Genetic Evidence Reveals the Indispensable Role of the rseC Gene for Autotrophy and the Importance of a Functional Electron Balance for Nitrate Reduction in Clostridium ljungdahlii

Abstract

For Clostridium ljungdahlii, the RNF complex plays a key role for energy conversion from gaseous substrates such as hydrogen and carbon dioxide. In a previous study, a disruption of RNF-complex genes led to the loss of autotrophy, while heterotrophy was still possible via glycolysis. Furthermore, it was shown that the energy limitation during autotrophy could be lifted by nitrate supplementation, which resulted in an elevated cellular growth and ATP yield. Here, we used CRISPR-Cas12a to delete: (1) the RNF complex-encoding gene cluster rnfCDGEAB; (2) the putative RNF regulator gene rseC; and (3) a gene cluster that encodes for a putative nitrate reductase. The deletion of either rnfCDGEAB or rseC resulted in a complete loss of autotrophy, which could be restored by plasmid-based complementation of the deleted genes. We observed a transcriptional repression of the RNF-gene cluster in the rseC-deletion strain during autotrophy and investigated the distribution of the rseC gene among acetogenic bacteria. To examine nitrate reduction and its connection to the RNF complex, we compared autotrophic and heterotrophic growth of our three deletion strains with either ammonium or nitrate. The rnfCDGEAB- and rseC-deletion strains failed to reduce nitrate as a metabolic activity in non-growing cultures during autotrophy but not during heterotrophy. In contrast, the nitrate reductase deletion strain was able to grow in all tested conditions but lost the ability to reduce nitrate. Our findings highlight the important role of the rseC gene for autotrophy, and in addition, contribute to understand the connection of nitrate reduction to energy metabolism.

Keywords: CRISPR; Clostridium ljungdahlii; RNF-gene cluster; acetogenic bacteria; autotrophy; nitrate reduction.

FIGURE 1

CRISPR-Cas12a-mediated rnfCDGEAB gene cluster deletion in C. ljungdahlii. (A) Modular CRISPR-Cas12a system established in the pMTL80000 shuttle-vector system (Heap et al., 2009). The final CRISPR-Cas12a plasmid for deletion of rnfCDGEAB contained the Fncas12a gene, homology-directed repair arms (HDRs), and a specific crRNA array comprising two directed repeats (DRs) and two sgRNA, which targeted the rnfC and rnfB genes. (B) Agarose gel with PCR-samples for the fdhA fragment (WT: 501 bp, deletion strain: 501 bp), rnfCDGEAB fragment (WT: 5,047 bp, deletion strain: no fragment), and for a fragment that was amplified with primers that bind ∼1,250 bp upstream and downstream of the rnfCDGEAB gene cluster locus (WT: 7,550 bp, deletion strain: 2,503 bp). DNA-template: gDNA of C. ljungdahlii ΔRNF (lane 1, 4, and 7); gDNA of C. ljungdahlii WT (lane 3, 6, and 9); and water (lane 2, 5, 8). M: Generuler™ 1 kb DNA ladder. (C) Growth of the wild type (WT) and reduced growth of the deletion strain (ΔRNF) with fructose in PETC medium. HDR1/2, homology-directed repair arm flanking the targeted gene; crRNA array, sequence containing FnCas12a-specific DRs and sgRNAs; sgRNA, guide RNA; repH, Gram-positive origin of replication; catP, antibiotic resistant cassette against chloramphenicol/thiamphenicol; colE1, Gram-negative origin of replication; traJ, conjugation gene; P_thl, promoter sequence of the thiolase gene in Clostridium acetobutylicum; AscI, FseI, PmeI, and SbfI are unique-cutting restriction sites, which were preserved during the cloning to maintain the modular functionality of the plasmid backbone.

FIGURE 2

Cultivation of C. ljungdahlii WT, C. ljungdahlii ΔRNF, and C. ljungdahlii ΔrseC in nitrate- or ammonium-containing medium with H₂ and CO₂. Cultures of C. ljungdahlii strain WT (●, ◦), ΔRNF (, ), and ΔrseC (, ) were grown in 100 mL PETC medium in 1 L bottles at 37°C and 150 rpm. The headspace consisted of H₂ and CO₂ (80/20 vol-%) and was set to 0.5 bar overpressure. The medium contained either 18.7 mM nitrate (NO₃^–) (filled circles) or 18.7 mM ammonium (NH₄⁺) (open circles) as nitrogen source. The cultivation times were 173 h for cultures of C. ljungdahlii WT and C. ljungdahlii ΔRNF and 186 h for cultures of C. ljungdahlii ΔrseC. All cultures were grown in biological triplicates, data is given as mean values, with error bars indicating the standard deviation. (A) Growth; (B) pH-behavior; (C) acetate concentrations; (D) ethanol concentration; (E) ammonium concentration; and (F) nitrate concentrations. WT, wild type; ΔRNF, RNF-gene cluster deletion; ΔrseC, rseC gene deletion.

