The alternative sigma factor sigma(E) is critical for envelope stress response and plays a role in pathogenicity of a variety of different bacteria. We previously identified several critical nucleotides in the Salmonella enterica serovar Typhimurium (S. Typhimurium) sigma(E)-dependent rpoEp3 promoter that corresponded to the most conserved nucleotides in the sigma(E) consensus sequence of the -10 and -35 promoter elements. In the present study, we exploited a previously established Escherichia coli (E. coli) two-plasmid system with an error-prone PCR mutagenesis to identify mutants in the rpoE gene that suppress the mutation of the most conserved residue A-30G of the rpoEp3 promoter. This analysis identified amino-acid changes in the conserved arginine residue (R171G, R171C) located in the conserved region 4.2 of sigma(E) that enabled efficient recognition of the mutated rpoEp3 promoter. However, the change of this conserved arginine to alanine (R171A) resulted in an almost complete loss of sigma(E) activity. The activity of the mutant sigma(E) factors in directing transcription of the wild-type (WT) and the A-30G mutated rpoEp3 promoters was investigated by S1-nuclease mapping using RNA isolated from the E. coli two-plasmid system. In addition to suppression of the A-30G mutated rpoEp3 promoter, both mutant sigma factors (R171G, R171C) also efficiently directed transcription from the WT rpoEp3 promoter and from the rpoEp3 promoter with other mutations in the -35 element, indicating relaxed recognition of the sigma(E)-dependent promoters by both mutants. The activity of both mutant sigma(E) factors was confirmed in vivo in S. Typhimurium. In conclusion, replacement of the conserved R171 residue in sigma(E) by different amino-acid residues exhibited intriguingly different phenotypes; R171A almost completely abolished sigma factor activity, whereas R171G and R171C impart a relaxed recognition phenotype to sigma(E).