Reconstructing genomes of carbon monoxide oxidisers in volcanic deposits including members of the class ktedonobacteria
Abstract
Microorganisms can potentially colonise volcanic rocks using the chemical energy in reduced gases such as methane, hydrogen (H2) and carbon monoxide (CO). In this study, we analysed soil metagenomes from Chilean volcanic soils, representing three different successional stages with ages of 380, 269 and 63 years, respectively. A total of 19 metagenome-assembled genomes (MAGs) were retrieved from all stages with a higher number observed in the youngest soil (1640: 2 MAGs, 1751: 1 MAG, 1957: 16 MAGs). Genomic similarity indices showed that several MAGs had amino-acid identity (AAI) values >50% to the phyla Actinobacteria, Acidobacteria, Gemmatimonadetes, Proteobacteria and Chloroflexi. Three MAGs from the youngest site (1957) belonged to the class Ktedonobacteria (Chloroflexi). Complete cellular functions of all the MAGs were characterised, including carbon fixation, terpenoid backbone biosynthesis, formate oxidation and CO oxidation. All 19 environmental genomes contained at least one gene encoding a putative carbon monoxide dehydrogenase (CODH). Three MAGs had form I coxL operon (encoding the large subunit CO-dehydrogenase). One of these MAGs (MAG-1957-2.1, Ktedonobacterales) was highly abundant in the youngest soil. MAG-1957-2.1 also contained genes encoding a [NiFe]-hydrogenase and hyp genes encoding accessory enzymes and proteins. Little is known about the Ktedonobacterales through cultivated isolates, but some species can utilise H2 and CO for growth. Our results strongly suggest that the remote volcanic sites in Chile represent a natural habitat for Ktedonobacteria and they may use reduced gases for growth.
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microorganisms Article Reconstructing Genomes of Carbon Monoxide Oxidisers in Volcanic Deposits Including Members of the Class Ktedonobacteria Marcela Hernández 1,2,3,* , Blanca Vera-Gargallo 4, Marcela Calabi-Floody 5, Gary M. King 6, Ralf Conrad 3and Christoph C. Tebbe 1 1Johann Heinrich von Thünen Institut, Institut für Biodiversität, 50, 38116 Braunschweig, Germany; [email protected] 2 School of Environmental Sciences, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, UK 3 Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany; [email protected] 4Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain; [email protected] 5BIOREN-UFRO, Universidad de La Frontera, Temuco 4811230, Chile; [email protected] 6Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA; [email protected] *Correspondence: mar[email protected] Received: 28 October 2020; Accepted: 24 November 2020; Published: 27 November 2020 Abstract: Microorganisms can potentially colonise volcanic rocks using the chemical energy in reduced gases such as methane, hydrogen (H 2 ) and carbon monoxide (CO). In this study, we analysed soil metagenomes from Chilean volcanic soils, representing three different successional stages with ages of 380, 269 and 63 years, respectively. A total of 19 metagenome-assembled genomes (MAGs) were retrieved from all stages with a higher number observed in the youngest soil (1640: 2 MAGs, 1751: 1 MAG, 1957: 16 MAGs). Genomic similarity indices showed that several MAGs had amino-acid identity (AAI) values >50% to the phyla Actinobacteria, Acidobacteria, Gemmatimonadetes, Proteobacteria and Chloroflexi. Three MAGs from the youngest site (1957) belonged to the class Ktedonobacteria (Chloroflexi). Complete cellular functions of all the MAGs were characterised, including carbon fixation, terpenoid backbone biosynthesis, formate oxidation and CO oxidation. All 19 environmental genomes contained at least one gene encoding a putative carbon monoxide dehydrogenase (CODH). Three MAGs had form I coxL operon (encoding the large subunit CO-dehydrogenase). One of these MAGs (MAG-1957-2.1, Ktedonobacterales) was highly abundant in the youngest soil. MAG-1957-2.1 also contained genes encoding a [NiFe]-hydrogenase and hyp genes encoding accessory enzymes and proteins. Little is known about the Ktedonobacterales through cultivated isolates, but some species can utilise H 2 and CO for growth. Our results strongly suggest that the remote volcanic sites in Chile represent a natural habitat for Ktedonobacteria and they may use reduced gases for growth. Keywords: Ktedonobacteria; carbon monoxide dehydrogenase; metagenome-assembled-genome; volcanic soils; shotgun sequencing 1. Introduction Volcanic eruptions provide a model for understanding soil-forming processes and the roles of pioneer bacteria during early biotic colonisation. Recently, it has been demonstrated that the structure of microbial communities can play a key role in the direction of plant community succession pathways [ 1 ]. This is due in part to bacterial contributions to weathering of volcanic rocks, which releases nutrients, resulting in some of the most fertile soils in the world. Microorganisms 2020,8, 1880; doi:10.3390/microorganisms8121880 www.mdpi.com/journal/microorganisms
