Investigating the Origins of Membrane Phospholipid Biosynthesis Genes Using Outgroup-Free Rooting

Abstract

One of the key differences between Bacteria and Archaea is their canonical membrane phospholipids, which are synthesized by distinct biosynthetic pathways with nonhomologous enzymes. This "lipid divide" has important implications for the early evolution of cells and the type of membrane phospholipids present in the last universal common ancestor. One of the main challenges in studies of membrane evolution is that the key biosynthetic genes are ancient and their evolutionary histories are poorly resolved. This poses major challenges for traditional rooting methods because the only available outgroups are distantly related. Here, we address this issue by using the best available substitution models for single-gene trees, by expanding our analyses to the diversity of uncultivated prokaryotes recently revealed by environmental genomics, and by using two complementary approaches to rooting that do not depend on outgroups. Consistent with some previous analyses, our rooted gene trees support extensive interdomain horizontal transfer of membrane phospholipid biosynthetic genes, primarily from Archaea to Bacteria. They also suggest that the capacity to make archaeal-type membrane phospholipids was already present in last universal common ancestor.

Keywords: lipid divide; lipid evolution; outgroup-free rooting; phylogenetics.

Fig. 1.

—(a) The canonical ether/ester biosynthetic pathways in Archaea and Bacteria and how they relate to glycerol metabolism. Based on figure 1 from Villanueva et al. (2017). Archaeal pathways in blue and yellow (blue = heterotrophic Archaea and yellow = autotrophic Archaea), bacterial pathway in red. Hypothetical biosynthetic pathway, as suggested by Villanueva et al. (2017), in dashed lines. (b) Composition of bacterial and archaeal phospholipids. In Archaea, glycerol-1-phosphate (G1P) is synthesized from dihydroxyacetone phosphate (DHAP) using the enzyme glycerol-1-phosphate dehydrogenase (G1PDH). The first and second isoprenoid chains (GGGPs) are added by geranylgeranylglyceryl phosphate synthase (GGGPS) and digeranylgeranylglyceryl phosphate synthase (DGGGPS), respectively. In Bacteria, glycerol-3-phosphate (G3P) is synthesized by glycerol-3-phosphate dehydrogenase (G3PDH) from DHAP. There are two forms of this enzyme, GpsA and GlpA/GlpD, encoded by the gps and glp genes, respectively. G3P may also be produced from glycerol by glycerol kinase (GlpK). In certain Bacteria, such as Gammaproteobacteria, the first fatty-acid chain is added by a version of glycerol-3-phosphate acyltransferase called PlsB. Other Bacteria, including most gram-positive bacteria, use a system which includes another glycerol-3-phosphate acyltransferase, PlsY, in conjunction with the enzyme PlsX (Yao and Rock 2013; Parsons and Rock 2013). The second fatty-acid chain is attached by 1-acylglycerol-3-phosphate O-acyltransferase (PlsC).

Fig. 2.

—Bayesian consensus trees of archaeal enzymes. Support values are Bayesian posterior probabilities. The black arrow and the white arrow indicate the modal root positions obtained using the RMC and MAD approaches, respectively. The dashed arrow indicates the RMC and MAD roots for the larger GGGPS subclade. Archaea in blue-tones and Bacteria in red/pink-tones. (a) G1PDH tree (111 sequences and 190 positions) inferred under the best-fitting LG + C60 model. (b) GGGPS tree (133 sequences and 129 positions) inferred under the best-fitting LG + C40 model. (c) DGGGPS tree (97 sequences and 119 positions) inferred under the best-fitting LG + C60 model. Terrabacteria are Firmicutes, Actinobacteria, Cyanobacteria, Chloroflexi, and related lineages. FCB are Fibrobacteres, Chlorobi, and Bacteroidetes and related lineages. PVC are Planctomycetes, Verrucomicrobia, and Chlamydiae and related lineages. TACK are Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota. DPANN include Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota, as well as several other lineages. For full trees, see supplementary figures 1–4, Supplementary Material online. For full unrooted trees, see supplementary figures 16–18, Supplementary Material online.

Fig. 3.

—Bayesian consensus trees of both G3PDH enzymes. Support values are Bayesian posterior probabilities. The black arrow and the white arrow indicate the modal root positions obtained using the RMC and MAD approaches, respectively. Archaea in blue-tones and Bacteria in red/pink-tones. (a) GpsA tree (84 sequences and 169 positions) inferred under the best-fitting LG + C60 model. (b) GlpA/GlpD tree (51 sequences and 199 positions) inferred under the best-fitting LG + C60 model. Terrabacteria are Firmicutes, Actinobacteria, Cyanobacteria, Chloroflexi, and related lineages. FCB are Fibrobacteres, Chlorobi, and Bacteroidetes and related lineages. PVC are Planctomycetes, Verrucomicrobia, and Chlamydiae and related lineages. TACK are Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota. DPANN include Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota, as well as several other lineages. For full trees, see supplementary figures 5 and 6, Supplementary Material online. For full unrooted trees, see supplementary figures 19 and 20, Supplementary Material online.

Fig. 4.

—Bayesian consensus trees of GlpK, PlsC, and PlsY enzymes. Support values are Bayesian posterior probabilities. The black arrow and the white arrow indicate the modal root positions obtained using the RMC and MAD approaches, respectively. Archaea in blue-tones and Bacteria in red/pink-tones. (a) GlpK tree (77 sequences and 363 positions) inferred under the best-fitting LG + C60 model. (b) PlsC tree (74 sequences and 57 positions) inferred under the best-fitting LG + C60 model. (c) PlsY tree (60 sequences and 104 positions) inferred under the best-fitting LG + C50 model. Terrabacteria are Firmicutes, Actinobacteria, Cyanobacteria, Chloroflexi, and related lineages. FCB are Fibrobacteres, Chlorobi, and Bacteroidetes and related lineages. PVC are Planctomycetes, Verrucomicrobia, and Chlamydiae and related lineages. TACK are Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota. DPANN include Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota, as well as several other lineages. For full trees, see supplementary figures 7–9, Supplementary Material online. For full unrooted trees, see supplementary figures 20–23, Supplementary Material online.