Figure 3. The C. crescentus genome encodes two functional citrate synthases.

(A) A schematic of the Krebs cycle and the corresponding gene products in C. crescentus. The two functional citrate synthases are indicated in blue. Essential gene products, as inferred from Tn-Seq (Christen et al., 2011), are highlighted in bold. (B) Spot dilutions (EOP assays) of the indicated WT and ∆gltA E. coli strains (eMB554 [WT], eMB556 [∆gltA + empty], eMB558 [∆gltA + citA], eMB560 [∆gltA + citB], eMB562 [∆gltA + citC] and eMB564 [∆gltA + gltA ] from top to bottom) on minimal medium containing glutamate or not. Only the strain carrying a functional citrate synthase can grow without glutamate. (C) LC-MS-based quantification of acetyl-CoA in extract of WT (MB1), citA::Tn (MB2622) and ∆citA (MB2559) cells grown in PYE liquid cultures. Error bars denote the standard deviation of the mean from three biological replicates. (D) ΦCR30-mediated generalized transduction frequencies of citA::Tn into WT (MB1) or ∆citBC double mutant cells (MB2679). For transduction, cells were normalized according to OD_600nm ~1 and infected with the same amount of phage lysates from citA::Tn cells or with phage lysates from cells with a transposon insertion in the hetN gene (encoding gentamycin resistance) as a control for transduction. The transductants were selected on PYE plates containing gentamycin. The numbers of transduced colonies were counted after 3 days of incubation at 30°C. Error bars denote the standard deviation of the mean for three independent experiments. Cells harboring the ∆citBC mutation are not able to accept the citA::Tn mutation. (E) Same as in panel (D) using the ∆citA::kan allele or a deletion in the spoT gene (encoding kanamycin resistance, ∆spoT::kan) delivered by ΦCR30-mediated generalized transduction. Transductants were selected on PYE plates containing kanamycin.

Figure 3—figure supplement 1. Primary structure alignment of CitA and homologs.

(A) Partial alignment of the active site of CitA (A0A0H3C985) with CitB (A0A0H3CCE2) and CitC (A0A0H3CD20) from C. crescentus, GltA (P0ABH7) from E. coli, CitA (P39119) and CitZ (P39120) from Bacillus subtilis. The histidine and aspartic acid catalytic residues are highlighted in red. An arrow indicates the alanine or tryptophan substitution that abolishes the catalytic activity of CitA (Figure 4). (B) Heatmap showing the changes in the level of various metabolites in WT and citA::Tn cells as measured by LC-MS. Cells were grown in PYE medium. Only the metabolites that were significantly increased or decreased in ∆citA compared to WT (p-value<0.05) are shown. Fold changes were calculated based on the mean of normalized ion counts from three biological replicates. (C) Distribution of citrate synthase paralogs encoded in various α-proteobacterial genomes. Blue and white boxes indicate the presence or absence, respectively, of a citrate synthase as identified by bi-directional BLASTP searches.