Microorganisms 2020,8, 1880 2 of 16 After lava and other volcanic deposits (i.e., ash and tephra) cool sufficiently, mineral surface areas become accessible for microbial colonisation [ 2 – 5 ]. In fact, microbes, and especially bacteria, are among the first colonisers of volcanic deposits and thereby initiate soil formation during the early stages of terrestrial ecosystem development [ 6 – 12 ]. While methane (CH 4 ), hydrogen sulphide (H 2 S), hydrogen (H 2 ) and carbon monoxide (CO) have been proposed to promote bacterial colonisation and support microbial life in these organic-carbon-deficient environments [ 13 , 14 ], the actual carbon and energy sources of the first colonisers remain elusive, but likely include a range of endogenous and exogenous sources, including reduced minerals and gases. The microbial ability to utilise these substrates for growth, and thereby initiate the formation of soil organic material, depends on specialised enzymes which may not be prevalent in many different microbial groups, thus representing a limited phylogenetic distribution. As a consequence, this could constrain the composition of pioneering microbial communities. CO is a potential source of carbon and energy for microbes pioneering the colonisation of volcanic substrates. CO utilisation under oxic conditions requires a molybdenum-dependent carbon monoxide dehydrogenase (Mo-CODH), which catalyses the oxidation of CO to CO 2 [ 15 ]. Surveys of genome databases (e.g., Integrated Microbial Genomes) reveal that Mo-CODHs occur in Proteobacteria, Actinobacteria, Firmicutes, Chloroflexi, Bacteroidetes, Deinococcus-Thermus, Halobacteria and Sulfolobales, among others. They were originally described as inducible enzymes and have subsequently been shown to be upregulated by carbon limitation during growth of Actinobacteria (e.g., Rhodococcus and Mycobacterium smegmatis [ 15 – 17 ]), and Chloroflexi (e.g., Thermogemmatispora and Thermomicrobium [ 18 ]). Mo-CODH has been previously targeted in molecular ecological studies of CO oxidisers in volcanic systems and other extreme environments [ 15 ]. These studies have revealed changes in community composition with the age and developmental status of individual sites [ 7 , 15 ]. The results suggest that CO-oxidising communities are not static, but that they change in response to changing environmental conditions, and possibly affect the direction of changes. In a preceding study, we identified the microbial communities involved in volcanic soil formation in different sites on Llaima Volcano (Chile). The bacterial communities of soils from 3 sites affected by lava deposition in 1640, 1751 and 1957 were analysed using 16S rRNA gene amplicon sequencing [ 19 ] and it was demonstrated that microbial diversity increased with the age of the soil deposits. Interestingly, bacterial phylotypes of the poorly studied Ktedonobacterales were among the predominant community members in the 1957 soil, representing 37% of all OTUs, as compared with 18% in the 1751 and 7% in the 1640 soils. Thus, we suspected that bacteria of this order could be instrumental for the initiation of soil formation, paving the way for soil organic carbon formation and preparing a substrate for microbial colonisation and plant growth. Some already cultivated Ktedonobacterales were found to be carboxydotrophs and hydrogenotrophs (i.e., carbon monoxide (CO) and hydrogen (H 2 ) oxidisers/consumers) [ 14 ]. Thus, this leads to the hypothesis that CO and H 2 are important carbon and energy sources for early stages of microbes colonising the Llaima Volcano soil. However, the ecophysiology of the few bacterial isolates assigned to Ktedonobacterales limits predictions about metabolic functions based on 16S rRNA gene sequences alone. Therefore, in this study, a metagenomic approach was chosen to identify microbial traits associated with early stages of colonisation and soil formation in a volcanic ecosystem. In particular, the presence of functional genes implicated in CO-oxidation (coxL genes) and H 2 -oxidation (hyd and hyp genes) was assessed from metagenome-assembled genomes (MAGs). These MAGs were