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.

FIG. 1

Plasmids used in this study. (A) Schematic representation of plasmids for expression of uppS genes from E. coli, H. influenzae, or S. pneumoniae as His-tag fusion protein and from E. coli uppS as untagged protein under the control of the T7 promoter. (B) Schematic representation of plasmids for generating pneumococcal mutants. For disrupting uppS and terminating expression of the downstream genes in the operon, a small internal fragment of the uppS gene was cloned into pJDC9, resulting in pKO1. To place the uppS operon under the control of the two tetracycline-regulatable promoters, p57opt and p57, an amino-terminal fragment of the uppS gene was cloned in the promoter vectors pRKO2* and pRKO1*, resulting in plasmids pKO2 and pKO3, respectively. To generate a disruption of the uppS gene while placing downstream genes under the control of the tetracycline-regulatable promoter p57opt, an internal fragment of uppS was cloned into the promoter vector pRKO2, resulting in plasmid pKO4. For details, see Materials and Methods. Numbers indicate nucleotides, staring with 1 at the putative initiation codon of each gene. Abbreviations for restriction enzymes: Ba, BamHI; Bs, BsaI; Kp, KpnI; Nc, NcoI; Nd, NdeI.

FIG. 2

SDS-PAGE of heparin column-purified Upp synthetase and radiolabeling with [³H]DAFTP-GDP. (A) Aliquots of the pool from the heparin column-purified Upp synthetase were radiolabeled with [³H]DAFTP-GDP and applied to an SDS-polyacrylamide gel, which then was stained with Coomassie blue. Lanes: 1, protein molecular size markers as indicated on the left; 2, radiolabeled pool in the presence of 20 μM FPP; 3, radiolabeled pool without FPP. (B) Autoradiograph of the same gel. (C) On a duplicate gel, the region between 20 and 40 kDa of lanes 2 and 3 was cut into 55 gel slices each, and the radioactivity in each slice was measured.

FIG. 3

Multiple amino acid sequence alignment of the 28 potential Upp synthetases which show homology to Upp synthetase from E. coli. Shaded areas represent residues that are identical in at least 21 of the 29 sequences (black) and similar amino acids (gray). Amino acid positions are indicated on the right. Sequences: E. coli (ECOLI; SWISS-PROT Q47675) and its homologues from H. influenzae (HAEIN; SWISS-PROT P44938), Helicobacter pylori (HELPY; SWISS-PROT P55984), S. pneumoniae (STRPN; ftp://ftp.tigr.org/pub/data/s_pneumoniae); Bacillus subtilis (BACSU; SWISS-PROT O31751), Aquifex aeolicus (AQUAE; TrEMBL O67291), Archaeoglobus fulgidus (ARCFU; TrEMBL O29049), Borrelia burgdorferi (BORBU; TrEMBL O51146), Mycobacterium tuberculosis (MYCTU; TrEMBL O53434), Mycobacterium leprae (MYCLE; SWISS-PROT P38119), Methanococcus jannaschii (METJA; SWISS-PROT Q58767), Streptomyces fradiae (STRFR; SWISS-PROT P20182), Pseudomonas aeruginosa (PSEAE; unannotated translation from EMBL entry D50811 for the cds gene), Pyrococcus horikoshii (PYRHO; TrEMBL O59258), Synechocystis (SYNY; SWISS-PROT Q55482), Chlamydia trachomatis (CHLTR; TrEMBL G3328883); Methanobacterium thermoauotrophicum (METTH; TrEMBL O26334), Neisseria gonorrhoeae (NEIGO; http://dna1.chem.ou.edu/gono.html), Saccharomyces cerevisiae (YEAST1; SWISS-PROT P35196), S. cerevisiae (YEAST2; SWISS-PROT Q03175), Caenorhabditis elegans (C.ELE; TrEMBL O18007) (amino acids 1 to 275 of 1893 amino acids), Enterococcus faecalis (ENTFA; ftp://ftp.tigr.org/pub/data/e_faecalis) (for this sequence, only the amino-terminal part is available), Corynebacterium glutamicum (CORGL; SWISS-PROT P38118), Treponema pallidum (TREPA; EMBL AE001235), Campylobacter jejuni (CAMJU; http://www.sanger.ac.uk/Projects/C_jejuni), Streptococcus pyogenes (STRPY; http://dna1.chem.ou.edu/strep.html), Brucella abortus (BRUAB; TrEMBL Q44626), Staphylococcus aureus (STAAU; (unreleased Hoffmann-La Roche data). The sequence information for the carboxy-terminal part is available for the last six sequences only. The highly conserved regions of Upp synthetase are indicated by bars and numbered I to V. The following consensus patterns can be derived for each region: I, H-x-x-x-x-M-D-G-N-(RG)-R-(WYF)-A; II, G-H-x-x-G; III, (TS)-x-x-A-F-S-(ST)-E-N-x-x-R-x-x-x-E-V-x-x-L-M-x-L; IV, A-x-x-Y-G-G-R-x-(DE)-(LIVM)-x-x-A; V, (DE)-L-x-I-R-T-(SAG)-G-E-x-R-x-S-N-F-(ML)-(LMP)-W-Q-x-x-Y-(SAT)-E-x-x-F-x-x-x-x-W-P-(DE)-F.

FIG. 4

Purification of the His-tagged Upp synthetase fusion proteins from overproducing E. coli strains. The protein was purified from E. coli BL21(DE3)(pLysS) carrying pUppS-His-EC, pUppS-His-HI, or pUppS-His-SP as indicated below. Samples from various stages of the purification procedure were analyzed by SDS-PAGE, and proteins were visualized after staining with Coomassie blue. Lanes: 1 to 3, pUppS-His-EC; 4 to 6, pUppS-His-HC; 7 and 8, pUppS-His-SP; 1, 4, and 7, soluble cell extract after IPTG induction; 2, 5, and 8, flowthrough from the Ni²⁺ column; 3, 6, and 9, specific elution from the Ni²⁺ column with 500 mM imidazole buffer.

FIG. 5

Purification of the E. coli Upp synthetase protein from the overproducing strain. The protein was purified from E. coli BL21(DE3)(pLysS)(pUppS-wt-EC) as described in the text. Samples from various stages of the purification procedure were analyzed on an SDS-polyacrylamide gel, and proteins were visualized after staining with Coomassie blue. Lanes: 1, protein standards as indicated on the left; 2, soluble cell extract after IPTG induction; 3, 150,000 × g pellet; 4; 150,000 × g supernatant; 5, 0 to 30% saturated (NH₄)₂SO₄ fraction; 6, 30 to 50% saturated (NH₄)₂SO₄ fraction; 7, 50 to 80% saturated (NH₄)₂SO₄ fraction; 8, pool after purification on Phospho-Ultrogel column; 9, pool after purification on Superdex 200 gel filtration column.

FIG. 6

Triton X-100 and MgCl₂ dependency of purified E. coli wt Upp synthetase. The coupled assay for Upp synthetase was performed as described in Materials and Methods. The Triton X-100 and MgCl₂ concentration was varied in the indicated range. The highest activity in each experiment represents 100%. Results of a representative experiment are shown. Triplicate values varied from 5 to 10%.

FIG. 7

Schematic representation of the expected chromosomal uppS mutants of S. pneumoniae R6 with various gene disruption plasmids. Numbers indicate the nucleotides, starting with 1 at the initiating codon of each gene. P1 to P4 indicate the primers used in the PCR to confirm the correct integration of the plasmids.