The ISME Journal volume 17, páginas 916–930 (2023)Cite este artigo
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Embora a decomposição da matéria orgânica pelas bactérias desempenhe um papel importante na ciclagem de nutrientes nos ecossistemas terrestres, o significado dos vírus permanece pouco compreendido. Aqui combinamos metagenômica e metatranscriptômica com amostragem temporal para estudar a importância de bactérias mesófilas e termofílicas e seus vírus na ciclagem de nutrientes durante a compostagem hipertermofílica (HTC) em escala industrial. Nossos resultados mostram que a dinâmica e a atividade da densidade vírus-bactéria estão fortemente acopladas, onde vírus específicos de bactérias mesófilas e termofílicas rastreiam as densidades de seus hospedeiros, desencadeando a sucessão da comunidade microbiana através do controle de cima para baixo durante a HTC. Além disso, os vírus específicos das bactérias mesófilas codificaram e expressaram vários genes metabólicos auxiliares (AMGs) ligados ao ciclo do carbono, impactando a renovação de nutrientes juntamente com as bactérias. A renovação de nutrientes correlacionou-se positivamente com a proporção vírus-hospedeiro, indicativo de uma relação positiva entre o funcionamento do ecossistema, a abundância viral e a atividade viral. Estes efeitos foram predominantemente impulsionados por vírus de ADN, uma vez que a maioria dos vírus de ARN detectados estavam associados a eucariotas e não associados à ciclagem de nutrientes durante a fase termofílica da compostagem. Nossas descobertas sugerem que os vírus de DNA poderiam impulsionar a ciclagem de nutrientes durante a HTC, reciclando a biomassa bacteriana através da lise celular e expressando os principais AMGs. Os vírus poderiam, portanto, ser potencialmente utilizados como indicadores do funcionamento do ecossistema microbiano para optimizar a produtividade dos sistemas biotecnológicos e agrícolas.
A decomposição da matéria orgânica é um processo chave do ecossistema, impactando a ciclagem de nutrientes e a produtividade nos ecossistemas terrestres [1]. Embora se saiba que as comunidades bacterianas e fúngicas desempenham papéis cruciais na reciclagem de nutrientes através da “alça microbiana” [1], o papel dos vírus bacterianos, ou seja, bacteriófagos (fagos), ainda é pouco compreendido [2]. Como a entidade biológica mais abundante na Terra, os vírus desempenham um papel crítico na condução da mortalidade microbiana através da lise celular [3, 4], impactando significativamente o ciclo dos elementos através da liberação de nutrientes e afetando a composição da comunidade microbiana, a diversidade e a necromassa microbiana [5,6, 7,8,9,10]. Embora a importância dos vírus para a ciclagem de nutrientes nos oceanos esteja bem estabelecida [11], estamos apenas começando a entender como os vírus modulam a renovação de nutrientes e a mineralização da matéria orgânica nos solos [9, 10, 12]. Demonstrou-se que genes metabólicos auxiliares (AMGs) codificados por vírus associados a hidrolases de glicosídeos e ao metabolismo do metano contribuem para a ciclagem de carbono nos ecossistemas do solo [4, 6, 13]. Por exemplo, 14 AMGs incluindo 9 famílias de glicosídeo hidrolases, como a endomananase com atividade funcional confirmada, indicam que os vírus têm a capacidade potencial de participar na complexa degradação do carbono [4]. Além de impulsionar a ciclagem de nutrientes por meio da lise de células bacterianas nos solos, foi recentemente demonstrado que os vírus melhoram a sobrevivência de suas bactérias hospedeiras sob estresse ambiental, codificando genes metabólicos auxiliares (AMGs) que aumentam a capacidade metabólica dos hospedeiros bacterianos [14]. Apesar desses avanços recentes, ainda temos uma compreensão limitada de como os vírus e as bactérias, juntos, conduzem a ciclagem de nutrientes e a decomposição da matéria orgânica nos ecossistemas terrestres [15].
