BACKGROUND: The search for new enzymes and microbial strains to degrade plant biomass is one of the most important strategies for improving the conversion processes in the production of environment-friendly chemicals and biofuels. In this study, we report a new Paenibacillus isolate, O199, which showed the highest efficiency for cellulose deconstruction in a screen of environmental isolates. Here, we provide a detailed description of the complex multi-component O199 enzymatic system involved in the degradation of lignocellulose. RESULTS: We examined the genome and the proteome of O199 grown on complex lignocellulose (wheat straw) and on microcrystalline cellulose. The genome contained 476 genes with domains assigned to carbohydrate-active enzyme (CAZyme) families, including 100 genes coding for glycosyl hydrolases (GHs) putatively involved in cellulose and hemicellulose degradation. Moreover, 31 % of these CAZymes were expressed on cellulose and 29 % on wheat straw. Proteomic analyses also revealed a complex and complete set of enzymes for deconstruction of cellulose (at least 22 proteins, including 4 endocellulases, 2 exocellulases, 2 cellobiohydrolases and 2 β-glucosidases) and hemicellulose (at least 28 proteins, including 5 endoxylanases, 1 β-xylosidase, 2 xyloglucanases, 2 endomannanases, 2 licheninases and 1 endo-β-1,3(4)-glucanase). Most of these proteins were secreted extracellularly and had numerous carbohydrate-binding domains (CBMs). In addition, O199 also secreted a high number of substrate-binding proteins (SBPs), including at least 42 proteins binding carbohydrates. Interestingly, both plant lignocellulose and crystalline cellulose triggered the production of a wide array of hydrolytic proteins, including cellulases, hemicellulases, and other GHs. CONCLUSIONS: Our data provide an in-depth analysis of the complex and complete set of enzymes and accessory non-catalytic proteins-GHs, CBMs, transporters, and SBPs-implicated in the high cellulolytic capacity shown by this bacterial strain. The large diversity of hydrolytic enzymes and the extracellular secretion of most of them supports the use of Paenibacillus O199 as a candidate for second-generation technologies using paper or lignocellulosic agricultural wastes.

Institute of Microbiology Ernst Moritz Arndt University of Greifswald Friedrich Ludwig Jahnstrasse 15 17487 Greifswald Germany

Laboratory of Environmental Microbiology Institute of Microbiology of the CAS v v i Průmyslová 595 252 42 Vestec Czech Republic

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Van Dyk JS, Pletschke BI. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes–factors affecting enzymes, conversion and synergy. Biotechnol Adv. 2012;30:1458–1480. doi: 10.1016/j.biotechadv.2012.03.002. PubMed DOI

Himmel ME, Xu Q, Luo Y, Ding S, Lamed R, Bayer EA. Microbial enzyme systems for biomass conversion: emerging paradigms. Biofuels. 2010;1:323–341. doi: 10.4155/bfs.09.25. DOI

Zhou Y, Pope PB, Li S, Wen B, Tan F, Cheng S, Chen J, Yang J, Liu F, Lei X, et al. Omics-based interpretation of synergism in a soil-derived cellulose-degrading microbial community. Sci Rep. 2014;4:5288. PubMed PMC

Rakotoarivonina H, Hermant B, Monthe N, Rémond C. The hemicellulolytic enzyme arsenal of Thermobacillus xylanilyticus depends on the composition of biomass used for growth. Microb Cell Fact. 2012;11:159. doi: 10.1186/1475-2859-11-159. PubMed DOI PMC

Song HY, Lim HK, Kim DR, Lee KI, Hwang IT. A new bi-modular endo-beta-1,4-xylanase KRICT PX-3 from whole genome sequence of Paenibacillus terrae HPL-003. Enzyme Microb Technol. 2014;54:1–7. doi: 10.1016/j.enzmictec.2013.09.002. PubMed DOI

Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014;42:D490–D495. doi: 10.1093/nar/gkt1178. PubMed DOI PMC

Brumm PJ. Bacterial genomes: what they teach us about cellulose degradation. Biofuels. 2013;4:669–681. doi: 10.4155/bfs.13.44. DOI

