Response of Bacillus subtilis to high osmolarity: uptake of carnitine, crotonobetaine and γ-butyrobetaine via the ABC transport system OpuC

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Abstract
Summary: It was found that low concentrations of the naturally occurring and structurally related betaines L-carnitine, crotonobetaine and γ-butyrobetaine conferred a high degree of osmotic tolerance to Bacillus subtilis. Kinetic analysis of L-[N-methyl −14C]carnitine uptake in cells grown in minimal medium revealed the presence of a high-affinity transport system with a K m value of 5 μM and a maximum rate of transport (V max) of 41 nmol min−1 (mg protein)−1. A rise in medium osmolarity moderately increased the maximum velocity [V max 71 nmol min−1 (mg protein)−1] of this transport system, but had little effect on its affinity. Growth and transport studies with a set of strains that carried defined mutations in the previously identified glycine betaine transport systems OpuA, OpuC and OpuD allowed the identification of the ATP-binding cassette (ABC) transport system OpuC as the only uptake route for L-carnitine in B. subtilis. Competition experiments with crotonobetaine and γ-butyrobetaine revealed that the OpuC system also exhibited a high affinity for these trimethylammonium compounds with K i values of 6.4 μM. Tracer experiments with radiolabelled L-carnitine and 13C-NMR tracings of cell extracts demonstrated that these betaines are accumulated by B. subtilis in an unmodified form. In contrast, the β-substituted acylcarnitine esters acetylcarnitine and octanoylcarnitine both functioned as osmoprotectants for B. subtilis but were found to be accumulated as carnitine by the cells. None of these trimethylammonium compounds were used as sole carbon or nitrogen sources. The results thus characterize L-carnitine, crotonobetaine and γ-butyrobetaine as effective compatible solutes for B. subtilis and establish a crucial role of the ABC transport system OpuC for the supply of B. subtilis with a variety of osmoprotectants.