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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 araPBAD promoter oriented outward at one end (Tn5-araPBAD). The transposon insertion mutants obtained were assayed for conditional lethal phenotypes (arabinose dependence or sensitivity), for growth at two temperatures (37 and 15°C) and in different media (rich and minimal medium). The Tn5-araPBAD-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°C) but not in the cold and in minimal medium. © 2004 Elsevier SAS. All rights reserved.