In bacteria, environmental challenges are often translated into a transcriptional response via the cognate response regulators (RRs) of specialized two-component systems (TCSs). A phylogenetic footprinting/shadowing approach was designed and used to identify many novel RR-specific operators for species of the Bacillus cereus group and related Gram-positives. Analysis of the operator sequences revealed characteristic traits for each RR subfamily. For instance, operators related to the largest subfamily (OmpR) typically consisted of direct repeats (e.g. TTAAGA-N5-TTAAGA), whereas operators related to the second largest family (NarL) consisted of inverted repeats (e.g. ATGACA-N2-TGTCAT). This difference indicates a fundamentally different organization of the bound RR dimers between the two subfamilies. Moreover, the identification of the specific operator motifs allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. Mostly, these regulons comprised genes encoding transport systems, suggesting a direct coupling of stimulus perception to the transport of target compounds. New biological roles could be attributed to various TCSs, including roles in cytochrome c biogenesis (HssRS), transport of carbohydrates, peptides and/or amino acids (YkoGH, LytSR), and resistance to toxic ions (LiaSR), antimicrobial peptides (BceRS) and beta-lactam antibiotics (BacRS, YcbLM). As more and more bacterial genome sequences are becoming available, the use of comparative analyses such as the approach applied in this study will further increase our knowledge of bacterial signal transduction mechanisms and provide directions for the assessment of their role in bacterial performance and survival strategies.