Jul 07, 2023
Un Sulfurimonas hydrogénotrophe est globalement abondant en profondeur.
Nature Microbiology volume 8, pages 651-665 (2023)Citer cet article 5048 Accès 2 Citations 401 Détails Altmetric Metrics Membres du genre bactérien Sulfurimonas (phylum Campylobacterota)
Nature Microbiology volume 8, pages 651-665 (2023)Citer cet article
5048 Accès
2 citations
401 Altmétrique
Détails des métriques
Les membres du genre bactérien Sulfurimonas (phylum Campylobacterota) dominent les communautés microbiennes dans les redoxclines marines et jouent un rôle important dans le cycle du soufre et de l'azote. Ici, nous avons utilisé la métagénomique et les analyses métaboliques pour caractériser un Sulfurimonas de la crête de Gakkel dans l'océan Arctique central et la crête sud-ouest de l'Inde, montrant que cette espèce est omniprésente dans les panaches hydrothermaux non flottants des dorsales médio-océaniques de l'ensemble de l'océan mondial. Une espèce de Sulfurimonas, USulfurimonas pluma, s'est avérée mondialement abondante et active dans les panaches hydrothermaux froids (<0−4 °C), saturés en oxygène et riches en hydrogène. Comparé à d’autres espèces de Sulfurimonas, US. pluma a un génome réduit (> 17 %) et des signatures génomiques d'un métabolisme chimiolithotrophique aérobie utilisant l'hydrogène comme source d'énergie, y compris l'acquisition d'oxydase de type A2 et la perte de nitrates et de nitrites réductases. La domination et la niche unique des États-Unis. Les plumes des panaches hydrothermaux suggèrent un rôle biogéochimique méconnu des Sulfurimonas dans les profondeurs océaniques.
Le genre Sulfurimonas appartient au phylum Campylobacterota (ancienne classe Epsilonproteobacteria). Elle a été initialement proposée après l’isolement de Sulfurimonas autotrophica à partir de sédiments collectés dans une source hydrothermale en eaux profondes1. Depuis lors, 12 espèces distinctes de Sulfurimonas ont été isolées dans des environnements pauvres en oxygène2,3,4,5,6,7,8,9,10,11. Basé sur des séquences génétiques d'ARNr 16S, ce genre bactérien mésophile et chimiolithoautotrophe est omniprésent et constitue un membre dominant des communautés microbiennes habitant les environnements rédoxclines12, y compris les environnements sulfurés des sources hydrothermales des grands fonds13,14,15,16,17. Les membres décrits du genre Sulfurimonas occupent des habitats définis par des températures modérées, des concentrations élevées de sulfure d'hydrogène et de faibles concentrations d'oxygène (<40 µM) par rapport aux habitats d'autres membres hydrothermaux de Campylobacterota (c'est-à-dire Sulfuruvum16) et d'oxydants de soufre marins (c'est-à-dire, SUP0518,19). Pourtant, d’abondantes séquences génétiques d’ARNr de Sulfurimonas 16S ont également été signalées dans des stades non flottants de panaches hydrothermaux14,20,21,22,23,24. Des panaches hydrothermaux se produisent partout où des fluides hydrothermaux anoxiques chauds émis par le fond marin se mélangent à de l'eau de mer froide oxygénée. Ils peuvent s’élever jusqu’à des centaines de mètres du fond marin et se disperser à des milliers de kilomètres de leur source25. Au stade non flottant, les panaches hydrothermaux sont principalement constitués d'eau de mer froide et saturée en oxygène avec des mélanges très dilués de fluide hydrothermal (<0,01%)25,26. Pour cette raison, les panaches hydrothermaux non flottants n'ont pas été considérés comme une niche et un habitat permanents pour les Sulfurimonas. La détection répétée de séquences de Sulfurimonas dans de tels panaches s’explique par le transport passif depuis les fonds marins et sous-marins26. Cependant, aucune étude n'a directement testé si les panaches non flottants offraient un environnement propice à la croissance de membres spécifiques de Sulfurimonas. Les panaches hydrothermaux contiennent des quantités importantes de gaz réduits inorganiques (H2S, CH4 et H2) et de métaux (Fe, Mn, Cu, Zn et Co)27, qui ont un impact considérable sur la chimie des océans28. Ainsi, l'identification et l'élucidation de la physiologie des micro-organismes se développant dans le panache sont cruciales pour comprendre la biogéochimie de l'océan.
