Isolation and characterization of temperature-sensitive pantothenate kinase (coaA) mutants of Escherichia coli

Abstract

Escherichia coli mutants conditionally defective in the conversion of pantothenate to coenzyme A were isolated and characterized. The gene was designated coaA and localized between argEH and rpoB near min 90 of the chromosome. The coaAl5(Ts) mutation caused a temperature-sensitive growth phenotype and temperature-dependent inactivation of pantothenate kinase activity assayed both in vivo and in vitro. At 30°C, coaA15(Ts) extracts contained less than 20% of the wild-type pantothenate kinase activity; the kinase had near normal kinetic constants for the substrates ATP and pantothenate and was inhibited by coenzyme A to the same degree as the wild-type enzyme. These data define the coaA gene as the structural gene for pantothenate kinase.Coenzyme A (CoA) is synthesized by a series of reactions beginning with the phosphorylation of the vitamin pantothenic acid (1). In Escherichia coli, pantothenate kinase catalyzes the rate-controlling step in the pathway (9,10

Acyl Carrier Protein Is a Cellular Target for the Antibacterial Action of the Pantothenamide Class of Pantothenate Antimetabolites

“…The pantothenate permease (PanF) of E. coli catalyzes the concentrative uptake of pantothenate by a sodium ion cotransport mechanism (6 -8). Strain DV1 is defective in PanF and has lost the ability to utilize extracellular pantothenate for growth (6). To determine whether the uptake of the pantothenamides was through the PanF permease, the MIC values of the compounds for strain DV1 were obtained and compared with the panF ϩ wild-type strain UB1005.…”

“…The E. coli strains used for minimal inhibitory concentration (MIC) determination were as follows: UB1005 (metB1, relA1, spoT1, R , Ϫ , gyrA216, F Ϫ ); ANS1 (tolC::Tn10, metB1, relA1, spoT1, R , Ϫ , gyrA216, F Ϫ ), a derivative of strain UB1005 that is defective in TolC-dependent type I secretion and efflux pumps (17); and DV1 (panF11, panD2, zad220::Tn10, metB1, relA1, spoT1, R , Ϫ , gyrA216, F Ϫ ), a ␤-alanine auxotroph that is also defective in pantothenate uptake (6). Strain SJ16 (panD2, zad220::Tn10, metB1, relA1, spoT1, R , Ϫ , gyrA216, F Ϫ ) is a ␤-alanine auxotroph (18) and was used in the CoA labeling experiment.…”

“…The last two steps are carried out by two separate enzymes, namely phosphopantetheine adenylyltransferase (CoaD) (3,4) followed by the addition of the 3Ј-ribose phosphate by dephospho-CoA kinase (CoaE) (5). E. coli is capable of de novo pantothenate biosynthesis (1) or can import pantothenate from the medium via a sodium-dependent active transport process (6)(7)(8). CoA is also required for the synthesis of ACP, the acyl group carrier in bacterial fatty acid synthesis.…”

Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity

“…(Epstein & Kim, 1971) were from our frozen stocks. MJF556 (Frag1, metB zij-2009 : : Tn10) was created by transducing Frag1 to tetracycline resistance using a P1 lysate grown on strain DV62 (metB zij-2009 : : Tn10) (Vallari & Rock, 1987). The co-transduction of the Tn10 marker and the metB allele was confirmed by demonstrating that MJF556 was auxotrophic for methionine.…”

Pantothenate Kinase Regulation of the Intracellular Concentration of Coenzyme A

“…E. coli is capable of de novo pantothenate biosynthesis, and additionally a sodium-dependent permease actively transports exogenous pantothenate into the cell (7)(8)(9). Metabolic labeling experiments established that the utilization, rather than the supply of pantothenate, controls the level of CoA (10).…”