The hidden diversity and functional potential of Chloroflexota genomes in arsenic and antimony co-contaminated soils

Highlights

	● A total of 170 middle- and high-quality Chloroflexota MAGs were reconstructed from As and Sb co-contaminated soils.
	● Many Chloroflexota MAGs have overlooked potential for C fixation and P solubilization.
	● The enriched As, Sb, P, C and N metabolism genes in the MAGs favor Chloroflexota to resist contamination and nutrient limitations.

Abstract

Microorganisms were reported to be the indicators and drivers of metal(loid)s-contaminated soils. Chloroflexota is a widely-distributed phylum in arsenic (As) and antimony (Sb) contaminated soils, but the diversity and functional potential of its genomes remain largely unknown. In this study, we collected As and Sb contaminated soils from smelting-affected agricultural soils and mining soils, with the latter exhibiting much higher concentrations of As (mean 19421.2 mg kg⁻¹) and Sb (mean 4953.5 mg kg⁻¹) as well as lower carbon and nitrogen levels. We reconstructed 170 medium- to high-quality metagenome-assembled genomes (MAGs) of Chloroflexota from these soils. A total of 11 MAGs were proposed as novel candidate species, including 3 novel candidate genera affiliated with the classes Ktedonobacteria, Limnocylindria, and Dormibacteria. Functional annotation reveals that many MAGs from Ktedonobacteria and Dormibacteria may have novel potential for carbon fixation through the Calvin–Benson–Bassham cycle. Additionally, many Chloroflexota MAGs harbored essential genes involved in enhancing soil phosphorus (P) availability. In Chloroflexota MAGs, the gene responsible for extracellular oxidation, dldH, rather than the intracellular oxidation gene arsO, was widespread for Sb(III) oxidation. Under heavy As and Sb contamination and nutrient limitation, Chloroflexota MAGs exhibited higher guanine-cytosine contents and smaller genome sizes. Moreover, MAGs derived from these conditions were enriched with a higher proportion of genes related to Sb oxidation, As/P transport, As reduction and methylation, as well as pathways involved in carbohydrate degradation and bioavailable nitrogen biosynthesis. These findings might be helpful for developing bioremediation strategy for Chloroflexota in As/Sb contaminated soils.

Graphical abstract

Keywords

Chloroflexota genomes / diversity / functional potential / arsenic and antimony pollution / nutrient limitation

References

[1] Abin, C.A., Hollibaugh, J.T., 2019. Transcriptional response of the obligate anaerobe Desulfuribacillus stibiiarsenatis MLFW-2^T to growth on antimonate and other terminal electron acceptors. Environmental Microbiology21, 618–630.

[2] Acker, M., Hogle, S.L., Berube, P.M., Hackl, T., Coe, A., Stepanauskas, R., Chisholm, S.W., Repeta, D.J., 2022. Phosphonate production by marine microbes: exploring new sources and potential function. Proceedings of the National Academy of Sciences of the United States of America119, e2113386119.

[3] An, Y.J., Kim, M., 2009. Effect of antimony on the microbial growth and the activities of soil enzymes. Chemosphere74, 654–659.

[4] Andres, J., Bertin, P.N., 2016. The microbial genomics of arsenic. FEMS Microbiology Reviews40, 299–322.

[5] Aramaki, T., Blanc-Mathieu, R., Endo, H., Ohkubo, K., Kanehisa, M., Goto, S., Ogata, H., 2020. KofamKOALA: KEGG ortholog assignment based on profile HMM and adaptive score threshold. Bioinformatics36, 2251–2252.

[6] Asnicar, F., Thomas, A.M., Beghini, F., Mengoni, C., Manara, S., Manghi, P., Zhu, Q.Y., Bolzan, M., Cumbo, F., May, U., Sanders, J.G., Zolfo, M., Kopylova, E., Pasolli, E., Knight, R., Mirarab, S., Huttenhower, C., Segata, N., 2020. Precise phylogenetic analysis of microbial isolates and genomes from metagenomes using PhyloPhlAn 3.0. Nature Communications11, 2500.

[7] Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics30, 2114–2120.

[8] Boraston, A.B., Bolam, D.N., Gilbert, H.J., Davies, G.J., 2004. Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochemical Journal382, 769–781.

[9] Bovio-Winkler, P., Cabezas, A., Etchebehere, C., 2024. Unveiling the hidden diversity and functional role of Chloroflexota in full-scale wastewater treatment plants through genome-centric analyses. ISME Communications4, ycae050.

