Pseudomonas aeruginosa dihydroorotases:: a tale of three pyrCs

被引:16
作者
Brichta, DM [1 ]
Azad, KN [1 ]
Ralli, P [1 ]
O'Donovan, GA [1 ]
机构
[1] Univ N Texas, Dept Biol Sci, Denton, TX 76203 USA
关键词
D O I
10.1007/s00203-004-0687-z
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 colipyrC 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.
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页码:7 / 17
页数:11
相关论文
共 40 条
[1]  
ALTSHUL SF, 1997, NUCLEIC ACIDS RES, V25, P3308
[2]   COORDINATION OF SYNTHESIS OF ENZYMES IN PYRIMIDINE PATHWAY OF E COLI [J].
BECKWITH, JR ;
PARDEE, AB ;
JACOB, F ;
AUSTRIAN, R .
JOURNAL OF MOLECULAR BIOLOGY, 1962, 5 (06) :618-&
[3]  
BRICHTA DM, 2003, THESIS U N TEXAS DEN
[4]   PYRIMIDINE BIOSYNTHETIC-PATHWAY OF PSEUDOMONAS-FLUORESCENS [J].
CHU, CP ;
WEST, TP .
JOURNAL OF GENERAL MICROBIOLOGY, 1990, 136 :875-880
[5]   PYRIMIDINE BIOSYNTHESIS IN SACCHAROMYCES-CEREVISIAE - THE URA2 CLUSTER GENE, ITS MULTIFUNCTIONAL ENZYME PRODUCT, AND OTHER STRUCTURAL OR REGULATORY GENES INVOLVED IN DENOVO UMP SYNTHESIS [J].
DENISDUPHIL, M .
BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE, 1989, 67 (09) :612-631
[6]   Electroporation of freshly plated Escherichia coli and Pseudomonas aeruginosa cells [J].
Enderle, PJ ;
Farwell, MA .
BIOTECHNIQUES, 1998, 25 (06) :954-+
[7]   CONSTRUCTION OF BROAD-HOST-RANGE PLASMID VECTORS FOR EASY VISIBLE SELECTION AND ANALYSIS OF PROMOTERS [J].
FARINHA, MA ;
KROPINSKI, AM .
JOURNAL OF BACTERIOLOGY, 1990, 172 (06) :3496-3499
[8]  
Galperin M.Y., 1999, ORG PROKARYOTIC GENO, P91
[9]   A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences:: application for isolation of unmarked Pseudomonas aeruginosa mutants [J].
Hoang, TT ;
Karkhoff-Schweizer, RR ;
Kutchma, AJ ;
Schweizer, HP .
GENE, 1998, 212 (01) :77-86
[10]   CHROMOSOMAL GENETICS OF PSEUDOMONAS [J].
HOLLOWAY, BW ;
KRISHNAPILLAI, V ;
MORGAN, AF .
MICROBIOLOGICAL REVIEWS, 1979, 43 (01) :73-102