Plant-derived compatible solutes proline betaine and betonicine confer enhanced osmotic and temperature stress tolerance to Bacillus subtilis

Abstract

l-Proline is a widely used compatible solute and is employed by Bacillus subtilis, through both synthesis and uptake, as an osmostress protectant. Here, we assessed the stress-protective potential of the plant-derived l-proline derivatives N-methyl-l-proline, l-proline betaine (stachydrine), trans-4-l-hydroxproline and trans-4-hydroxy-l-proline betaine (betonicine) for cells challenged by high salinity or extremes in growth temperature. l-Proline betaine and betonicine conferred salt stress protection, but trans-4-l-hydroxyproline and N-methyl-l-proline was unable to do so. Except for l-proline, none of these compounds served as a nutrient for B. subtilis. l-Proline betaine was a considerably better osmostress protectant than betonicine, and its import strongly reduced the l-proline pool produced by B. subtilis under osmotic stress conditions, whereas a supply of betonicine affected the l-proline pool only modestly. Both compounds downregulated the transcription of the osmotically inducible opuA operon, albeit to different extents. Mutant studies revealed that l-proline betaine was taken up via the ATP-binding cassette transporters OpuA and OpuC, and the betaine-choline-carnitine-transporter-type carrier OpuD; betonicine was imported only through OpuA and OpuC. l-Proline betaine and betonicine also served as temperature stress protectants. A striking difference between these chemically closely related compounds was observed: l-proline betaine was an excellent cold stress protectant, but did not provide heat stress protection, whereas the reverse was true for betonicine. Both compounds were primarily imported in temperature-challenged cells via the high-capacity OpuA transporter. We developed an in silico model for the OpuAC–betonicine complex based on the crystal structure of the OpuAC solute receptor complexed with l-proline betaine.