Dans cette étude, nous avons étudié la distribution et la fonction des Sulfurimonas dans les panaches hydrothermaux. Nous avons étudié ses ribotypes, ses génotypes et son métabolisme dans deux panaches de ventilation de la crête de Gakkel et dans un panache de la crête sud-ouest de l'Inde (SWIR), et nous les avons comparés avec des données accessibles au public provenant d'autres panaches de ventilation des dorsales médio-océaniques et d'autres environnements hébergeant Sulfurimonas sp. Notre hypothèse est que les panaches hydrothermaux non flottants constituent un environnement approprié pour des membres spécifiques de Sulfurimonas.
99%) in the non-buoyant hydrothermal plumes of Gakkel Ridge and in seawater from a ridge valley of the SWIR belonged to the genus Sulfurimonas (Supplementary Table 1 and Extended Data Fig. 2). In addition, more than 97% of the Sulfurimonas sequences of these three remote sites on ultraslow spreading ridges belonged to two closely related operational taxonomic units (OTU1 and OTU2), with a similarity of 99.5%. Fluorescence in situ hybridization using both a Campylobacterota-specific rRNA probe and tailored highly specific probes for the two detected Sulfurimonas OTUs confirmed these results (Extended Data Fig. 1b–f)./p>99.5% identity) dominated hydrothermal plumes across the ridge systems of the Central Arctic, Atlantic and Indian/Southern Oceans (Fig. 1a). The same ribotype was also found in the plume and the surrounding water column of the Guaymas Basin in the Gulf of California34, but with low proportions to the total bacterial community (Fig. 1a)./p>40 kpb). These results excluded that the observed genome reduction was an artefact of assembly and binning procedures./p>13 to >500 times higher expressed than genes for sulfur oxidation suggests that hydrogen is a critical energy source to sustain the growth of US. pluma in the Aurora plume (Fig. 2), where it was most abundant and active (Supplementary Table 1 and Extended Data Fig. 2). Laboratory experiments with cultures of S. denitrificans also found that this species grows more efficiently when supplied with hydrogen than with thiosulfate as electron donor38, suggesting that hydrogen can be an important energy substrate for the genus Sulfurimonas./p>20%), the cbb3-type oxidase becomes inefficient, resulting in impaired growth9,12. In fact, the cultured Sulfurimonas strains grow optimally at an O2 concentration of 1–8%, and become inactive at O2 concentrations higher than 20%1,2,3,4,5,9,11. Moreover, previous studies found Sulfurimonas predominantly in environments subject to strong fluctuations in O2 concentrations (that is, benthic and pelagic redoxclines12; Supplementary Table 3). The cold polar waters studied here are oxygen-saturated and the diluted hydrothermal fluids do not substantially lower their oxygen contents. Hence, US. pluma is permanently exposed to high oxygen concentrations (ca. 300 µM; Supplementary Table 3). We hypothesize that the acquisition of caa3-type (A2-type) cytochrome c oxidase allows an efficient respiration of US. pluma in this fully oxic environments. This cytochrome c oxidase is present in many aerobic bacteria and it has strong homology to the mitochondrial cytochrome oxidase (A1-type)43. Of note, within the phylum of Campylobacterota, we found all four subunits of caa3-type oxidase in the genome of Sulfurovum sp. AR derived from aerobic Arctic sediments44. This oxidase has an amino acid identity of 70% to that of US. pluma. However, this caa3-type oxidase cannot be misassembled in the US. pluma MAGs because Sulfurovum sequences are rare in the Gakkel seawater (Supplementary Table 1), and the synteny analysis of contigs encoding for this enzyme points toward an acquisition by horizontal gene transfer (Supplementary Fig. 2)./p>99.5% 16S rRNA gene sequence similarity) in hydrothermal plumes across the globe (Fig. 1) suggests that the