The ptsN gene of Pseudomonas putida encodes IIA(Ntr), a protein of the phosphoenol pyruvate:sugar phosphotransferase (PTS) system which is required for the C source inhibition of the sigma(54)-dependent promoter Pu of the TOL (toluate degradation) plasmid pWW0. Using two-dimensional gel electrophoresis, we have examined the effect of ptsN disruption on the general expression pattern of P. putida. To this end, cells were grown in the presence or absence of glucose, and a 1,117-spot subset of the P. putida proteome was used as a reference for comparisons. Among all gene products whose expression was lowered by this carbon source (247 spots [about 22%]), only 6 behaved as Pu (i.e., were depressed in the ptsN background). This evidenced only a minor role for IIA(Ntr) in the extensive inhibition of gene expression in P. putida caused by glucose. However, the same experiments revealed a large incidence of glucose-independent effects brought about by the ptsN mutation. As many as 108 spots (ca. 9% of the cell products analyzed) were influenced, positively or negatively, by the loss of IIA(Ntr). By matching this pattern with that of an rpoN::OmegaKm strain of P. putida, which lacks the sigma(54) protein, we judge that most proteins whose expression was affected by ptsN were unrelated to the alternative sigma factor. These data suggest a role of IIA(Ntr) as a general regulator, independent of the presence of repressive carbon sources and not limited to sigma(54)-dependent genes.