Genetic and functional properties of uncultivated MCG archaea assessed by metagenome and gene expression analyses

Abstract

The Miscellaneous Crenarchaeota group (MCG) Archaea is one of the predominant archaeal groups in anoxic environments and may have significant roles in the global biogeochemical cycles. However, no isolate of MCG has been cultivated or characterized to date. In this study, we investigated the genetic organization, ecophysiological properties and evolutionary relationships of MCG archaea with other archaeal members using metagenome information and the result of gene expression experiments. A comparison of the gene organizations and similarities around the 16S rRNA genes from all available MCG fosmid and cosmid clones revealed no significant synteny among genomic fragments, demonstrating that there are large genetic variations within members of the MCG. Phylogenetic analyses of large-subunit+small-subunit rRNA, concatenated ribosomal protein genes and topoisomerases IB gene (TopoIB) all demonstrate that MCG constituted a sister lineage to the newly proposed archaeal phylum Aigarchaeota and Thaumarchaeota. Genes involved in protocatechuate degradation and chemotaxis were found in a MCG fosmid 75G8 genome fragment, suggesting that this MCG member may have a role in the degradation of aromatic compounds. Moreover, the expression of a putative 4-carboxymuconolactone decarboxylase was observed when the sediment was supplemented with protocatechuate, further supporting the hypothesis that this MCG member degrades aromatic compounds.

Figure 1

The phylogenetic tree of uncultivated MCG discussed in the text. The tree was constructed from the alignment of >900 unambiguously aligned base pairs using MAFFT followed by Maximum likelihood method by RAxML with the GTRGAMMA model. The stability of the topology was evaluated by bootstrapping (100 replicates). The resulting bootstrap values are indicated at each node in the tree. The names of MCG groups (MCG-A to -G, and class 1–17) were modified based on Jiang et al.'s classification (Jiang et al., 2011) and Kubo et al.'s classification (Kubo et al., 2012), respectively.

Figure 2

Comparison of gene organization. The gene organizations of the genomic fragment from six MCG fosmid/cosmid clones were compared with each other. The genes are colored according to Clusters of Orthologous Groups (COG) category, and 16S rRNAs are linked in gray.

Figure 3

The maximum likelihood tree based on the LSU-SSU sequences from archaea and bacteria. All sequences were retrieved from whole genomes or from environmental genomic fragments that contain LSU-SSU operon. In the tree, 75G8 indicates the genomic fragment obtained in this study. The sequences of LSU-SSU operon were aligned using MAFFT with L-INS-i strategy. The maximum likelihood tree was computed by RAxML program using the general time-reversible (GTR) model of sequence evolution, by including a gamma-correction. The numbers at the nodes represent the non-parametric bootstrap values that were computed by RAxML.

Figure 4

Unrooted maximum likelihood phylogenetic tree of TopoIB. TopoIB sequences from Thaumarchaeota, Virus and Euryotes. The numbers at the branches represent the bootstrap proportions. The scale bar represents the average number of substitutions per site.

Figure 5

(a) A photo of the syringe filled with sediment and protocatechuate after 45 days' culturing. The labels from top to bottom (L1, L2, L3 and L4) correspond to the colors of the sediment layers. (b) Reverse transcription PCR (RT-PCR) analysis of 75G8_CDS16 and 75G8_CDS 17 from the RNA extracted from different layers of a culturing syringe (L1–L4), original sediment (B1) and a control sample (B2). L1–L4 indicate the different layers of the syringe sediment. B1 represents the original sediment sample without any treatment and B2 represents the control sample that was cultured under the same conditions but without protocatechuate. P indicates the positive control that used 75G8 fosmid DNA as the PCR template, and N indicates the negative control that used water as template.