Involvement of Pseudomonas putida RpoN sigma factor in regulation of various metabolic functions

Abstract

The RpoN protein was originally identified in Escherichia coli as a sigma (sigma) factor essential for the expression of nitrogen regulons. In the present study we cloned the Pseudomonas putida rpoN gene and identified its gene product as a protein with an apparent molecular weight of 78,000. A mutant rpoN gene was constructed by in vitro insertion mutagenesis with a kanamycin cassette. A P. putida rpoN mutant was then isolated by replacement of the intact chromosomal rpoN gene by the mutant rpoN gene through homologous recombination. Examination of the phenotypes of the P. putida rpoN mutant thus obtained allowed the identification of a series of metabolic functions whose expression depends upon the RpoN sigma factor. The rpoN mutation in P. putida affected the utilization by this organism of nitrate, urea, and uncharged amino acids, namely, alanine, glycine, isoleucine, leucine, and serine, as nitrogen sources. The mutation also affected the utilization of the above-mentioned amino acids, as well as lysine, C4-dicarboxylates (succinate, fumarate), and alpha-ketoglutarate, as carbon sources. In contrast to the P. putida wild-type strain, the rpoN mutant was nonmotile. The colony morphology of the mutant strain was different from that of the wild-type strain. Studies on the expression of the TOL plasmid catabolic operons in the mutant strain demonstrated that transcription from the upper-operon promoter and from the xylS gene promoter requires the RpoN sigma factor.

Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

“…They can also utilize lysine as a nitrogen source though not as a carbon source. In P. putida, the inability of the rpoN mutant to grow on lysine is probably due to the fact that lysine decarboxylase is under 54 control (38). The major phenotypic difference that we observed between the P. putida and the P. syringae rpoN mutants was that the P. syringae rpoN mutant could not utilize proline and histidine as nitrogen sources, whereas the P. putida rpoN mutant could.…”

“…The amino acid sequence of the ES4326 54 protein is most closely related to the P. putida 54 protein, and the phenotype of the ES4326 rpoN mutant resembles the phenotype of P. putida rpoN mutants (38). Both P. syringae and P. putida rpoN mutants grow more slowly than their wild-type counterparts in all media tested and were unable to utilize several uncharged amino acids as substrates.…”

“…Carbon and nitrogen source utilization tests for ES4326 rpoN mutants were performed in M9 salts minimal medium by providing a (23) carbon source at 10 mM and by replacing ammonium chloride with an alternative nitrogen source at 5 mM when required. Bacterial motility was tested on "swarm plates" consisting of 0.3% agar, 0.5% NaCl and 0.5% tryptone (38). Antibiotic concentrations for E. coli and P. syringae strains were as follows: streptomycin, 150 g/ml; kanamycin, 25 g/ml; tetracycline, 12 g/ml; gentamicin, 20 g/ml; and spectinomycin, 20 g/ml.…”

The IIANtr (PtsN) Protein of Pseudomonas putida Mediates the C Source Inhibition of the ς54-dependent Pu Promoter of the TOL Plasmid

“…A 3.2-kb SalI-ScaI fragment from plasmid pNTR1 (spanning rpoN and 1.5 kb downstream; Ref. 40) was cloned in vector pBlueScript KS ϩ digested with SmaI and SalI. To generate a Km insertion in ORF102, the resulting plasmid (pBSNtr) was digested with HindIII, and the overhanging ends were filled in with dNTPs using the Klenow fragment of DNA polymerase (35) and ligated to an equally filled in 0.8-kb segment of pUCKm bearing the promoterless kanamycin cassette mentioned above.…”

“…For constructing pJM154 (a ptsN ϩ broad host range plasmid), a 0.78-kb PstI fragment from pARG5.1 (spanning the corresponding chromosomal region of P. putida; Ref. 40) was cloned in pNot18. This vector is a variant of pUC18 in which the entire ␣-lac segment and the Plac promoter of the plasmid are flanked by NotI sites (41).…”