retrieved from volcanic soils of different ages, representing sites 1640, 1751 and 1957. During this period, the soil formation evolved as indicated by their different levels of soil organic matter ranging from 65.33% in the most recent soil (1957) to 9.33% in both medium (1751) and oldest soils (1640) [20]. Especially, the most recent soil (youngest soil) was suspected to reveal microbial adaptations to the challenging environmental conditions and thus to unveil the metabolic processes which initiate microbial colonisations. Therefore, functional metabolic modules annotated in the environmental genomes were analysed, with a main focus on the poorly characterised class of the Ktedonobacteria
Microorganisms 2020,8, 1880 3 of 16 (Chloroflexi). Three Ktedonobacteria MAGs were obtained and all contained genes encoding CO and H 2 oxidation. Additional MAGs from other phyla were also found to contain these genes. Our study advances the understanding of the ecology of Ktedonobacteria and their potential to act as early colonisers in volcanic soils. 2. Materials and Methods 2.1. Sequencing The DNA from the volcanic soils used in our study had been previously extracted [ 20 ]. The soil physico-chemical characteristics have been published [ 20 ] showing a pH of 5.6 in both the medium and oldest soil and 4.7 in the youngest soil, and nitrogen (mg/kg) of 25 (1640), 26 (1751) and 36 (1957). Briefly, the soil samples originated from three different sites of different ages according to the latest lava eruption (1640, 1751, 1957, map in Reference [ 20 ]). A total of nine samples (triplicate per site) were sequenced on an Illumina MiSeq at the Max-Planck-Genome Centre, Köln, Germany. The metagenome was analysed on a high-performance computer using 650 GB RAM and 64 cores at the Thünen Institute of Biodiversity, Braunschweig, Germany. 2.2. Quality Control The sequence reads were checked using FastQC version 0.11.8 [ 21 ]. Low-quality reads were discarded using BBDuk version 38.68, quality-trimming to Q15 using the Phred algorithm [ 22 ]. A schematic overview of the steps and programs used are shown in Figure 1. Microorganisms2020,8,xFORPEERREVIEW3of17 period,thesoilformationevolvedasindicatedbytheirdifferentlevelsofsoilorganicmatterranging from65.33%inthemostrecentsoil(1957)to9.33%inbothmedium(1751)andoldestsoils(1640)[20]. Especially,themostrecentsoil(youngestsoil)wassuspectedtorevealmicrobialadaptationsto thechallengingenvironmentalconditionsandthustounveilthemetabolicprocesseswhichinitiate microbialcolonisations.Therefore,functionalmetabolicmodulesannotatedintheenvironmental genomeswereanalysed,withamainfocusonthepoorlycharacterisedclassoftheKtedonobacteria (Chloroflexi).ThreeKtedonobacteriaMAGswereobtainedandallcontainedgenesencodingCOand H2oxidation.AdditionalMAGsfromotherphylawerealsofoundtocontainthesegenes.Ourstudy advancestheunderstandingoftheecologyofKtedonobacteriaandtheirpotentialtoactasearly colonisersinvolcanicsoils. 2.MaterialsandMethods 2.1.Sequencing TheDNAfromthevolcanicsoilsusedinourstudyhadbeenpreviouslyextracted[20].Thesoil physico‐chemicalcharacteristicshavebeenpublished[20]showingapHof5.6inboththemedium andoldestsoiland4.7intheyoungestsoil,andnitrogen(mg/kg)of25(1640),26(1751)and36(1957). Briefly,thesoilsamplesoriginatedfromthreedifferentsitesofdifferentagesaccordingtothelatest lavaeruption(1640,1751,1957,mapinReference[20]).Atotalofninesamples(triplicatepersite) weresequencedonanIlluminaMiSeqattheMax‐Planck‐GenomeCentre,Köln,Germany.The metagenomewasanalysedonahigh‐performancecomputerusing650GBRAMand64coresatthe ThünenInstituteofBiodiversity,Braunschweig,Germany. 2.2.QualityControl ThesequencereadswerecheckedusingFastQCversion0.11.8[21].Low‐qualityreadswere discardedusingBBDukversion38.68,quality‐trimmingtoQ15usingthePhredalgorithm[22].A schematicoverviewofthestepsandprogramsusedareshowninFigure1. Figure1.Workflowofthemetagenomeanalysisandprogramsusedinthepresentstudy.Binning wasrunbyusingtheMetaWrappackage.AAI:AminoAcidIdentity. 2.3.MetagenomeAssemblyandBinning AlltrimmedIlluminareadsweremergedintolongercontiguoussequences(scaffolds)usingde novoassemblersMegahitversion1.2.8[23]withk‐mers21,29,39,59,79,99,119and141,and MetaSPAdes(SPAdesforco‐assembly)version3.13.1[24,25]withk‐mers21,31,41,51,61,71and81. Qualitycontrol BBDuk,FastQC Assembly Megahit,MetaSpades MetaQuast Binning Concoct,Maxbin2,Metabat2 Binsconsolidation/qualitycontrol Completeness>70% Contamination<5% CheckM Functionalannotation Cognizer GenePrediction Prodigal Binclassification Metawrap,RAST Functionalprofile RAST,KEGG AAI Enveomics Phylogenetic tree PhyloSift,FastTree MEGAX Binabundance Blast Figure 1. Workflow of the metagenome analysis and programs used in the present study. Binning was run by using the MetaWrap package. AAI: Amino Acid Identity. 