Consequences of reduced intracellular coenzyme A content in Escherichia coli

Abstract

Escherichia coli 0-alanine auxotrophs (panD2) were used to manipulate the specific cellular content of coenzyme A (CoA) and assess the associated physiological effects. Growth-limiting concentrations of CoA resulted in an increase in phospholipid/protein ratio in reUA mutants, but not in their rel+ counterparts4 indicating that protein biosynthesis was more severely affected by CoA deprivation than phospholipid biosynthesis. Acetyl-CoA was the dominant component (79.8%) of the CoA pool in cells exponentially growing in glucose-minimal medium, with significant concentrations of CoA (13.8%) and succinyl-CoA (5.9%) also detected. Malonyl-CoA was a minor species (0.5%), and the mixed digulfide of CoA and glutathione was not present. Acetyl-CoA was also the major constituent in cells depleted of CoA. On the other hand, succinyl-CoA was absent, suggesting that the protein synthesis defect may be due to the inability to generate sufficient quantities of precursors via the tricarboxylic acid cycle to support amino acid biosynthesis. PIroduction of acyl carrier protein was controlled in part by the availability of CoA, and the lower concentration of acyl carrier protein in CoA-depleted cells was associated with a concomitant decrease in the saturated/unsaturated fatty acid ratio.Coenzyme A (CoA) and acyl carrier protein (ACP) are the major cellular constituents in Escherichia coli that are biosynthesized from the vitatnii pantothenic acid (1,22). Both compounds function as coenzymes in the metabolism of acyl moieties which are bound as thioesters to the terminal sulfhydryl of the 4'-phosphopantetheine portion of these molecules. These two cofactors are interrelated, in that CoA serves as the donor of 4'-phosphopantetheine to apo-ACP (8), and the prosthetic group of ACP turns over (13,21), thus releasing 4'-phosphopantetheine that is either reused for CoA biosynthesis (14, 21) or irreversibly excreted from the cell (14). Several investigators have observed that the intracellular concentration of CoA can be manipulated by varying the amount of pantothenate (1, 11) or 3-alanine (11,12,14,21) Showe and DeMoss (24). Cells were washed once with 50 mM Tris hydrochloride, pH 7.5, and 1 mM MgCl2 and then resuspended in 0.4 ml of the same buffer containing 100 ,ug of lysozyme per ml and 4 ,ug DNase I per ml. The suspension was freeze-thawed three times with a dry ice and ethanol bath and a 37°C water bath, followed by sonication in a cuphorn attached to a Heat Systems sonicator for three 10-s intervals at an output setting of 10. The resulting sample was then centrifuged for 15 min in a Beckman Microfuge-12 (Beckman Instruments, Inc., Fullerton, Calif.). The pellet contained less than 2% of the ACP plus CoA in the total extract. The amount of CoA and ACP,in the P-alanine-labeled cell extracts was determined by thin-layer chromatography (11).Protein was determined by using the microbiuret method (20), with bovine serum albumin as the standard. Cell pellets were dissolved in 1 N NaOH at 60°C prior to protein measurements. Phosphol...

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

“…The unchanged CoA level was consistent with the continued protein synthesis (Fig. 4B), because the primary physiological effect of reduced CoA is the inhibition of protein synthesis (26).…”

“…However, the requirement for CoA in rich medium is very low. The primary physiological effect of reduced CoA levels is the inhibition of protein biosynthesis (26), which is primarily due to the inability to produce glutamate when cells are grown in minimal medium (27). This limitation occurs when the CoA drops to 5% of the normal level in cells grown in minimal medium, and the CoA requirement is even lower in rich broth (28).…”

“…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.…”

Changes in the Intracellular Concentration of Acetyl-CoA and Malonyl-CoA in Relation to the Carbon and Energy Metabolism of Escherichia coli K12

“…However, few measurements of the intracellular concentration of the acyl thioesters have been reported, except for recent papers by Bockner & Ames (1982), Jackowski & Rock (1986) and Vallari et al (1987). The procedures used in these studies for determination of CoA-thioesters are complicated and insufficiently sensitive to define rapid changes of intracellular acyl-CoAs.…”

Acyl Carrier Protein (ACP) Import into Chloroplasts

“…Labeled CoA was obtained using an E. coli strain, SJ16 (panD2 zad-220: :TnlO relAl metBl spoTl gyr A216 lumbar F-), kindly provided by Dr John E. Cronan (University of Illinois), that is auxotrophic for p-alanine, essentially as described (Jackowski and Rock, 1986). The cells were incubated at 37°C overnight in minimal medium E (Vogel and Bonner, 1956) with 0.4% glucose, 0.001% thiamin, 0.01% methionine, and 1 pM unlabelled p-alanine; 30 p1 of this culture was used to inoculate 3 ml of the same medium without p-alanine, and the culture was incubated at 37°C for 2 h to deplete the unlabeled CoA.…”