[10] Buchfink, B., Reuter, K., Drost, H.G., 2021. Sensitive protein alignments at tree-of-life scale using DIAMOND. Nature Methods18, 366–368.

[11] Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V., Henrissat, B., 2009. The carbohydrate-active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Research37, D233–D238.

[12] Chaumeil, P.A., Mussig, A.J., Hugenholtz, P., Parks, D.H., 2020. GTDB-Tk: a toolkit to classify genomes with the genome taxonomy database. Bioinformatics36, 1925–1927.

[13] Chen, L., Li, K.K., Shang, J.Y., Wu, Y., Chen, T., Wanyan, Y., Wang, E.T., Tian, C.F., Chen, W.F., Chen, W.X., Mi, G.H., Sui, X., 2021. Plant growth–promoting bacteria improve maize growth through reshaping the rhizobacterial community in low-nitrogen and low-phosphorus soil. Biology and Fertility of Soils57, 1075–1088.

[14] Chen, Q.Y., Costa, M., 2021. Arsenic: a global environmental challenge. Annual Review of Pharmacology and Toxicology61, 47–63.

[15] Chen, S.C., Sun, G.X., Yan, Y., Konstantinidis, K.T., Zhang, S.Y., Deng, Y., Li, X.M., Cui, H.L., Musat, F., Popp, D., Rosen, B.P., Zhu, Y.G., 2020. The great oxidation event expanded the genetic repertoire of arsenic metabolism and cycling. Proceedings of the National Academy of Sciences of the United States of America117, 10414–10421.

[16] Chklovski, A., Parks, D.H., Woodcroft, B.J., Tyson, G.W., 2023. CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning. Nature Methods20, 1203–1212.

[17] Chuckran, P.F., Flagg, C., Propster, J., Rutherford, W.A., Sieradzki, E.T., Blazewicz, S.J., Hungate, B., Pett-Ridge, J., Schwartz, E., Dijkstra, P., 2023. Edaphic controls on genome size and GC content of bacteria in soil microbial communities. Soil Biology and Biochemistry178, 108935.

[18] Freches, A., Fradinho, J.C., 2024. The biotechnological potential of the Chloroflexota phylum. Applied and Environmental Microbiology90, e0175623.

[19] Garritano, A.N., Song, W.Z., Thomas, T., 2022. Carbon fixation pathways across the bacterial and archaeal tree of life. PNAS Nexus1, pgac226.

[20] Geng, H.H., Wang, F., Yan, C.C., Ma, S., Zhang, Y.Y., Qin, Q.Z., Tian, Z.J., Liu, R.P., Chen, H.L., Zhou, B.H., Yuan, R. F., 2022. Rhizosphere microbial community composition and survival strategies in oligotrophic and metal(loid) contaminated iron tailings areas. Journal of Hazardous Materials436, 129045.

[21] Guo, G.H., Chen, S.Q., Zhang, D.G., Wang, J., Lei, M., Ju, T.N., Wei, H., 2024. Influence of biochar on the arsenic phytoextraction potential of Pteris vittata in soils from an abandoned arsenic mining site. Chemosphere352, 141389.

[22] Han, J.L., Liang, L.C., Zhu, Y.R., Xu, X.H., Wang, L., Shang, L.H., Wu, P., Wu, Q.X., Qian, X.L., Qiu, G.L., Feng, X.B., 2022. Heavy metal(loid)s in farmland soils on the Karst Plateau, Southwest China: an integrated analysis of geochemical baselines, source apportionment, and associated health risk. Land Degradation & Development33, 1689–1703.

[23] He, M.C., Wang, N.N., Long, X.J., Zhang, C.J., Ma, C.L., Zhong, Q.Y., Wang, A.H., Wang, Y., Pervaiz, A., Shan, J., 2019. Antimony speciation in the environment: Recent advances in understanding the biogeochemical processes and ecological effects. Journal of Environmental Sciences75, 14–39.

[24] He, M.C., Wang, X.Q., Wu, F.C., Fu, Z.Y., 2012. Antimony pollution in China. Science of the Total Environment 421–422, 41–50.

[25] Ihaka, R., Gentleman, R., 1996. R: a language for data analysis and graphics. Journal of Computational and Graphical Statistics5, 299–314.

[26] Konstantinidis, K.T., Rosselló-Móra, R., Amann, R., 2017. Uncultivated microbes in need of their own taxonomy. The ISME Journal11, 2399–2406.

