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

The cellular levels of the alternative sigma factor [[sigma].sup.54] 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 [[sigma].sup.54] were also monitored in P. putida strains with knockout mutations in ptsO or ptsN, known to be required for the C-source control of the [[sigma].sup.54]-dependent Pu promoter of the TOL plasmid. Our results show that ~80 [+ or -] 26 molecules of [[sigma].sup.54] exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of [[sigma].sup.54] determine adaptation and differentiation when cells face starvation, [[sigma].sup.54] 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 [[sigma].sup.54] molecules per cell in P. putida is barely above the predicted number of [[sigma].sup.54]-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other [[sigma].sup.54]-dependent systems) may become amenable to physiological control.