Plasmids used in this study are listed in Table 2. Plasmids pMP146 and pMP147 were constructed in E. coli STBL2, whereas pMP198 was constructed in E. coli DH10B. Plasmid DNA was purified using Qiagen columns (Qiagen Inc., Chatsworth, CA, USA) and DNA fragments were isolated using Gene Clean (Bio101, Vista, CA, USA). The wild-type murZ, murB, and murC genes were amplified from E. coli HB101 genomic DNA using Vent polymerase (NEB, Beverly, MA, USA). Oligonucleotides used for DNA amplification were synthesized by The University of Rochester Functional Genomics Center (Rochester, NY, USA) (Pv77, 78, 79, 80) and Invitrogen (Carlsbad, CA, USA) (Pv147, 149). The primers used for murZ amplification were Pv77 (5′-ggttcaagatctgataaatttcgtgttcagg-3′), Pv78 (5′-tcgacagaattcagttgatgcgtag-3′); murB Pv79 (5′-ggttcaagatctaaccactccttaaaaccctgg-3′), Pv80 (5′-agagtggaattcaccgttcgctaacagg-3′); and murC Pv147 (5′-atgtgcctcgagaatacacaacaattggcaaaactgcgttc-3′), Pv149(5′-cagaattcagagaagcttcccgctca-3′). Underlined sequences denote engineered restriction endonuclease sites used for cloning into the pTrcHis expression vectors (Invitrogen, Carlsbad, CA, USA) (Pv77 BglII, Pv78 EcoRI, Pv79 BglII, Pv80 EcoRI, Pv147 XhoI, Pv149 HindIII). All primers were based upon reported E. coli genomic DNA sequences: murZ (accession number M92358) , murB (accession number AAA24185) , murC (accession number AAB60787) . Amplification reaction mixtures contained 100 ng of HB101 genomic DNA, 50 pmol of each primer, dNTPs at a concentration of 0.2 mM each, and 2 mM MgSO4. Reactions were carried out in a Perkin Elmer 2400 cycler (Norwalk, CT, USA) with the following parameters: 94°C for 5 min (1 cycle) and 94°C for 1 min, 55°C for 1.5 min, 72°C for 1.5 min (35 cycles). The amplified products were cloned into the appropriate pTrcHis expression vector. Plasmids were confirmed by restriction mapping and sequencing by ACGT (Northbrook, IL, USA) and The University of Rochester Functional Genomics Center (Rochester, NY, USA).