Aqui usamos uma compostagem hipertermofílica (HTC) como sistema modelo para estudar o papel das bactérias mesófilas e termofílicas e seus vírus na decomposição da matéria orgânica. HTC é uma tecnologia de tratamento de resíduos utilizada na degradação da fração orgânica de resíduos sólidos urbanos ou agrícolas, atingindo temperaturas extremamente altas (até 90 °C) sem aquecimento exógeno devido à atividade da comunidade bacteriana termofílica [16,17,18]. HTC contém três fases principais de temperatura: hipertermofílica (>80 °C), termofílica (>50 °C) e fase de maturação (temperatura ambiente). Durante o processo, substâncias poliméricas enriquecidas com carbono e nitrogênio (lignocelulose, proteínas, polissacarídeos e lipídios) são degradadas durante as fases termofílicas da compostagem, enquanto os compostos enriquecidos com húmicos de degradação lenta são degradados durante a fase de maturação. A composição das comunidades microbianas que impulsionam a degradação da matéria orgânica muda dinamicamente após a temperatura de compostagem durante HTC [19], e táxons termofílicos e resistentes ao calor (Firmicutes; Bacillus e Deinococcota; Thermus) são importantes para a decomposição da matéria orgânica durante o fase termofílica [16]. Embora os efeitos da temperatura, das matérias-primas e das propriedades físico-químicas da compostagem tenham sido extensivamente estudados em relação à montagem da comunidade microbiana e à degradação da matéria orgânica [20, 21], muito pouco se sabe sobre o papel dos vírus durante a HTC.
90 °C), thermophilic phase (from day 10 to 26: >55 °C) and maturation phase (from day 27 to 45: <45 °C). To cover changes during the whole composting process, eight samples from five compost piles were collected at different phases of hyperthermophilic composting on days 0 (D0), 4 (D4), 7 (D7), 9 (D9), 15 (D15), 21 (D21), 27 (D27), 33 (D33) and 45 (D45). To obtain well-distributed and homogenized samples, each pile was diagonally divided into five domains, and each domain was sampled from the same location at a depth of 40–50 cm at different sampling time points. Within each pile, five subsamples (5000 g each) per domain were collected, and then mixed into a single composite sample, which was further divided into two aliquots. One replicate aliquot was stored in liquid nitrogen for biological analyses and the other was kept at 4 °C for physicochemical analyses. An automatic temperature controller was used to determine temperature changes during the composting./p>30 and length >36 bases) [35]. All high-quality sequences were co-assembled using SPAdes v3.13.1 with the parameters “-k 33, 55, 77, 99, 111,127 --meta” [36]. We also assembled reads generated at each thermal phase of composting separately (composting phase-specific assemblies) using SPAdes with the same parameters. All assembled scaffolds longer than 2.0 kb were binned using metawrap [37] based on MetaBAT2 [38], MaxBin2 [39], and Concoct [40] with default parameters. Bins were further manually curated to obtain high-quality genomes using Bin_refinement module in Metawrap [37]. The completeness and contamination of genome bins were assessed using CheckM v1.0.13 [41], and metagenome-assembled genomes (MAGs) with more than 50% completeness and less than 10% contamination level were retained for further analyses. Bins from different samples were dereplicated to produce medium to high quality genomes using dRep v.2.3.2 [42] and assigned to taxonomic classifications based on the Genome Taxonomy Database (GTDB; release 03-RS86) using the GTDB-Tk toolkit (v.0.3.2) with the classify workflow [43]. To construct bacterial MAGs, genes were called using Prodigal with parameters “-p meta” [44] and annotated against the KEGG and Pfam databases using the Diamond tool [45]. The predicted proteins were screened for candidate CAZymes using hmmscan module from HMMER v3.2.1 and dbCAN database (cutoffs: coverage fraction: 0.40; e-value:1e-18) [46]. Genes encoding proteases and peptidases were identified using Diamond against the MEROPS database release 12.0 (cutoffs: e-value 1e-20 -accel 0.8). Ribosomal RNAs were predicted using RNAmmer v1.2 [47]. The optimal growth temperature (OGT) of MAGs was predicted by the machine learning method using the Tome v1.1 [48]. Thermophilic MAGs were defined as ones with OGT ≥ 50 °C, while MAGs were assigned as mesophilic when their OGT < 50 °C. To build phylogenetic MAG trees, the “classify” workflow in GTDB-Tk (v.0.3.2; default settings) was used to identify 120 bacterial marker genes, which were used for tree construction based on multiple sequence alignment. The resulting FASTA files containing multiple sequence alignments of the submitted genomes were used for maximum likelihood phylogenetic tree inference using FastTree v.2.1.10 with the default parameters [49]. Newick tree output files were visualized with iTOL v.5 [50]./p>1000 