Identification of the purC gene product of Escherichia coli

Abstract

The purC region of the Escherichia coli chromosome was isolated from in vivo-derived lambda transducing bacteriophages and cloned in high-copy-number plasmids. The product of the purC gene, phosphoribosylaminoimidazolesuccinocarboxamide synthetase, was identified as a protein with an Mr of ca. 27,000. The level of the protein is increased by more than 60-fold in strains carrying the gene on a highcopy-number plasmid. Purine addition represses the enzyme level in both plasmid-and non-plasmidcontaining strains.The de novo biosynthesis of AMP and GMP involves 13 enzymes. In the enteric bacteria the structural genes for 12 of the enzymes have been mapped. Genes purJ, purH, and purD form an operon, as do genes guaB and guaA. The rest of the genes are not tightly linked, although the gene encoding the enzyme catalyzing the third step in purine synthesis has not been located (7).Studies on the regulation of these genes have been slowed by the difficulty of assaying the enzymes and the complexity of metabolic interconversions. It is known that the expression of the pur genes is repressed upon the addition of purines, with hypoxanthine and guanine being corepressors (9). Results by Benson and Gots (2) and Gots et al. (7) indicate that the pur genes, but not the gua operon, may be controlled by a common aporepressor, the product of the purR gene. A protein which binds to different pur gene promoter regions in the presence of either ATP or GTP has been isolated (11). However, the exact nature of the purR gene and its product is still unknown.Identification of the purine biosynthetic enzymes, cloning of structural genes, and formation of gene fusions (13, 31) promise to be of help in the elucidation of regulatory mechanisms. In this paper I describe the isolation and cloning of purC, the structural gene for phosphoribosylaminoimidazolesuccinocarboxamide synthetase (EC 6.3.2.6; PurC), and the identification of this protein on two-dimensional polyacrylamide gels. MATERIALS AND METHODSBacterial, viral, and plasmid strains. The bacterial strains used in this work are described in Table 1. Lysogens of strain JF298 containing X c1857 Sam7 were prepared by Poul J0rgensen. The plasmid cloning vectors used were pBR322 (3) and pBR328 (26). The recombinant plasmids used, pSIU103, pSIU104, pSIU107, and pSIU111, are described below.Media and growth conditions. The complex medium used was L-broth (12) supplemented with 0.2% (wt/vol) glucose or 0.2% maltose when growing cells as recipients for X transduction. The defined medium was the MOPS medium of Neidhardt et al. (17) supplemented with 0.4% glucose, 10 ,ug of thiamine per ml, and, when necessary, 50 ,ug of L-amino acids and 20 ,ug of purine bases per ml. Cultures were grown aerobically in Erlenmeyer flasks with rotary shaking. Growth was monitored with a Zeiss spectrophotometer.Isolation of transducing phage. X d purC transducing phages were isolated by the general procedure described by 712 Schrenk and Weisberg (24) from attA deletion strains lysogenic for A c1857 Sam7. A lysate pr...

A molecular analysis of the 53.3 minute region of the Escherichia coli linkage map

“…This analysis clearly indicates that all four pLC plasmids possess overlapping segments of DNA from the 53 min region, although pLC46-7 could still contain some additional DNA from the 83 min region. A purC + plasmid (pSIU103) containing part of the 53 min region also expresses five proteins with mobilities that match some of the pLC-encoded proteins (Parker, 1984). Two of these proteins have been identified as products of the purC (Parker, 1984) and dap A (Richaud et al ., 1986) genes, and a third migrated like BCP ().…”

“…A purC + plasmid (pSIU103) containing part of the 53 min region also expresses five proteins with mobilities that match some of the pLC-encoded proteins (Parker, 1984). Two of these proteins have been identified as products of the purC (Parker, 1984) and dap A (Richaud et al ., 1986) genes, and a third migrated like BCP (). It is important to note that the vertical arrangement of the groups of proteins boxed in reflects the linkage map of the corresponding genes.…”

“…Restriction map for the segment of bacterial DNA in pLC25-14 and the structures of several pUC derivative plasmids. The positions and polarities of previously characterized and unidentified genes, purC , dap A and urf (Parker, 1984; Richaud et al ., 1986), are indicated, as are those of orfl , 2 ( bcp ), and 3 . Plasmid pSIU103 is a pBR322 derivative that contains the same insert as pGS211 and pGS212 (Parker, 1984).…”

Protein Cages: From Fundamentals to Advanced Applications

“…While some bacteriophages infect a range of bacterial strains, the majority infect only selected species. − The most common strategy to overcome this specificity issue is to use a combination of phages with different host ranges. , However, the concoction of phage cocktails requires laborious performance optimization and poses a challenge for reproducible manufacturing. Engineered phages with broader antibacterial spectra − and optimized efficacy − are a promising alternative. Other concerns with phage therapy include inactivation by neutralizing antibodies and clearance by the reticuloendothelial system. − Thus, phages have been engineered to provide them with greater capacity to remain in the circulatory system. , To overcome the release of toxic bacterial components during infection, lysis-deficient bacteriophages have been developed for efficient killing of bacteria with minimized toxin release. − …”

Different Ways of Doing the Same: Variations in the Two Last Steps of the Purine Biosynthetic Pathway in Prokaryotes

“…Additionally, the enzymes that catalyze their synthesis are important targets for antimicrobial and anticancer compounds (Kirsch and Whitney 1991). The enzymatic reactions involved in the de novo biosynthesis of purines were elucidated in the 1950s (Buchanan and Hartman 1959) and all genes that encode these enzymes were identified and their products biochemically characterized by the year 2000 (Zalkin 1983; Parker 1984; Schrimsher et al. 1986; Aiba and Mizobuchi 1989; Watanabe et al.…”