Koeck DE, Pechtl A, Zverlov VV, Schwarz WH. Genomics of cellulolytic bacteria. Curr Opin Biotechnol. 2014;29:171–183. doi: 10.1016/j.copbio.2014.07.002. PubMed DOI

Sukharnikov LO, Cantwell BJ, Podar M, Zhulin IB. Cellulases: ambiguous nonhomologous enzymes in a genomic perspective. Trends Biotechnol. 2011;29:473–479. doi: 10.1016/j.tibtech.2011.04.008. PubMed DOI PMC

Dos Santos Castro L, Ramos Pedersoli W, Campos Antoniêto AC, Stecca Steindorff A, Silva-Rocha R, Martinez-Rossi NM, Rossi A, Brown NA, Goldman GH, Faça VM, et al. Comparative metabolism of cellulose, sophorose and glucose in Trichoderma reesei using high-throughput genomic and proteomic analyses. Biotechnol Biofuels. 2014;7:41. doi: 10.1186/1754-6834-7-41. PubMed DOI PMC

Mori T, Kamei I, Hirai H, Kondo R. Identification of novel glycosyl hydrolases with cellulolytic activity against crystalline cellulose from metagenomic libraries constructed from bacterial enrichment cultures. SpringerPlus. 2014;3:365. doi: 10.1186/2193-1801-3-365. PubMed DOI PMC

Maki M, Leung KT, Qin W. The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int J Biol Sci. 2009;5:500–516. doi: 10.7150/ijbs.5.500. PubMed DOI PMC

Yang JK, Zhang JJ, Yu HY, Cheng JW, Miao LH. Community composition and cellulase activity of cellulolytic bacteria from forest soils planted with broad-leaved deciduous and evergreen trees. Appl Microbiol Biotechnol. 2014;98:1449–1458. doi: 10.1007/s00253-013-5130-4. PubMed DOI

Gastelum-Arellanez A, Paredes-Lopez O, Olalde-Portugal V. Extracellular endoglucanase activity from Paenibacillus polymyxa BEb-40: production, optimization and enzymatic characterization. World J Microbiol Biotechnol. 2014;30:2953–2965. doi: 10.1007/s11274-014-1723-z. PubMed DOI

Mihajlovski KR, Carević MB, Dević ML, Šiler-Marinković S, Rajilić-Stojanović MD, Dimitrijević-Branković S. Lignocellulosic waste material as substrate for Avicelase production by a new strain of Paenibacillus chitinolyticus CKS1. Inter Biodeter Biodegr. 2015;104:426–434. doi: 10.1016/j.ibiod.2015.07.012. DOI

Hwang IT, Lim HK, Song HY, Cho SJ, Chang JS, Park NJ. Cloning and characterization of a xylanase, KRICT PX1 from the strain Paenibacillus sp. HPL-001. Biotechnol Adv. 2010;28:594–601. doi: 10.1016/j.biotechadv.2010.05.007. PubMed DOI

Park IH, Chang J, Lee YS, Fang SJ, Choi YL. Gene cloning of endoglucanase Cel5A from cellulose-degrading Paenibacillus xylanilyticus KJ-03 and purification and characterization of the recombinant enzyme. Protein J. 2012;31:238–245. doi: 10.1007/s10930-012-9396-7. PubMed DOI

Pason P, Kosugi A, Waeonukul R, Tachaapaikoon C, Ratanakhanokchai K, Arai T, Murata Y, Nakajima J, Mori Y. Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6. Appl Microbiol Biotechnol. 2010;85:573–580. doi: 10.1007/s00253-009-2117-2. PubMed DOI

van Dyk JS, Sakka M, Sakka K, Pletschke BI. Identification of endoglucanases, xylanases, pectinases and mannanases in the multi-enzyme complex of Bacillus licheniformis SVD1. Enzyme Microb Technol. 2010;47:112–118. doi: 10.1016/j.enzmictec.2010.05.004. DOI

Baldrian P, López-Mondéjar R. Microbial genomics, transcriptomics and proteomics: new discoveries in decomposition research using complementary methods. Appl Microbiol Biotechnol. 2014;98:1531–1537. doi: 10.1007/s00253-013-5457-x. PubMed DOI

