Transcriptomic profiles of Clostridium ljungdahlii during lithotrophic growth with syngas or H2 and CO2 compared to organotrophic growth with fructose

Clostridium ljungdahlii derives energy by lithotrophic and organotrophic acetogenesis. C. ljungdahlii was grown organotrophically with fructose and also lithotrophically, either with syngas - a gas mixture containing hydrogen (H2), carbon dioxide (CO2), and carbon monoxide (CO), or with H2 and CO2. Gene expression was compared quantitatively by microarrays using RNA extracted from all three conditions. Gene expression with fructose and with H2/CO2 was compared by RNA-Seq. Upregulated genes with both syngas and H2/CO2 (compared to fructose) point to the urea cycle, uptake and degradation of peptides and amino acids, response to sulfur starvation, potentially NADPH-producing pathways involving (S)-malate and ornithine, quorum sensing, sporulation, and cell wall remodeling, suggesting a global and multicellular response to lithotrophic conditions. With syngas, the upregulated (R)-lactate dehydrogenase gene represents a route of electron transfer from ferredoxin to NAD. With H2/CO2, flavodoxin and histidine biosynthesis genes were upregulated. Downregulated genes corresponded to an intracytoplasmic microcompartment for disposal of methylglyoxal, a toxic byproduct of glycolysis, as 1-propanol. Several cytoplasmic and membrane-associated redox-active protein genes were differentially regulated. The transcriptomic profiles of C. ljungdahlii in lithotrophic and organotrophic growth modes indicate large-scale physiological and metabolic differences, observations that may guide biofuel and commodity chemical production with this species.

Salvaging and detoxification of urea cycle intermediates. As an alternative to arginase, C. ljungdahlii possesses a set of genes for ammonia-producing, ATP-yielding fermentation of arginine to ornithine (Figure 2), and a partial second set -lacking arginine deiminase -for fermentation of citrulline to ornithine ( Figure 2). Of the first set, the genes for arginine deiminase (arcA CLJU_RS04570), ornithine carbamyltransferase (arcB-1 CLJU_RS04575), the arginine/ornithine antiporter (arcD-1 CLJU_RS04580) with 48% protein sequence identity to that of Pseudomonas aeruginosa 1 , and carbamate kinase (arcC-1 CLJU_RS04585) were downregulated with H 2 /CO 2 ( Figure 2). Of the second set, the putative citrulline/ornithine antiporter gene (arcD-2 CLJU_RS13825) with 46% protein sequence identity to the arginine/ornithine antiporter of P. aeruginosa 1 was not upregulated, but the ornithine carbamyltransferase (arcB-2 CLJU_RS13835) and carbamate kinase (arcC-2 CLJU_RS13830) genes were upregulated with syngas and with H 2 /CO 2 ( Figure 2; as previously reported with H 2 /CO 2 2 ). These enzymes may be important if the urea cycle stalls for lack of aspartate or if too much ammonia has been sequestered, because they can revert citrulline to ornithine plus carbamyl-phosphate, recover one-third of the expended ATP, and release ammonia ( Figure 2).
The gene for cyanate lyase (CLJU_RS07235) was upregulated with syngas and with H 2 /CO 2 ( Figure 2), indicating that carbamyl-phosphate may accumulate under lithotrophic conditions and undergo nonenzymatic base-catalyzed elimination of phosphate, producing cyanate that must be detoxified by cyanate lyase. Supplementary discussion S2.

Upregulation of molybdopterin-containing enzymes under lithotrophic conditions.
Among the molybdopterin biosynthesis genes are encoded two aldehyde:ferredoxin oxidoreductases (aor-1 CLJU_RS09865 and aor-2 CLJU_RS09915, sharing 79% protein sequence identity), which are molybdopterin cofactor-containing enzymes. Both genes were upregulated with H 2 /CO 2 , and the aor-1 gene was also upregulated with syngas ( Figure 1). With H 2 as the electron donor and without the hypothetical coupled reduction of methylene-THF and ferredoxin with NADH 1 , the Wood-Ljungdahl pathway ending with acetyl-CoA, acetylphosphate, and acetate has a predicted net energy yield of 0.41 ATP, while the pathway ending with reduction of acetyl-CoA with NADH to acetaldehyde and reoxidation to acetate by aldehyde:ferredoxin oxidoreductase has a predicted net energy loss of 0.05 ATP. C. ljungdahlii may sacrifice a fraction of the flux through the Wood-Ljungdahl pathway in this manner to maintain a pool of reduced ferredoxin sufficient to favour the reaction catalyzed by carbon monoxide dehydrogenase. However, this fraction is likely to be small because the enzymes that could reduce acetyl-CoA with NADH were generally downregulated ( Figure 1).
In addition to formate dehydrogenase, C. ljungdahlii possesses a formate hydrogen-lyase, the Hfn or Hyt complex ( Figure 1) Table S1). This is consistent with earlier reports that these genes were not differentially regulated with H 2 /CO 2 4 and upregulated 1.8-fold or less with CO/CO 2 5 .

Phosphate uptake and nucleotide dephosphorylation. Consistent with diminished nucleic
acid synthesis under lithotrophic conditions, genes for phosphate uptake transporters were downregulated (Supplementary Table S12). In contrast, the surE gene (CLJU_RS17130), encoding a nucleoside-3'/5'-monophosphate phosphatase and short-chain exopolyphosphatase, was upregulated, suggesting that C. ljungdahlii under lithotrophic conditions may recycle intracellular phosphate instead of taking up more from its surroundings.
Surprisingly, of the two genes encoding isozymes of deoxyuridine-5'-triphosphate pyrophosphohydrolase, an enzyme required for conversion of uracil into thymine, transcripts were more abundant and microarray signals were more intense for CLJU_RS04975, which was not differentially regulated; the other gene CLJU_RS20710 was only downregulated with H 2 /CO 2 , not with syngas ( Figure 6). An understanding of these two isozymes could be conducive to control of biomass production by limiting DNA replication in C. ljungdahlii to maximize the output of organic end products.

Supplementary discussion S5.
Downregulation of threonine biosynthesis genes, high-threonine-content genes, and cell surface proteins. Two genes for biosynthesis of threonine were strongly downregulated (Supplementary Table S13, as previously observed with H 2 /CO 2 versus fructose 1 ), suggesting that this amino acid may be especially important for organotrophic growth. The presence of a Tbox antiterminator riboswitch on the 5' side of these genes suggests that their expression during growth on fructose may increase in response to deaminoacylated tRNA-Thr. Consistent with the idea that threonyl-tRNA is limiting during growth on fructose, threonyl-tRNA synthetase was downregulated under lithotrophic conditions (Supplementary Table S13). The gene on the 5' side of the T-box (CLJU_RS03515), encoding an ACT domain protein of unknown function, was also downregulated (Supplementary Table S13); ACT domains often sense amino acids.
Threonine is a precursor of isoleucine and cobamide. There were no indications of increased isoleucine biosynthesis during growth with fructose (Supplementary Table S1), but the genes for enzymes that activate threonine for cobamide biosynthesis were downregulated for lithotrophic growth (Supplementary Table S13).
To explore the idea that C. ljungdahlii might need to synthesize proteins with high threonine content to grow organotrophically, the threonine content of every predicted protein of C.
ljungdahlii was computed. Of those whose threonine content is at least 8.6%, which is two standard deviations (1.9%) above the mean (4.8%), most were not downregulated (data not shown). High-threonine-content protein genes that were downregulated with both syngas and H 2 /CO 2 (Supplementary Table S14 Table S15), interspersed with adenosylcobamide-responsive riboswitches (some of which are transcribed with CO/CO 2