The energy metabolism of Cupriavidus necator in different trophic conditions

Abstract

The "knallgas" bacterium Cupriavidus necator is attracting interest due to its extremely versatile metabolism. C. necator can use hydrogen or formic acid as an energy source, fixes CO₂ via the Calvin-Benson-Bassham (CBB) cycle, and grows on organic acids and sugars. Its tripartite genome is notable for its size and duplications of key genes (CBB cycle, hydrogenases, and nitrate reductases). Little is known about which of these isoenzymes and their cofactors are actually utilized for growth on different substrates. Here, we investigated the energy metabolism of C. necator H16 by growing a barcoded transposon knockout library on succinate, fructose, hydrogen (H₂/CO₂), and formic acid. The fitness contribution of each gene was determined from enrichment or depletion of the corresponding mutants. Fitness analysis revealed that (i) some, but not all, molybdenum cofactor biosynthesis genes were essential for growth on formate and nitrate respiration. (ii) Soluble formate dehydrogenase (FDH) was the dominant enzyme for formate oxidation, not membrane-bound FDH. (iii) For hydrogenases, both soluble and membrane-bound enzymes were utilized for lithoautotrophic growth. (iv) Of the six terminal respiratory complexes in C. necator H16, only some are utilized, and utilization depends on the energy source. (v) Deletion of hydrogenase-related genes boosted heterotrophic growth, and we show that the relief from associated protein cost is responsible for this phenomenon. This study evaluates the contribution of each of C. necator's genes to fitness in biotechnologically relevant growth regimes. Our results illustrate the genomic redundancy of this generalist bacterium and inspire future engineering strategies.IMPORTANCEThe soil bacterium Cupriavidus necator can grow on gas mixtures of CO₂, H₂, and O₂. It also consumes formic acid as carbon and energy source and various other substrates. This metabolic flexibility comes at a price, for example, a comparatively large genome (6.6 Mb) and a significant background expression of lowly utilized genes. In this study, we mutated every non-essential gene in C. necator using barcoded transposons in order to determine their effect on fitness. We grew the mutant library in various trophic conditions including hydrogen and formate as the sole energy source. Fitness analysis revealed which of the various energy-generating iso-enzymes are actually utilized in which condition. For example, only a few of the six terminal respiratory complexes are used, and utilization depends on the substrate. We also show that the protein cost for the various lowly utilized enzymes represents a significant growth disadvantage in specific conditions, offering a route to rational engineering of the genome. All fitness data are available in an interactive app at https://m-jahn.shinyapps.io/ShinyLib/.

Keywords: Cupriavidus necator; RB-TnSeq; Ralstonia eutropha; barcoded library; chemostat; energy metabolism; gene fitness; knockout library; protein cost; substrate limitation; transposon.

Fig 1

Creation of the C. necator transposon library and screening workflow. (A) C. necator H16 was conjugated with an E. coli donor strain carrying a transposon integration construct [sketch of the vector was adopted from reference (23)]. The construct contains two inverted repeat (IR) elements that serve as recognition sites for the transposase, an R6K plasmid replication origin, a kanamycin resistance cassette (kanR), two universal priming sites (U1 and U2), and the 20 nt random barcode (N20). Transposon integration sites were mapped to barcodes using the TnSeq workflow. Genomic DNA was isolated, shear-fragmented, and end-repaired. Transposon-containing fragments were PCR amplified including the barcode and a portion of genomic DNA and then subjected to NGS. (B) Library competition experiments were mostly performed in chemostat bioreactors. Samples were taken after 0, 8, and 16 generations, genomic DNA was extracted, and barcodes were sequenced using NGS. The changes in barcode abundance over time were used to calculate a fitness score for each gene.