bp, composting phase-specific assemblies) were compared against the database containing all available viral RdRp gene sequences in NCBI/GenBank (37, 441 genes, downloaded on February 2023) and previous published studies [77, 78] using Diamond BLASTx (coverage ≥ 70%, E-value≤1e-10 and score ≥ 70). Sequences that had hits in the RdRp database with the RdRp core domain were considered as the potential RNA viruses [80]. This analysis identified 109 contigs with RdRp gene. These potential RNA virus contigs were clustered with CD-HIT using 95% average nucleotide identity across 85% alignment fraction, resulting in a total of 83 potential RNA viruses./p>5 kb) were obtained from the metatranscriptomic assemblies. After clustering (95% nucleotide similarity and over 85% coverage), a total of 41 dsDNA viral operational taxonomic units (dsvOTUs) were retained. By comparing the viruses’ contigs derived from transcriptomic data and metagenomic data, only 7 of 68 dsvOTUs could be assembled from the metagenomic data. This is not surprising as the DNA was removed during RNA library preparation and very few DNA sequences was retained in transcriptome./p>80 °C for 9 days (“hyperthermophilic phase”), after gradually declining to 55 °C (“thermophilic phase”) and ambient temperature by day 27 (“maturation phase”, Fig. 1a). The organic matter (OM) decomposition, carbon, and nitrogen turnover followed closely different phases of HTC (Fig. 1a). Compared to the initial composting raw materials, total carbon (TC, F3,23 = 33.6, p < 0.0001) and nitrogen (TN, F3,23 = 19.8, p < 0.0001) contents significantly decreased by 32% and 28% by the end of HTC, respectively (Fig. S1a). Similarly, the OM content that showed the highest degradation rate at the hyperthermophilic phase declined from 51.3% to 38.7% (F3,23 = 68.3, p < 0.0001), while the concentration of water-soluble carbon (WSC, F3,23 = 19.8, p < 0.0001) and water-soluble nitrogen (WSN, F3,23 = 26.4, p < 0.0001) increased during HCT, reaching peak concentrations at the hyperthermophilic phase (Fig. 1a). The degradation rate of OM correlated positively with temperature, WSC, and WSN (Fig. S1b), indicative of efficient nutrient cycling during HTC./p>5 kb) were obtained from the metagenomic assemblies. After clustering (95% nucleotide similarity and over 85% coverage), a total of 1297 viral operational taxonomic units (vOTUs) were retained (Table S1), which mainly belonged to double-stranded DNA viruses (97%) and were predicted to be mostly lytic (66.2%). The genome quality of vOTUs consisted of 0.7% of high-quality, 2.4% of medium-quality, and 85.6% low-quality vOTUs, while the quality of remaining 11.3% of vOTUs could not be determined. Overall, 78.6% of vOTUs were detected during non-thermophilic phases (D0 and D27), while 21.3% occurred during thermophilic phases (D4 and D15, Table S1). Only 7.7% of vOTUs could be clustered with taxonomically known viruses in RefSeq database (v216, released in February 2023), while only 35 vOTUs (2.6%) clustered with known viruses in IMG/VR (v3) database, suggesting that most of the composting viruses were novel. Primarily, they belonged to Dividoviricota (88%) and Uroviricota (2%) phyla and Mesyanzhinovviridae (27.2%), Herelleviridae (18.2%), Salasmaviridae (16.4%), Autographiviridae (5.4%), Vilmaviridae (5.4%) and Matshushitaviridae (3.6%) families (Table S1 and Fig. S4). Similar to bacteria, the richness (F3.8 = 4.7, p = 0.0359) and composition (R2 = 0.78, p < 0.001, PERMANOVA test) of viral communities followed different phases of HTC (Fig. 1d). While Vilmaviridae (37.8%) and Autographiviridae (14.5%) were dominant viruses in the composting raw material (D0), Vilmaviridae abundances significantly decreased to 1.5% by the maturation phase (D27; F3,8 = 9.7, p = 0.0047, Fig. S4). In contrast, relative abundance of Matshushitaviridae family under Dividoviricota phylum (consisting mainly of thermophile-associated Thermus phages) increased from 1.4% at D0 to 66.3% at D15 (F3,8 = 5.7, p = 0.0245, Fig. S4). As most of viruses could not be classified, viral abundances were also investigated based on their predicted host taxonomy (see Methods). The viral taxa abundances followed bacterial taxa abundances (Fig. 1d), and for example, the abundance of viruses infecting Deinococcota clearly increased with rising composting temperature by D15. Moreover, Matshushitaviridae viral abundances correlated positively with their Firmicute (R2 = 0.34, p = 0.028) and Deinococcota (R2 = 0.53, p = 0.0042, Fig. S5) host abundances. Overall, changes in viral community richness (R2 = 0.50, p = 0.0058) and composition (beta-dissimilarity, R2 = 0.71, p < 0.0001) correlated positively with changes in bacterial community richness and composition (Fig. 2a, b)./p> 50 °C) bacteria (upper panel) and 180 mesophilic and 47 thermophilic MAGs (lower panel) during HTC. b Box plots and heatmap representing