Promoter-creating mutations in Pseudomonas putida : A model system for the study of mutation in starving bacteria

A novel experimental system to study mutation in starving bacteria was designed, relying on the activation of a promoterless phenol degradation operon of Pseudomonas putida. The Phe+ (phenol-utilizing) mutants accumulated in the starving culture of P. putida in the presence of phenol but not in the absence of it. We ruled out the possibility that the absence of phenol eliminates Phe+ mutants from the starving population. Sequence analysis of the Phe+ mutants revealed that base substitutions, deletions, and insertion of Tn4652 can result in creation of a sequence similar to the sigma70-specific promoter consensus. One particular C --> A transversion was predominant in the Phe+ mutants that arose in the starving population under selection for phenol use. In contrast, various deletions were the most frequent Phe+ mutants occurring in a culture growing without selection. The accumulation rate of the Phe+ mutants on selective plates was found to be higher for bacteria plated from stationary-phase culture than that from exponentially growing cells. This suggests that some specific processes, occurring predominantly in stationary-phase cells, facilitate generation and/or fixation of such mutations.