a, Growth rate of wild-type P. denitrificans DSM 413 on the BHAC substrates glycolate and glyoxylate. The middle line and box are the median and interquartile range of n = 6 independent experiments and the whiskers indicate the maximum range of the dataset. b, c, Representative growth curves of wild-type P. denitrificans DSM 413 (grey) and bhc deletion strains (coloured) grown in the presence of 60 mM glycolate (b) or 60 mM glyoxylate (c). Deletion of any single gene in the bhc gene cluster is sufficient to completely abolish growth in the presence of glycolate and glyoxylate. These experiments were repeated three times independently with similar results. d–f, Growth rates (μ) of wild-type P. denitrificans DSM 413 (grey) and BHAC deletion strains (coloured) grown in the presence of 60 mM acetate (d), 30 mM succinate (e) or 20 mM glucose (f). Deletion of any single gene in the bhc gene cluster, or of the whole bhc gene cluster, still permits growth on acetate, succinate or glucose with comparable growth rates as for the wild type. Data are the mean ± s.d. of n = 3 independently grown cultures. g, Analysis of the proteome of glycolate-grown compared to succinate-grown P. denitrificans DSM 413. All proteins that were quantified by at least three unique peptides are shown. The 15 proteins that showed the strongest increase in abundance are marked in the volcano plot. The four enzymes of the BHAC are marked in red, the three subunits of glycolate oxidase in orange, the proteins of a putative operon for lactate utilization in white and the proteins directly downstream of the bhc gene cluster in light red. h, The abundance of these proteins, given as the percentage of the intensity-based absolute quantification (iBAQ) value. Data are the mean ± s.d. of n = 4 independently grown cultures. i, Specific activities of BHAC enzymes in cell-free extracts of glycolate-grown P. denitrificans DSM 413, as measured spectrophotometrically. Note that the activity of BhcD is plotted on the right y axis and consists of the actual iminosuccinate reductase activity (iminosuccinate to l-aspartate) as well as endogenous malate dehydrogenase activity (oxaloacetate to l-malate). j, Ratio of malate to aspartate determined by LC–MS during the enzyme assay for BhcD activity. The ratio remains approximately constant at 12:1, indicating that only approximately 8% of the activity (around 1.3 U mg−1) shown in i can be ascribed to iminosuccinate reductase. i, j, Data are the mean ± s.d. of n = 3 independently grown cultures; each data point represents the mean of n = 3 technical replicates. k, DNA-binding properties of BhcR. Left, a fluorescently labelled DNA fragment carrying the putative promoter region of the bhc gene cluster (P bhc ) was incubated with increasing amounts of purified BhcR protein and subsequently separated by electrophoresis to visualize DNA bound to BhcR and free DNA; a DNA fragment derived from the coding region of bhcA was used as a negative control. BhcR specifically forms a complex with the DNA fragment containing the putative promoter region of the bhc gene cluster. Right, the P bhc –BhcR complex was incubated with increasing concentrations of glyoxylate and subsequently separated by electrophoresis to assess the effect of glyoxylate on complex formation; the bhcA DNA fragment together with BhcR was used as a negative control. Increasing concentrations of glyoxylate decrease the binding of BhcR to the P bhc DNA fragment. For gel source data, see Supplementary Fig. 1. Source data