Pseudomonas aeruginosa dihydroorotases: a tale of three pyrCs
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.
KeywordsOrotate Orotate Phosphoribosyl Transferase Xylella Fastidiosa Potential Promoter Region Pseudomonas Isolation Agar
We thank John Houghton and Tim Brown for kind assistance with DNA sequence analysis of pyrC, and the members of our laboratory for helpful discussions. We also appreciate the helpful suggestions and comments on the manuscript made by Tim Hoover, Rod Kelln and two anonymous reviewers. This work was supported by a faculty research grant from the College of Arts and Sciences at UNT (G.A.O’D).
- Altshul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein data base search programs. Nucleic Acids Res 25:3308–3402Google Scholar
- Brichta DM (2003) Construction of a Pseudomonas aeruginosa dihydroorotase mutant and the discovery of a novel link between pyrimidine intermediates and virulence response. PhD dissertation. University of North Texas, DentonGoogle Scholar
- Galperin MY, Tatusov RL, Koonin EV (1999) Comparing microbial genomes: how the gene set determines the lifestyle. In: Charlebois RL (ed) Organization of the prokaryotic genome. American Society for Microbiology, Washington, pp 91–108Google Scholar
- Miller JH (1992) A short course in bacterial genetics; A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 72–74, 438Google Scholar
- Neuhard J, Kelln RA (1996) Biosynthesis and conversions of pyrimidines. In: Neidhardt FC, Curtiss R, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella: cellular and molecular biology, 2nd edn. American Society for Microbiology, Washington, pp 580–599Google Scholar
- Sambrook J, Russell DW (2000) Molecular cloning: a laboratory handbook, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
- Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1:784–791Google Scholar
- Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959–964CrossRefPubMedGoogle Scholar
- Vickrey JF (1993) Characterization of the aspartate transcarbamoylase in Pseudomonas aeruginosa. PhD thesis. University of North Texas, DentonGoogle Scholar