Genomic and in-situ Transcriptomic Characterization of the Candidate Phylum NPL-UPL2 From Highly Alkaline Highly Reducing Serpentinized Groundwater

Abstract

Serpentinization is a process whereby water interacts with reduced mantle rock called peridotite to produce a new suite of minerals (e.g., serpentine), a highly alkaline fluid, and hydrogen. In previous reports, we identified abundance of microbes of the candidate phylum NPL-UPA2 in a serpentinization site called The Cedars. Here, we report the first metagenome assembled genome (MAG) of the candidate phylum as well as the in-situ gene expression. The MAG of the phylum NPL-UPA2, named Unc8, is only about 1 Mbp and its biosynthetic properties suggest it should be capable of independent growth. In keeping with the highly reducing niche of Unc8, its genome encodes none of the known oxidative stress response genes including superoxide dismutases. With regard to energy metabolism, the MAG of Unc8 encodes all enzymes for Wood-Ljungdahl acetogenesis pathway, a ferredoxin:NAD⁺ oxidoreductase (Rnf) and electron carriers for flavin-based electron bifurcation (Etf, Hdr). Furthermore, the transcriptome of Unc8 in the waters of The Cedars showed enhanced levels of gene expression in the key enzymes of the Wood-Ljungdahl pathway [e.g., Carbon monoxide dehydrogenase /Acetyl-CoA synthase complex (CODH/ACS), Rnf, Acetyl-CoA synthetase (Acd)], which indicated that the Unc8 is an acetogen. However, the MAG of Unc8 encoded no well-known hydrogenase genes, suggesting that the energy metabolism of Unc8 might be focused on CO as the carbon and energy sources for the acetate formation. Given that CO could be supplied via abiotic reaction associated with deep subsurface serpentinization, while available CO₂ would be at extremely low concentrations in this high pH environment, CO-associated metabolism could provide advantageous approach. The CODH/ACS in Unc8 is a Bacteria/Archaea hybrid type of six-subunit complex and the electron carriers, Etf and Hdr, showed the highest similarity to those in Archaea, suggesting that archaeal methanogenic energy metabolism was incorporated into the bacterial acetogenesis in NPL-UPA2. Given that serpentinization systems are viewed as potential habitats for early life, and that acetogenesis via the Wood-Ljungdahl pathway is proposed as an energy metabolism of Last Universal Common Ancestor, a phylogenetically distinct acetogen from an early earth analog site may provide important insights in primordial lithotrophs and their habitat.

Keywords: acetogen; alkaliphile ecology; carbon monoxide dehydrogenase; last universal common ancestor; metagenome; metatranscriptome; serpentinization; subsurface microbial community.

FIGURE 1

Phylogenetic relationships among NPL-UPA2 16S rRNA genes. Tree topologies are supported by bootstrap values for 100 replicates. Red font denotes the Unc8 from The Cedars and blue font denotes phylotypes recovered from the serpentinization sites; Lost City hydrothermal field (LCHF) and Prony Bay hydrothermal field (PBHF). Accession numbers of OTU included in the triangle region are JQ816227, GU553753, JQ817159, JN676031, JX000976, JQ817187, JQ817075, JQ816462, JQ817138, JQ816442, JN230011, EU385719, JQ816456 and JQ818038. Ca. Desulforudis audaxviator was root of the tree.

FIGURE 2

MAG of Unc8 with key genes highlighted. The inner scales designate the coordinates (in kbp). The first (inner-most) circle shows the GC-skew. The second and third circles show the blastn analysis against the Prony Bay metagenome with different similarity cutoff. The lower and upper identity threshold in the blastn analysis is shown in the circle. The fourth circle shows the gene expression level (RNA reads normalized by the DNA reads) in BS5sc. The locations of some genes of interest are indicated.

FIGURE 3

Predicted energy metabolism for Unc8. Proteins and complexes are colored based on the functional categories. Abbreviations: CODH-Carbon monoxide dehydrogenase, Fdh-Formate dehydrogenase, Fhs-Formyl-THF synthase, Fch-Formyl-THF cyclohydrolase, FolD-Methylene-THF dehydrogenase, MetFV-Methylene-THF reductase, AcsE-Methyltransferase, CODH/ACS-Carbon monoxide dehydrogenase/Acetyl-CoA synthase complex, AcyP-Acylphosphatase, Acd-ADP forming Acetyl-CoA synthetase, Adh-Alcohol dehydrogenase, Nuo-NADH-quinone oxidoreductase, ETF-Electron transfer flavoprotein, HdrABC-Heterodisulfide reductase, Mrp-Multiple resistance and pH antiporter, A₁A_O-ATPase,-an Archaeal type ATPase, Rnf-proton/sodium-translocating ferredoxin-NAD:oxidoreductase complex, MgtE-Magnesium transporter, Trk-Potassium uptake system, YrbG-Ca²⁺/Na⁺ antiporter, Pst-ABC-type Phosphate transporter, Fep-Iron transporter, Tup-Tungstate transporter, Cbi-Cobalt/Nickel transporter.

FIGURE 4

Scatter plots of in situ gene expression of Unc8 in The Cedars springs. Scatter plots of all CDS in Unc8 responses as measured by mRNA frequency (mRNA-RPKM) between GPS1 and BS5sc. Key genes for acetogenesis are shown as unique colors, all other groups are shown in gray.

FIGURE 5

Alignment of NtpC amino acid sequences from Unc8 to determine the interactive cations (i.e., sodium or proton) (Mulkidjanian et al., 2008). N-end (A) and C-end (B) of NtpC that include active sites are shown. Colors in the species names indicate the genomes encoding proton (blue) or sodium (red) coupled A-ATPases. Active site residues are shown with black/white dots at the top of each alignment and white dots indicate specific residues that are conserved in sodium-ligands and are partly absent in proton binding subunit (Mulkidjanian et al., 2008).

FIGURE 6

Comparison of gene clusters for CODH and ACS. Green colored genes are archaeal in origin and red colored genes are bacterial in origin. In general, CODH/ACS is a five-subunits enzyme but Unc8, Chloroflexi bacterium RGB 13-51-36 and Ca. Desulforudis audaxviator encode a bacterial and archaeal hybrid type of six-subunits enzyme. Ca. Desulforudis audaxviator have two different clusters of gene sets for CODH/ACS on the MAG. Respective homologs of cdhA, cdhC, cdhD, and cdhE in Archaea are acsA, acsB, acsD and acsC in Bacteria (Adam et al., 2018). cdhB is exclusive to Archaea and acsE is exclusive to Bacteria.

FIGURE 7

Protein phylogeny of CODH/ACS complex. Phylogenetic trees were constructed for CdhA (A), CdhB (B), CdhC (C), AcsD/CdhD (D), AcsC/CdhE (E) and AcsE (F). Detailed trees are shown in Supplementary Figure S2.