The structural gene (purB) for succinyl-AMP (S-AMP) lyase and three additional ORFs are on the same DNA strand of the chromosome of Escherichia coli. Cassette mutagenesis and primer extension mapping demonstrated that purl? is co-transcribed with an upstream gene (ORF23, or ycfC) encoding a 22-9 kDa membrane-associated protein of non-essential, but unknown, function unrelated to purine biosynthesis. The purB operon lies between phoP and an ORF expressing an essential function which may correspond to asu€ (frmU). S-AMP lyase was purified to near homogeneity. The purified enzyme is a homotetramer of 50 kDa subunits, has a Km for S-AMP of 3.7 pM and a pH optimum of 74-706.Keywords : Escherichia coli, purl3 gene, succinyl-AMP lyase, internal promoter, phoP geneThe purine nucleotides AMP and GMP are synthesized through a branched multi-enzyme pathway. In Escberichia coli, the structural genes (pur and gtla) for these enzymes have been mapped on the chromosome (Berlyn e t al., 1996), and occur at various loci either individually (ptlrT, pwL, pztrC, ptlrA) or collectively within operons (PtlrF, ptlrHD, ptlrMN, pzrrEK, gztaBA). The nucleotide sequences for all the genes are known but the previously published sequence and organization of the purB region (He e t al., 1992) differ significantly from those reported here. pztrB maps at 25.57' on the E. coli chromosome between astlE (trmU), the site of an antisuppressor mutation that affects tRNA modification (Sullivan et al., 1985; Bjork, 1995), andpboP (Groisman etal., 1992 He e t al., 1992; Kasahara e t al., 1992), encoding a regulatory protein involved in stress responses (Miller, 1991). pztrB encodes succinyl-AMP (S-AMP) lyase (EC 4,3.2,2), a bifunctional enzyme that converts succinyl-AMP to AMP (the last reaction of AMP biosynthesis) and succinyl aminoimidazole carboxamide ribotide to aminoimidazole carboxamide ribotide (a precursor of both AMP and GMP).

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The concentration and pressure dependence of dual-pump coherent anti-Stokes Raman spectroscopy (CARS) signals from nitrogen and methane was investigated. CARS spectra were acquired from a gas cell at pressures of 0.007 to 2.24 MPa and methane concentrations of 0.5 to 50%. The square root of the methane signal intensity divided by the nitrogen signal intensity was found to have a near-linear dependence on methane concentration at all pressures investigated. The pressure dependence of this integrated intensity ratio decreased with increasing pressure and became negligible at the highest pressures tested. The shot-to-shot variation at concentrations determined from single-laser-shot measurements was less than 7%. Single-laser-shot CARS spectra of nitrogen and methane were obtained from the cylinder of a firing direct-injection natural gas engine.

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