sigma54 is unique among the bacterial sigma factors. Besides not being related in sequence with the rest of such factors, its mechanism of transcription initiation is completely different and requires the participation of a transcription activator. In addition, whereas the rest of the alternative sigma factors use to be involved in transcription of somehow related biological functions, this is not the case for sigma54 and many different and unrelated genes have been shown to be transcribed from sigma54-dependent promoters, ranging from flagellation, to utilization of several different carbon and nitrogen sources, or alginate biosynthesis. These genes have been characterized in many different bacterial species and, only until recently with the arrival of complete genome sequences, we have been able to look at the sigma54 functional role from a genomic perspective. Aided by computational methods, the sigma54 regulon has been studied both in Escherichia coli, Salmonella typhimurium and several species of the Rhizobiaceae. Here we present the analysis of the sigma54 regulon (sigmulon) in the complete genome of Pseudomonas putida KT2440. We have developed an improved method for the prediction of sigma54-dependent promoters which combines the scores of sigma54-RNAP target sequences and those of activator binding sites. In combination with other evidence obtained from the chromosomal context and the similarity with closely related bacteria, we have been able to predict more than 80% of the sigma54-dependent promoters of P. putida with high confidence. Our analysis has revealed new functions for sigma54 and, by means of comparative analysis with the previous studies, we have drawn a potential mechanism for the evolution of this regulatory system.