Sigma 54 Levels and Physiological Control of the Pseudomonas putida Pu Promoter

Abstract

The cellular levels of the alternative sigma factor 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 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 54 -dependent Pu promoter of the TOL plasmid. Our results show that ϳ80 ؎ 26 molecules of 54 exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of 54 determine adaptation and differentiation when cells face starvation, 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 54 molecules per cell in P. putida is barely above the predicted number of 54 -dependent promoters. These figures impose a framework on the mechanism by which Pu (and other 54 -dependent systems) may become amenable to physiological control.Bacterial RNA polymerase (RNAP) holoenzymes are assembled by a common catalytic core enzyme that associates with a polypeptide () conferring promoter recognition specificity. The majority of bacteria have alternative factors, most of which show homology with the major factor of Escherichia coli ( 70 ) (34). A different class is composed of a unique member ( 54 , encoded by rpoN) that differs both in amino acid sequence and mechanism of transcription activation (5). In essence, 54 -RNAP holoenzyme forms a stable closed complex at the target promoter that is activated by a specialized family of regulators (20, 25) in a nucleotide (nucleoside triphosphate) hydrolysis-dependent manner (12,26,32).

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…A similar role in adaptation to changes in growth conditions can be attributed to EI Ntr of P. aeruginosa and L. pneumophila, considering that ptsP mutants of these pathogens grow normally in rich medium but grow very slowly after infection of a host cell. 54 present in the cell does not seem to be controlled by PTS Ntr , as it is always low and is affected neither by growth on glucose nor by ptsN or npr mutations (388). Many effects of the ptsN, npr, or ptsP mutations, such as those on melanin production, poly-␤-hydroxybutyrate accumulation, Pu promoter expression, or era activity, seem to be unrelated to nitrogen metabolism.…”

“…In P. putida, EIIA Ntr affects the regulation of gene expression from the TOL plasmid. The TOL plasmid harbors the genes necessary for the metabolism of toluene and xylenes and contains four transcription units: a so-called upper pathway and a meta pathway operon flanked by the promoters Pu and P M , respectively, and the regulator-encoding genes xylS and xylR connected to the promoters P S and P R , respectively (28,388). Transcription from Pu and P S1 is 54 dependent and is subject to CCR: it is low when cells grow exponentially on rich medium and is also down-regulated by glucose or gluconate but not by fructose (22,101,103).…”

The Upstream-activating Sequences of the σ54 Promoter Pu of Pseudomonas putida Filter Transcription Readthrough from Upstream Genes

“…Second, the key factors involved in Pu functioning ( 54 and the m-xylene responsive regulator XylR) are in very short supply in P. putida. It has been calculated that there are ϳ50 -60 54 -dependent promoters in the genome of this bacterium (58), whereas the number of 54 molecules is as small as 70 -80 per cell (59). Similarly, the number of XylR multimers available for activation of Pu and Ps (a second 54 promoter of the TOL plasmid) may not be above 10 per bacterium (60).…”

m -Xylene-Responsive Pu - PnifH Hybrid σ 54 Promoters That Overcome Physiological Control in Pseudomonas putida KT2442

“…Growth phase control of Po has been attributed to the ability of the alarmone ppGpp to facilitate the access of 54 to the core RNAP during sigma factor competition at stationary phase (33,37). Such competition is critical in the case of 54 , given that only a few molecules of the factor (ϳ80) are present in the cell at any growth stage (34). In this context, it is possible that 54 promoters with a better Ϫ12/Ϫ24 region can function even at the low concentrations of 54 -RNAP available prior to stationary phase.…”