Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. 1993 Jun; 175(11): 3401–3407.
PMCID: PMC204738

The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels.


The widely used and closely related Escherichia coli "wild types" W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene. This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low. After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E. coli K-12, E. coli B, and Salmonella typhimurium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda 2A6. DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110. This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E. coli strains, as confirmed by analysis of protein synthesis in minicells. The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.5M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • An G, Justesen J, Watson RJ, Friesen JD. Cloning the spoT gene of Escherichia coli: identification of the spoT gene product. J Bacteriol. 1979 Mar;137(3):1100–1110. [PMC free article] [PubMed]
  • Andersen JT, Poulsen P, Jensen KF. Attenuation in the rph-pyrE operon of Escherichia coli and processing of the dicistronic mRNA. Eur J Biochem. 1992 Jun 1;206(2):381–390. [PubMed]
  • Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed]
  • Bonekamp F, Andersen HD, Christensen T, Jensen KF. Codon-defined ribosomal pausing in Escherichia coli detected by using the pyrE attenuator to probe the coupling between transcription and translation. Nucleic Acids Res. 1985 Jun 11;13(11):4113–4123. [PMC free article] [PubMed]
  • Bonekamp F, Clemmesen K, Karlström O, Jensen KF. Mechanism of UTP-modulated attenuation at the pyrE gene of Escherichia coli: an example of operon polarity control through the coupling of translation to transcription. EMBO J. 1984 Dec 1;3(12):2857–2861. [PMC free article] [PubMed]
  • Clemmesen K, Bonekamp F, Karlström O, Jensen KF. Role of translation in the UTP-modulated attenuation at the pyrBI operon of Escherichia coli. Mol Gen Genet. 1985;201(2):247–251. [PubMed]
  • Craven MG, Henner DJ, Alessi D, Schauer AT, Ost KA, Deutscher MP, Friedman DI. Identification of the rph (RNase PH) gene of Bacillus subtilis: evidence for suppression of cold-sensitive mutations in Escherichia coli. J Bacteriol. 1992 Jul;174(14):4727–4735. [PMC free article] [PubMed]
  • Dandanell G, Hammer K. Two operator sites separated by 599 base pairs are required for deoR repression of the deo operon of Escherichia coli. EMBO J. 1985 Dec 1;4(12):3333–3338. [PMC free article] [PubMed]
  • Daniels DL, Plunkett G, 3rd, Burland V, Blattner FR. Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. Science. 1992 Aug 7;257(5071):771–778. [PubMed]
  • Deutscher MP, Marshall GT, Cudny H. RNase PH: an Escherichia coli phosphate-dependent nuclease distinct from polynucleotide phosphorylase. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4710–4714. [PMC free article] [PubMed]
  • Gaal T, Gourse RL. Guanosine 3'-diphosphate 5'-diphosphate is not required for growth rate-dependent control of rRNA synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5533–5537. [PMC free article] [PubMed]
  • GERHART JC, PARDEE AB. The enzymology of control by feedback inhibition. J Biol Chem. 1962 Mar;237:891–896. [PubMed]
  • Jensen KF. Apparent involvement of purines in the control of expression of Salmonella typhimurium pyr genes: analysis of a leaky guaB mutant resistant to pyrimidine analogs. J Bacteriol. 1979 Jun;138(3):731–738. [PMC free article] [PubMed]
  • Jensen KF. Hyper-regulation of pyr gene expression in Escherichia coli cells with slow ribosomes. Evidence for RNA polymerase pausing in vivo? Eur J Biochem. 1988 Aug 15;175(3):587–593. [PubMed]
  • Jensen KF, Andersen JT, Poulsen P. Overexpression and rapid purification of the orfE/rph gene product, RNase PH of Escherichia coli. J Biol Chem. 1992 Aug 25;267(24):17147–17152. [PubMed]
  • Jensen KF, Larsen JN, Schack L, Sivertsen A. Studies on the structure and expression of Escherichia coli pyrC, pyrD, and pyrF using the cloned genes. Eur J Biochem. 1984 Apr 16;140(2):343–352. [PubMed]
  • Jensen KF, Neuhard J, Schack L. RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of a fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to a mutation in rpoBC. EMBO J. 1982;1(1):69–74. [PMC free article] [PubMed]
  • Jin DJ, Gross CA. Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J Mol Biol. 1988 Jul 5;202(1):45–58. [PubMed]
  • Jin DJ, Gross CA. Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli. J Bacteriol. 1989 Sep;171(9):5229–5231. [PMC free article] [PubMed]
  • Jin DJ, Gross CA. Three rpoBC mutations that suppress the termination defects of rho mutants also affect the functions of nusA mutants. Mol Gen Genet. 1989 Apr;216(2-3):269–275. [PubMed]