2.3. Metagenome Assembly and Binning All trimmed Illumina reads were merged into longer contiguous sequences (scaffolds) using de novo assemblers Megahit version 1.2.8 [ 23 ] with k-mers 21, 29, 39, 59, 79, 99, 119 and 141, and MetaSPAdes (SPAdes for co-assembly) version 3.13.1 [ 24 , 25 ] with k-mers 21, 31, 41, 51, 61, 71 and 81. Triplicate samples were co-assembled in order to improve the assembly of low-abundance organisms. Assembly quality was checked with MetaQuast version 5.0.2 [ 26 ], showing that the best quality was obtained with SPAdes for our samples (data not shown). Downstream analysis was carried out using the scaffolds retrieved from SPAdes. Krona charts [ 27 ] were recovered from MetaQuast runs
Microorganisms 2020,8, 1880 4 of 16 to identify taxonomic profiles. Downstream binning analysis was performed with two sets of scaffolds: full-size scaffolds and scaffolds larger than 1000 bp. Metagenomic binning of the assembled scaffolds was carried out with the metaWRAP version 1.2.1 pipeline [ 28 ], in which binning module employs three binning software programs: MaxBin2 [ 29 ], metaBAT2 [ 30 ], and CONCOCT [ 31 ]. Completion and contamination metrics of the extracted bins were estimated using CheckM [ 32 ]. The resulting bins were collectively processed to produce consolidated metagenome-assembled genomes (MAGs) using the bin_refinement module (criterion: completeness >70%; contamination <5%). Both sets of MAGs (18 from scaffolds larger than 1000 bp and 17 from full-size scaffolds) were aggregated, visualised with VizBin [ 33 ] and then dereplicated using dRep [ 34 ]. Only the highest scoring MAG from each secondary cluster was retained in the dereplicated set. The abundance of each MAG in the different sites was calculated using BLASTN version 2.5.0+[ 35 ], keeping only hits with >95% identity and e-value 1e-5 for the analysis [ 36 ]. A final heatmap was constructed using the function heatmap.2 from the gplots package version 3.0.4 [ 37 ] in R version 4.0.2 (https://www.r-project.org). 2.4. Functional Annotation The open reading frames (ORFs) in all scaffolds of each MAG were predicted using Prodigal (v2.6.3) [ 38 ]. Functions were annotated using Cognizer [ 39 ] and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation framework [40]. The annotations of the predicted proteins from KEGG were used to confirm protein functional assignment and identify pathways. Complete pathways were identified using KEGG BRITE pathway mapping [ 40 ]. Aerobic carbon-monoxide dehydrogenases and hydrogen dehydrogenase were also identified using KEGG ortholog annotations. CODH was further distinguished as form I and form II (putative CODH) based on active site motifs present in coxL genes (e.g., Reference [41]). 2.5. Phylogenomic Analysis Taxonomic classification of MAGs was performed using the classify_bins module from metaWRAP whichrelies ontheNCBI_ntdatabase. MAGswerealsoscreened using theRASTServer(RapidAnnotations using Subsystems Technology [ 42 , 43 ]), which also allowed to retrieve information regarding close relative genomes in order to construct the phylogenetic tree. To estimate intergenomic similarity, amino-acid comparisons between MAGs and their closest relative genomes present in the databases were calculated based on reciprocal best hits (two-way AAI) using the enveomics collection (http://enve-omics.gatech.edu/[44]). The phylogenetic affiliation of MAGs was determined by constructing a genomic tree using FastTree version 2.1.11 [ 45 ]. Reference genomes were manually downloaded from the National Center for Biotechnology Information (NCBI) Refseq database (Supplementary Table S1). Conserved genes from the extracted bins and the reference genomes were concatenated using Phylosift version 1.0.1 [ 46 ]. Phylogenetic analysis of the large sub-unit CO dehydrogenase gene (coxL) using the Maximum Likelihood method with a JTT matrix-based model [ 47 ] was performed. Bootstrap values (100 replicates) are shown where support ≥ 70 percent. The scale bar indicates substitutions per site. All gapped positions were deleted, resulting in 420 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [48,49]. 2.6. Accession Number Raw metagenomic data and environmental genomes derived from binning processes were deposited in the Sequence Read Archive (SRA) under the bioproject accession number PRJNA602600 for raw data and PRJNA602601 for metagenome-assembled genomes.
