title: Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene.
abstract: The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.
authors.affiliation: Pharmaceutical Research Preclinical Infectious Diseases, F. Hoffmann- La Roche Ltd., CH-4070 Basel, Switzerland. christina.apfel@roche.com
authors.forename: C M
authors.initials: CM
authors.lastname: Apfel
authors.forename: B
authors.initials: B
authors.lastname: Takács
authors.forename: M
authors.initials: M
authors.lastname: Fountoulakis
authors.forename: M
authors.initials: M
authors.lastname: Stieger
authors.forename: W
authors.initials: W
authors.lastname: Keck
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D017173:Caenorhabditis elegans / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C023266:undecaprenyl pyrophosphate synthetase