[27] Kuypers, M.M.M., Marchant, H.K., Kartal, B., 2018. The microbial nitrogen-cycling network. Nature Reviews Microbiology16, 263–276.

[28] Langmead, B., Salzberg, S.L., 2012. Fast gapped-read alignment with bowtie 2. Nature Methods9, 357–359.

[29] Letunic, I., Bork, P., 2024. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Research52, W78–W82.

[30] Li, D.H., Liu, C.M., Luo, R.B., Sadakane, K., Lam, T.W., 2015. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics31, 1674–1676.

[31] Li, H.J., Shen, Y.Y., He, Y.Q., Gao, T.P., Li, G.W., Zuo, M.B., Ji, J., Li, C.M., Li, X.K., Chen, Y.L., Yin, Z.X., Li, X.X., 2022. Effects of heavy metals on bacterial community structures in two lead–zinc tailings situated in northwestern China. Archives of Microbiology204, 78.

[32] Li, J.X., Wang, Q., Oremland, R.S., Kulp, T.R., Rensing, C., Wang, G.J., 2016. Microbial antimony biogeochemistry: enzymes, regulation, and related metabolic pathways. Applied and Environmental Microbiology82, 5482–5495.

[33] Li, S.J., Hua, Z.S., Huang, L.N., Li, J., Shi, S.H., Chen, L.X., Kuang, J.L., Liu, J., Hu, M., Shu, W.S., 2014. Microbial communities evolve faster in extreme environments. Scientific Reports4, 6205.

[34] Li, Y.B., Zhang, M.M., Xu, R., Lin, H.Z., Sun, X.X., Xu, F.Q., Gao, P., Kong, T.L., Xiao, E.Z., Yang, N., Sun, W.M., 2021. Arsenic and antimony co-contamination influences on soil microbial community composition and functions: relevance to arsenic resistance and carbon, nitrogen, and sulfur cycling. Environment International153, 106522.

[35] Liang, J.L., Liu, J., Jia, P., Yang, T.T., Zeng, Q.W., Zhang, S.C., Liao, B., Shu, W.S., Li, J.T., 2020. Novel phosphate-solubilizing bacteria enhance soil phosphorus cycling following ecological restoration of land degraded by mining. The ISME Journal14, 1600–1613.

[36] Liu, R.L., Wei, X., Song, W.Z., Wang, L., Cao, J.W., Wu, J.X., Thomas, T., Jin, T., Wang, Z.X., Wei, W.X., Wei, Y.L., Zhai, H.F., Yao, C., Shen, Z.Y., Du, J.T., Fang, J.S., 2022. Novel Chloroflexi genomes from the deepest ocean reveal metabolic strategies for the adaptation to deep-sea habitats. Microbiome10, 75.

[37] Mawia, A.M., Hui, S.Z., Zhou, L., Li, H.J., Tabassum, J., Lai, C.K., Wang, J.X., Shao, G.N., Wei, X.J., Tang, S.Q., Luo, J., Hu, S.K., Hu, P.S., 2021. Inorganic arsenic toxicity and alleviation strategies in rice. Journal of Hazardous Materials408, 124751.

[38] Mehrshad, M., Salcher, M.M., Okazaki, Y., Nakano, S.I., Šimek, K., Andrei, A.S., Ghai, R., 2018. Hidden in plain sight—highly abundant and diverse planktonic freshwater Chloroflexi. Microbiome6, 176.

[39] Okkenhaug, G., Zhu, Y.G., He, J.W., Li, X., Luo, L., Mulder, J., 2012. Antimony (Sb) and arsenic (As) in Sb mining impacted paddy soil from Xikuangshan, China: differences in mechanisms controlling soil sequestration and uptake in rice. Environmental Science & Technology46, 3155–3162.

[40] Olm, M.R., Brown, C.T., Brooks, B., Banfield, J.F., 2017. dRep: a tool for fast and accurate genomic comparisons that enables improved genome recovery from metagenomes through de-replication. The ISME Journal11, 2864–2868.

[41] Peng, J.J., Zhou, X., Rensing, C., Liesack, W., Zhu, Y.G., 2024. Soil microbial ecology through the lens of metatranscriptomics. Soil Ecology Letters6, 230217.