Microorganisms 2020,8, 1880 5 of 16 3. Results 3.1. MAGs Recovery A total of ~3–4 million scaffolds were recovered from the soil metagenomes in each site. Even though all the sites underwent similar sequencing efforts (between 2.3 GB in 1640 and 1957 to 2.4 GB in 1751), the youngest soil had the largest number of scaffolds with 499 sequences >50 kb (N50 length of 700) compared to the oldest soil with only 12 scaffolds with a size >50 kb (N50 length of 914) (Table 1). A total of 19 MAGs with a completeness of >70% and a contamination <5% (2 from 1640, 1 from 1751 and 16 from 1957) were retrieved and characterised. Table 1. Summary report for the assembly quality assessment using MetaQuast. Statistics 1640 1751 1957 Number of scaffolds (>= 0 bp) 3,631,380 4,047,900 3,138,527 Number of scaffolds (>= 500 bp) 1,488,437 1,609,020 1,578,887 Number of scaffolds (>= 1000 bp) 326,980 333,885 447,418 Number of scaffolds (>= 25,000 bp) 172 195 1551 Number of scaffolds (>= 50,000 bp) 12 40 499 Total length (>= 0 bp) 2,320,437,372 2,460,323,814 2,396,069,091 Total length (>= 500 bp) 1,385,386,471 1,404,094,641 1,717,252,425 Total length (>= 1000 bp) 631,179,105 570,404,360 964,085,094 Total length (>= 25,000 bp) 5,697,241 8,152,100 8,236,5310 Total length (>= 50,000 bp) 686,375 2,983,958 46,419,897 N50 914 850 700 L50 391,387 477,193 348,604 N50—length such that scaffolds of this length or longer include half the bases of the assembly; L50—number of scaffolds that are longer than, or equal to, the N50 length and therefore include half the bases of the assembly (https://www.ncbi.nlm.nih.gov/assembly/help/#globalstats). 3.2. MAG Identification MAGs were affiliated to the phyla Actinobacteria, Proteobacteria, Acidobacteria, Gemmatimonadetes, Chloroflexi, Firmicutes and Verrucomicrobia (Figure 2). In the oldest soil, two environmental genomes were retrieved related to Actinomycetales (Actinobacteria) and Rhodospirillales (Proteobacteria). The only MAG retrieved from the middle soil was related to Acidobacteria. MAGs binned from the youngest soil included six assigned to Acidobacteria, one to Proteobacteria, one to Firmicutes, three to Actinobacteria, one to the phylum Gemmatimonadetes, one to Verrucomicrobia and three to the phylum Chloroflexi (Figure 2). The abundance of the MAGs in each site was calculated by using BLASTN (Figure 3). MAGs were more abundant from the soil they were recovered. MAGs with a total abundance >1% were found only in the young soil (1957). MAG 1957-2.1 (Ktedonobacteria, 1.21% ± 0.82%), MAG 1957-5.1 (Actinomycetales, 1.02% ± 0.84%), MAG 1957-13.1 (Verrucomicrobiales, 1.58% ± 1.1%) and MAG 1957-16.1 (Acidobacteria, 1.14% ±0.87%) were the most abundant MAGs (Figure 3).
