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Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization.

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  • 1. Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
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    • Link AJ 1
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Journal of Bacteriology, 01 Oct 1997, 179(20):6228-6237
https://doi.org/10.1128/jb.179.20.6228-6237.1997 PMID: 9335267 PMCID: PMC179534

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Abstract 

We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli. Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome. Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA. These deletions are placed in the E. coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted. Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach. When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions. When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen. Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ. The two genes proved to be nonessential in both rich and glucose-minimal media. In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ. These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli. Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.

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J Bacteriol. 1997 Oct; 179(20): 6228–6237.
PMCID: PMC179534
PMID: 9335267

Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization.

A J Link, D Phillips, and G M Church
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Abstract

We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli. Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome. Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA. These deletions are placed in the E. coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted. Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach. When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions. When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen. Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ. The two genes proved to be nonessential in both rich and glucose-minimal media. In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ. These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli. Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.

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Selected References

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  • Albertini AM, Hofer M, Calos MP, Miller JH. On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions. Cell. 1982 Jun;29(2):319–328. [Abstract] [Google Scholar]
  • Bassford PJ, Jr, Silhavy TJ, Beckwith JR. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol. 1979 Jul;139(1):19–31. [Europe PMC free article] [Abstract] [Google Scholar]
  • Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. [Europe PMC free article] [Abstract] [Google Scholar]
  • Blomfield IC, Vaughn V, Rest RF, Eisenstein BI. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol. 1991 Jun;5(6):1447–1457. [Abstract] [Google Scholar]
  • Boeke JD, Trueheart J, Natsoulis G, Fink GR. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. [Abstract] [Google Scholar]
  • Bollen A, Lathe R, Herzog A, Denicourt D, Lecocq JP, Desmarez L, Lavallé R. A conditionally lethal mutation of Escherichia coli affecting the gene coding for ribosomal protein S2 (rpsB). J Mol Biol. 1979 Aug 5;132(2):219–233. [Abstract] [Google Scholar]
  • Brown ED, Vivas EI, Walsh CT, Kolter R. MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli. J Bacteriol. 1995 Jul;177(14):4194–4197. [Europe PMC free article] [Abstract] [Google Scholar]
  • Chang SY, McGary EC, Chang S. Methionine aminopeptidase gene of Escherichia coli is essential for cell growth. J Bacteriol. 1989 Jul;171(7):4071–4072. [Europe PMC free article] [Abstract] [Google Scholar]
  • Church GM, Kieffer-Higgins S. Multiplex DNA sequencing. Science. 1988 Apr 8;240(4849):185–188. [Abstract] [Google Scholar]
  • Ciampi MS, Schmid MB, Roth JR. Transposon Tn10 provides a promoter for transcription of adjacent sequences. Proc Natl Acad Sci U S A. 1982 Aug;79(16):5016–5020. [Europe PMC free article] [Abstract] [Google Scholar]
  • Gay P, Le Coq D, Steinmetz M, Berkelman T, Kado CI. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol. 1985 Nov;164(2):918–921. [Europe PMC free article] [Abstract] [Google Scholar]
  • Gutterson NI, Koshland DE., Jr Replacement and amplification of bacterial genes with sequences altered in vitro. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4894–4898. [Europe PMC free article] [Abstract] [Google Scholar]
  • Hamilton CM, Aldea M, Washburn BK, Babitzke P, Kushner SR. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. [Europe PMC free article] [Abstract] [Google Scholar]
