Characterization of a Sinorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible sol utes glycine betaine and proline betaine for both protection against osmoti c stress and, at low osmolarities, as an energy source. A PCR strategy base d on conserved domains in components of the glycine betaine uptake systems from Escherichia coil (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding prote in-dependent transporter in S. meliloti, This system was encoded by three g enes (hutXWV) of an operon which also contained a fourth gene (hutH2) encod ing a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the pe riplasmic binding protein (hutX) and of the gene encoding the cytoplasmic A TPase (hutV) was done to study the substrate specificity of this transporte r and its contribution in betaine uptake, These mutants showed a 50% reduct ion in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histi dine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2), Expression analysis of the hut operon det ermined using a hutX-lacZ fusion revealed induction by histidine, but not b y salt stress, suggesting this uptake system has a catabolic role rather th an being involved in osmoprotection. To our knowledge, Hut is the first cha racterized histidine ABC transporter also involved in proline and betaine u ptake.