News Release

Widespread Bathyarchaeia encode a novel methyltransferase utilizing lignin-derived aromatics

Peer-Reviewed Publication

Beijing Zhongke Journal Publising Co. Ltd.

Figure 1 Microbial composition of enrichment cultures and growth curves of the cultured Ca. B. ligniniphilus.           Figure 2 An overview of the central metabolic pathway of Ca. B. ligniniphilus.


A. The relative abundance of microbial populations based on 16S rRNA gene-tag sequencing analysis before purification by adding antibiotics. B.Growth curves of Ca. B. ligniniphilus in anaerobic medium supplemented with lignin. The error bars were obtained from triplicate Q-PCR reactions. C.The relative abundance of microbial populations based on 16S rRNA gene-tag sequencing analysis after purification by adding antibiotics.     

The process of O-demethylation of guaiacol is accompanied by the fixation of carbon dioxide and the production of acetate. Yellow rectangles of varied shades exhibit different gene transcription levels, and blue rectangles of different shades show whether the relevant proteins were detected in the proteomics. Below the pathway is the Gibbs free energy (ΔG) estimated for the overall equation.


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Credit: Beijing Zhongke Journal Publising Co. Ltd.

This study wasled by Prof.Fengping Wang (School of Oceanography, Shanghai Jiao Tong University). In 2015, The researchers enriched Bathyarchaeia from coastal sediment samples by adding lignin as the sole organic carbon source, followed by one year of incubation. The enrichment culture was transferredto artificial seawater medium supplemented with 5g/lkraft lignin and 50 mM sodium bicarbonate; then, it was incubated at 35℃. The growth of Bathyarchaeia was confirmed by quantitative PCR(Q-PCR) after two monthsofincubation. Subsequently, the growth of Bathyarchaeia was maintained in theirlab for more than five years by consecutive 1:10 transfers at 2–3 months intervals. By December 2021, one archaeon of Bathyarchaeia named CandidatusBaizosediminiarchaeumligniniphilus DL1YTT001 (Ca. B.ligniniphilus) predominated the enrichment culture, accounting for ~60% of the totalabundance of 16S rRNA gene sequences (Fig.1). The enrichment culture was transferred to fresh media supplemented with theantibiotics as well as an additive of metabolites prepared from supernatant of the same enrichment culture. After three transfers, Ca. B. ligniniphilus reached ~90% sequence abundance of all microbes in the enrichment (Fig. 1).

Metabolomes targeting low molecular weight (LMW) aromatic compounds on day 0 and day 30 of the incubation were analyzed by GC-MS. On day 0, a variety of lignin-derived ArOCH3, including guaiacol, vanillin, acetovanillone, vanillic acid, homovanillic acid, and syringic acid, were detected. These monomers all dramatically decreased during the 30-day incubation, and there was a substantial accumulation of their demethylated products, including catechol and protocatechoic acid; this confirmed the occurrence of O-demethylation from ArOCH3 during the incubation of Ca. B. ligniniphilus.

One gene cluster that may carry out the O-demethylation/methyltransferase process on ArOCH3 was found within the Ca. B. ligniniphilus MAG. This putative methyltransferase complex includes two copies of putative methyltransferase 1 (MT1, MtgB_1 and MtgB_2), one copy of putative methyltransferase 2 (MT2, MtgA), and one copy of putative corrinoid protein (CP,MtgC) (Fig. 2). In themethyltransferase complex, MT1 is the key enzyme responsible for substrate selectivity and determines the type of methyl substrate.Neither of putative MT1s (MtgB_1 and MtgB_2) of Ca. B. ligniniphilus had homology with known MT1, which indicated that these two MT1s (MtgB_1 and MtgB_2) are novel.To verify the function of the novel MT1s of Ca. B. ligniniphilus in O-demethylation and methyl transfer, theMtgB_2 was expressed and purified in Escherichia coli and its O-demethylation activity was confirmed (Fig. 3).

The genes encoding MtgB were only found in genomes of Bathyarchaeia. In 297bathyarchaeial MAGs, 60 MAGs were found to contain the Bathyarchaeia-specific MtgBgene or its gene cluster;intriguingly, all 60 MAGs clustered in the orderBaizomonadales(Fig. 4). Thus, the evolution model of Bathyarchaeia-specific MtgBgenes may be vertical inheritance because they likely evolved from the common ancestor of order Baizomonadales.

The MtgB genes were identified in metagenomic datasets from a wide range of coastal sediments, and they were high expressed in coastal sediments from the East China Sea.These findings suggest that Bathyarchaeia capable of O-demethylation via theirnovelandspecificmethyltransferases are ubiquitous in coastal sediments.

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