Berlemont R, Martiny AC. Phylogenetic distribution of potential cellulases in bacteria. Appl Environ Microbiol. 2013;79:1545–1554. doi: 10.1128/AEM.03305-12. PubMed DOI PMC

Mba Medie F, Davies GJ, Drancourt M, Henrissat B. Genome analyses highlight the different biological roles of cellulases. Nat Rev Microbiol. 2012;10:227–234. doi: 10.1038/nrmicro2729. PubMed DOI

Wilson DB. Processive and nonprocessive cellulases for biofuel production—lessons from bacterial genomes and structural analysis. Appl Microbiol Biotechnol. 2012;93:497–502. doi: 10.1007/s00253-011-3701-9. PubMed DOI

Takasuka TE, Book AJ, Lewin GR, Currie CR, Fox BG. Aerobic deconstruction of cellulosic biomass by an insect-associated Streptomyces. Sci Rep. 1030;2013:3. PubMed PMC

Maqbool A, Horler RS, Muller A, Wilkinson AJ, Wilson KS, Thomas GH. The substrate-binding protein in bacterial ABC transporters: dissecting roles in the evolution of substrate specificity. Biochem Soc Trans. 2015;43:1011–1017. doi: 10.1042/BST20150135. PubMed DOI

Harris PV, Xu F, Kreel NE, Kang C, Fukuyama S. New enzyme insights drive advances in commercial ethanol production. Curr Opin Chem Biol. 2014;19:162–170. doi: 10.1016/j.cbpa.2014.02.015. PubMed DOI

Chua P, Yoo H-S, Gan HM, Lee S-M. Draft genome sequences of two cellulolytic Paenibacillus sp. strains, MAEPY1 and MAEPY2, from Malaysian landfill leachate. Genome Announc. 2014;2:e00065-14. doi: 10.1128/genomeA.00065-14. PubMed DOI PMC

Dhar H, Swarnkar MK, Gulati A, Singh AK, Kasana RC. Draft genome sequence of a cellulase-producing psychrotrophic Paenibacillus strain, IHB B 3415, isolated from the cold environment of the Western Himalayas, India. Genome Announc. 2015;3:e01581-14. doi: 10.1128/genomeA.01581-14. PubMed DOI PMC

Yuki M, Oshima K, Suda W, Oshida Y, Kitamura K, Iida T, Hattori M, Ohkuma M. Draft genome sequence of Paenibacillus pini JCM 16418T, isolated from the rhizosphere of pine tree. Genome Announc. 2014;2:e00210–e00214. PubMed PMC

Shin SH, Kim S, Kim JY, Song HY, Cho SJ, Kim DR, Lee KI, Lim HK, Park NJ, Hwang IT, Yang KS. Genome sequence of Paenibacillus terrae HPL-003, a xylanase-producing bacterium isolated from soil found in forest residue. J Bacteriol. 2012;194:1266. doi: 10.1128/JB.06668-11. PubMed DOI PMC

Eastman AW, Heinrichs DE, Yuan Z. Comparative and genetic analysis of the four sequenced Paenibacillus polymyxa genomes reveals a diverse metabolism and conservation of genes relevant to plant-growth promotion and competitiveness. BMC Genom. 2014;15:851. doi: 10.1186/1471-2164-15-851. PubMed DOI PMC

Adams AS, Jordan MS, Adams SM, Suen G, Goodwin LA, Davenport KW, Currie CR, Raffa KF. Cellulose-degrading bacteria associated with the invasive woodwasp Sirex noctilio. ISME J. 2011;5:1323–1331. doi: 10.1038/ismej.2011.14. PubMed DOI PMC

Dam P, Kataeva I, Yang SJ, Zhou F, Yin Y, Chou W, Poole FL, 2nd, Westpheling J, Hettich R, Giannone R, et al. Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725. Nucleic Acids Res. 2011;39:3240–3254. doi: 10.1093/nar/gkq1281. PubMed DOI PMC

Berlemont R, Allison SD, Weihe C, Lu Y, Brodie EL, Martiny JB, Martiny AC. Cellulolytic potential under environmental changes in microbial communities from grassland litter. Front Microbiol. 2014;5:639. doi: 10.3389/fmicb.2014.00639. PubMed DOI PMC

