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J Bacteriol. 1985 December; 164(3): 1124–1135.
PMCID: PMC219306

Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable.

Abstract

The lon gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in the intracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, such as UV sensitivity and overproduction of capsular polysaccharide. We have isolated lambda transducing phage carrying the lon gene and used the lon phage as a target for insertional mutagenesis by a defective transposon Tn10 to produce lon::delta 16 delta 17Tn10 derivatives. The delta 16 delta 17Tn10 (hereafter called delta Tn10) elements were inserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distance from the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different lambda lon::delta Tn10 phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. The lon::delta Tn10 mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis of intact Lon protein, as assayed by antibody on Western blots. An analysis of the protein-degradative ability of lon::delta Tn10 cells suggests that although the insertions in lon caused a reduction in ATP-dependent protein degradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::delta Tn10 mutations and a lon deletion retaining only the amino-terminal 25% of the gene did not affect the energy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in the ATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provide clear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli.

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Selected References

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  • Baker TA, Grossman AD, Gross CA. A gene regulating the heat shock response in Escherichia coli also affects proteolysis. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6779–6783. [PubMed]
  • Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. [PubMed]
  • Charette MF, Henderson GW, Markovitz A. ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4728–4732. [PubMed]
  • Chung CH, Goldberg AL. The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4931–4935. [PubMed]
  • Chung CH, Waxman L, Goldberg AL. Studies of the protein encoded by the lon mutation, capR9, in Escherichia coli. A labile form of the ATP-dependent protease La that inhibits the wild type protease. J Biol Chem. 1983 Jan 10;258(1):215–221. [PubMed]
  • Enquist LW, Weisberg RA. A genetic analysis of the att-int-xis region of coliphage lambda. J Mol Biol. 1977 Apr;111(2):97–120. [PubMed]
  • Foster TJ, Davis MA, Roberts DE, Takeshita K, Kleckner N. Genetic organization of transposon Tn10. Cell. 1981 Jan;23(1):201–213. [PubMed]
  • Gayda RC, Stephens PE, Hewick R, Schoemaker JM, Dreyer WJ, Markovitz A. Regulatory region of the heat shock-inducible capR (lon) gene: DNA and protein sequences. J Bacteriol. 1985 Apr;162(1):271–275. [PMC free article] [PubMed]
  • Gayda RC, Yamamoto LT, Markovitz A. Second-site mutations in capR (lon) strains of Escherichia coli K-12 that prevent radiation sensitivity and allow bacteriophage lambda to lysogenize. J Bacteriol. 1976 Sep;127(3):1208–1216. [PMC free article] [PubMed]
  • Goff SA, Casson LP, Goldberg AL. Heat shock regulatory gene htpR influences rates of protein degradation and expression of the lon gene in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6647–6651. [PubMed]
  • Goldberg AL. Degradation of abnormal proteins in Escherichia coli (protein breakdown-protein structure-mistranslation-amino acid analogs-puromycin). Proc Natl Acad Sci U S A. 1972 Feb;69(2):422–426. [PubMed]
  • Goldberg AL, Swamy KH, Chung CH, Larimore FS. Proteases in Escherichia coli. Methods Enzymol. 1981;80(Pt 100):680–702. [PubMed]
  • Goldberg AL, St John AC. Intracellular protein degradation in mammalian and bacterial cells: Part 2. Annu Rev Biochem. 1976;45:747–803. [PubMed]
