Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

Abstract

Propionyl coenzyme A (propionyl-CoA) assimilation by Rhodobacter sphaeroides proceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway, propionyl-CoA carboxylase (PCC), was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway, acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of the pccB gene, which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation of pccB transcript levels and, consequently, PCC activity: growth substrate-dependent regulation was lost when pccR was inactivated by an in-frame deletion. In the pccR mutant, lacZ expression from a 215-bp plasmid-borne pccB upstream fragment including 27 bp of the pccB coding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR, together with homologs from other organisms, formed a distinct clade within the family of short-chain fatty acyl coenzyme A regulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR).

Importance: Short-chain acyl-CoAs are intermediates in essential biosynthetic and degradative pathways. The regulation of their accumulation is crucial for appropriate cellular function. This work identifies a regulator (PccR) that prevents the accumulation of propionyl-CoA by controlling expression of the gene encoding propionyl-CoA carboxylase, which is responsible for propionyl-CoA consumption by Rhodobacter sphaeroides. Many other Proteobacteria and Actinomycetales contain one or several PccR homologs that group into distinct clades on the basis of the pathway of acyl-CoA metabolism that they control. Furthermore, an upstream analysis of genes encoding PccR homologs allows the prediction of conserved binding motifs for these regulators. Overall, this study evaluates a single regulator of propionyl-CoA assimilation while expanding the knowledge of the regulation of short-chain acyl-CoAs in many bacterial species.

FIG 1

Comparison of pccB transcript abundance (A) and the PCC activity (B) of R. sphaeroides 2.4.1 and RsΔpccRMC12 cells grown with succinate (filled symbols), acetate (open symbols), and propionate-HCO₃⁻ (hatched symbols). Expression levels were relative to the average value of recA and rpoZ transcript abundance. Error bars represent the standard deviations from at least three biological replicates. RsΔpccRMC12 did not grow with propionate-HCO₃⁻.

FIG 2

(A) Genomic context of pccB (encoding the β subunit of propionyl-CoA carboxylase), pccR, and the consensus sequence (open boxes) initially identified upstream of pccR genes within several alphaproteobacteria. Striped arrows, genes annotated to encode a multidrug-resistant transporter; checkered arrows, conserved coding regions of unknown function; open arrows, coding regions with no significant sequence identity to the other genes shown. (B) Illustration of the mutated pccB upstream regions that are present on each plasmid in the pMC85 series. Each plasmid contains a derivative of a 215-bp fragment of the pccB upstream fragment including 27 bp of the pccB coding region fused to lacZ. Regulation of expression from each putative promoter is presented in Table 2.

FIG 3

Photoheterotrophic growth of R. sphaeroides 2.4.1 (open circles), RsΔpccRMC12 (filled circles), RsΔpccRMC12(pBBRsm2MCS5) (crosses), and RsΔpccRMC12(pMC66) (stars) with succinate, acetate, and propionate-HCO₃⁻.

FIG 4

Photoheterotrophic growth of R. sphaeroides 2.4.1 (left) and RsΔpccRMC12 (right) that carry no plasmid (circles), pMC85 (diamonds), pMC85Δ1 (triangles), pMC85Δ2 (inverted triangles), or pMC85Δ12 (squares) with succinate, acetate, or propionate-HCO₃⁻.

FIG 5

Neighbor-joining tree of selected ScfR proteins. Nodes with bootstrap values greater than 50% are labeled. Each entry includes the class name assigned to the given protein on the basis of the genomic context of its respective gene. A question mark indicates that the corresponding genetic context does not contain any genes of the indicated metabolic gene clusters. An asterisk indicates a protein that clusters in the tree with proteins of a different class. Following the protein name is the locus tag for the given protein and the species in which the protein is found. To the right of the tree is an illustration of the general genome neighborhood of the genes that encode proteins from the corresponding clade. See Fig. S2 in the supplemental material for the extended tree. A DNA logo (50) is included if a conserved motif was identified upstream of the genes responsible for the proteins in the corresponding clade. The indicated motifs may have occurred multiple times in the queried sequences. Gene annotations are as follows: pccB, propionyl-CoA carboxylase β subunit; pccA, propionyl-CoA carboxylase α subunit; mcm, methylmalonyl-CoA mutase; prpB, 2-methylcitrate lyase; prpC, 2-methylcitrate synthase; prpD, 2-methylcitrate dehydratase; prpE, propionyl-CoA synthetase; mmsA, methylmalonate semialdehyde dehydrogenase; hibA, acyl-CoA dehydrogenase; hibB, enoyl-CoA hydratase; mmsB, 3-hydroxyisobutyrate dehydrogenase; aceA, isocitrate dehydrogenase; aceB, malate synthase.