The methylaspartate cycle in haloarchaea and its possible role in carbon metabolism

Abstract

Haloarchaea (class Halobacteria) live in extremely halophilic conditions and evolved many unique metabolic features, which help them to adapt to their environment. The methylaspartate cycle, an anaplerotic acetate assimilation pathway recently proposed for Haloarcula marismortui, is one of these special adaptations. In this cycle, acetyl-CoA is oxidized to glyoxylate via methylaspartate as a characteristic intermediate. The following glyoxylate condensation with another molecule of acetyl-CoA yields malate, a starting substrate for anabolism. The proposal of the functioning of the cycle was based mainly on in vitro data, leaving several open questions concerning the enzymology involved and the occurrence of the cycle in halophilic archaea. Using gene deletion mutants of H. hispanica, enzyme assays and metabolite analysis, we now close these gaps by unambiguous identification of the genes encoding all characteristic enzymes of the cycle. Based on these results, we were able to perform a solid study of the distribution of the methylaspartate cycle and the alternative acetate assimilation strategy, the glyoxylate cycle, among haloarchaea. We found that both of these cycles are evenly distributed in haloarchaea. Interestingly, 83% of the species using the methylaspartate cycle possess also the genes for polyhydroxyalkanoate biosynthesis, whereas only 34% of the species with the glyoxylate cycle are capable to synthesize this storage compound. This finding suggests that the methylaspartate cycle is shaped for polyhydroxyalkanoate utilization during carbon starvation, whereas the glyoxylate cycle is probably adapted for growth on substrates metabolized via acetyl-CoA.

Figure 1

Anaplerotic pathways of acetyl-CoA assimilation functioning in haloarchaea. (a) The glyoxylate cycle and the tricarboxylic acid cycle (Kornberg and Krebs, 1957); (b) the methylasparate cycle (Khomyakova et al., 2011). The key reactions of the anaplerotic pathways are shown in red. Note that the methylaspartate cycle is also tightly linked to the tricarboxylic acid cycle, as both cycles share enzymes converting succinyl-CoA to 2-oxoglutarate. The names of the genes encoding key enzymes of the methylaspartate cycle are shown: mamAB, glutamate mutase (hah_1337/hah_1338); mal, methylaspartate ammonia lyase (hah_1339); mct, mesaconate CoA-transferase (hah_1336); mch, mesaconyl-CoA hydratase (hah_1340); mcl, β-methylmalyl-CoA lyase (hah_1341).

Figure 2

Genomic region of Haloarcula marismortui and H. hispanica coding for the enzymes of the methylaspartate cycle (Baliga et al., 2004; Liu et al., 2011b). MamAB, glutamate mutase; Mal, methylaspartate ammonia lyase; Mct, mesaconate CoA-transferase; Mch, mesaconyl-CoA hydratase; Mcl, β-methylmalyl-CoA lyase.

Figure 3

Conversion of mesaconate to propionyl-CoA and glyoxylate by cell extracts of acetate-grown H. hispanica. The experiment was started by the addition of succinyl-CoA, and the samples after 0 (a), 5 (b) and 10 min (c) of incubation were analyzed by reversed phase C₁₈ UPLC to follow CoA-esters (at 260 nm) and glyoxylate phenylhydrazine (at 324 nm). (d) Control without mesaconate after 10 min of incubation. Increase of the CoA peak in the control is due to succinyl-CoA hydrolysis. Abs₂₆₀, absorption at 260 nm; Abs₃₂₄, absorption at 324 nm; Gl-Ph, glyoxylate phenylhydrazone.

Figure 4

Growth of Haloarcula hispanica wild-type strain (a), ΔmamB (Δhah_1338) (b), Δmal (Δhah_1339) (c), Δmct (Δhah_1336) (d), Δmch (Δhah_1340) (e) on the medium with acetate (), pyruvate () or acetate and pyruvate (). The experiment was done in triplicate, the error bars represent the standard deviations. Note that the mutants did not grow on the medium with acetate alone even after 21 days of incubation. The Δmct mutant grows slowly on the medium with pyruvate compared with the wild-type strain. The reason for that is unknown.

Figure 5

Phylogeny of the class Halobacteria based on the analysis of RNA polymerase subunit B' (RpoB') protein sequences. The tree shows the pattern of characteristic enzymes involved in haloarchaeal carbon metabolism (according to the data of the genome analysis), that is, the presence of the characteristic enzymes of the glyoxylate cycle (isocitrate lyase) and of the methylaspartate cycle (glutamate mutase, methylaspartate ammonia lyase, succinyl-CoA:mesaconate CoA-transferase, mesaconyl-CoA hydratase and β-methylmalyl-CoA lyase) as well as of the homologs of haloarchaeal polyhydroxyalkanoate synthase (Han et al., 2010). Growth on the substrates metabolized via acetyl-CoA (acetate, fatty acids, tween, leucine and/or lysine) is according to descriptions of the corresponding strains/species. Numbers at nodes indicate the percentage bootstrap values for the clade of this group in 1000 replications. The branches with the values below 50% are drawn as unresolved. The accession numbers of the sequences used for the construction of the tree are listed in Supplementary Table 3.

Figure 6

Diagram with the distribution of genes for the pathways of acetate assimilation and haloarchaeal polyhydroxyalkanoate synthase (dotted part of each sector) in sequenced haloarchaeal genomes.