Microorganisms 2020,8, 1880 6 of 16 Microorganisms2020,8,xFORPEERREVIEW6of17 Figure2.Phylogenomictreeofthebacterialgenomicbins.ThetreewasbuiltwithPhyloSiftagainst referencegenomesdownloadedfromNCBI.MAGsareindicatedinboldtogetherwiththeir respectivecompletenessandcontamination.FastTreeconfidencevaluesofMAGbranchesareshown. Thehorizontalbarrepresents10%sequencedivergence. TheabundanceoftheMAGsineachsitewascalculatedbyusingBLASTN(Figure3).MAGs weremoreabundantfromthesoiltheywererecovered.MAGswithatotalabundance>1%were foundonlyintheyoungsoil(1957).MAG1957‐2.1(Ktedonobacteria,1.21%0.82%),MAG1957‐5.1 Figure 2. Phylogenomic tree of the bacterial genomic bins. The tree was built with PhyloSift against reference genomes downloaded from NCBI. MAGs are indicated in bold together with their respective completeness and contamination. FastTree confidence values of MAG branches are shown. The horizontal bar represents 10% sequence divergence.
Microorganisms 2020,8, 1880 7 of 16 Microorganisms2020,8,xFORPEERREVIEW7of17 (Actinomycetales,1.02%0.84%),MAG1957‐13.1(Verrucomicrobiales,1.58%1.1%)andMAG 1957‐16.1(Acidobacteria,1.14%0.87%)werethemostabundantMAGs(Figure3). Figure3.HeatmaprepresentingtheabundanceofMAGsineachmetagenome.Theanalysiswasdone byblastandonlyhitsgreaterthan95%identityande‐value1×10 −5 wereused. 3.3.MetabolicCharacterisationofMAGs Genesencodingenzymesinvolvedincarbohydrateandenergymetabolism,suchascarbon fixation,sulphurmetabolism,ATPsynthesisandnitrogenmetabolism,aswellasterpenoidbackbone biosynthesis,werefoundinalltheMAGs(Figure4).Otherfunctions,includingxenobiotic biodegradation,fattyacidmetabolism,nucleotidemetabolismandvitaminmetabolism,among others,werealsofound(SupplementaryTableS2). Figure 3. Heatmap representing the abundance of MAGs in each metagenome. The analysis was done by blast and only hits greater than 95% identity and e-value 1 ×10−5were used. Microorganisms2020,8,xFORPEERREVIEW8of17 Figure4.Summaryofcompletecellularfunctionsandotherhigh‐levelfeaturesintheMAGs recoveredformsites1640,1751and1957retrievedfromKEGGanalysis.Referencegenomesfor Ketedonobacteria:DSM45816T[50],DSM44963T[51]andNBRC113551T[52],(K:KEGGorthology; M:KEGGMode).AsterisksindicatetheMAGsisolatedfromtheclassKtedonobacteria.“X”indicates MAGscontaininggenesencodingformIoftheCoxL. 3.3.1.CharacterisationofCODHandHydrogenaseGenesinMAGs ThreeMAGs(MAG‐1640‐1.1,MAG‐1751‐1.1andMAG‐1957‐2.1)encodedformIoftheCO‐ dehydrogenaselargesubunit(coxL).TheseMAGswereeachassociatedwithaparticularsoil,with lowabundanceinthemetagenomesoftheothersites(Figure5A).Inadditiontothesethreeform‐I coxL‐encodingMAGs,15otherscaffoldsfromMAGscontainingformIIcoxL‐likegeneswere recovered(datanotshown),butthefunctionofformIICoxLisnotyetknown.Thearrangementof genesencodingformICODHineachoftheMAGsisshowninFigure5B.Itshouldbenotedthatall ofthesethreeMAGsshowthecanonicalarrangementforthethreestructuralgenesofCODH,thatis theMSL(coxM‐coxS‐coxL)genes.Thegenesencodingthe[NiFe]‐hydrogenaseanditsaccessory proteinswereonlyidentifiedinMAG‐1957‐2.1,andinstead,onlysomeoftheaccessoryhypgenes werefoundintheothertwoMAGs(Figure5B).AphylogeneticanalysisoftheformIcoxLgeneswas performed,showingtheyareaffiliatedwithActinobacteria(MAG‐1640‐1.1),NitrospiraeCandidatus Manganitrophusnoduliformans(MAG‐1751‐1.1)andChloroflexi(MAG‐1957‐2.1)(Figure6).This groupingisconsistentwiththeresultsofPhyloSift(Figure2),exceptforMAG‐1751‐1.1,whereitwas 1751 Cellular functions MAG-1.1 MAG-2.1 MAG-1.1 MAG-1.1 