Efficient Sulfide Assimilation in Methanosarcina acetivorans Is Mediated by the MA1715 Protein

Abstract

Conserved genes essential to sulfur assimilation and trafficking in aerobic organisms are missing in many methanogens, most of which inhabit highly sulfidic, anaerobic environmental niches. This suggests that methanogens possess distinct pathways for the synthesis of key metabolites and intermediates, including cysteine, homocysteine, and protein persulfide groups. Prior work identified a novel tRNA-dependent two-step pathway for cysteine biosynthesis and a new metabolic transformation by which sulfur is inserted into aspartate semialdehyde to produce homocysteine. Homocysteine biosynthesis requires two of the three proteins previously identified in our laboratory by a comprehensive bioinformatics approach. Here, we show that the third protein identified in silico, the ApbE-like protein COG2122, facilitates sulfide assimilation in Methanosarcina acetivorans. Knockout strains lacking the gene encoding COG2122 are severely impaired for growth when sulfide is provided as the sole sulfur source. However, rapid growth is recovered upon supplementation with cysteine, homocysteine, or cystathionine, suggesting that COG2122 is required for efficient biosynthesis of both cysteine and homocysteine. Deletion of the gene encoding COG2122 does not influence the extent of sulfur modifications in tRNA or the prevalence of iron-sulfur clusters, indicating that the function of COG2122 could be limited to sulfide assimilation for cysteine and homocysteine biosynthesis alone. IMPORTANCEWe have found that the conserved M. acetivorans ma1715 gene, which encodes an ApbE-like protein, is required for optimal growth with sulfide as the sole sulfur source and supports both cysteine and homocysteine biosynthesis in vivo. Together with related functional-genomics studies in methanogens, these findings make a key contribution to elucidating the novel pathways of sulfide assimilation and sulfur trafficking in anaerobic microorganisms that existed before the advent of oxygenic photosynthesis. The data suggest that the MA1715 protein is particularly important to sustaining robust physiological function when ambient sulfide concentrations are low. Phylogenetic analysis shows that MA1715 and other recently discovered methanogen sulfur-trafficking proteins share an evolutionary history with enzymes in the methanogenesis pathway. The newly characterized genes thus likely formed an essential part of the core metabolic machinery of the ancestral euryarchaeote. Genes that function in sulfate assimilation and sulfur trafficking in aerobic organisms are often not identifiable in the genomes of methanogens and other anaerobes, suggesting that these ancient organisms may retain metabolic vestiges characteristic of life on the early earth (1). In aerobes, sulfur is imported into the cell as sulfate, which is reduced to sulfide in an ATP-dependent pathway (2). In turn, sulfide is incorporated directly into activated serine and homoserine derivatives to yield cysteine (Cys) and homocysteine (Hcy), respectively (Fig. 1, left) (3). Cys and Hcy can al...

Filling Gaps in Bacterial Amino Acid Biosynthesis Pathways with High-throughput Genetics

“…Of the 11 genuine gaps, we identified genes to fill nine of them; these novel enzymes explain the biosynthesis of four different amino acids by bacteria from six different genera. And we found that homocysteine synthesis in Desulfovibrio vulgaris Miyazaki F required DUF39, NIL/ferredoxin, and COG2122 proteins, as expected from studies of this pathway in other organisms (Rauch et al 2014;Rauch and Perona 2016;Kuehl et al 2014) . Our genetic data imply that, in contrast to all other known pathways, homoserine is not an intermediate in homocysteine synthesis in D. vulgaris.…”

“…This strongly suggests that the COG2122 protein is also required for homocysteine formation. In contrast, (Rauch and Perona 2016) found that the orthologous protein from Methanosarcina acetivorans ( MA1715) was important for growth in defined media at low sulfide concentrations, but that mutants could be rescued by either higher sulfide concentrations or by added cysteine or homocysteine. They proposed that COG2122 aids in the formation of an unknown intermediate in sulfide assimilation for both methionine and cysteine.…”

“…First, in Shewanella oneidensis MR-1, SO3749 is the missing acetylornithine deacetylase for arginine synthesis (Deutschbauer et al 2011) . Second, the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F contains recently-discovered genes for the biosynthesis of homocysteine, which is a precursor to methionine (DUF39, NIL/ferredoxin, and COG2122; (Rauch et al 2014) ) (Rauch and Perona 2016) ). Unfortunately, the information from these studies has not made its way into the annotation databases.…”

Homocysteine Editing, Thioester Chemistry, Coenzyme A, and the Origin of Coded Peptide Synthesis †

“…Hcy has also been shown to be an intermediate in Met and Cys metabolism in Archaea methanogens [43]. Because Hcy is a non-coded amino acid, living organisms must have evolved the ability to prevent its access to the Genetic Code.…”

Biosynthesis of Sulfur-Containing tRNA Modifications: A Comparison of Bacterial, Archaeal, and Eukaryotic Pathways

“…The K M of Na 2 S is ~1 mM, close to the estimated intracellular concentrations of free sulfide in methanococci (~1–3 mM) [47]; this suggests that sulfide is a physiologically relevant sulfur donor. Furthermore, free l -cysteine is not a sulfur donor for the biosynthesis of Fe–S cluster [64] and tRNA thionucleosides [65] in methanogens; this suggests that a cysteine desulfurase is not required as a central sulfur donor for the biosynthesis of sulfur containing compounds. Notably, the methanogenic archaeal ThiI homologs have three conserved Cys residues (two from a CXXC motif) in the putative catalytic domain [46,47].…”