Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate

Abstract

3-Hydroxypropionate is a product or intermediate of the carbon metabolism of organisms from all three domains of life. However, little is known about how carbon derived from 3-hydroxypropionate is assimilated by organisms that can utilize this C(3) compound as a carbon source. This work uses the model bacterium Rhodobacter sphaeroides to begin to elucidate how 3-hydroxypropionate can be incorporated into cell constituents. To this end, a quantitative assay for 3-hydroxypropionate was developed by using recombinant propionyl coenzyme A (propionyl-CoA) synthase from Chloroflexus aurantiacus. Using this assay, we demonstrate that R. sphaeroides can utilize 3-hydroxypropionate as the sole carbon source and energy source. We establish that acetyl-CoA is not the exclusive entry point for 3-hydroxypropionate into the central carbon metabolism and that the reductive conversion of 3-hydroxypropionate to propionyl-CoA is a necessary route for the assimilation of this molecule by R. sphaeroides. Our conclusion is based on the following findings: (i) crotonyl-CoA carboxylase/reductase, a key enzyme of the ethylmalonyl-CoA pathway for acetyl-CoA assimilation, was not essential for growth with 3-hydroxypropionate, as demonstrated by mutant analyses and enzyme activity measurements; (ii) the reductive conversion of 3-hydroxypropionate or acrylate to propionyl-CoA was detected in cell extracts of R. sphaeroides grown with 3-hydroxypropionate, and both activities were upregulated compared to the activities of succinate-grown cells; and (iii) the inactivation of acuI, encoding a candidate acrylyl-CoA reductase, resulted in a 3-hydroxypropionate-negative growth phenotype.

Fig 1

Growth of wild-type Rhodobacter sphaeroides 2.4.1 in defined medium containing 3-hydroxypropionate as a sole carbon source under photoheterotrophic (anaerobic/light) (A) and chemoheterotrophic (aerobic/dark) (B) growth conditions. Experiments were carried out with triplicate cultures. Growth was monitored by measuring the OD₅₇₈ (solid line). The extracellular 3-hydroxypropionate concentration (dotted line) was determined by an enzymatic endpoint assay using recombinant propionyl-CoA synthase from C. aurantiacus. Standard deviations are indicated by the error bars.

Fig 2

Photoheterotrophic (anaerobic/light) growth of wild-type Rhodobacter sphaeroides 2.4.1 (○), the Δccr MC4 mutant (▵), the ΔccrMC4(pMC19_2) ccr-complemented mutant (×), and the ΔccrMC4(pBBRsm2MCS5) mutant (□) in defined medium containing succinate (A), acetate (B), or 3-hydroxypropionate (C) as a sole carbon source (10 mM final concentration). pBBRsm2MCS5 was the vector used for the construction of ccr complementation plasmid pMC19_2.

Fig 3

HPLC analysis of CoA-thioesters formed during the reductive conversion of acrylate by cell extracts of R. sphaeroides grown photoheterotrophically with 3-hydroxypropionate as the carbon source. Shown are data for the reaction mix before the addition of acrylate and NADPH (A), 5 min after the addition of acrylate but not NADPH (B), 2 min after the addition of NADPH to the reaction mixture (C), and after 5 min (D) and 10 min (E) of additional incubation in the presence of NADPH and acrylate. The identity of compound X is unknown but is also formed when acrylyl-CoA is synthesized using acyl-CoA synthetase from S. tokodaii and may represent an adduct between acrylyl-CoA and free CoA.

Fig 4

Alignment of AcuI-like protein sequences and acuI-like genes. (A) Amino acid sequence alignment of AcuI-like proteins. The proposed acrylyl-CoA reductase from R. sphaeroides strain 2.4.1, AcuI (GenBank accession number ABA77575), was aligned with acrylyl-CoA reductase from Sulfolobus tokodaii strain 7 (accession number ACJ71675) and partially aligned with the acrylyl-CoA reductase domain of propionyl-CoA synthase from Chloroflexus aurantiacus strain J-10-f (accession number AAL47820) and 1914, a protein with unknown function from Ruegeria pomeroyi strain DSS-3 (accession number AAV95191). Red boxes indicate residues that are identical between two proteins, blue boxes indicate residues that are identical between three proteins, and green boxes indicate residues that are the same between all four proteins (and therefore apply only to the region where Pcs is coaligned) and contain an NAD(P)H binding domain, as indicated. (B) Genomic context of genes encoding AcuI-like proteins. acuR encodes a transcriptional regulator (40), acuI encodes the proposed acrylyl-CoA reductase (this work), and dddL encodes DMSP lyase (14). acr encodes an acrylyl-CoA reductase (42); the functions of the gene products from genes next to acr are unknown. pcs encodes propionyl-CoA synthase (1); the three domains refer to the acyl-CoA synthetase domain (pink), the acrylyl-CoA hydratase domain (blue), and the acrylyl-CoA reductase domain (brown; the region that was not aligned is shown in a fainter color). dmdA encodes DMSP demethylase (23), and SPO_1914 encodes a protein of unknown function.

Fig 5

Photoheterotrophic (anaerobic/light) growth of wild-type (○), ΔacuI::kan mutant (▵), ΔacuI::kan(pMA5_1) acuI-complemented mutant (▴), and ΔacuI::kan(pBBRsm2MCS5) mutant (♢) strains in minimal medium containing succinate (10 mM) (A) or 3-hydroxypropionate (10 mM) (B) as a sole carbon source. pBBRsm2MCS5 is the vector used for the construction of acuI complementation plasmid pMA5-1.

Fig 6

Proposed scheme for 3-hydroxypropionate assimilation by R. sphaeroides. The reductive conversion of 3-hydroxypropionate to propionyl-CoA involving AcuI is essential for the growth of R. sphaeroides with 3-hydroxypropionate as the sole carbon source. The ethylmalonyl-CoA pathway involving Ccr is not required for 3-hydroxypropionate assimilation. The dotted line represents an oxidative path that cannot be ruled out based on the results presented here and might be used for the biosynthesis of cell constituents derived directly from acetyl-CoA. However, in principle, acetyl-CoA may also be formed from succinyl-CoA oxidation via pyruvate.