Exploration of the humus-respiring mechanism was prompted by previous findings that the surrogate for humus, AQDS, facilitates the membrane-bound electron transport chain and energy conservation in M. acetivorans14. We first tested how humus impacts the growth and methane production in M. acetivorans. The addition of 0.5 and 1 mM humus significantly enhanced methane production at the mid-log phase of cultures that were amended with either methanol or acetate as a carbon source (Figs. 1A and 1D), and the amounts of cellular protein that were cultured for 100 hours and 200 hours for methanol-grown and acetate-grown, respectively, were 2-fold greater when the medium was supplemented with humus (Figs. 1C and 1F). The enhancing effect was also confirmed by monitoring the consumption of methanol and acetate, which indicated that humus stimulated both methylotrophic and acetoclastic methanogenesis in M. acetivorans (Figs. 1B and 1E). Prominent and abundant coccoid-shaped M. acetivorans cells that were attached to humus were detected by scanning electron microscopy (Figs. 1G and 1H). In addition, FTIR analysis of the cellular surface showed peaks at 3,430 cm-1, which were ascribed to O-H stretching and were wider in cells cultured with humus than in those not cultured with humus (Extended Fig. 1A). When the humus in the medium was substituted with AQDS, we observed a reduction in AQDS accompanied by cellular growth, which validated the previous findings that the quinone groups in humus constitute the primary electron-accepting moiety (Extended Fig. 1B).
Identification of cell surface quinoproteins involved in humus respiration
To gain insights into how humus respiration stimulates methanogenic pathways, we performed transcriptomic analyses of cells that were cultured with or without humus. The differentially expressed genes were screened with univariate statistical significance (log2 (FC) > 1.0 or < 1.0, and p < 0.05). Among the 4,462 genes expressed in M. acetivorans, a total of 79 and 340 genes were upregulated in the methanol- and acetate-grown cultures with humus respiration, respectively. Totals of 60 and 16 genes were downregulated in the methanol- and acetate-grown cultures with humus respiration, respectively (Extended Fig. 2). However, none of the genes directly involved in the methanogenic pathway, such as the genes encoding methyl-CoM reductase that catalyzes the production of methane in both methylotrophic and acetoclastic methanogenesis, were significantly regulated (Extended table 1). In addition, those genes encoding membrane-bound Rnf and Fpo complexes that have been known to participate in energy conservation for the growth of M. acetivorans, were also not significantly regulated (Extended table 1).
Remarkably, a gene cluster ranging from MA4284 to MA4315 was significantly upregulated in both methanol- and acetate-grown cells with humus respiration (Fig. 2C). The gene cluster contains a total of 32 genes, 19 of which were obtained for RNA sequence reads. The remaining 13 genes were not obtained for transcript reads, consistent with the information of these genes in the NCBI database, i.e., these are pseudogenes missing a functional N- or C-terminus (Fig. 2A). A total of 17 expressed genes have conserved N-terminal signal peptide sequences, and the predicted encoding products of these genes in the NCBI database are cell surface proteins. We are particularly interested in the cell surface proteins that were upregulated in humus-respiratory cells because close extracellular interactions between humus and M. acetivorans were observed. The detailed domain architectures of all expressed genes in the gene cluster are shown in Fig. 2B, and a brief overview is provided in Extended table 2. A total of 11 expressed genes (e.g., MA4284, MA4290-91, MA4294, MA4297, MA4305, MA4309-10, MA4312, and MA4314-15) were annotated as pyrroloquinoline quinone (PQQ)-binding β-propeller repeat proteins, which are composed of multiple β-propeller repeats with corresponding numbers of PQQ as cofactors and thus are also called quinoproteins 20. Three expressed genes (e.g., MA4285, MA4289 and MA4292) were annotated as leucine-rich repeat (LRR) proteins, which have multiple LRR domains that are frequently involved in the formation of protein–protein interactions at the cell surface21. Most PQQ-binding β-propeller repeat proteins and LRR proteins contain multiple polycystic kidney disease (PKD) domains that are usually found in the extracellular segments of archaeal surface-layer proteins with ambiguous functions22. Five expressed genes (e.g., MA4291, MA4305, MA4309, MA4312 and MA4315) contain multiple parallel beta-helix repeats (PbH1) that behave as a stack of parallel beta strands. Proteins containing PbH1 most often are enzymes with polysaccharide substrates23. None of the above stated domains are conserved in another five expressed genes, of which MA4293, MA4298 and MA4302 were annotated as hypothetical proteins, while MA4295 and MA4304 were annotated as a cobaltochelatase subunit and DUF4430 domain-containing protein, respectively. According to the domain composition of the gene cluster, we tentatively named the encoding products of the gene cluster cell surface quinoproteins, hereafter referred to as CSQs.
