Structural and functional metagenomic analyses of a tropical agricultural soil


Understanding the intricate link between the soil microbiota and their metabolic functions is important for agricultural and ecological processes and could be used as a biomarker of soil health. To understand the relationship between soil microbial community structure and functions, a soil microcosm designated 2S (agricultural soil) was set up. Metagenomic DNA was extracted from the soil microcosm and sequenced using Miseq Illumina next generation sequencing and analysed for their structural and functional properties. Structural analysis of the soil microcosm by MG-RAST revealed 40 phyla, 78 classes, 157 orders, 273 families and 750 genera. Actinobacteria (54.0%) and Proteobacteria (17.5%) are the dominant phyla while Conexibacter (8.38%), Thermoleophilum (7.40%), and Streptomyces (4.14%) are the dominant genera. Further assignment of the metagenomics using Cluster of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), GhostKOALA, and NCBI’s CDD revealed diverse metabolic pathways utilized by the microbial community for the metabolism of carbohydrates, amino acids, lipids, biosynthesis of secondary metabolites and resistance to antibiotics. Taxonomic analysis of the annotated genes also revealed the preponderance of members of Actinobacteria and Proteobacteria. This study has established that members of the phyla Actinobacteria and Proteobacteria are the key drivers of the majority of important metabolic activities in the soil ecosystem and are thus an integral part of the soil microbial community.


Aislabie J, Deslippe JR. 2013. Soil microbes and their contribution to soil services. In: Dymond JR, editor. Ecosystem services in New Zealand-conditions and trends. Lincoln: Manaaki Whenua Press. p. 143-161.

Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davis J, Handelsman J. 2010. Call of the wild: Antibiotic resistance genes in natural environments. Nat Rev Microbiol. 8(4):251-259.

Ara I, Kudo T. 2007. Luedemannella gen. nov., a new member of the family Micromonosporaceae and description of Luedemannella helvata sp. nov. and Luedemannella flava sp. nov. J Gen Appl Microbiol. 53:39-51.

Arias ME, González-Pérez JA, González-Vila FJ, Ball AS. 2005. Soil health-a new challenge for microbiologists and chemists. Int Microbiol. 8:13-21.

Asadu CLA, Nwafor IA, Chibuike GU. 2015. Contributions of microorganisms to soil fertility in adjacent forest, fallow and cultivated land use Types in Nsukka, Nigeria. Int J Agric Forest 5(3):199-204.

Azman A-S, Othman I, Velu SS, Chan K-G, Lee L-H. 2015. Mangrove rare actinobacteria: taxonomy, natural compound, and discovery of bioactivity. Front Microbiol. 6:856. doi:10.3389/fmicb.2015.00856.

Bashir Y, Singh SP, Konwar BK. 2014. Metagenomics: an application-based perspective. Chinese J Biol. doi:10-1155/2014/146030 1-7.

Bossio DA, Scow KM, Gunapala N, Graham KJ. 1998. Determinants of soil microbial communities: effects of agricultural management, season, and soil type on phospholipid fatty acid profiles. Microb Ecol. 36:1-12.

Calderón FJ, Jackson LE, Scow KM, Rolston DE. 2001. Short-term dynamics of nitrogen, microbial activity, and phospholipid fatty acids after tillage. Soil Sci Soc Am J. 65:118-126.

Castaneda LE, Barbosa O. 2017. Metagenomic analysis exploring taxonomic and functional diversity of soil microbial communities in Chilean vineyards and surrounding native forests. Peer J. 5:e3098. doi:10.7717/peerj.3098.

Chater KF. 2006. Streptomyces inside-out: a new perspective on the bacteria that provide us with antibiotics. Phil Trans Royal Society B. 361:761-768.

Cheema S, Lavania M, Lal B. 2015. Impact of petroleum hydrocarbon contamination on the indigenous microbial community. Ann Microbiol. 66:359-369.

Cheung P-Y, Kinkle BK. 2001. Mycobacterium diversity and pyrene mineralisation in petroleum-contaminated soil. Appl Environ Microbiol. 67:2222-2229.

Costa PS, Reis MP, Ávila MP, Leite LR, de Araújo FMG, Salim ACM, Oliveira G, Barbosa F, Chartone-Souza E, Nascimiento AMA. 2015. Metagenome of a microbial community inhabiting a metal-rich tropical stream sediment. Plos ONE 10(3):e0119465. doi:10.1371/journal.pone.0119465.

