Phylogenomics of an uncultivated, aerobic and thermophilic, photoheterotrophic member of Chlorobia sheds light into the evolution of the phylum Chlorobi

Abstract

All cultivated members of the phylum Chlorobi are classified under the two classes Chlorobia and Ignavibacteria. The recently-reported, uncultivated genome-species of Chlorobi have not suggested any alteration in the dichotomy of the two classes, but have hypothesized the existence of a distinct, aerobic and photoheterotrophic, order/family level lineage within Chlorobia, which otherwise was considered to be a monophyletic group of anaerobic sulfur-photolithoautotrophs. Here we report the discovery of a novel population genome bin (named Chlorobi-445) from the combined metagenomes of three spatially-contiguous but visually-distinct microbial mats growing along the 65-41 °C hydrothermal gradient of a boron-rich microbialite spring located in the Puga geothermal area of Eastern Ladakh, India. 1.3, 8.2 and 3.8% metagenomic reads from the mat communities located at 65 °C, 52 °C and 41 °C sample-sites respectively, were found to map-back to the 2,809,852 bp genome of Chlorobi-445. Phylogenomically, and therefore in terms of potential metabolic attributes, Chlorobi-445 showed close relationship with Ca. Thermochlorobacter aerophilum. Gene content suggested Chlorobi-445 to be an aerobic photoorganoheterotroph. Although this new lineage encodes all the proteins necessary for the biosynthesis of bacteriochlorophylls and the photosynthetic reaction centre, it is potentially devoid of genes concerned with lithotrophic sulfur oxidation and carbon-fixation. Individual Chlorobi phylogenies based on the sequence similarities of 16S rRNA genes, 22 ribosomal proteins, and 56 conserved marker-proteins that are encoded from single-copy genes, unanimously suggested that the class Chlorobia encompasses two major branches/clades. Whereas the Clade-I is a homogeneous cluster of culturable, anaerobic sulfur-/iron-oxidizing photolithoautotrophs, Clade-II harbors (i) Chloroherpeton species, and (ii) uncultivated aerobic photoheterotrophs such as Chlorobi-445, Chlorobium sp. GBChlB &Ca. T. aerophilum, in its two sub-clades. Distribution of bioenergetic attributes over the different branches of Chlorobi, together with the aerobic chemoorganoheterotrophic nature of the deepest-branching genome-species NICIL-2, indicated that the early Chlorobi were aerobic chemoorganoheterotrophs, while anaerobicity, phototrophy, lithotrophy, and autotrophy were all potentially added in the course of evolution.