Fig 2

Clustering of genes with significantly changed fitness. (A) Fitness scores for 354 genes exceeding a threshold f ≤ −2 or f ≥ 2. Genes were clustered based on similarity of fitness scores for the various growth conditions (dendrogram colored by cluster number). A cluster number of 7 was used after testing cluster separation by silhouette width, see Fig. S1. P, pulsed feeding; C, continuous feeding. (B) t-SNE dimensionality reduction of genes, color coded and labeled according to the clusters in A. (C) Gene enrichment for KEGG pathways. The top three pathways by P-value were selected. Colors and numbers in brackets correspond to the clusters in A.

Fig 3

Fitness related to formate dehydrogenases and their cofactors. (A) Fitness score for transposon insertion mutants of the MoCo biosynthesis pathway. (B) Fitness score for transposon insertion mutants of various formate dehydrogenase genes. A detailed description can be found in the Results section. The fitness of the fdoHI genes was not quantified. The scale bar indicates the gene fitness score after eight generations of growth. Inset numbers in genome plots represent the number of transposon insertions per gene. cGTP, cyclic GTP; cPMP, cyclic pyranopterin monophosphate; MPT, molybdopterin; MoCo, molybdopterin cofactor; GMP-MoCo, GMP modified MoCo; SFDH, soluble formate dehydrogenase; MFDH, membrane-bound formate dehydrogenase.

Fig 4

Fitness related to hydrogenases and their cofactors. (A) Fitness score for the genes involved in biosynthesis of the Ni-Fe-(CN)₂-CO cofactor (hyp genes). (B) Fitness score for the genes forming MBH, soluble hydrogenase (SH), RH, and accessory genes for maturation of hydrogenases. For details, see the Results section. The scale bar indicates gene fitness scores after eight generations of growth. Inset numbers in genome plots represent the number of transposon insertions per gene. Asterisk: note that HoxJ is inactive in the H16 strain and cannot regulate HoxA activity. CN⁻, cyanide; Ni, nickel; Fe, iron; UQ, ubiquinone; UQH₂, ubiquinol; NDH, NADH dehydrogenase (complex I).

Fig 5

Fitness related to electron transport chain complexes. Fitness score for the genes encoding subunits of different respiratory complexes in the electron transport chain. For details, see the Results section. The scale bar indicates gene fitness scores after eight generations of growth. Inset numbers in genome plots represent the number of transposon insertions per gene. UQ, ubiquinone; UQH₂, ubiquinol; e⁻, electrons.

Fig 6

Protein cost explains the growth advantage of hydrogenase mutants. (A) Estimated growth rate µ of hydrogenase-related transposon mutants. The growth rate was calculated from the log₂ fold change in mutant abundance over time compared to the population average. The dashed line marks the nominal growth rate for bioreactor experiments. (B) Corresponding protein cost for the knockout mutants in A in percent of the total protein mass. Open symbols, individual cost. Closed symbols, protein cost when disruption of downstream gene expression is taken into account. (C) Organization of the various hydrogenase-related genes. Membrane-bound hydrogenase MBH (purple), accessory hyp genes (orange), and regulatory hox genes (blue) are located sequentially on megaplasmid pHG1. Genes for soluble hydrogenase (SH) are located around 60 kb downstream of the MBH operon. Expression of the promoters P_MBH and P_SH is controlled by the HoxA master regulator. Inset table: effect on transcription of the selected gene knockouts. It is assumed that knockout of any of the primary hyp genes (orange) also disrupts hoxA expression. (D) Biomass yield of selected, in-frame knockout mutants for batch cultures in fructose-supplemented minimal medium. Stars indicate the significance level of a two-sided t-test of each mutant against wild type. *, P ≤ 0.05. **, P ≤ 0.01. ***, P ≤ 0.001. (E) Growth rate determined from optical density measurement for the same cultures as in D. (F) Gas chromatography measurement in the headspace of batch cultures of wild-type C. necator H16 (green), and a mutant lacking all known hydrogenases (∆hoxG, ∆hoxH, ∆hoxC, and ∆hofG, in red). Open symbols, O₂ measurement. Closed symbols, H₂ measurement. All subfigures: points and bars represent the mean of at least four biological replicates except (F) where three replicates were used. Error bars represent SD.