Characterization of a Sinorhizobium meliloti ATP-Binding Cassette Histidine Transporter Also Involved in Betaine and Proline Uptake

Abstract

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine 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 determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

Rhizobium leguminosarumHas a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family

“…A histidine transporter (HutXWV) of the quaternary amine uptake transporter family has been identified in S. meliloti (6). However, this permease is not present in all rhizobia as no apparent orthologue of hutXWV is present in the complete genome sequence of M. loti.…”

Proline Betaine Accumulation and Metabolism in Alfalfa Plants under Sodium Chloride Stress. Exploring Its Compartmentalization in Nodules

“…Pro betaine and Pro uptake by purified symbiosomes and free bacteroids was measured under aerobic conditions, at 25°C, according to the method described for S. meliloti (Boncompagni et al, 2000). All assays were carried out in 1 mL MES-Tris buffer (25 mM, pH 7.4) containing 3.0 kBq of [ 14 C]Pro betaine or [ 14 C]Pro, at final concentrations from 0.5 to 5 mM.…”

“…Pro betaine, which is released by alfalfa seeds during germination (Phillips et al, 1995), is an inducer of nodulation (nod) genes in Sinorhizobium meliloti, the alfalfa-symbiotic species (Phillips et al, 1992). Interestingly, S. meliloti transports this betaine by a high-affinity Na 1 -coupled transporter, BetS (Boscari et al, 2002), and via an ABC (ATP-binding cassette) protein-dependent transporter, Hut (Boncompagni et al, 2000). In addition, S. meliloti uses Pro betaine as a carbon and nitrogen source or as an osmoprotectant (Gloux and Le Rudulier, 1989;Goldmann et al, 1991;Burnet et al, 2000).…”

Characterization and Molecular Mechanism of AroP as an Aromatic Amino Acid and Histidine Transporter in Corynebacterium glutamicum

“…Histidine transport has been intensively studied in the Gramnegative bacteria Escherichia coli, Salmonella enterica serovar Typhimurium, and Sinorhizobium meliloti, which adopt similar ABC transporters for histidine uptake (9)(10)(11). The well-understood high-affinity ABC-type histidine transporter of E. coli and S. Typhimurium consists of four components: the histidine-binding protein HisJ, the two membrane-bound proteins HisQ and HisM, and the ATPase HisP (10).…”