Identification of an acetoacetyl coenzyme A synthetase-dependent pathway for utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium meliloti

Abstract

D-(-)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of Sinorhizobium meliloti. In contrast, wild-type S. meliloti is not able to utilize the L-(+) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the S. meliloti acsA2 gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in S. meliloti is AcsA2 dependent.

FIG. 1.

Growth kinetics of the wild-type strain and representative mutants on different carbon sources. Strains used were Rm1021 (wild type) (▪), Rm11172 (age-1::Tn5-Tp) (⧫), Rm11107 (bdhA₁::Tn5) (•), and Rm11281 (bdhA₁::Tn5 age-1::Tn5-Tp) (▴). Carbon sources used were 10 mM glucose(A), 30 mM acetate (B), 15 mM AA (C), and 15 mM DLHB (D).

FIG. 2.

Genetic map of the S. meliloti chromosome, showing the locations of the acsA2 gene, required for utilization of AA, and age-1, an insertion in which results in an increased growth rate on AA and HB. Arrows indicate positions and orientations of Tn5-mob insertions. The positions of the auxotrophic markers are from the genetic map of the chromosome (17). The positions of the mapped loci are approximate and are based on conjugation and transduction linkage data (8). The relative order of the Tn5-233 insertions and the suppressor alleles depicted is arbitrary. Cotransduction frequencies are given as percentages below the arrows. The tail of each arrow indicates the donor marker and the head represents the recipient in transduction.

FIG. 3.

Growth kinetics of Rm1021 (wild type) and Rm11107 (bdhA₁::Tn5) containing multiple copies of acsA2 (pGQ105) or parent vector plasmid (pSP329) on different carbon sources. Strains used were Rm1021(pSP329) (▪), Rm1021(pGQ105) (□), Rm11107(pSP329) (•), and Rm11107(pGQ105) (○). Carbon sources were 10 mM glucose (A), 30 mM acetate (B), 15 mM AA (C), 15 mM DLHB (D), 15 mM DHB (E), and 15 mM LHB (F).