[42] Petriglieri, F., Kondrotaite, Z., Singleton, C., Nierychlo, M., Dueholm, M.K., Nielsen, P.H., 2023. A comprehensive overview of the Chloroflexota community in wastewater treatment plants worldwide. mSystems8, e0066723.

[43] Shu, W.S., Huang, L.N., 2022. Microbial diversity in extreme environments. Nature Reviews Microbiology20, 219–235.

[44] Song, X.W., Li, Y.Q., Stirling, E., Zhao, K.K., Wang, B.H., Zhu, Y.G., Luo, Y.M., Xu, J.M., Ma, B., 2022. AsgeneDB: a curated orthology arsenic metabolism gene database and computational tool for metagenome annotation. NAR Genomics and Bioinformatics4, lqac080.

[45] Steely, S., Amarasiriwardena, D., Xing, B.S., 2007. An investigation of inorganic antimony species and antimony associated with soil humic acid molar mass fractions in contaminated soils. Environmental Pollution148, 590–598.

[46] Sun, W.M., Sun, X.X., Li, B.Q., Xu, R., Young, L.Y., Dong, Y.R., Zhang, M.M., Kong, T.L., Xiao, E.Z., Wang, Q., 2020. Bacterial response to sharp geochemical gradients caused by acid mine drainage intrusion in a terrace: Relevance of C, N, and S cycling and metal resistance. Environment International138, 105601.

[47] Sun, W.M., Xiao, E.Z., Häggblom, M., Krumins, V., Dong, Y.R., Sun, X.X., Li, F.B., Wang, Q., Li, B.Q., Yan, B., 2018. Bacterial survival strategies in an alkaline tailing site and the physiological mechanisms of dominant phylotypes as revealed by metagenomic analyses. Environmental Science & Technology52, 13370–13380.

[48] Tang, S.T., Song, X.W., Chen, J., Shen, J., Ma, B., Rosen, B.P., Zhang, J., Zhao, F.J., 2023. Widespread distribution of the arsO gene confers bacterial resistance to environmental antimony. Environmental Science & Technology57, 14579–14588.

[49] Tong, H., Zheng, C.J., Li, B., Swanner, E.D., Liu, C.S., Chen, M.J., Xia, Y.F., Liu, Y.H., Ning, Z.P., Li, F.B., Feng, X.B., 2021. Microaerophilic oxidation of Fe(II) coupled with simultaneous carbon fixation and As(III) oxidation and sequestration in karstic paddy soil. Environmental Science & Technology55, 3634–3644.

[50] Tu, Q.C., Lin, L., Cheng, L., Deng, Y., He, Z.L., 2019. NCycDB: a curated integrative database for fast and accurate metagenomic profiling of nitrogen cycling genes. Bioinformatics35, 1040–1048.

[51] Uritskiy, G.V., DiRuggiero, J., Taylor, J., 2018. MetaWRAP-a flexible pipeline for genome-resolved metagenomic data analysis. Microbiome6, 158.

[52] Von Meijenfeldt, F.A.B., Hogeweg, P., Dutilh, B.E., 2023. A social niche breadth score reveals niche range strategies of generalists and specialists. Nature Ecology & Evolution7, 768–781.

[53] Wang, L.Y., Ye, L., Yin, Z.P., Zhang, L.X., Jing, C.Y., 2022. Antimonite oxidation by microbial extracellular superoxide in Pseudomonas sp. SbB1. Geochimica et Cosmochimica Acta 316, 122–134.

[54] Wiegand, S., Sobol, M., Schnepp-Pesch, L.K., Yan, G., Iqbal, S., Vollmers, J., Müller, J.A., Kaster, A.K., 2023. Taxonomic re-classification and expansion of the phylum Chloroflexota based on over 5000 genomes and metagenome-assembled genomes. Microorganisms11, 2612.

[55] Williams, T.J., Allen, M.A., Ray, A.E., Benaud, N., Chelliah, D.S., Albanese, D., Donati, C., Selbmann, L., Coleine, C., Ferrari, B.C., 2024. Novel endolithic bacteria of phylum Chloroflexota reveal a myriad of potential survival strategies in the antarctic desert. Applied and Environmental Microbiology90, e0226423.

[56] Wu, X.J., Cui, Z.L., Peng, J.J., Zhang, F.S., Liesack, W., 2022. Genome-resolved metagenomics identifies the particular genetic traits of phosphate-solubilizing bacteria in agricultural soil. ISME Communications2, 17.

