  Background: Previous studies have used different methods in an effort to
extract the modular organization of transcriptional regulatory networks (TRNs).
However, these approaches are not natural, as they try to cluster highly
connected genes into a module or locate known pleiotropic transcription factors
in lower hierarchical layers. Here, we unravel the TRN of Escherichia coli by
separating it into its key elements, thus revealing its natural organization.
We also present a mathematical criterion, based on the topological features of
the TRN, to classify the network elements into one of two possible classes:
hierarchical or modular genes.
  Results: We found that modular genes are clustered into physiologically
correlated groups validated by a statistical analysis of the enrichment of the
functional classes. Hierarchical genes encode transcription factors responsible
for coordinating module responses based on general interest signals.
Hierarchical elements correlate highly with the previously studied global
regulators, suggesting that this could be the first mathematical method to
identify global regulators. We identified a new element in TRNs never described
before: intermodular genes. These are structural genes that integrate, at the
promoter level, signals coming from different modules, and therefore from
different physiological responses. Using the concept of pleiotropy, we have
reconstructed the hierarchy of the network and discuss the role of feedforward
motifs in shaping the hierarchical backbone of the TRN.
  Conclusions: This study sheds new light on the design principles underpinning
the organization of TRNs, showing a novel nonpyramidal architecture composed of
independent modules globally governed by hierarchical transcription factors,
whose responses are integrated by intermodular genes.