Dantas G, Sommer MOA, Oluwasegun RD, Church GM. 2008. Bacteria subsisting on antibiotics. Science 320(5872):100-103.

de Menezes AB, Lockhart RJ, Cox MJ, Allison HE, McCarthy AJ. 2008. Cellulose degradation by Micromonospora recovered from freshwater lakes and classification of these actinomycetes by DNA gyrase B gene sequencing. Appl Environ Microbiol. 74:7080-7084. doi:10.1128/AEM. 01092-08.

de Menezes AB, McDonald JE, Allison HE, McCarthy AJ. 2012. Importance of Micromonospora spp. as colonizers of cellulose in freshwater lakes as demonstrated by quantitative reverse transcriptase PCR of 16S rRNA. Appl Environ Microbiol. 78:3495-3499.

Durso LM, Miller DN, Wienhold B. 2012. Distribution and quantification of antibiotic resistant genes and bacteria across agricultural and non-agricultural metagenomes. PLoS ONE 7(11):e48325. doi:10.1371/journal.pone.0048325.

Fierer N, Leff JW, Adams BJ, Nielsen UN, Bates ST, Lauber CL, Owens S, Gilbert JA, Wall DH, Caporaso G. 2012. Cross-biome metagenomic analyses of soil microbial communities and their functional attributes. Proc Nat Acad Sci USA. 109:21390-21395.

Garrity GM, Bell JA, Lilburn TG. 2005. Class III. Gammaproteobacteria class. nov. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM, editors. Bergey’s manual of systematic bacteriology, 2nd ed., Vol. 2. New York, NY: Springer. p. 1.

Handelsman J. 2004. Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev. 68:669-668.

Ilori MO, Amund OO, Obayori OS, Omotayo AE. 2015. Microbial population and physico-chemical dynamics of a soil ecosystem upon petroleum contamination. J Sci Res Dev. 15:25-33.

Islam MR, Trivedi P, Palaniappan P, Reddy MS, Sa T. 2009. Evaluating the effect of fertilizer application on soil microbial community structure in rice-based cropping system using fatty acid methyl esters (FAME) analysis. World J Microbiol Biotechnol. 25:1115-1117.

Jiang Y, Xiong X, Danska J, Parkinson J. 2016. Metatranscriptomic analysis of diverse microbial communities reveals core metabolic pathways and microbiome-specific functionality. Microbiome 4:2. doi:10.1186/s40168-015-0146x.

Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol. 428(4):726-731.

Kertesz MA, Fellows E, Schmalenberger A. 2007. Rhizobacteria and plant sulfur supply. Adv Appl Microbiol. 62:235-268.

Kertesz MA, Mirleau R. 2004. The role of soil microbes in plant sulfur nutrition. J Exp Botany 55:1939-1945.

Köberl M, Müller H, Ramadan EM, Berg G. 2011. Desert farming benefits from microbial potential in arid soils and promotes diversity and plant health. PLoS ONE 6(9):e24452. doi:10.1371/journal.pone.0024452.

Kunihiro T, Veuger B, Vasquez-Cardenas D, Pozzato L, Le Guitton M, Moriya K, Kuwae M, Omori K, Boschker HTS, van Oevelen D. 2014. Phospholipid-derived fatty acids and quinones as markers for bacterial biomass and community structure in marine sediments. PLoS ONE 9(4):e96219. doi:10.1371/journal.pone.0096219.

Lauber CL, Hamady M, Knight R, Fierer N. 2009. Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl Environ Microbiol. 75(15):5111-5120.

Li X, Rui J, Xiong J, Li J, He Z, Zhou J, Yannarell AC, Mackie RI. 2014. Functional potential of soil microbial communities in the maize rhizosphere. PLoS ONE 9(11):e112609.

Lipson DA, Schmidt SK. 2004. Seasonal changes in alpine soil bacterial community in the Colorado Rocky Mountains. Appl Environ Microbiol. 70(5):2867-2879.

Liu WT, Marsh TL, Cheng H, Forney LJ. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol. 63:4516-4522.

Lizarraga-Guerra R, López MG. 1996. Content of free amino acids in Huitlacoche (Ustilago maydis). J Agric Food Chem. 44(9):2556-2559.