  • Hasty P, Ramírez-Solis R, Krumlauf R, Bradley A. Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells. Nature. 1991 Mar 21;350(6315):243–246. [Abstract] [Google Scholar]
  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. [Abstract] [Google Scholar]
  • Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. [Abstract] [Google Scholar]
  • Ito K, Bassford PJ, Jr, Beckwith J. Protein localization in E. coli: is there a common step in the secretion of periplasmic and outer-membrane proteins? Cell. 1981 Jun;24(3):707–717. [Abstract] [Google Scholar]
  • Jasin M, Schimmel P. Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments. J Bacteriol. 1984 Aug;159(2):783–786. [Europe PMC free article] [Abstract] [Google Scholar]
  • Kendrick KE, Reznikoff WS. Transposition of IS50L activates downstream genes. J Bacteriol. 1988 Apr;170(4):1965–1968. [Europe PMC free article] [Abstract] [Google Scholar]
  • Kleckner N, Bender J, Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991;204:139–180. [Abstract] [Google Scholar]
  • Link AJ, Robison K, Church GM. Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrophoresis. 1997 Aug;18(8):1259–1313. [Abstract] [Google Scholar]
  • Murray V. Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res. 1989 Nov 11;17(21):8889–8889. [Europe PMC free article] [Abstract] [Google Scholar]
  • Ponce MR, Micol JL. PCR amplification of long DNA fragments. Nucleic Acids Res. 1992 Feb 11;20(3):623–623. [Europe PMC free article] [Abstract] [Google Scholar]
  • Prentki P, Krisch HM. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. [Abstract] [Google Scholar]
  • Roth JR, Lawrence JG, Rubenfield M, Kieffer-Higgins S, Church GM. Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium. J Bacteriol. 1993 Jun;175(11):3303–3316. [Europe PMC free article] [Abstract] [Google Scholar]
  • Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. [Abstract] [Google Scholar]
  • Rudd KE, Miller W, Werner C, Ostell J, Tolstoshev C, Satterfield SG. Mapping sequenced E.coli genes by computer: software, strategies and examples. Nucleic Acids Res. 1991 Feb 11;19(3):637–647. [Europe PMC free article] [Abstract] [Google Scholar]
  • Scherer S, Davis RW. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. [Europe PMC free article] [Abstract] [Google Scholar]
  • Senecoff JF, Cox MM. Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing. J Biol Chem. 1986 Jun 5;261(16):7380–7386. [Abstract] [Google Scholar]
  • Shen P, Huang HV. Homologous recombination in Escherichia coli: dependence on substrate length and homology. Genetics. 1986 Mar;112(3):441–457. [Europe PMC free article] [Abstract] [Google Scholar]
  • Shen P, Huang HV. Effect of base pair mismatches on recombination via the RecBCD pathway. Mol Gen Genet. 1989 Aug;218(2):358–360. [Abstract] [Google Scholar]
  • Shevell DE, Abou-Zamzam AM, Demple B, Walker GC. Construction of an Escherichia coli K-12 ada deletion by gene replacement in a recD strain reveals a second methyltransferase that repairs alkylated DNA. J Bacteriol. 1988 Jul;170(7):3294–3296. [Europe PMC free article] [Abstract] [Google Scholar]
  • Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8794–8797. [Europe PMC free article] [Abstract] [Google Scholar]
  • Slater S, Maurer R. Simple phagemid-based system for generating allele replacements in Escherichia coli. J Bacteriol. 1993 Jul;175(13):4260–4262. [Europe PMC free article] [Abstract] [Google Scholar]
  • Smith GR. Mechanism and control of homologous recombination in Escherichia coli. Annu Rev Genet. 1987;21:179–201. [Abstract] [Google Scholar]
  • Smith GR. Homologous recombination in procaryotes. Microbiol Rev. 1988 Mar;52(1):1–28. [Europe PMC free article] [Abstract] [Google Scholar]
  • Sternberg N. Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs. Proc Natl Acad Sci U S A. 1990 Jan;87(1):103–107. [Europe PMC free article] [Abstract] [Google Scholar]
  • Wang A, Roth JR. Activation of silent genes by transposons Tn5 and Tn10. Genetics. 1988 Dec;120(4):875–885. [Europe PMC free article] [Abstract] [Google Scholar]
  • Winans SC, Elledge SJ, Krueger JH, Walker GC. Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1219–1221. [Europe PMC free article] [Abstract] [Google Scholar]
  • Yoshida T, Ueguchi C, Mizuno T. Physical map location of a set of Escherichia coli genes (hde) whose expression is affected by the nucleoid protein H-NS. J Bacteriol. 1993 Dec;175(23):7747–7748. [Europe PMC free article] [Abstract] [Google Scholar]
  • Yoshida T, Ueguchi C, Yamada H, Mizuno T. Function of the Escherichia coli nucleoid protein, H-NS: molecular analysis of a subset of proteins whose expression is enhanced in a hns deletion mutant. Mol Gen Genet. 1993 Feb;237(1-2):113–122. [Abstract] [Google Scholar]
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