PQQ was isolated from the membrane fraction of M. acetivorans.
The gene cluster encoding CSQs in methanogenic archaea has not been reported to have a specific function, and it is completely unknown whether these CSQs contain PQQ as cofactors. Therefore, we followed a previously commonly used procedure for isolating quinones from the microbial membrane fraction and determined the isolated products by LC–MS based on a previously reported method24,25. As illustrated in Figs. 3A-3C, our isolated products from the methanol- and acetate-grown membrane fractions exhibited the same molecular ion peaks at m/z equal to 329, 285, 241 and 197 as the PQQ standard. According to previous reports26,27, the precursor ion of PQQ is m/z 329 [M-H]-, while the PQQ ions that were fragmented by different carboxyl groups are m/z 285 [M-H-CO2]-, m/z 241 [M-H-2CO2]-, and m/z 197 [M-H-3CO2]-. Our results clearly indicated that PQQ does exist in the membrane fraction of M. acetivorans. We then quantified the relative PQQ abundances in the membrane fractions by normalizing the total peak areas of the four ions to the known concentration of the PQQ standard. Figure 3D shows that the PQQ abundances in the membranes isolated from methanol- and acetate-grown cultures respiring with humus were significantly higher than those in normal membrane fractions (p < 0.05 or 0.01).
Upregulated CSQs facilitated extracellular electron transfer.
Because there is no documented method to directly monitor humus reduction, we used ferrihydrite, a natural form of Fe3+, as an extracellular acceptor to quantify the rates of extracellular electron transfer from normal resting cells and resting cells with upregulated CSQs. The resting cells were washed three times to remove residual humus. Figures 4B and 4C show two representative time courses for the reduction of ferrihydrite catalyzed by methanol- and acetate-grown resting cells, respectively. The electron transfer rates catalyzed by the resting cells with upregulated CSQs were ~ 2-fold greater than those catalyzed by the same amounts of normal resting cells. Additions of 2-hydroxyphenazine (2-HP), an analog of MP, increased the rates by ~ 2-fold when catalyzed by normal resting cells but by ~ 4-fold when catalyzed by resting cells with upregulated CSQs, which suggested a role of 2-HP as an electron mediator to interact with CSQs. These results establish that the CSQs that are abundant on the cell surface of M. acetivorans facilitate extracellular electron transfer, in which MP was likely involved.
CSQs are widely distributed in methanogens
A survey within the archaeal domain retrieved 2,684 predicted PQQ-binding β-propeller repeat proteins. Intriguingly, they are primarily clustered in Halobacteria (59.2%) and methanogens (19.3%). Of particularly interest, 6.7% of all PQQ-binding proteins were predicted in genus Methanosarcina, although only a handful of species were identified in this genus (Fig. 5A). A further search for CSQ homologous proteins in a nonredundant protein database confirmed the finding that PQQ-binding proteins are most abundant in the genus Methanosarcina among methanogens. For the MA4284 homologous that have multiple PQQ domains and PKD domains, 90.6% were present in the phylum Euryarchaeota, of which 62.6% are present in Methanosarcina (Fig. 5B). For the MA4290 homologous proteins that have only three PQQ domains, over 50% were found in the phylum Euryarchaeota, of which 34.6% were present in Methanosarcina (Fig. 5C). The analysis for all the other PQQ-containing CSQs were consistent with those for MA4284 and MA4290 (Extended Fig. 3). Taken together, these results clearly suggest the evolutionary enrichment of PQQ-binding and CSQ-homologous proteins in methanogens, particularly in Methanosarcina, which confirms the key role of PQQ in these archaeal groups.