Ludwig W, Euzeby J, Schumann P, Busse H-J, Trujillo ME, Kämpfer P, Whitman WB. 2012. Roadmap of the phylum Actinobacteria. In: Goodfellow M, Kämpfer P, Busse H-J, Trujillo ME, Suzuki K-I, Ludwig W, Whitman WB, editors. Bergey’s Manual of Systematic Bacteriology. 2nd Ed. New York, NY: Springer. p. 1-28.

Maier RM, Pepper IL. 2009. Earth environment. In: Maier RM, Pepper IL, Gerba CP, editors. Environmental Microbiology. 2nd Ed. Burlington: Academic Press. p. 57-82.

Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Bryant SH. 2015. CDD: NCBI’s conserved domain database. Nucleic Acids Res. 43(D):222-226.

Matsumoto A, Kawaguchi Y, Nakashima T, Iwatsuki M, Omaura S. Takashi Y. 2014. Rhizocola hellebori gen. nov., sp. nov., an actinomycete of the family Micromonosporaceae containing 3,4-dihydroxydiaminopimelic acid in the cell wall peptidoglycan. Int J Syst Microbiol. 64:2706-2711.

Mellouli L, Mehdi RB, Sioud S, Salem M, Bejar S. 2003. Isolation, purification and partial characterization of antibacterial activities produced by a newly isolated Streptomyces sp. US24 strain. Res Microbiol. 154:345-352.

Moats GA, Foster JW, Spector MP. 2002. Microbial Physiology, 4th Edition. New York: Wiley-Liss Inc. 714 p.

Mocali S, Benedetti A. 2010. Exploring research frontiers in microbiology: the challenges of metagenomics in soil microbiology. Res Microbiol. 161(6):497-505. doi:10.1016/j.resmic.2010.04.010.

Monciardini P, Cavaletti, L, Ranghetti A, Schumann P, Rohde M, Bamonte R, Sosio M, Mezzelani A, Donadio S. 2009. Novel members of the family Micromonosporaceae, Rugosimonospora acidiphila gen. nov., sp. nov. and Rugosimonospora africana sp. nov. Int J Syst Evol Microbiol. 59:2752-2758.

Monciardini P, Cavaletti L, Schumann P, Rohde M, Donadio S. 2003. Conexibacter woesei gen. nov., sp. nov., a novel representative of a deep evolutionary line of descent within the class Actinobacteria. Int J Syst Evol Microbiol. 53(Pt 2):569-576.

Montecchia MS, Tosi M, Soria MA, Vogrig JA, Sydorenko O, Correa OS. 2015. Pyrosequencing reveals changes in soil bacterial communities after conversion of Yungas forests to agriculture. PLoS One 10:e0119426. doi:10.1371/journal.pone.0119426.

Muyzer G, de Waal EC, Uitterlinden AG. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 59:695-700.

Myrold DD, Zeglin LH, Jansson JK. 2013. The potential of metagenomics approaches for understanding soil microbial processes. Soil Sci Soc Am J. 78:3-10.

Nannipieri, P, Ascher J, Ceccherini MT, Landi L, Pietramellara G, Renella G. 2003. Microbial diversity and soil functions. Eur J Soil Sci. 54:655-670.

Newby DT, Marlowe EM, Maier RM. 2009. Nucleic acid-based methods of analysis. In: Maier RM, Pepper IL, Gerba CP, editors. Environmental Microbiology. 2nd Ed. Burlington: Academic Press. p. 243-28.

Ogbulie TE, Nwaokorie FO. 2016. Molecular diversity of microbes with probable degradative genes in agriculture soil contaminated with Bonny Light crude oil. J Ecosyst Ecograph S5:002. doi:10.4172/2157-7625.S5-002.

Oulas A, Pavloudi G, Polymanakou P, Pavlopoulus GA, Papanikolaou N, Kotoulas G, Arvanitidis C, Iliopoulus I. 2015. Metagenomics: Tools and insights for analyzing next-generation sequencing data derived from biodiversity studies. Bioinform Biol Insights 9:75-88.

Pacchioni RG, Carvalho FM, Thompson CE, Faustino AL, Nicolini F, Pereira TS, Silva RC, Cantão ME, Gerber A, Vasconcelos AT, Agnez-Lima LF. 2014. Taxonomic and functional profiles of soil samples from Atlantic forest and Caatinga biomes in northeastern Brazil. MicrobiologyOpen 3(3):299-315.

Pan JJ, Baumgarten AM, May G. 2008. Effects of host plant environment and Ustilago maydis infection on the fungal endophyte community of maize (Zea mays). New Phytologist 178:147-156.

