Pseudomonas aeruginosa dihydroorotases: a tale of three pyrCs

Abstract

Pseudomonas aeruginosa PAO1 was shown to contain three pyrC sequences. Two of these genes, designated pyrC (PA3527) and pyrC2 (PA5541), encode polypeptides with dihydroorotase (DHOase) activity, while the third, pyrC' (PA0401), encodes a DHOase-like polypeptide that lacks DHOase activity, but is necessary for the structure and function of ATCase. Both pyrC and pyrC2 were cloned and complemented an Escherichia coli pyrC mutant. In addition, pyrC and pyrC2 were individually inactivated in P. aeruginosa by homologous exchange with a mutated allele of each. The resulting mutant strains were prototrophic. A pyrC, pyrC2 double mutant was also constructed, and this strain had an absolute requirement for pyrimidines. The transcriptional activity of pyrC and pyrC2 was measured using lacZ promoter fusions. While pyrC was found to be constitutively expressed, pyrC2 was expressed only in the pyrC mutant background. An in vitro transcriptional/translational system was used to estimate the size of the pyrC2 gene product. The expressed polypeptide was approximately 47 kDa, which is in keeping with the theoretical molecular mass of 48 kDa, making it the largest prokaryotic DHOase polypeptide identified to date. To our knowledge, this is the first report of a true DHOase mutant in P. aeruginosa and also the first confirmation that pyrC2 encodes a polypeptide with DHOase activity.

Nutritional Cues ControlPseudomonas aeruginosaMulticellular Behavior in Cystic Fibrosis Sputum

“…For SCFM, amino acids were added from 100-mM stocks to a buffered base (6.5 ml 0.2 M NaH 2 PO 4 , 6.25 ml 0.2 M Na 2 HPO 4 , 0.348 ml 1 M KNO 3 , 0.122 g NH 4 Cl, 1.114 g KCl, 3.03 g NaCl, 10 mM MOPS, 779.6 ml deionized water) in the following volumes: L-aspartate, 8.27 ml; L-threonine, 10.72 ml; L-serine, 14.46 ml; L-glutamate ⅐ HCl, 15.49 ml; L-proline, 16.61 ml; L-glycine, 12.03 ml; Lalanine, 17.8 ml; L-cysteine ⅐ HCl, 1.6 ml; L-valine, 11.17 ml; L-methionine, 6.33 ml; L-isoleucine, 11.2 ml; L-leucine, 16.09 ml; L-tyrosine, 8.02 ml; L-phenylalanine, 5.3 ml; L-ornithine ⅐ HCl, 6.76 ml; L-lysine ⅐ HCl, 21.28 ml; L-histidine ⅐ HCl, 5.19 ml; L-tryptophan, 0.13 ml; and L-arginine ⅐ HCl, 3.06 ml. SCFM was adjusted to pH 6.8 and filter sterilized through a 0.2-m-pore-size filter.…”

A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life

“…Similar to PyrC', the zinc-binding residues are not conserved in the PyrC paralog whose gene we predict to be regulated by Zur. However, unlike PyrC', this PyrC paralog has previously been shown to display dihydroorotase activity [ 84 ]. For porphobilinogen synthase, the existence of zinc binding and non-zinc binding variants is documented in the literature [ 85 ].…”

The effect of zinc limitation on the transcriptome ofPseudomonas protegens Pf‐5

“…For example, in Methanosarcina thermophila, g-class carbonic anhydrase could function with iron as a cofactor in addition to zinc (Tripp et al, 2004;Macauley et al, 2009). The P. aeruginosa homologue (PA5541) of the Pf-5 non-zinc-requiring PyrC (PFL_6183) was found to be functional (Brichta et al, 2004). Likewise, the zinc-dependent and non-zincrequiring paralogues of FolE2 were found to be functionally redundant (Sankaran et al, 2009).…”