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

被引:753
作者
Link, AJ
Phillips, D
Church, GM
机构
[1] HARVARD UNIV, SCH MED, DEPT GENET, BOSTON, MA 02115 USA
[2] HARVARD UNIV, SCH MED, HOWARD HUGHES MED INST, BOSTON, MA 02115 USA
关键词
D O I
10.1128/jb.179.20.6228-6237.1997
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
引用
收藏
页码:6228 / 6237
页数:10
相关论文
共 47 条
  • [1] ON THE FORMATION OF SPONTANEOUS DELETIONS - THE IMPORTANCE OF SHORT SEQUENCE HOMOLOGIES IN THE GENERATION OF LARGE DELETIONS
    ALBERTINI, AM
    HOFER, M
    CALOS, MP
    MILLER, JH
    [J]. CELL, 1982, 29 (02) : 319 - 328
  • [2] [Anonymous], METHOD ENZYMOL
  • [3] [Anonymous], 1996, ESCHERICHIA COLI SAL
  • [4] BASSFORD PJ, 1979, J BACTERIOL, V139, P19, DOI 10.1128/JB.139.1.19-31.1979
  • [5] Berg C. O., COMMUNICATION
  • [6] BERG CM, 1996, ESCHERICHIA COLI SAL, P2588
  • [7] BIRNBOIM HC, 1979, NUCLEIC ACIDS RES, V7, P1513
  • [8] ALLELIC EXCHANGE IN ESCHERICHIA-COLI USING THE BACILLUS-SUBTILIS SACB GENE AND A TEMPERATURE-SENSITIVE PSC101 REPLICON
    BLOMFIELD, IC
    VAUGHN, V
    REST, RF
    EISENSTEIN, BI
    [J]. MOLECULAR MICROBIOLOGY, 1991, 5 (06) : 1447 - 1457
  • [9] BOEKE JD, 1987, METHOD ENZYMOL, V154, P164
  • [10] CONDITIONALLY LETHAL MUTATION OF ESCHERICHIA-COLI AFFECTING THE GENE CODING FOR RIBOSOMAL PROTEIN-S2 (RPSB)
    BOLLEN, A
    LATHE, R
    HERZOG, A
    DENICOURT, D
    LECOCQ, JP
    DESMAREZ, L
    LAVALLE, R
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1979, 132 (02) : 219 - 233