled decreased self-osmoprotection by this bacterium. Interestingly, at these salt concentrations, exogenous glycine betaine led to the greatest beneficial effect. Glycine betaine or choline added to the medium was actively taken up by the cells in an osmolality-dependent manner, and the resulting betaine accumulation caused a sharp decrease in intracellular ectoine content. Radiolabelling of ectoine occurred only when L-[U-14C]glutamate was used as a precursor; de novo synthesis was dependent on the external osmolality and was strongly inhibited by exogenously supplied glycine betaine. These results imply that ectoine may play a major role in counteracting the effects of osmotic stress in media of osmotic pressure equivalent to ⋜ 1 M-NaCl.