changes in the transcriptional activity of viruses associated with mesophilic and thermophilic bacteria (upper panel) and individual vOTUs (lower panel) during HTC. Box plots and heatmaps representing changes in the transcriptional activity of mesophilic (OGT < 50 °C) and thermophilic (OGT > 50 °C) bacteria during HTC based on mean (upper panel) and individual (lower panel) MAGs (including 180 mesophilic and 47 thermophilic MAGs) in association to carbon (CAZyme) (c) and nitrogen metabolism genes (d). e Box plot and heatmap representing changes in the transcriptional activity of virus-associated carbon (CAZyme) metabolism genes linked with mesophilic MAGs (OGT < 50 °C). In all (a–e), the mean transcriptional activity (MAGs and vOTUs) shown in boxplots is based on transcript abundances (transcripts per million, TPM) normalized by MAG and vOTU abundances. Box plots encompass 25–75th percentiles, whiskers show the minimum and maximum values, and the midline shows the median (dots present the biologically independent samples, asterisks denote for significant differences (*p < 0.05, **p < 0.01. n.s, no significant differences). Heatmaps show the transcriptional activity (MAGs or vOTUs) based on non-normalized transcripts abundances (transcripts per million, TPM). In (c and d), selected CAZymes include GHs, GTs, PLs, CEs, CBMs, and AAs. Nitrogen metabolic pathways include assimilatory nitrate reduction, dissimilatory nitrate reduction, nitrification, and nitrogen fixation pathways. More detail about the functional genes included can be found in Supplementary Data 6 and 7, respectively./p>5 kb) were obtained from the metatranscriptomic assemblies. After clustering (95% nucleotide similarity and over 85% coverage), a total of 41 dsDNA viruses were retained. By comparing the dsDNA viruses contigs derived from transcriptomic data and metagenomic data, 89% viruses (61 of 68) could be assembled from both transcriptomic and metagenomic data, suggesting that very few dsDNA viruses exclusively exist in metatranscriptomic dataset. As a result, the RNA viral community abundance (based on beta-dissimilarity of abundance matrix) did not correlate with changes in composting properties (Mantel statistic r = 0.0173, p = 0.35), which suggests that they did not contribute to the nutrient cycling during composting./p>90 °C) and consistently surpassed bacterial abundances in terms of virus-host abundance ratio. Mesophilic and thermophilic bacteria and their viruses showed clear microbial community succession, where the initial phase of composting was dominated by mesophiles, which were subsequently replaced by thermophiles and subsequently by mesophiles towards the end of the HTC. Although similar compositional succession of bacterial and fungal communities have been observed in previous composting experiments [19, 90], this is the first evidence demonstrating that viruses can also drive ecological succession in microbial communities during HTC. These findings are also indicative of “Kill-the-Winner” hypothesis, where viruses target and regulate the most abundant group of host bacteria, reducing the dominance effects and evening out competition between different bacterial taxa [4, 89]. Such dynamics could explain the observed community shift between thermophilic and maturation phases of HTC, where thermophilic viruses likely drove down the abundances of thermophilic bacteria, giving rise to mesophilic bacteria and their phages. For example, Thermus and Planifilum bacterial genera play important role in heat production during HTC [17] and several lytic phages that infected Thermus thermophilus (T_bin.227) and Planifilum fulgidum (T_bin.201) were identified, including five potentially novel Thermus viruses that had genome sizes about 5 kbp similar to hyperthermophilic phage φOH3 isolated from Obama hot spring [91]. While 61% of detected phages were predicted to be lytic, it is possible that some of the correlations between bacterial and viral taxa were also driven by lysogenic phages or prophages because unfiltered DNA samples were used for metagenomics. As a result, our dataset likely underestimates phage diversity, and phage enrichment [92] should be used in future studies. Moreover, future work should also consider the potential role of RNA viruses for HTC, which we did not explore in detail as compost-associated bacteria are most often associated with DNA viruses [19]. Nevertheless, our results suggest that a small portion of thermophilic viruses played a key role in microbial activity during the thermophilic phase of HTC, indicating that compost ecosystem functioning was at least temporally driven by low-diversity microbial communities. Terrestrial phages could hence be important drivers of biogeochemical cycling in soil ecosystems via “viral shunt” akin to marine phages [11, 93]./p>
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