Sulfurimonas cluster, including US. pluma, is part of the ocean microbial seed bank, and therefore that background seawater might be the source of US. pluma. On the other hand, it may be that US. pluma enters into the hydrothermal plumes from populations living on seafloor vent-associated environments, which due to oxygen tolerance have a higher dispersal potential than benthic Sulfurimonas species, resulting in higher global connectivity17. Future studies on uncultivated Sulfurimonas species described here will be needed to verify these hypotheses, and to shed light on environmental and ecological forces that shape the connections and composition of microbial communities between different environments such as subsurface aquifers, diffusive flow and hydrothermal plumes./p>99%), representing the Sulfurimonas OTU1 and OTU2 identified by the analysis of 16S rRNA gene amplicon sequences (described in the section ‘Illumina 16S rRNA gene sequencing’). We designed specific probes for OTU1 (SLFM-A484 5’–GCTTATTCATAGGCTACC–3’; 15% formamide) and OTU2 (SLFM-B484 5’–GCTTATTCATATGCTACC–3’; 20% formamide), both synthetized by Biomers. Due to the high similarity between these two oligonucleotides (one mismatch for G and T), each probe was used in a mix together with the other (non-labelled) probe as competitor oligonucleotide. To check the coverage and specificity of US. pluma’s probes in the environmental samples, double CARD-FISH hybridizations were carried out using the Campylobacterota probe (EPSY914) as a positive control./p>50,000 reads per sample (CeBiTec), following the standard instructions of the 16S metagenomic sequencing library preparation protocol (Illumina). The workflow and scripts used in this study for the quality cleaning, merging, clustering and annotation of the sequences can be found in ref. 67. Briefly, only reverse and forward reads with quality score higher than 20 (applying a sliding window of 4) were merged, clustering of sequences into OTUs was done using the programme swarm (v2.2.2)68, and the taxonomic classification was based on the SILVA rRNA reference database (release 132)65./p>7 were used for sequencing. The TruSeq Stranded Total RNA kit (Illumina) was used for RNA library preparation. The rRNA depletion step was omitted. Of the total RNA, 80 ng (in 5 μl volume) was combined with 13 μl of ‘Fragment, Prime and Finish mix’ for the RNA fragmentation step according to the Illumina TruSeq stranded mRNA sample preparation guide. Subsequent steps were performed as described in the sample preparation guide. The library was sequenced on a HiSeq1500 platform (Illumina) in a 1 × 150 bp single-end run generating >20 million reads per sample. The resulting reads were pre-processed, including removal of adaptors and quality trimming (slidingwindow:4:21 minlen:100) using bbduk v34 from the BBMAP package69 and Trimmomatic software v0.3570, respectively. The trimmed reads were sorted into ribosomal RNA (rRNA) and non-ribosomal RNA (non-rRNA) reads using SortMeRNA software v2.071. A random subset of 1 million rRNA reads per sample was taxonomically classified with phyloFlash software v3.0 beta 172 based on the SILVA database (release 132)65./p>50 kpb), completeness (>75%) and redundancy (<25%) filtering, and a total of 19 de-replicated bins (ANI > 99%) were obtained. Sulfurimonas bins were identified and refined using Anvi’o interactive interface (v6.2)84 after the Anvi’o contig database was built to calculate k-mer frequencies to identify open reading frames using Prodigal (v2.6.3)85 and single-copy genes using HMMER (v3.2.1)86, and to classify the bins on the basis of single-copy gene taxonomy of GTDB87 using DIAMOND (v0.9.14)88. Sequences of 16S rRNA genes were extracted with RNAmmer (v1.2)89. Refined Sulfurimonas bins were repeatedly re-assembled using BBmap (99% similarity) and SPAdes, removing contigs smaller than 1 kb after each re-assembly