The leuX gene encoding the minor tRNA5(Leu) is important for the expression of several virulence factors of pathogenic Escherichia coli strains. The differential usage of minor codons to control the expression of specialized genes has been proposed to be a general mechanism of bacteria to regulate gene expression at the posttranscriptional level. The minor codon usage theory foots on the biased codon usage of bacterial genes and the selective availability of tRNA isoacceptors. We aimed at the further investigation of the regulatory role of the tRNA5(Leu) for gene expression in pathogenic E. coli. For this purpose, the molecular mechanism underlying the tRNA5(Leu)-dependent regulation of different virulence-associated genes of pathogenic E. coli as well as the regulation of leuX transcription under various growth conditions were investigated in detail. The global impact of the presence or absence of the leuX-encoded tRNA on gene expression of the uropathogenic E. coli strain 536 was studied by proteome analysis. The obtained results argue for a general importance of the tRNA5(Leu) for gene expression of E. coli and the involvement of this tRNA in global regulatory networks.