MAG-2.1 * MAG-3.1 * MAG-4.1 MAG-5.1 MAG-6.1 * MAG-7.1 MAG-8.1 MAG-9.1 MAG-10.1 MAG-11.1 MAG-12.1 MAG-13.1 MAG-14.1 MAG-15.1 MAG-16.1 DSM45816 T DSM44963 T NBRC113551 T Carbon monoxide oxidation K03518 Carbon monoxide dehydrogenase small subunit coxS K03519 Carbon monoxide dehydrogenase medium subunit coxM K03520 Carbon monoxide dehydrogenase large subunit coxL (Form-II) K03520 Carbon monoxide dehydrogenase large subunit coxL (Form-I) XXX Formate oxidation K00122 Formate dehydrogenase K00123 Formate dehydrogenase major subunit Hydrogen oxidation: H 2 dehydrogenase K00436: NAD-reducing hydrogenase large subunit Central carbohydrate metabolism M00001 Glycolysis (Embden-Meyerhof pathway), glucose => pyruvate M00002 Glycolysis, core module involving three-carbon compounds M00003 Gluconeogenesis, oxaloacetate => fructose-6P M00004 Pentose phosphate pathway (Pentose phosphate cycle) M00005 PRPP biosynthesis, ribose 5P => PRPP M00006 Pentose phosphate pathway, oxidative phase, glucose 6P => ribulose 5P M00007 Pentose phosphate pathway, non-oxidative phase, fructose 6P => ribose 5P M00009 Citrate cycle (TCA cycle, Krebs cycle) M00010 Citrate cycle, first carbon oxidation, oxaloacetate => 2-oxoglutarate M00011 Citrate cycle, second carbon oxidation, 2-oxoglutarate => oxaloacetate M00307 Pyruvate oxidation, pyruvate => acetyl-CoA M00580 Pentose phosphate pathway, archaea, fructose 6P => ribose 5P Other carbohydrate metabolism M00012 Glyoxylate cycle M00061 D-Glucuronate deg., D-glucuronate => pyruvate + D-glyceraldehyde 3P M00081 Pectin degradation M00549 Nucleotide sugar biosynthesis, glucose => UDP-glucose M00552 D-galactonate deg., D. L-Doudoroff pathway, D-galactonate => glycerate-3P M00554 Nucleotide sugar biosynthesis, galactose => UDP-galactose M00631 D-Galacturonate deg., D-galacturonate => pyruvate + D-glyceraldehyde 3P M00632 Galactose degr., Leloir pathway, galactose => alpha-D-glucose-1P M00741 Propanoyl-CoA metabolism, propanoyl-CoA => succinyl-CoA M00761 Undecaprenylphosphate &-L-Ara4N biosynthesis M00854 Glycogen biosynthesis, glucose-1P => glycogen/starch M00855 Glycogen degradation, glycogen => glucose-6P M00909 UDP-N-acetyl-D-glucosamine, prokaryotes, glucose => UDP-GlcNAc Carbon fixation M00166 Reductive pentose phosphate cycle, ribulose-5P => glyceraldehyde-3P M00168 CAM (Crassulacean acid metabolism), dark M00169 CAM (Crassulacean acid metabolism), light M00172 C4-dicarboxylic acid cycle, NADP - malic enzyme type M00579 Phosphate acetyltransferase-acetate kinase pathway, acetyl-CoA => acetate ATP synthesis M00144 NADH:quinone oxidoreductase, prokaryotes M00149 Succinate dehydrogenase, prokaryotes M00151 Cytochrome bc1 complex respiratory unit M00155 Cytochrome c oxidase, prokaryotes M00156 Cytochrome c oxidase, cbb3-type M00157 F-type ATPase, prokaryotes and chloroplasts Terpenoid backbone biosynthesis M00096 C5 isoprenoid biosynthesis, non-mevalonate pathway M00364 C10-C20 isoprenoid biosynthesis, bacteria M00365 C10-C20 isoprenoid biosynthesis, archaea 1640 Reference1957 Figure 4. Summary of complete cellular functions and other high-level features in the MAGs recovered form sites 1640, 1751 and 1957 retrieved from KEGG analysis. Reference genomes for Ketedonobacteria: DSM45816T [ 50 ], DSM44963T [ 51 ] and NBRC 113551T [ 52 ], (K: KEGG orthology; M: KEGG Mode). Asterisks indicate the MAGs isolated from the class Ktedonobacteria. “X” indicates MAGs containing genes encoding form I of the CoxL.
