Scanning the Escherichia coli chromosome by random transposon mutagenesis and multiple phenotypic screening.
Analysis of the complete DNA sequences of many microbial genomes available reveals a fair number of putative ORFs without an identified function. A systematic scan of the Escherichia coli chromosome was achieved by random transposition with a newly developed Tn5 minitransposon derivative carrying the arabinose-inducible araP(BAD) promoter oriented outward at one end (Tn5-araP(BAD)). The transposon insertion mutants obtained were assayed for conditional lethal phenotypes (arabinose dependence or sensitivity), for growth at two temperatures (37 and 15 degrees C) and in different media (rich and minimal medium). The Tn5-araP(BAD)-tagged genes were identified by sequencing the transposon insertion points. In this way we found a new essential gene cluster (yhbN-yhbG), produced conditional lethal (arabinose-dependent) mutations in already known essential genes (folD, frr, plsC, thiL, serS, thrS, and trpS) and provided a new phenotype (cold sensitivity) to other known genes (holD, ahpC, and tolA). Moreover, we identified eight putative ORFs (kch, ycaM, ycbQ, yddA, yddB, ydeK, ydeX, and yliF) that appear to be required in optimum growth conditions (rich medium at 37 degrees C) but not in the cold and in minimal medium.
Serina
Stefania
S
Dipartimento di Scienze biomolecolari e Biotecnologie, Università degli Studi di Milano, Milan, Italy.
Nozza
Francesca
F
Nicastro
Giovanna
G
Faggioni
Federico
F
Mottl
Harald
H
Dehò
Gianni
G
Polissi
Alessandra
A
D002876:Chromosomes, Bacterial / Q000737:chemistry* / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics*; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic
D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins