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Characterization of a Snorhizobium 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.

SUBMITTER: Boncompagni E

PROVIDER: S-EPMC94542 | biostudies-literature | 2000 Jul

REPOSITORIES: biostudies-literature

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

Boncompagni E E Dupont L L Mignot T T Osteräs M M Lambert A A Poggi M C MC Le Rudulier D D

Journal of bacteriology 20000701 13

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 syst ...[more]

PMID: 10850986

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Project description:Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.

Project description:By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently. The frcBCA genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (K(m) of 6 microM) fructose uptake system in S. meliloti. The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family. The expression of S. meliloti frcBCAK in Escherichia coli, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter. The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein. However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in S. meliloti. The expression of the frcBC genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose. Neither Nod nor Fix phenotypes were impared in the TnphoA mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is expressed in bacteroids isolated from alfalfa nodulated by S. meliloti wild-type strains.

Dataset Information

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

Publications

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

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