Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: Cloning, expression, and characterization of the essential uppS gene

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly- cis-decaprenylcistransfer EC 2.5.1.31) was purified from the soluble fracti on of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Supe rdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E,E)-[1-H- 3]-(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, tryps in digested, and subjected to matrix-assisted laser desorption ionization m ass spectrometric analysis. Comparison of the experimental data with comput er-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 w ere identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans, The homologous genes (uppS) were cloned from E. co li, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. c oli as amino-terminal His-tagged fusion proteins, and purified over a Ni2affinity column. An untagged version of the E. coli uppS gene was also clon ed and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Furt her, biochemical characterization revealed no differences between the recom binant untagged E. coli Upp synthetase and the three His-tagged fusion prot eins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With th e use of a regulatable gene disruption system,we demonstrated that uppS is essential for growth in S. pneumoniae R6.