step to extend contigs and reduce the size of genome gaps. Completeness and redundancy of the final Sulfurimonas MAGs were evaluated using CheckM (v1.2.1; based on 104 bacterial single-copy genes)90, CheckM2 (v0.1.3; based on machine learning algorithm)91 and BUSCO (v5.2.2; based on 628 Campylobacterales single-copy genes)92. The number of transfer RNAs was identified using ARAGORN (v1.2.36)93. We obtained two almost complete Sulfurimonas MAGs, named MAG-1 and MAG-2 (Supplementary Table 2). These two MAGs represent consensus MAGs, which are based on 16 individual bins produced from different environmental samples. Proteins from the final Sulfurimonas MAGs were predicted and annotated using Prokka (v1.11)94. The Prokka-predicted proteins were additionally annotated with Pfam (release 30)95 and TIGRFAM (release 14)96 profiles using HMMER searches (v3.1b2)86 and by the identification of KEGG Orthology numbers with the GhostKOALA webserver97. The proteins were also assigned to clusters of orthologous groups (COGs)98 using the software COGsoft (v4.19.2012)99 and transmembrane motifs were identified using TMHMM (v2.0)100. On the basis of the various annotation tools, the annotation of proteins of specific interest was manually refined. The sequences of hydrogenases were classified using HydDB101. Iron-related genes were identified using FeGenie’s tool and database102. RedoxyBase103 and SORGOds104 were used to identify classes of peroxidase and types of superoxide reductase, respectively./p>98 and coverage >97%: JN874148.1 and JN874176.1; GeneBank nucleotide; accessed May 2020). The sequences of Sulfuricurvum kujiense from SILVA SSU r138 RefNR (n = 3) were used as outgroup. Sequences were aligned with MAFFT using the L-INS-i method with default settings114, and the alignment was cleaned with BMGE with default setting115. Both programmes were used on the Galaxy platform116. A maximum-likelihood-based tree was constructed using W-IQ-TREE117, first searching for the best substitution model118 before evaluating branch support using 1,000 ultrafast boostrap (UFBoot) and SH-aLRT branch test replicates. Evolutionary placement algorithm (EPA) in RAxML (v8.2.4)119 was applied to add 253 partial Sulfurimonas 16S rRNA gene sequences (250−1,400 bp retrieved from GenBank nucleotide database; data accessed May 2020) to the tree without changing its topology. Further partial 16S rRNA gene sequences of Sulfurimonas sp. obtained from previous next-generation sequencing studies conducted in deep-sea hydrothermal fluids (JAH_MCR_Bv6_MCR_CTD03_08; JAH_AXV_Bv6v4_FS788; downloaded from vamps.mbl.edu) and plumes (PRJEB36848; SRP016119; PRJNA638507) were likewise added to the tree./p>85% and redundancy <5%) from GenBank (accessed January 2020). Supplementary Table 9a reports information for each isolate genome and MAG. DNA and amino acid sequences of the genomes, including US. pluma MAG-1 and MAG-2, were stored in an Anvi’o’s genome database (programme ‘anvi-gen-genomes-storage’). From the genome database, we computed the pangenome to identify the gene clusters (programme ‘anvi-pan-genome’) representing sequences of one or more predicted open reading frames (Prodigal v2.6.3)85 grouped together on the basis of their homology at the translated DNA sequence level. For multiple sequences alignments, Anvi’o used MUSCLE (v3.8.1551)121, the MCL algorithm to identify clusters in amino acid sequence similarity122 and the programme ‘anvi-run-ncbi-cogs’ to annotate genes with functions by searching them against the COG database (October 2019 release)98 using blastp v2.9.0+123. ANI was computed for all Sulfurimonas species and MAGs representative for different environments (that is, hydrothermal vent and plume, marine pelagic, marine oxic aquifer, costal and terrestrial) with the anvi’o programme ‘anvi-compute-genome-similarity’./p>
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