The β-Oxidation Systems ofEscherichia coliandSalmonella entericaAre Not Functionally Equivalent

Abstract

Based on its genome sequence, the pathway of ␤-oxidative fatty acid degradation in Salmonella enterica serovar Typhimurium LT2 has been thought to be identical to the well-characterized Escherichia coli K-12 system. We report that wild-type strains of S. enterica grow on decanoic acid, whereas wild-type E. coli strains cannot. Mutant strains (carrying fadR) of both organisms in which the genes of fatty acid degradation (fad) are expressed constitutively are readily isolated. The S. enterica fadR strains grow more rapidly than the wild-type strains on decanoic acid and also grow well on octanoic and hexanoic acids (which do not support growth of wild-type strains). By contrast, E. coli fadR strains grow well on decanoic acid but grow only exceedingly slowly on octanoic acid and fail to grow at all on hexanoic acid. The two wild-type organisms also differed in the ability to grow on oleic acid when FadR was overexpressed. Under these superrepression conditions, E. coli failed to grow, whereas S. enterica grew well. Exchange of the wild-type fadR genes between the two organisms showed this to be a property of S. enterica rather than of the FadR proteins per se. This difference in growth was attributed to S. enterica having higher cytosolic levels of the inducing ligands, long-chain acyl coenzyme As (acyl-CoAs). The most striking results were the differences in the compositions of CoA metabolites of strains grown with octanoic acid or oleic acid. S. enterica cleanly converted all of the acid to acetyl-CoA, whereas E. coli accumulated high levels of intermediate-chain-length products. Exchange of homologous genes between the two organisms showed that the S. enterica FadE and FadBA enzymes were responsible for the greater efficiency of ␤-oxidation relative to that of E. coli.

Stress sigma factor RpoS degradation and translation are sensitive to the state of central metabolism

“…Addition of other carbon sources that do not directly improve acetyl-CoA synthesis (succinate and oleate) did not mimic the effect of acetate in reversing RpoS stability (Fig. S6 C and D) (28). These results are consistent with a depletion of acetyl-CoA or its products as the major defect in the aceE mutant.…”

Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

“…The CoA pools were extracted from strain SI192 after overnight growth in minimal medium supplemented with ß-[3-3 H]alanine and the extracted compounds were dephosphorylated and subsequently re-phosphorylated using [γ-33 P]ATP as described in Materials and Methods. The reaction mixtures were then separated via HPLC [10]. The CoA and CoA ester peaks were collected and the levels of 3 H and 33 P were determined by dual channel scintillation counting.…”

“…The CoA and CoA esters were separately dephosphorylated then a portion of each compound was rephosphorylated with DPCK-Hs as per the standard reaction conditions. The products of both the dephosphorylation and re-phosphorylation reactions were chromatographed on a C18 HPLC column as described previously [10]. The absorbance at 254 nm was measured over time.…”

“…Extraction with chaotrophic acids has long been known to completely release the intracellular contents of E. coli cells [7;14;15;16]. The protocol for biosynthetic labeling was that of Iram and Cronan [10] E. coli strain SI92 was grown on minimal E plates supplemented with 20 μg/ml chloramphenicol, 0.01% methionine, 0.2% glucose, and 0.5 μM β-alanine overnight at 37°C. The cells were then plated on minimal E plates supplemented as above, but lacking β-alanine to deplete the preexisting CoA pools.…”

“…Strain SI92, a ΔpanD derivative of Escherichia coli K-12 strain lacking asparate-1-decarboxylase was provided by Dr. S. Iram [10]. The E. coli and Aquifex aeolicus DPCK genes (DPCK E and DPCK A , respectively) were cloned into vector pET28b and expressed in strain E. coli BL21(DE3).…”