Wilson DB. Microbial diversity of cellulose hydrolysis. Curr Opin Microbiol. 2011;14:259–263. doi: 10.1016/j.mib.2011.04.004. PubMed DOI

Sanchez MM, Irwin DC, Pastor FI, Wilson DB, Diaz P. Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca. Biotechnol Bioeng. 2004;87:161–169. doi: 10.1002/bit.20099. PubMed DOI

Berger E, Zhang D, Zverlov VV, Schwarz WH. Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically. FEMS Microbiol Lett. 2007;268:194–201. doi: 10.1111/j.1574-6968.2006.00583.x. PubMed DOI

Vardakou M, Katapodis P, Topakas E, Kekos D, Macris BJ, Christakopoulos P. Synergy between enzymes involved in the degradation of insoluble wheat flour arabinoxylan. Innov Food Sci Emerg. 2004;5:107–112. doi: 10.1016/S1466-8564(03)00044-4. DOI

Ozdemir I, Blumer-Schuette SE, Kelly RM. S-layer homology domain proteins Csac_0678 and Csac_2722 are implicated in plant polysaccharide deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus. Appl Environ Microbiol. 2012;78:768–777. doi: 10.1128/AEM.07031-11. PubMed DOI PMC

Chow V, Kim YS, Rhee MS, Sawhney N. St John FJ, Nong G, Rice JD, Preston JF: A 1,3-1,4-β-Glucan Utilization Regulon in Paenibacillus sp. Strain JDR-2. Appl Environ Microbiol. 2016;82:1789–1798. doi: 10.1128/AEM.03526-15. PubMed DOI PMC

Fukuda M, Watanabe S, Yoshida S, Itoh H, Itoh Y, Kamio Y, Kaneko J. Cell surface xylanases of the glycoside hydrolase family 10 are essential for xylan utilization by Paenibacillus sp. W-61 as generators of xylo-oligosaccharide inducers for the xylanase genes. J Bacteriol. 2010;192:2210–2219. doi: 10.1128/JB.01406-09. PubMed DOI PMC

Waeonukul R, Kyu KL, Sakka K, Ratanakhanokchai K. Effect of Carbon Sources on the Induction of Xylanolytic-Cellulolytic Multienzyme Complexes in Paenibacillus curdlanolyticus Strain B-6. Biosci Biotechnol Biochem. 2008;72:321–328. doi: 10.1271/bbb.70333. PubMed DOI

Wegmann U, Louis P, Goesmann A, Henrissat B, Duncan SH, Flint HJ. Complete genome of a new Firmicutes species belonging to the dominant human colonic microbiota (‘Ruminococcus bicirculans’) reveals two chromosomes and a selective capacity to utilize plant glucans. Environ Microbiol. 2014;16:2879–2890. doi: 10.1111/1462-2920.12217. PubMed DOI

Nataf Y, Bahari L, Kahel-Raifer H, Borovok I, Lamed R, Bayer EA, Sonenshein AL, Shoham Y. Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors. Proc Natl Acad Sci USA. 2010;107:18646–18651. doi: 10.1073/pnas.1012175107. PubMed DOI PMC

Zhang H, Hutcheson SW. Complex expression of the cellulolytic transcriptome of Saccharophagus degradans. Appl Environ Microbiol. 2011;77:5591–5596. doi: 10.1128/AEM.00464-11. PubMed DOI PMC

Xu C, Huang R, Teng L, Wang D, Hemme CL, Borovok I, He Q, Lamed R, Bayer EA, Zhou J, Xu J. Structure and regulation of the cellulose degradome in Clostridium cellulolyticum. Biotechnol Biofuels. 2013;6:73. doi: 10.1186/1754-6834-6-73. PubMed DOI PMC

Yokoyama H, Yamashita T, Morioka R, Ohmori H. Extracellular secretion of noncatalytic plant cell wall-binding proteins by the cellulolytic thermophile Caldicellulosiruptor bescii. J Bacteriol. 2014;196:3784–3792. doi: 10.1128/JB.01897-14. PubMed DOI PMC

