Propionyl coenzyme A is a common intermediate in the 1,2-propanediol and propionate catabolic pathways needed for expression of the prpBCDE operon during growth of Salmonella enterica on 1,2-propanediol

Abstract

The studies reported here identify propionyl coenzyme A (propionyl-CoA) as the common intermediate in the 1,2-propanediol and propionate catabolic pathways of Salmonella enterica serovar Typhimurium LT2. Growth on 1,2-propanediol as a carbon and energy source led to the formation and excretion of propionate, whose activation to propionyl-CoA relied on the activities of the propionate kinase (PduW)/phosphotransacetylase (Pta) enzyme system and the CobB sirtuin-controlled acetyl-CoA and propionyl-CoA (Acs, PrpE) synthetases. The different affinities of these systems for propionate ensure sufficient synthesis of propionyl-CoA to support wild-type growth of S. enterica under low or high concentrations of propionate in the environment. These redundant systems of propionyl-CoA synthesis are needed because the prpE gene encoding the propionyl-CoA synthetase enzyme is part of the prpBCDE operon under the control of the PrpR regulatory protein, which needs 2-methylcitrate as a coactivator. Because the synthesis of 2-methylcitrate by PrpC (i.e., the 2-methylcitrate synthase enzyme) requires propionyl-CoA as a substrate, the level of propionyl-CoA needs to be raised by the Acs or PduW-Pta system before 2-methylcitrate can be synthesized and prpBCDE transcription can be activated.

FIG. 1.

In vivo evidence that proteins with propionate kinase activity restore the growth of a cobB mutant strain on propionate. In the genotypes described below, pBAD refers to cloning vector pBAD30 (18), cobB represents allele cobB1176::Tn10d(tet⁺), and pduN represents allele pduN8::MudJ(kan⁺). In both panels, when added to the medium, arabinose was at 200 μM. (A) Complementation by pduW⁺. Strains (genotype; symbol): JE4175 (cobB⁺ pdu⁺ pta⁺/pBAD; open squares), JE5913 (cobB pduN/pduW⁺; solid circles), JE5945, (cobB pduN/pBAD; solid triangles), JE6954 (cobB pduN pta pduW⁺; solid squares), and JE6953 (cobB pduN pta/pBAD; open circles). The growth behavior of strains JE5913 and JE6954 in the absence of arabinose is represented by open triangles. (B) Complementation by tdcD⁺. Strains (genotype; symbol): JE4175 (open squares); JE6176 (cobB pduN tdcD⁺; solid squares), JE5945 (cobB pduN/pBAD; solid triangles), JE6955 (cobB pduN pta tdcD⁺; solid squares), JE6953 (open circles). The growth behavior of strains JE6176 and JE6955 in the absence of arabinose is represented by open triangles.

FIG. 2.

In vitro evidence that pduW encodes a protein with propionate kinase enzyme activity. Cells were grown in the presence or absence of arabinose (500 μM) and/or 1,2-PDL (12 mM), as indicated. Data shown as solid bars were obtained with extracts of strain JE6925, data shown as open bars were obtained with cell extracts of strain JE6924, and data shown as light gray bars were obtained with cell extracts of strain JE6923. Growth conditions are shown at the bottom. Error bars show the standard deviation of determinations performed in triplicate.

FIG. 3.

In vivo evidence that the PduW-Pta and Acs systems are needed during growth on 1,2-PDL. (A) Strains (symbols): TR6383 (solid squares), JE4597 (open squares), JE4718 (open triangles), JE2445 (solid triangles), and JE2506 (solid diamonds). (B) Strains (symbols): TR6583 (solid squares), JE4597 (open squares), JE6290 (open circles), JE4313 (solid circles), and JE2506 (solid diamonds). Cultures (5 ml of each) were grown in tubes (16 by 150 mm) with shaking at 37°C. Growth was monitored at 650 nm. The culture medium contained: NCE buffer salts, glycerol (1 mM), 1,2-PDL (30 mM), vitamin B₁₂ (150 nM), l-methionine (0.5 mM), and trace minerals.

FIG. 4.

Pro-CoA synthesis during growth on 1,2-PDL. PduCDE, coenzyme B₁₂-dependent propanediol dehydratase; PduP, putative propionaldehyde dehydrogenase; PduW, propionate kinase; Pta, phosphotransacetylase; PrpE, Pro-CoA synthetase; Acs, Ac-CoA synthetase; PrpC, 2-methylcitrate synthase; PrpD, 2-methylcitrate dehydratase; AcnA and AcnB, aconitases A and B, respectively; PrpB, 2-methylisocitrate lyase; TCA, tricarboxylic acid; *, under the control of the CobB sirtuin system; §, the chromosomal level does not compensate for the lack of PduW.