The study of protein function mostly relies on perturbing regulatory networks by acting upon protein expression levels or using transdominant negative agents. Here, we used the E.coli global transcription regulator Fur (ferric uptake regulator) as a case study to compare the perturbations exerted by a gene knockout, the expression of a dominant negative allele of a gene and the expression of peptide aptamers that bind a gene product. These three perturbations caused phenotypes that differed quantitatively and qualitatively from one another. The Fur peptide aptamers inhibited the activity of their target to various extents and reduced the virulence of a pathogenic E.coli strain in Drosophila. A genome-wide transcriptome analysis revealed that the “penetrance” of a peptide aptamer was comparable to that of a dominant-negative allele but lower than the “penetrance” of the gene knockout. Our work shows that comparative analysis of phenotypic and transcriptome responses to different types of perturbation can help decipher complex regulatory networks that control various biological processes.