Carbamoylphosphate synthetase (CPS) genes of Neisseria gonorrhoeae and other Neisseria species: Novel gene organisation, variable intergenic sequences, characterisation of naturally occurring mutants, and evolution of CPS genes.
Creators: name: Lawson, Fiona S.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 1996
Subjects: subject: Biology, Molecular.

description: Carbamoylphosphate synthetase (CPS) catalyses the formation of carbamoylphosphate from CO$\sb2$, ATP, and glutamine; the first committed step in the arginine and pyrimidine biosynthetic pathways. Initial interest in the CPS enzyme of Neisseria gonorrhoeae stemmed from the natural occurrence of CPS deficiencies in 10-20% of clinical isolates. This property, causing a concurrent citrulline and uracil auxotrophy, is employed in a typing scheme and there have been some indications that this auxotrophy may be beneficial to the gonococcus. In all of the prokaryotes examined, CPS is a heterodimer encoded by two genes commonly called carA and carB. I have now determined the complete sequence of the carA and car B genes from N. gonorrhoeae CH811. carA (1125 bp) and carB (3237 bp) are similar in size to other prokaryotic CPS genes, and encode all the highly conserved regions present in other CPS's. However, these genes in strain CH811 are separated by a 3287 bp intergenic sequence. To determine whether the intervening sequence observed in N. gonorrhoeae CH811 was typical of gonococcal isolates, the sequence between carA and carB was PCR amplified from isolates of N. gonorrhoeae, N. meningitidus, and 9 other Neisseria species. The intervening sequence was found to vary in size, from approximately 2.2 to 3.7 kb, although the carA and carB genes themselves did not vary in size in isolates with functional CPS. The presence of a variable sequence near carA and carB, and the numerous repetitive sequences found in this area in strain CH811, suggests that this region may be a hotspot for recombination and may in part explain the high frequency of occurrence of CPS mutants. PCR amplification of carA and carB from CPS deficient isolates indicate that more than one mutation is responsible for this phenotype. Transformation of a gonococcal CPS mutant with the cloned gonococcal CPS genes has demonstrated that other mutations are present in the arginine and pyrimidine biosynthetic pathways of this isolate, which are hidden by the CPS deficiency. This study also suggests that isolates of auxotype CUH and OUH are closely related. I also report an analysis of the first CPS gene sequence from an archaeon (Sulfolobus solfataricus P2; supplied by R. L. Charlebois). This analysis indicates that the archaeal CPS is similar in function, size and gene organization to other prokaryotic CPS's. A comprehensive phylogenetic analysis of all known CPS genes, including the gonococcal and archaeal sequences, was performed. Using the internal duplication as a root for phylogenetic tree construction, I was able to determine a branching order for the tree of life and provide support for the theory that this archaeon is more related to eukaryotes than bacteria. This is the first report of confirmation of this theory using a metabolic gene and is the first use of an internal duplication within a gene to root the tree of life. This analysis also showed that at least two, and possibly even three, separate gene duplication events led to the formation of two CPS enzymes present in Gram-positive bacteria and most eukaryotes. Comparisons of the branching order with the organization of CPS genes in different organisms further indicated that the evolution of CPS has been complex, involving multiple deletions and insertions. (Abstract shortened by UMI.)