Paul S, Cortez Y, Vera N, Villena GK, Gutiérrez-Correa M. 2016. Metagenomic analysis of microbial community of an Amazonian geothermal spring in Peru. Genomic Data 9:63-66.

Qin S, Li W-J, Dastager SG, Hozzein WN, editors. 2016. Actinobacteria in special and extreme habitats: diversity, function, roles and environmental adaptations. Lausanne: Frontiers Media. doi:10.3389/978-2-88945-013-8.

Rashid MI, Mujawar LH, Shahzad T, Almeelbi T, Ismail IMI, Oves M. 2016. Bacteria and fungi can contribute to nutrients bioavailability and aggregate formation in degraded soils. Microbiol Res. 183:26-41.

Rashid MH, Young JP, Everall I, Clercx P, Willems A, Santhosh Braun M, Wink M. 2015. Average nucleotide identity of genome sequences supports the description of Rhizobium lentis sp. nov., Rhizobium bangladeshense sp. nov., and Rhizobium binae sp. nov., from lentil (Len culinaris) nodules. Int J Syst Evol Microbiol. 65(9):3037-3045.

Reddy BV, Kallifidas D, Kim JH, Charlop-Powers Z, Feng Z, Brady SF. 2012. Natural product biosynthetic gene diversity in geographically distinct soil microbiomes. Appl Environ Microbiol. 78(10):3744-3752.

Rho M, Tang H, Ye Y. 2010. FragGeneScan: predicting genes in short and error-prone reads. Nucleic Acid Res. 38:20-191.

Salam LB, Ilori MO, Amund OO, Numata M, Horisaki T, Nojiri H. 2014. Carbazole angular dioxygenation and mineralization by bacteria isolated from hydrocarbon-contaminated tropical African soil. Environ Sci Pollut Res. 21(15):9311-9324. doi:10.1007/s11356-014-2855-2.

Salam LB, Obayori OS, Nwakorie FO, Suleiman A, Mustapha R. 2017. Metagenomic insight into effects of spent oil perturbation on the microbial community composition and function in a tropical agricultural soil. Environ Sci Pollut Res. 24:7139-7159. doi:10.1007/s11356-017-8364-3.

Sathya A, Vijayabharathi R, Gopalakrishnan S. 2017. Plant growth-promoting actinobacteria: a new strategy for enhancing sustainable production and protection of grain legumes. 3 Biotech 7(2):102. doi:10.1007/s13205-017-0736-3.

Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF. 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 75(23):7537-7541.

Segata N, Boernigen D, Tickle TL, Morgan XC, Garrett WS, Huttenhower C. 2013. Computational meta’omics for microbial community studies. Mol Syst Biol. 9:666. doi:10.1038/msb.2013.22.

Seipke RF, Kaltenpoth M, Huchings MI. 2011. Streptomyces as symbionts: an emerging and widespread theme? FEMS Microbiol Rev. 36 (2012):862-876.

Seki T, Matsumoto A, Omura S, Takahashi Y. 2012. Conexibacter arvalis sp. nov. isolated from cultivated a field sample. Int J Syst Evol Microbiol. 62(10):2400-2404.

Selvin J, Sathiyanarayanan G, Lipton AN, Al-Dhabi NA, Valan Arasu M, Kiran GS. 2016. Ketide Synthase (KS) domain prediction and analysis of iterative Type II PKS Gene in Marine Sponge-Associated Actinobacteria producing biosurfactants and antimicrobial agents. Front Microbiol. 7:63. doi:10.3389/fmicb.2016.00063.

Sharma SB, Sayyed RZ, Trivedi MH, Gobi TA. 2013. Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus 2:587. doi:10.1186/2193-1801-2-587.

Shivlata L, Satyanarayana T. 2015. Thermophilic and alkaliphilic Actinobacteria: biology and potential applications. Front Microbiol. 6:1014. doi:10.3389/fmicb.2015.01014.

Silva MS, Sales AN, Magalhaes-Guedes KT, Dias DR, Scwan RF. 2013. Brazillian cerrado soil actinobacterial ecology. BioMed Research International, Vol 2013, Article ID 503805.10 p. doi:10.1155/2013/503805.

Soil Survey Division Staff. 1993. Soil Survey Manual. USDA Handbook No. 18. Washington, D.C., USA: US Government Printing Office. p. 63-65.