  • Jin DJ, Walter WA, Gross CA. Characterization of the termination phenotypes of rifampicin-resistant mutants. J Mol Biol. 1988 Jul 20;202(2):245–253. [PubMed]
  • Kelly KO, Deutscher MP. The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli. J Bacteriol. 1992 Oct;174(20):6682–6684. [PMC free article] [PubMed]
  • Kelly KO, Reuven NB, Li Z, Deutscher MP. RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells. J Biol Chem. 1992 Aug 15;267(23):16015–16018. [PubMed]
  • Kohara Y, Akiyama K, Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Lundberg LG, Thoresson HO, Karlström OH, Nyman PO. Nucleotide sequence of the structural gene for dUTPase of Escherichia coli K-12. EMBO J. 1983;2(6):967–971. [PMC free article] [PubMed]
  • Neuhard J, Stauning E, Kelln RA. Cloning and characterization of the pyrE gene and of PyrE::Mud1 (Ap lac) fusions from Salmonella typhimurium. Eur J Biochem. 1985 Feb 1;146(3):597–603. [PubMed]
  • O'Callaghan CH, Morris A, Kirby SM, Shingler AH. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother. 1972 Apr;1(4):283–288. [PMC free article] [PubMed]
  • Poulsen P, Andersen JT, Jensen KF. Molecular and mutational analysis of three genes preceding pyrE on the Escherichia coli chromosome. Mol Microbiol. 1989 Mar;3(3):393–404. [PubMed]
  • Poulsen P, Bonekamp F, Jensen KF. Structure of the Escherichia coli pyrE operon and control of pyrE expression by a UTP modulated intercistronic attentuation. EMBO J. 1984 Aug;3(8):1783–1790. [PMC free article] [PubMed]
  • Poulsen P, Jensen KF. Effect of UTP and GTP pools on attenuation at the pyrE gene of Escherichia coli. Mol Gen Genet. 1987 Jun;208(1-2):152–158. [PubMed]
  • Poulsen P, Jensen KF, Valentin-Hansen P, Carlsson P, Lundberg LG. Nucleotide sequence of the Escherichia coli pyrE gene and of the DNA in front of the protein-coding region. Eur J Biochem. 1983 Sep 15;135(2):223–229. [PubMed]
  • Roland KL, Liu CG, Turnbough CL., Jr Role of the ribosome in suppressing transcriptional termination at the pyrBI attenuator of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7149–7153. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Schrenk WJ, Weisberg RA. A simple method for making new transducing lines of coliphage lambda. Mol Gen Genet. 1975;137(2):101–107. [PubMed]
  • Schwartz M, Neuhard J. Control of expression of the pyr genes in Salmonella typhimurium: effects of variations in uridine and cytidine nucleotide pools. J Bacteriol. 1975 Mar;121(3):814–822. [PMC free article] [PubMed]
  • Silhavy TJ, Beckwith J. Isolation and characterization of mutants of Escherichia coli K12 affected in protein localization. Methods Enzymol. 1983;97:11–40. [PubMed]
  • Singer M, Baker TA, Schnitzler G, Deischel SM, Goel M, Dove W, Jaacks KJ, Grossman AD, Erickson JW, Gross CA. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. [PMC free article] [PubMed]
  • Tsui HC, Arps PJ, Connolly DM, Winkler ME. Absence of hisT-mediated tRNA pseudouridylation results in a uracil requirement that interferes with Escherichia coli K-12 cell division. J Bacteriol. 1991 Nov;173(22):7395–7400. [PMC free article] [PubMed]
  • Uhlin BE, Nordström K. A runaway-replication mutant of plasmid R1drd-19: temperature-dependent loss of copy number control. Mol Gen Genet. 1978 Oct 4;165(2):167–179. [PubMed]
  • VanBogelen RA, Neidhardt FC. The gene-protein database of Escherichia coli: edition 4. Electrophoresis. 1991 Nov;12(11):955–994. [PubMed]
  • Yura T, Mori H, Nagai H, Nagata T, Ishihama A, Fujita N, Isono K, Mizobuchi K, Nakata A. Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region. Nucleic Acids Res. 1992 Jul 11;20(13):3305–3308. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Conserved Domains
    Conserved Domains
    Conserved Domain Database (CDD) records that cite the current articles. Citations are from the CDD source database records (PFAM, SMART).
  • Gene
    Gene records that cite the current articles. Citations in Gene are added manually by NCBI or imported from outside public resources.
  • GEO Profiles
    GEO Profiles
    Gene Expression Omnibus (GEO) Profiles of molecular abundance data. The current articles are references on the Gene record associated with the GEO profile.
  • MedGen
    Related information in MedGen
  • Nucleotide
    Primary database (GenBank) nucleotide records reported in the current articles as well as Reference Sequences (RefSeqs) that include the articles as references.
  • Pathways + GO
    Pathways + GO
    Pathways and biological systems (BioSystems) that cite the current articles. Citations are from the BioSystems source databases (KEGG and BioCyc).
  • Protein
    Protein translation features of primary database (GenBank) nucleotide records reported in the current articles as well as Reference Sequences (RefSeqs) that include the articles as references.
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...