Sigma 54 levels and physiological control of the Pseudomonas putida Pu promoter

Abstract

The cellular levels of the alternative sigma factor σ54 of Pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins. The levels of σ54 were also monitored in P. putida strains with knockout mutations inptsO orptsN, known to be required for the C-source control of the σ54-dependent Pu promoter of the TOL plasmid. Our results show that ∼80 ± 26 molecules of σ54 exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of σ54 determine adaptation and differentiation when cells face starvation, σ54 in P. putida remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the ptsO and ptsN mutant strains analyzed. The number of σ54 molecules per cell in P. putida is barely above the predicted number of σ54-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other σ54-dependent systems) may become amenable to physiological control.