Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase

Abstract

The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-ion. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30°C and 5% of that at 40°C. At 42°C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30°C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30°C and almost the same in both strains at 42°C. The mutant had no obvious phenotype regarding its growth properties.It is well known that 3-hydroxy-3-methylglutaryl coenzyme A reductase is a rate-limiting enzyme in the biosyntheses of isoprenoid compounds, undergoing multivalentfeedback regulation (4). However, a branch point of the synthetic pathway of various isoprenoids seems to be a reaction utilizing isopentenyl diphosphate (IPP) or farnesyl diphosphate (FPP) or both. For elucidation of the mechanism by which the syntheses of various isoprenoids are regulated differently, many researchers have studied the regulation of activities of the enzymes, utilizing IPP or FPP or both as a substrate (1,(5)(6)(7)11).Escherichia coli includes isoprenoid quinones (ubiquinone-8, menaquinone-8, and demethylmenaquinone-8) having an all-E-octaprenyl side chain (8) and sugar carrier lipid (decaprenyl phosphate, undecaprenyl phosphate, and dodecaprenyl phosphate) containing a Z,E-mixed isoprenoid chain (27). Because the composition of isoprenoids in E. coli is simpler than that in eucaryotes, E. coli is a useful system as a model for studying the biosynthesis of nonsterol isoprenoids and its regulation. We have studied the biosynthetic enzymes, utilizing IPP as a substrate in E. coli, and separated IPP isomerase and three prenyltransferases, namely, FPP synthase, octaprenyl diphosphate synthase, and undecaprenyl diphosphate synthase, from each other (13). FPP synthase catalyzes the condensation of IPP with dimethylallyl diphosphate (C5) or geranyl diphosphate (C1O) to give FPP (C15). The latter two enzymes catalyze the sequential condensation of IPP with FPP to give long-chain polyprenyl diphosphates which are precursors of isoprenoid quinones and sugar carrier lipid.One effective approach to the elucidation of the role of a certain enzyme in vivo is to isolate the mutant containing the defective enzyme and to characterize it. We screened a collection of temperature-sensitive mutants of E. coli (24) for defects in isoprenoid synthesis by measuring 14C incorpora-* Corresponding author. Screening of temperature-sensitive mut...

Genetic and biochemical analyses of the biosynthesis of the yellow carotenoid 4,4'-diaponeurosporene of Staphylococcus aureus

“…Indeed, it was shown that E. coli possesses the gene encoding farnesyl diphosphate synthase (ispA) (11).…”

A Defective Undecaprenyl Pyrophosphate Synthase Induces Growth and Morphological Defects That Are Suppressed by Mutations in the Isoprenoid Pathway of Escherichia coli

“…Given their fundamental importance, it is not surprising that mutations in isoprenoid pathways can affect bacterial growth, but it is intriguing that some of these mutations also alter cell shape (34)(35)(36)(37)(38)(39). The usual explanation for either phenotype is that interfering with isoprenoid synthesis alters the rate of production of Und-P, which in turn may affect cell wall synthesis (37,38,40,41). In a similar vein, sequestering Und-P in unusable intermediates also makes it less available for PG synthesis, leading to assorted defects in growth and cell shape (42)(43)(44).…”

Evaluation of risedronate as an antibiofilm agent

“…The mutant contains less than 13 and 18 %, respectively, of ubiquinone-8 and menaquinone-8 and 40-70 % of the WT levels of undecaprenyl phosphate, although a low level of geranyl diphosphate synthase activity has been reported in ispA null mutants, which may be due to the activity of other prenyltransferases (Fujisaki et al, 1989(Fujisaki et al, , 2005Saito et al, 2007). The effects of these changes on biofilm formation have not been studied.…”