The CTP:phosphocholine cytidylyltransferase encoded by the licC gene of Streptococcus pneumoniae: cloning, expression, purification, and characterization11The sequence described herein has been deposited in GenBank with accession number AF402777.

Campbell, Heidi A.; Kent, Claudia

doi:10.1016/s1388-1981(01)00174-3

Cited by 26 publications

(24 citation statements)

References 35 publications

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“…(ii) CDP-ethanolamine and CDP-choline are well known as key intermediates of mammalian pathways for the synthesis of phosphatidylethanolamine and phosphatidylcholine; although its operation in bacteria is far from general, there is at least one well-documented case of use of the CDP-choline pathway by Treponema denticola (23). (iii) CDP-choline is the putative precursor for the "decoration" of cell surface lipopolysaccharides or (lipo)teichoic acids with phosphocholine (9,37). Any of these CDP-alcohol-dependent processes could be affected by UshA enzymes with CDP-alcohol hydrolase activity, like UshA Ec and UshA Yi .…”

Section: Discussionmentioning

confidence: 99%

CDP-Alcohol Hydrolase, a Very Efficient Activity of the 5′-Nucleotidase/UDP-Sugar Hydrolase Encoded by the ushA Gene of Yersinia intermedia and Escherichia coli

et al. 2008

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Nucleoside 5-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDPalcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25°C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshA Yi ) of the Y. intermedia ushA gene (ushA Yi ) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli (UshA Ec ). Therefore, UshA Ec was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshA Ec and UshA Yi behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with k cat /K m ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshA Ec conformers and the respective homology models constructed for UshA Yi , and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis, which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations.

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Section: Discussionmentioning

confidence: 99%

CDP-Alcohol Hydrolase, a Very Efficient Activity of the 5′-Nucleotidase/UDP-Sugar Hydrolase Encoded by the ushA Gene of Yersinia intermedia and Escherichia coli

et al. 2008

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“…Depending on the pneumococcal strain, each repeat unit of TA contains one or two PCho residues (18,39). Following the identification of a genetic locus (lic) required for PCho metabolism in S. pneumoniae, it has been proposed that choline is transported into the cytoplasm by LicB (40), converted to PCho by LicA (38), and activated to CDP-choline by LicC (4,27). In addition, LicD2 is thought to mediate the incorporation of choline (presumably from CDP-choline) into lipoteichoic acid and perhaps also into TA (40).…”

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Mutations in thetacFGene of Clinical Strains and Laboratory Transformants ofStreptococcus pneumoniae:Impact on Choline Auxotrophy and Growth Rate

et al. 2008

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The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.

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“…The protein products of licB, licA, and licC catalyze cellular uptake (5) and conversion of intracellular choline to phosphorylcholine in an ATP-dependent reaction (28) followed by the conversion of phosphorylcholine in a CTP-dependent reaction to CDP-choline (2,14,17), which is assumed to be the substrate of an additional enzyme(s) that catalyzes the incorporation of P-choline residues (one or two, depending on the particular S. pneumoniae strain) into the teichoic acid chains. The genetic determinants involved appear to be part of a second operon (lic2) adjacent to lic1 on the pneumococcal chromosome (11,12,31).…”

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Different Pathways of Choline Metabolism in Two Choline-Independent Strains ofStreptococcus pneumoniaeand Their Impact on Virulence

et al. 2008

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The two recently characterized Streptococcus pneumoniae strains-R6Chi and R6Cho؊ -that have lost the unique auxotrophic requirement of this bacterial species for choline differ in their mechanisms of choline independence. In strain R6Chi the mechanism is caused by a point mutation in tacF, a gene that is part of the pneumococcal lic2 operon, which is essential for growth and survival of the bacteria. Cultures of lic2 mutants of the encapsulated strain D39Chi growing in choline-containing medium formed long chains, did not autolyze, had no choline in their cell wall, and were completely avirulent in the mouse intraperitoneal model. In contrast, while the Cho ؊ strain carried a complete pneumococcal lic2 operon and had no mutations in the tacF gene, deletion of the entire lic2 operon had no effect on the growth or phenotype of strain Cho ؊ . These observations suggest that the biochemical functions normally dependent on determinants of the pneumococcal lic2 operon may also be carried out in strain Cho ؊ by a second set of genetic elements imported from Streptococcus oralis, the choline-independent streptococcal strain that served as the DNA donor in the heterologous transformation event that produced strain R6Cho؊ . The identification in R6Cho ؊ of a large (20-kb) S. oralis DNA insert carrying both tacF and licD genes confirms this prediction and suggests that these heterologous elements may represent a "backup" system capable of catalyzing P-choline incorporation and export of teichoic acid chains under conditions in which the native lic2 operon is not functional.The cell wall and membrane teichoic acid of the bacterial pathogen Streptococcus pneumoniae is unusual in that it contains in its structure phosphorylcholine residues, which are involved with a wide variety of physiological and ecological functions (5, 6). S. pneumoniae-as a species-is also unique in its dependence on an exogenous source of choline for growth (15).Choline is taken up from the culture medium (or from the in vivo environment) and converted to CDP-choline by the sequential activity of proteins encoded by determinants organized into the lic1 operon (31). The protein products of licB, licA, and licC catalyze cellular uptake (5) and conversion of intracellular choline to phosphorylcholine in an ATP-dependent reaction (28) followed by the conversion of phosphorylcholine in a CTP-dependent reaction to CDP-choline (2, 14, 17), which is assumed to be the substrate of an additional enzyme(s) that catalyzes the incorporation of P-choline residues (one or two, depending on the particular S. pneumoniae strain) into the teichoic acid chains. The genetic determinants involved appear to be part of a second operon (lic2) adjacent to lic1 on the pneumococcal chromosome (11,12,31).While the unique auxotrophic requirement for choline can be fulfilled by other structurally different amino alcohols (24, 27), the normal physiological properties of the bacterium require the trimethylamino group of choline. S. pneumoniae strains growing in medium in which choline...

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The CTP:phosphocholine cytidylyltransferase encoded by the licC gene of Streptococcus pneumoniae: cloning, expression, purification, and characterization11The sequence described herein has been deposited in GenBank with accession number AF402777.

Cited by 26 publications

References 35 publications

CDP-Alcohol Hydrolase, a Very Efficient Activity of the 5′-Nucleotidase/UDP-Sugar Hydrolase Encoded by the ushA Gene of Yersinia intermedia and Escherichia coli

CDP-Alcohol Hydrolase, a Very Efficient Activity of the 5′-Nucleotidase/UDP-Sugar Hydrolase Encoded by the ushA Gene of Yersinia intermedia and Escherichia coli

Mutations in thetacFGene of Clinical Strains and Laboratory Transformants ofStreptococcus pneumoniae:Impact on Choline Auxotrophy and Growth Rate

Different Pathways of Choline Metabolism in Two Choline-Independent Strains ofStreptococcus pneumoniaeand Their Impact on Virulence

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