[57] Xiao, E.Z., Krumins, V., Xiao, T.F., Dong, Y.R., Tang, S., Ning, Z.P., Huang, Z.Y., Sun, W.M., 2017. Depth-resolved microbial community analyses in two contrasting soil cores contaminated by antimony and arsenic. Environmental Pollution221, 244–255.

[58] Xu, M.Z., Cui, Y.X., Beiyuan, J., Wang, X., Duan, C.J., Fang, L.C., 2021. Heavy metal pollution increases soil microbial carbon limitation: evidence from ecological enzyme stoichiometry. Soil Ecology Letters3, 230–241.

[59] Xu, R., Li, B.Q., Xiao, E.Z., Young, L.Y., Sun, X.X., Kong, T.L., Dong, Y.R., Wang, Q., Yang, Z.H., Chen, L., Sun, W.M., 2020a. Uncovering microbial responses to sharp geochemical gradients in a terrace contaminated by acid mine drainage. Environmental Pollution261, 114226.

[60] Xu, R., Sun, X.X., Lin, H.Z., Han, F., Xiao, E.Z., Li, B.Q., Qiu, L., Song, B.R., Yang, Z.H., Sun, W.M., 2020b. Microbial adaptation in vertical soil profiles contaminated by an antimony smelting plant. FEMS Microbiology Ecology96, fiaa188.

[61] Xu, Y., Ma, K., Huang, S.W., Liu, L.M., Lu, Y.H., 2012. Diel cycle of methanogen mcrA transcripts in rice rhizosphere. Environmental Microbiology Reports4, 655–663.

[62] Yu, X.L., Zhou, J.Y., Song, W., Xu, M.Z., He, Q., Peng, Y.S., Tian, Y., Wang, C., Shu, L.F., Wang, S.Q., Yan, Q.Y., Liu, J.H., Tu, Q.C., He, Z.L., 2021. SCycDB: a curated functional gene database for metagenomic profiling of sulphur cycling pathways. Molecular Ecology Resources21, 924–940.

[63] Zeng, J.X., Tu, Q.C., Yu, X.L., Qian, L., Wang, C., Shu, L.F., Liu, F., Liu, S.W., Huang, Z.J., He, J.G., Yan, Q.Y., He, Z.L., 2022. PCycDB: a comprehensive and accurate database for fast analysis of phosphorus cycling genes. Microbiome10, 101.

[64] Zhang, M.M., Xiong, Y.Q., Sun, H.C., Xiao, T.F., Xiao, E.Z., Sun, X.X., Li, B.Q., Sun, W.M., 2024. Selective pressure of arsenic and antimony co-contamination on microbial community in alkaline sediments. Journal of Hazardous Materials464, 132948.

[65] Zhang, Y.D., O'Loughlin, E.J., Kwon, M.J., 2022. Antimony redox processes in the environment: a critical review of associated oxidants and reductants. Journal of Hazardous Materials431, 128607.

[66] Zhao, Y., Liu, P.F., Rui, J.P., Cheng, L., Wang, Q., Liu, X., Yuan, Q., 2020. Dark carbon fixation and chemolithotrophic microbial community in surface sediments of the cascade reservoirs, Southwest China. Science of the Total Environment698, 134316.

[67] Zheng, Y., Saitou, A., Wang, C.M., Toyoda, A., Minakuchi, Y., Sekiguchi, Y., Ueda, K., Takano, H., Sakai, Y., Abe, K., Yokota, A., Yabe, S., 2019. Genome features and secondary metabolites biosynthetic potential of the class Ktedonobacteria. Frontiers in Microbiology10, 893.

Acknowledgements

This study was financially supported by the National Key Research and Development Program of China (Grant No. 2020YFC1807700); by complementary fund from the Guizhou Provincial Department of Science and Technology; by Guizhou Provincial 2021 Science and Technology Subsidies (Grant No. GZ2021SIG); by the Chinese Academy of Sciences "Light of West China" Program; by Guizhou Provincial Science and Technology Projects (Grant No. ZK[2022]328).

Data availability statement

The genomic drafts of the 170 Chloroflexota MAGs is available on the Figshare platform (https://doi.org/10.6084/m9.figshare.26207045.v1). The genomic information of MAGs and the result of AAI calculation were provided in the Supplementary Table.

Conflicts of interest

The authors of this study declare no conflicts of interest.

Electronic supplementary material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s42832-024-0266-y and is accessible for authorized users.

RIGHTS & PERMISSIONS

2024 Higher Education Press