RESPONSE OF BACILLUS-SUBTILIS TO HIGH OSMOLARITY - UPTAKE OF CARNITINE, CROTONOBETAINE AND GAMMA-BUTYROBETAINE VIA THE ABC TRANSPORT-SYSTEMOPUC

It was found that low concentrations of the naturally occurring and st ructurally related betaines L-carnitine, crotonobetaine and gamma-buty robetaine conferred a high degree of osmotic tolerance to Bacillus sub tilis. Kinetic analysis of L-[N-methyl-C-14]carnitine uptake in cells grown in minimal medium revealed the presence of a high-affinity trans port system with a K-m value of 5 mu M and a maximum rate of transport (V-max) of 41 nmol min(-1) (mg protein)(-1) A rise in medium osmolari ty moderately increased the maximum velocity [V-max 71 nmol min(-1) (m g protein)(-1)] of this transport system, but had little effect on its affinity. Growth and transport studies with a set of strains that car ried defined mutations in the previously identified glycine betaine tr ansport systems OpuA, OpuC and OpuD allowed the identification of the ATP-binding cassette (ABC) transport system OpuC as the only uptake ro ute for L-carnitine in B. subtilis. Competition experiments with croto nobetaine and gamma-butyrobetaine revealed that the OpuC system also e xhibited a high affinity for these trimethylammonium compounds with K- i values of 6.4 mu M. Tracer experiments with radiolabelled L-carnitin e and C-13-NMR tracings of cell extracts demonstrated that these betai nes are accumulated by B. subtilis in an unmodified form. In contrast, the beta-substituted acylcarnitine esters acetylcarnitine and octanoy lcarnitine both functioned as osmoprotectants for B. subtilis but were found to be accumulated as carnitine by the cells. None of these trim ethylammonium compounds were used as sole carbon or nitrogen sources. The results thus characterize L-carnitine, crotonobetaine and gamma-bu tyrobetaine as effective compatible solutes for B. subtilis and establ ish a crucial role of the ABC transport system OpuC for the supply of B. subtilis with a variety of osmoprotectants.