  • Gottesman S, Gottesman M, Shaw JE, Pearson ML. Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability. Cell. 1981 Apr;24(1):225–233. [PubMed]
  • Gottesman S, Trisler P, Torres-Cabassa A. Regulation of capsular polysaccharide synthesis in Escherichia coli K-12: characterization of three regulatory genes. J Bacteriol. 1985 Jun;162(3):1111–1119. [PMC free article] [PubMed]
  • Gottesman S, Zipser D. Deg phenotype of Escherichia coli lon mutants. J Bacteriol. 1978 Feb;133(2):844–851. [PMC free article] [PubMed]
  • Gulletta E, Das A, Adhya S. The pleiotropic ts15 mutation of E. coli is an IS1 insertion in the rho structural gene. Genetics. 1983 Oct;105(2):265–280. [PubMed]
  • HOWARD-FLANDERS P, SIMSON E, THERIOT L. A LOCUS THAT CONTROLS FILAMENT FORMATION AND SENSITIVITY TO RADIATION IN ESCHERICHIA COLI K-12. Genetics. 1964 Feb;49:237–246. [PubMed]
  • Huisman O, D'Ari R, Gottesman S. Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4490–4494. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Maurer R, Osmond BC, Shekhtman E, Wong A, Botstein D. Functional interchangeability of DNA replication genes in Salmonella typhimurium and Escherichia coli demonstrated by a general complementation procedure. Genetics. 1984 Sep;108(1):1–23. [PubMed]
  • Mizusawa S, Gottesman S. Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein. Proc Natl Acad Sci U S A. 1983 Jan;80(2):358–362. [PubMed]
  • Mizusawa S, Ward DF. A bacteriophage lambda vector for cloning with BamHI and Sau3A. Gene. 1982 Dec;20(3):317–322. [PubMed]
  • Mosteller RD, Goldstein RV, Nishimoto KR. Metabolism of individual proteins in exponentially growing Escherichia coli. J Biol Chem. 1980 Mar 25;255(6):2524–2532. [PubMed]
  • Murakami K, Voellmy R, Goldberg AL. Protein degradation is stimulated by ATP in extracts of Escherichia coli. J Biol Chem. 1979 Sep 10;254(17):8194–8200. [PubMed]
  • Rupprecht KR, Markovitz A. Conservation of capR (lon) DNA of Escherichia coli K-12 between distantly related species. J Bacteriol. 1983 Aug;155(2):910–914. [PMC free article] [PubMed]
  • Schoemaker JM, Markovitz A. Identification of the gene lon (capR) product as a 94-kilodalton polypeptide by cloning and deletion analysis. J Bacteriol. 1981 Jul;147(1):46–56. [PMC free article] [PubMed]
  • Shineberg B, Zipser D. The ion gene and degradation of beta-galactosidase nonsense fragments. J Bacteriol. 1973 Dec;116(3):1469–1471. [PMC free article] [PubMed]
  • Simon LD, Gottesman M, Tomczak K, Gottesman S. Hyperdegradation of proteins in Escherichia coli rho mutants. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1623–1627. [PubMed]
  • Simon LD, Randolph B, Irwin N, Binkowski G. Stabilization of proteins by a bacteriophage T4 gene cloned in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2059–2062. [PubMed]
  • Simon LD, Tomczak K, St John AC. Bacteriophages inhibit degradation of abnormal proteins in E. coli. Nature. 1978 Oct 5;275(5679):424–428. [PubMed]
  • Trisler P, Gottesman S. lon transcriptional regulation of genes necessary for capsular polysaccharide synthesis in Escherichia coli K-12. J Bacteriol. 1984 Oct;160(1):184–191. [PMC free article] [PubMed]
  • Walker JR, Ussery CL, Allen JS. Bacterial cell division regulation: lysogenization of conditional cell division lon - mutants of Escherichia coli by bacteriophage. J Bacteriol. 1973 Mar;113(3):1326–1332. [PMC free article] [PubMed]
  • Ward DF, Murray NE. Convergent transcription in bacteriophage lambda: interference with gene expression. J Mol Biol. 1979 Sep 15;133(2):249–266. [PubMed]
  • Wray LV, Jr, Jorgensen RA, Reznikoff WS. Identification of the tetracycline resistance promoter and repressor in transposon Tn10. J Bacteriol. 1981 Aug;147(2):297–304. [PMC free article] [PubMed]
  • Zehnbauer BA, Foley EC, Henderson GW, Markovitz A. Identification and purification of the Lon+ (capR+) gene product, a DNA-binding protein. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2043–2047. [PubMed]

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