Long-Term Transcriptional Activity at Zero Growth of a Cosmopolitan Rare Biosphere Member

Abstract

Microbial diversity in the environment is mainly concealed within the rare biosphere (all species with <0.1% relative abundance). While dormancy explains a low-abundance state very well, the mechanisms leading to rare but active microorganisms remain elusive. We used environmental systems biology to genomically and transcriptionally characterize "Candidatus Desulfosporosinus infrequens," a low-abundance sulfate-reducing microorganism cosmopolitan to freshwater wetlands, where it contributes to cryptic sulfur cycling. We obtained its near-complete genome by metagenomics of acidic peat soil. In addition, we analyzed anoxic peat soil incubated under in situ-like conditions for 50 days by Desulfosporosinus-targeted qPCR and metatranscriptomics. The Desulfosporosinus population stayed at a constant low abundance under all incubation conditions, averaging 1.2 × 10⁶ 16S rRNA gene copies per cm³ soil. In contrast, transcriptional activity of "Ca. Desulfosporosinus infrequens" increased at day 36 by 56- to 188-fold when minor amendments of acetate, propionate, lactate, or butyrate were provided with sulfate, compared to the no-substrate-control. Overall transcriptional activity was driven by expression of genes encoding ribosomal proteins, energy metabolism, and stress response but not by expression of genes encoding cell growth-associated processes. Since our results did not support growth of these highly active microorganisms in terms of biomass increase or cell division, they had to invest their sole energy for maintenance, most likely counterbalancing acidic pH conditions. This finding explains how a rare biosphere member can contribute to a biogeochemically relevant process while remaining in a zero-growth state over a period of 50 days.IMPORTANCE The microbial rare biosphere represents the largest pool of biodiversity on Earth and constitutes, in sum of all its members, a considerable part of a habitat's biomass. Dormancy or starvation is typically used to explain the persistence of low-abundance microorganisms in the environment. We show that a low-abundance microorganism can be highly transcriptionally active while remaining in a zero-growth state for at least 7 weeks. Our results provide evidence that this zero growth at a high cellular activity state is driven by maintenance requirements. We show that this is true for a microbial keystone species, in particular a cosmopolitan but permanently low-abundance sulfate-reducing microorganism in wetlands that is involved in counterbalancing greenhouse gas emissions. In summary, our results provide an important step forward in understanding time-resolved activities of rare biosphere members relevant for ecosystem functions.

Keywords: cryptic sulfur cycle; growth arrest; keystone species; maintenance; metatranscriptome; peatland.

FIG 1

Metabolic model of Desulfosporosinus sp. MAG SbF1. Gene expression stimulated by specific substrates in combination with sulfate is indicated by colored points. Paralogous genes are indicated by an underscore followed by a number. Plus signs indicates proposed protein complexes. Details for all genes are given in Table S1a, and transcription patterns are shown in Fig. 4. For the citric acid cycle and anaplerotic reactions, carriers of reducing equivalents and further by-products are not shown. The following abbreviations were used: X, unknown reducing equivalent carrier, e.g., NAD⁺ or ferredoxin; WL, Wood-Ljungdahl pathway consisting of enzymes encoded by the acs operon, MetF, FolD, FchA, and Fhs; TCA, citric acid cycle; FDH, formate dehydrogenase; Hase, hydrogenase; NDH-1, NADH dehydrogenase 1; LDH, lactate dehydrogenase.

FIG 2

(a) Time‐resolved absolute abundance of the Desulfosporosinus population (black circles) compared to all Bacteria and Archaea (gray triangles) in anoxic peat soil microcosms under various in situ-like conditions as determined by quantitative PCR (modified from reference 5). Error bars represent 1 standard deviation of the mean (n = 3; n = 2 for propionate with sulfate stimulation, all days, and butyrate with sulfate stimulation, day 50). (b) Corresponding overall transcriptional changes (mRNA of all CDS) of Desulfosporosinus sp. MAG SbF1 in the same anoxic microcosms. Error bars represent 1 standard deviation of the mean (n = 3; n = 2 for propionate with sulfate stimulation).

FIG 3

Time-resolved transcriptional changes of selected genes representing the sulfate reduction pathway (sat, dsrA), ribosomal proteins of the large (rplA) and small (rpsC) subunit, the GroEL chaperon (groL), cell division (ftsZ), DNA replication (gyrB), and peptidoglycan synthesis (murA). Panels represent the various substrate incubations: initial, initial peat soil to set up peat microcosms; +/−S, incubations with or without external sulfate, respectively. The size and color of the dots represent average FPKM values of the respective normalized gene expression.

FIG 4

Transcription patterns of whole pathways and central enzyme complexes involved in the carbon and energy metabolism of Desulfosporosinus sp. MAG SbF1 under in situ-like conditions. In addition, transcription patterns of general stress response proteins are shown. Mean abundance for the native soil (–) and each incubation treatment and time point is shown. Supplemented substrates are indicated by initials, and addition of external sulfate is depicted by −S/+S (columns). Abundance values are normalized variance-stabilized counts x, which were scaled from 0 to 1 for each CDS using the formula [x − min(x)]/max[x − min(x)]. Incompletely assembled genes are indicated by _a, _b, and _c.