Microorganisms 2020,8, 1880 8 of 16 3.3. Metabolic Characterisation of MAGs Genes encoding enzymes involved in carbohydrate and energy metabolism, such as carbon fixation, sulphur metabolism, ATP synthesis and nitrogen metabolism, as well as terpenoid backbone biosynthesis, were found in all the MAGs (Figure 4). Other functions, including xenobiotic biodegradation, fatty acid metabolism, nucleotide metabolism and vitamin metabolism, among others, were also found (Supplementary Table S2). 3.3.1. Characterisation of CODH and Hydrogenase Genes in MAGs Three MAGs (MAG-1640-1.1, MAG-1751-1.1 and MAG-1957-2.1) encoded form I of the CO-dehydrogenase large subunit (coxL). These MAGs were each associated with a particular soil, with low abundance in the metagenomes of the other sites (Figure 5A). In addition to these three form-I coxL-encoding MAGs, 15 other scaffolds from MAGs containing form II coxL-like genes were recovered (data not shown), but the function of form II CoxL is not yet known. The arrangement of genes encoding form I CODH in each of the MAGs is shown in Figure 5B. It should be noted that all of these three MAGs show the canonical arrangement for the three structural genes of CODH, that is the MSL (coxM-coxS-coxL) genes. The genes encoding the [NiFe]-hydrogenase and its accessory proteins were only identified in MAG-1957-2.1, and instead, only some of the accessory hyp genes were found in the other two MAGs (Figure 5B). A phylogenetic analysis of the form I coxL genes was performed, showing they are affiliated with Actinobacteria (MAG-1640-1.1), Nitrospirae Candidatus Manganitrophus noduliformans (MAG-1751-1.1) and Chloroflexi (MAG-1957-2.1) (Figure 6). This grouping is consistent with the results of PhyloSift (Figure 2), except for MAG-1751-1.1, where it was loosely associated with Acidobacteria (although with an amino-acid identity of only 40%) rather than Nitrospirae. 3.3.2. Complete Metabolic Characterisation of Ktedonobacterales MAGs Here, we focused on the Ktedonobacterales MAGs because of their apparent importance in early soil formation. Three Ktedonobacterales MAGs were identified in the 1957 soil metagenomes but were not found in the older soils (Figures 2and 3). Two of the MAGs (MAGs 1957-2.1 and 1957-3.1), affiliated to the class Ktedonobacteria (phylum Chloroflexi), contained genes for the complete electron transport chain, citric acid metabolism, nitrogen metabolism, sulphur metabolism, several transporters, the complete gene set for carbon monoxide oxidation (CO dehydrogenase), herbicide degradation and degradation aromatics, as well as the major subunit of the formate dehydrogenase, and also a hydrogenase. MAG 1957-6.1 (Ktedonobacteria) had very similar pathways as the other Chloroflexi MAGs, except a step for CO-oxidation and the electron transport chain were absent (Figure 4, Supplementary Table S2, Figure 7).
Microorganisms 2020,8, 1880 9 of 16 Microorganisms2020,8,xFORPEERREVIEW9of17 looselyassociatedwithAcidobacteria(althoughwithanamino‐acididentityofonly40%)ratherthan Nitrospirae. Figure5.(A)RelativeabundanceofMAG‐1640‐1.1,MAG‐1751‐1.1andMAG‐1957‐2.1insites1640, 1751and1957.Barsindicatestandarderroroftriplicates.(B)Genearrangementofthecarbon monoxidedehydrogenase(CODH)andmembrane‐bound[NiFe]‐hydrogenaseinMAG‐1640‐1.1, Figure 5. ( A ) Relative abundance of MAG-1640-1.1, MAG-1751-1.1 and MAG-1957-2.1 in sites 1640, 1751 and 1957. Bars indicate standard error of triplicates. ( B ) Gene arrangement of the carbon monoxide dehydrogenase (CODH) and membrane-bound [NiFe]-hydrogenase in MAG-1640-1.1, MAG-1751-1.1 and MAG-1957-2.1 (* indicates hypothetical proteins; small indicates NiFe-hydrogenase small subunit and large indicates NiFe-hydrogenase large subunit).
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