Evidence of multiple regulatory functions for the PtsN (IIA(Ntr)) protein of Pseudomonas putida

Abstract

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.

FIG. 1

2D gels of protein extracts from P. putida MAD2 (wt), P. putida MAD2 ptsN::Km, and P. putida rpoN::ΩKm. Cultures of each strain were grown in M9-AA medium (supplemented or not with 10 mM glucose, as indicated) until early stationary phase (OD₆₀₀ of ∼1.5). Cultures were then labeled with [³⁵S]methionine as explained in Materials and Methods. Protein extracts were first electrofocused in a pH 4 to 7 gradient and then run across a 12.5% denaturing PAGE system. The autoradiographs of a subset of dried 2D gel results used for the scanning are shown here. A selection of spots whose intensity changes depending on the strain is indicated for reference: boxed spots (types I to V) correspond to proteins affected by the lack of ptsN (further examined in Fig. 3); circled spots (affected by glucose) coincide with the proteins whose expression is shown in Fig. 2.

FIG. 2

Expression of selected glucose-repressible spots in P. putida MAD2 (wt), P. putida MAD2 ptsN::Km, and P. putida rpoN::ΩKm. Cultures of each of these strains were grown, labeled, and resolved in 2D gels as explained in the legend to Fig. 1. Expression levels are plotted as a percentage of those of the wild-type strain in the absence of glucose. An expression level of zero indicates levels below detection by our experimental setup. The six spots under scrutiny (numbered 60, 91, 276, 1424, 1428, and 1500 in Fig. 1) are down-regulated by glucose only if the wild-type ptsN gene is present, but they are fully expressed in the ptsN-negative mutant, regardless of the added C source. Expression of these six proteins whose inhibition by glucose was dependent on IIA^Ntr was reexamined in the proteome of the ς⁵⁴-negative strain. Note that spots 60 and 91 are derepressed in the rpoN::ΩKm strain as they are in the ptsN::Km background, thereby indicating that their down-regulation by C source is IIA^Ntr dependent but ς⁵⁴ independent (see text for explanation).

FIG. 3

Types of spots in 2D protein gels of P. putida KT2442 and its ptsN::Km and rpoN::ΩKm variants. The photographs to the left show representative spots from the 2D gels (numbered as shown in Fig. 1), whereas the bars to the right are a quantification of the intensities of the selected proteins that fall under the various categories, as a percentage of the full expression levels, as indicated. (A) Spots whose expression is lessened in the ptsN-negative strain. Proteins of this kind do appear whose expression is either reduced in both the ptsN::Km strain and the rpoN::ΩKm strain (type I) or whose level declines in the ptsN mutant but not in the rpoN strain (type II). (B) Products whose expression is increased in the ptsN-negative mutant. Among these, some proteins are ς⁵⁴-dependent products (type III), others are ς⁵⁴ independent (type IV; expression is increased both in the ptsN-negative and rpoN-negative strains), and yet another class of spots (type V) augment their expression in the ptsN-negative mutant but are missing in the rpoN strain. Photographs and quantification values shown are from the cultures not supplemented with glucose. Similar patterns were detected in extracts from cells grown with glucose. (See text for interpretation.) WT, wild type.

FIG. 4

Connections between IIA^Ntr, ς⁵⁴, and glucose-repressible expression in P. putida as revealed by 2D gel analysis of ptsN::Km and rpoN::ΩKm mutants. Among the 1,117-spot subset of the P. putida proteome, as many as 247 products were repressible by glucose (i.e., had expression ≥2-fold lower in the presence of the sugar), while 93 proteins were dependent on ς⁵⁴ (i.e., were entirely missing in extracts of the rpoN strains under all conditions). The ptsN-regulated spots considered for this representation include only those whose changes (≥5-fold greater or lesser than the wild-type levels) can be complemented by a functional ptsN copy. The areas of the circles are approximately proportional to the number of spots included in the categories represented.