FIGURE 3

Gene expression change of the rnfCDGEAB cluster genes and the rseC gene in the ΔRNF and ΔrseC deletion strains. (A) Gene expression change for the genes rnfC, rnfD, rnfG, rnfE, rnfA, and rnfB in strain C. ljungdahlii ΔrseC after 3 h cultivation time; (B) gene expression change for the gene rseC in strain C. ljungdahlii ΔRNF after 3 h cultivation time; (C) gene expression change for the genes rnfC, rnfD, rnfG, rnfE, rnfA, and rnfB in strain C. ljungdahlii ΔrseC after 20 h cultivation time; and (D) gene expression change for the gene rseC in strain C. ljungdahlii ΔRNF after 20 h cultivation time. RNA samples were purified from cultures that were cultivated either autotrophically with hydrogen and carbon dioxide (blue bars) or heterotrophically with fructose (orange bars). cDNA was synthesized from the purified RNA samples and used as template for qRT-PCR analyses. The individual gene expression profiles of each gene was calculated using the wild-type strain as reference, which was grown under the same conditions. The rho gene was used as “housekeeping” gene. The fold change in gene expression was determined with the 2^–ΔΔCT method (Livak and Schmittgen, 2001). ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; *ns, not significant (P > 0.05). We defined log₂ (fc) ≤ –2 as downregulated genes and ≥ + 2 as upregulated genes.

FIGURE 4

Location and orientation of rseC genes in model microbes that possess RNF complex gene clusters. The conserved protein domain RseC_MucC (pfam04246) was identified in the rseC protein sequence of C. ljungdahlii and used to search for putative rseC genes in the genome of C. autoethanogenum, A. woodii, E. limosum, C. carboxidovorans, C. kluyveri, T. maritima, R. capsulatus, and E. coli. All sequence analyses and gene arrangements were adapted from the JGI platform and the NCBI database (03/2021). The type strains are listed in Table 3. In red, putative rseC genes; in red pattern fill, rseC-associated genes in E. coli; in blue, RNF-complex gene cluster; in blue pattern fill, rsx genes, which are homologous to the rnf genes in R. capsulatus.

FIGURE 5

Growth, pH behavior, nitrate reduction of C. ljungdahlii Δnar with H₂ and CO₂. Cultures were grown in 100 mL PETC medium in 1 L bottles at 37°C and 150 rpm for 185 h. The headspace consisted of H₂ and CO₂ (80/20 vol-%) and was set to 0.5 bar overpressure. The medium contained either 18.7 mM nitrate (NO₃^–) () or 18.7 mM ammonium (NH₄⁺) () as nitrogen source. The C. ljungdahlii WT data (●, ◦) from Figure 1 is given for comparison. All cultures were grown in biological triplicates, data is given as mean values, with error bars indicating the standard deviation. (A) Growth; (B) pH-behavior; (C) acetate concentrations; (D) ethanol concentration; (E) ammonium concentration; and (F) nitrate concentrations. Δnar, deletion of nitrate reductase gene cluster; rpm, revolutions per minute; CO₂, carbon dioxide; and H₂, hydrogen.

FIGURE 6

Schematic model of RNF-gene regulation and nitrate reduction in the deletion strains C. ljungdahlii ΔRNF and C. ljungdahlii ΔrseC during autotrophy and heterotrophy. In both deletion strains, nitrate reduction is not possible in non-growing cells during autotrophy with carbon dioxide and hydrogen due to the lack of a functional RNF complex, and thus the missing regeneration of reducing equivalents such as NADH. On the contrary, nitrate reduction can proceed in C. ljungdahlii ΔRNF during heterotrophy with NADH, which is provided by glycolysis of fructose. In C. ljungdahlii ΔrseC, the RNF complex genes are repressed during autotrophy but not during heterotrophy, which indicates a further unknown regulation mechanism during heterotrophy. Thus, a functional RNF complex is formed, and nitrate reduction can proceed such as proposed for the wild type. H₂, hydrogen; H⁺, proton, CO₂, carbon dioxide; NO₃^–, nitrate; NO₂^–, nitrite; NH₄⁺, ammonium; ATP, adenosine triphosphate; ADP + P_i, adenosine diphosphate + phosphate; Fd_red/ox, reduced/oxidized ferredoxin; NADH/NAD⁺, reduced/oxidize nicotinamide adenine dinucleotide; NADPH/NADP⁺, reduced/oxidized nicotinamide adenine dinucleotide phosphate; RnfCDGEAB, RNF-complex subunits; Nar, nitrate reductase; Nir, nitrite reductase; Hcp, hydroxylamine reductase; H₂-ase, bifurcating hydrogenase/lyase; Nfn, bifurcating transhydrogenase; e^–, electron; ΔRNF, C. ljungdahlii ΔRNF; and ΔrseC, C. ljungdahlii ΔrseC. The model was adapted from Emerson et al. (2019).