Sulfolobus acidocaldarius synthesizes UMP via a standard de novo pathway: results of biochemical-genetic study

Abstract

A genetic approach was used to establish the route of UMP biosynthesis in Sulfolobus acidocaldarius, a member of the hyperthermophilic division (the Crenarchaeota) of the Archaea domain. Pyrimidine auxotrophs of S. acidocaldarius DG6 were isolated by direct selection and by brute-force methods. Enzymatic assay of extracts from wild-type S. acidocaldarius, from pyrimidine auxotrophs, and from phenotypic revertants demonstrated that S. acidocaldarius synthesizes UMP via orotate in six enzymatic steps corresponding to the de novo pathway of other organisms. The results also show that a single carbamoyl phosphate synthetase supplies both the pyrimidine and arginine pathways of this organism. To gain similar insight into pyrimidine salvage pathway(s), prototrophic mutants resistant to toxic pyrimidine analogs were also isolated and characterized. The results suggest that a single class of mutants which had acquired elevated resistance to four different 5-fluoropyrimidines had been isolated. These fluoropyrimidine-resistant mutants appear to have a regulatory defect leading to overproduction of one or more endogenous pyrimidine compounds.

Genetic fidelity under harsh conditions: Analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius

“…It has 13% residual OPRTase activity and a low level of FOA resistance and can grow to a limited extent in the absence of uracil supplementation (7). The fact that the A 7 3 A 6 frameshift is both leakier and also much more frequent than most pyrE alleles explains prior observations that low-stringency FOA selection yields two phenotypically distinct classes of pyr mutants in approximately equal numbers (7). As deduced from the nucleotide sequence, this Ϫ1 frameshift modifies the carboxyl terminus of OPRTase by replacing the last 14 residues with SSYLIIW.…”

“…The remaining mutants, which contain intragenic additions and deletions, exhibited the tight auxotrophic phenotype associated with complete loss of enzyme function (7). Those from sets I and II therefore can be pooled for analysis.…”

“…The laboratory designation for the resulting strain is DG185. Unless otherwise noted, growth media (pH 3.5) consisted of a mineral mixture supplemented with xylose, tryptone, and uracil; plates contained gellan gum for solidification (7). Incubation was at 75°C, which is near the midpoint of the temperature range for growth for this species (6).…”

Photoreactivation in an archaeon from geothermal environments

“…These two wildtype strains exhibit the same profile of restriction fragments (Grogan, 1989;McAfee et al, 1995) but have different histories (Grogan, 1991) and yield colonies of slightly different opacity on some solid media (unpublished results). Cultures were grown from single colonies in xylose/tryptone medium (Grogan & Gunsalus, 1993) at 80 "C with continuous aeration. Numbers of viable cells were determined by serial 1 : 10 dilution of cell suspensions in Sdil buffer (Grogan, 1996) (D,,,), where the concentration of cells in a reference culture was determined by this study, stationary-phase cultures have a cell density of about 9 x lo8 cells m1-l.…”

Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications

“…Strains used in this study are listed in Table 1. S. acidocaldarius pyrE mutants were cultured in uracilsupplemented xylose-tryptone medium (Grogan & Gunsalus, 1993) and were electroporated as described previously (Grogan & Rockwood, 2010). Recombinants were selected on xylose-tryptone plates lacking uracil, streaked for isolation on the same medium, and cryo-preserved.…”