The ATP-binding cassette transporter ChoVWX is one of several choline import systems operating in Sinorhizobium meliloti. Here fluorescence-based ligand binding assays were used to quantitate substrate binding by the periplasmic ligandbinding protein ChoX. These data confirmed that ChoX recognizes choline and acetylcholine with high and medium affinity, respectively. We also report the crystal structures of ChoX in complex with either choline or acetylcholine. These structural investigations revealed an architecture of the ChoX binding pocket and mode of substrate binding similar to that reported previously for several compatible solute-binding proteins. Additionally the ChoX-acetylcholine complex permitted a detailed structural comparison with the carbamylcholinebinding site of the acetylcholine-binding protein from the mollusc Lymnaea stagnalis. In addition to the two liganded structures of ChoX, we were also able to solve the crystal structure of ChoX in a closed, substrate-free conformation that revealed an architecture of the ligand-binding site that is superimposable to the closed, ligand-bound form of ChoX. This structure is only the second of its kind and raises the important question of how ATP-binding cassette transporters are capable of distinguishing liganded and unligandedclosed states of the binding protein.

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3 end of a tRNA Ser gene. Phage mv4 DNA integration into the tRNA Ser gene preserved an intact tRNA Ser gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA Ser gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present.Gram-positive Lactobacillus strains are extensively used as preservatives in food products and as lactic acid producers in dairy fermentations, but development of bacteriophages is the main cause of fermentation failures. Taxonomic studies on phages from Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis led to the determination of two genetic groups (21, 32). The phage representative of the most widespread group, bacteriophage mv4, has been well characterized (13). Phage mv4 is a temperate phage which infects and lysogenizes L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains. The 36-kb genome of the phage has been mapped physically, and its DNA is circularly permuted (20). Several genes have been characterized, such as those encoding structural proteins (52) or genes involved in cell lysis (6). The phage attachment site (attP) has previously been located on the mv4 genome, and several attachment sites on the chromosome of independently isolated lysogens have been identified (20).Most temperate bacteriophages integrate their DNA into the host chromosome by a site-specific recombination process following the Campbell model (10). This mechanism involves two specific attachment sites, one on the bacterial chromosome (attB) and the other one on the phage genome (attP). The recombination process is catalyzed by a phage-encoded integrase. There are many well-characterized examples of sitespecific recombination in gram-negative bacteriophages, and the best-studied system is that of bacteriophage (for a review, see reference 22). The integration system of phages of grampositive bacteria is less well documented, but data on sitespecific recombination are available for phages L54a, 11, and 13 of Staphylococcus aureus (14,25,55,56), for mycobacteriophage L5 (26), and for phages of...

In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant. Moreover, choline is an efficient energy source which supports growth. Using a PCR strategy, we identified three chromosomal genes (choXWV) which encode components of an ABC transporter: ChoX (binding protein), ChoW (permease), and ChoV (ATPase). Whereas the best homology scores were obtained with components of betaine ProU-like systems, Cho is not involved in betaine transport. Site-directed mutagenesis of choX strongly reduced (60 to 75%) the choline uptake activity, and purification of ChoX, together with analysis of the ligand-binding specificity, showed that ChoX binds choline with a high affinity (K D , 2.7 M) and acetylcholine with a low affinity (K D , 145 M) but binds none of the betaines. Uptake competition experiments also revealed that ectoine, various betaines, and choline derivatives were not effective competitors for Cho-mediated choline transport. Thus, Cho is a highly specific high-affinity choline transporter. Choline transport activity and ChoX expression were induced by choline but not by salt stress. Western blotting experiments with antibodies raised against ChoX demonstrated the presence of ChoX in bacteroids isolated from nitrogen-fixing nodules obtained from Medicago sativa roots. The choX mutation did not have an effect on growth under standard conditions, and neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that the remaining choline uptake system(s) still present in the mutant strain can compensate for the lack of Cho transporter.Choline is a common constituent of eukaryotic membranes in the form of phosphatidylcholine (PC) and therefore should be widespread in different environments, including the soil and the rhizosphere. Indeed, significant amounts of choline are readily liberated into the environment from plant and animal residues (15). Sinorhizobium meliloti, a plant root-associated bacterium, possesses distinct transport activities for choline uptake (27) and has the ability to oxidize choline to glycine betaine via the bet operon (34,24). In contrast to Escherichia coli and Bacillus subtilis (25, 2), S. meliloti can use choline for growth. This depends on a functional bet locus (34, 24) associated with catabolism of glycine betaine which is absent in E. coli and B. subtilis. This catabolism is reduced under hyperosmotic conditions, and under these conditions glycine betaine accumulation is favored (34). Moreover, due to the presence of a PC synthase in S. meliloti, which directly condenses choline to CDP-diacylglyceride, choline is a direct precursor of PC, as recently demonstrated for other bacteria, including Agrobacterium, Brucella, and Pseudomonas (6,19). In addition to this PC synthase pathway, S. meliloti possesses a methylation pathway for PC biosynthesis which functions by threefold methylation of phosphatidylethanolamine with S-adenosylmethionine as a methy...