Spain AM, Krumholz LR, Elshahed MS. 2009. Abundance, composition, diversity and novelty of soil Proteobacteria. ISME Journal 9:992-1000.

Stach JEM, Burns RG. 2002. Enrichment versus biofilm culture: a functional and phylogenetic comparison of polycyclic aromatic hydrocarbon-degrading microbial communities. Environ Microbiol. 4(3):159-182.

Streit WR, Schmitz RA. 2004. Metagenomics- key to the uncultured microbes. Curr Opin Microbiol. 7:492-498.

Suzuki M, Rappe MS, Giovanoni SJ. 1998. Kinetic bias in estimates of coastal picoplankton community structure obtained by measurement of small subunit rRNA gene PCR amplicon length heterogeneity. Appl Environ Microbiol. 64:4522-4529.

Tatusov RL, Natale DA, Garkavtsev IV, Tatusova TA, Shankavaram UT, Rao BS, Kiryutin B, Galperin MY, Fedorova ND, Koonin EV. 2001. The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res. 29(1):22-28.

Thakur D, Yadav A, Gogoi BK, Bora TC. 2007. Isolation and screening of Streptomyces in soil of protected forest areas from the states of Assam and Tripura, India, for antimicrobial metabolites. Journal de Mycologie Médicale 17:242-249.

Trivedi P, Delgado-Baquerizo M, Anderson IC, Singh BK. 2016. Response of soil properties and microbial communities to agriculture: implications for primary productivity and health indicators. Front Plant Sci. 7:990. doi:10.3389/fpls.2016.00990.

Trujillo ME, Riesco R, Benito P, Carro L. 2015. Endophytic Actinobacteria and the interaction of Micromonospora and nitrogen-fixing plants. Front Microbiol. 6:1341. doi:10:3389/fmicb.2015.01341.

Vandova GA, O’Brien RV, Lowry B, Robbins TF, Fischer CR, Davis RW, Khosla C, Harvey CJB, Hillenmeyer ME. 2017. Heterologous expression of diverse propionyl-CoA carboxylases affects polyketide production in Escherichia coli. The Journal of Antibiotics 70:859-863.

Wang X, Jia F, Liu C, Zhao J, Wang L, Shen Y, Wang J, Zhang J, Li C, Xiang W. 2013. Xiangella phaseoli gen. nov., sp. nov., a member of the family Micromonosporaceae. Int J Syst Evol Microbiol. 63:2138-2145.

Wardle DA, Bardgett RD, Klironomos JN, Seta¨la¨ H, van der Putten WH, Wall DH. 2004. Ecological linkages between aboveground and belowground biota. Science 304(5677):1629-1633.

Wawrik B, Kerkhof L, Zylstra GJ, Kukor JJ. 2005. Identification of unique type II polyketide synthase genes in soil. Appl Environ Microbiol. 71(5):2232-2238.

Wibberg D, Al-Dilaimi A, Busche T, Wedderhoff I, Schrempf H, Kakinowski J, Ortiz de Orué Lucana D. 2016. Complete genome sequence of Streptomyces reticuli, an efficient degrader of crystalline cellulose. J Biotechnol. 222:13-14.

Williams KP, Gillespie JJ, Sobral BW, Nordberg EK, Snyder EE, Shallom JM, Dickerman AW. 2010. Phylogeny of gammaproteobacteria. J Bacteriol. 192(9):2305-2314.

Williams KP, Sobral BW, Dickerman AW. 2007. A robust species tree for the Alphaproteobacteria. J Bacteriol. 189:4578-4586.

Xiong W, Sun Y, Ding X, Zhang Y, Zeng Z. 2014. Antibiotic resistance genes occurrence and bacterial community composition in the Liuxi River. Front Environ Sci. 2:61. doi:10.3389/fenvs.2014.00061.

Yin C, Mueth N, Hulbert S. 2017. Bacterial community on wheat grown under long-term conventional tillage and no-till in the Pacific Northwest of the United States. Phytobiomes J. 1(2):83-90.

Yousuf B, Kumar R, Mishra A, Jha B. 2014. Unravelling the carbon and sulphur metabolism in coastal soil ecosystems using comparative cultivation-independent genome-level characterisation of microbial communities. PLoS ONE 9(9):e107025. doi:10.1371/journal.pone.0107025.

Zubay GL, Parson WW, Vance DE. 1995. Principles of Biochemistry. Dubuque: Wm. C. Brown Publishers. 863 p.