Šnajdr J, Cajthaml T, Valášková V, Merhautová V, Petranková M, Spetz P, Leppanen K, Baldrian P. Transformation of Quercus petraea litter: successive changes in litter chemistry are reflected in differential enzyme activity and changes in the microbial community composition. FEMS Microbiol Ecol. 2011;75:291–303. doi: 10.1111/j.1574-6941.2010.00999.x. PubMed DOI

Šnajdr J, Valášková V. Merhautová Vr, Herinková J, Cajthaml T, Baldrian P: Spatial variability of enzyme activities and microbial biomass in the upper layers of Quercus petraea forest soil. Soil Biol Biochem. 2008;40:2068–2075. doi: 10.1016/j.soilbio.2008.01.015. DOI

López-Mondéjar R, Voříšková J, Větrovský T, Baldrian P. The bacterial community inhabiting temperate deciduous forests is vertically stratified and undergoes seasonal dynamics. Soil Biol Biochem. 2015;87:43–50. doi: 10.1016/j.soilbio.2015.04.008. DOI

Valášková V, Šnajdr J, Bittner B, Cajthaml T, Merhautová V, Hofrichter M, Baldrian P. Production of lignocellulose-degrading enzymes and degradation of leaf litter by saprotrophic basidiomycetes isolated from a Quercus petraea forest. Soil Biol Biochem. 2007;39:2651–2660. doi: 10.1016/j.soilbio.2007.05.023. DOI

Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, editors. Nucleic acids techniques in bacterial systematics. Chichester: Wiley; 1991. pp. 115–147.

Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, et al. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol. 2012;62:716–721. doi: 10.1099/ijs.0.038075-0. PubMed DOI

Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–829. doi: 10.1101/gr.074492.107. PubMed DOI PMC

Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, et al. The RAST server: rapid annotations using subsystems technology. BMC Genom. 2008;9:75. doi: 10.1186/1471-2164-9-75. PubMed DOI PMC

Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, et al. The SEED and the rapid annotation of microbial genomes using subsystems technology (RAST) Nucleic Acids Res. 2014;42:D206–D214. doi: 10.1093/nar/gkt1226. PubMed DOI PMC

Yin Y, Mao X, Yang J, Chen X, Mao F, Xu Y. dbCAN: a web resource for automated carbohydrate-active enzyme annotation. Nucleic Acids Res. 2012;40:W445–W451. doi: 10.1093/nar/gks479. PubMed DOI PMC

Grube M, Cernava T, Soh J, Fuchs S, Aschenbrenner I, Lassek C, Wegner U, Becher D, Riedel K, Sensen CW, Berg G. Exploring functional contexts of symbiotic sustain within lichen-associated bacteria by comparative omics. ISME J. 2015;9:412–424. doi: 10.1038/ismej.2014.138. PubMed DOI PMC

Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn M. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J Proteome Res. 2006;5:2339–2347. doi: 10.1021/pr060161n. PubMed DOI

Saeed A, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003;34:374–378. PubMed

Schneider T, Schmid E, de Castro JV, Jr, Cardinale M, Eberl L, Grube M, Berg G, Riedel K. Structure and function of the symbiosis partners of the lung lichen (Lobaria pulmonaria L. Hoffm.) analyzed by metaproteomics. Proteomics. 2011;11:2752–2756. doi: 10.1002/pmic.201000679. PubMed DOI

Edgar RC. MUSCLE: multiple sequence alignment with high accurance and high throughput. Nucleic Acids Res. 2004;32:1792–1797. doi: 10.1093/nar/gkh340. PubMed DOI PMC

Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59:307–321. doi: 10.1093/sysbio/syq010. PubMed DOI

Vizcaíno JA, Deutsch EW, Wang R, Csordas A, Reisinger F, Ríos D, Dianes JA, Sun Z, Farrah T, Bandeira N, et al. ProteomeXchange provides globally co-ordinated proteomics data submission and dissemination. Nat Biotechnol. 2014;30:223–226. doi: 10.1038/nbt.2839. PubMed DOI PMC

Burton RA, Gidley MJ, Fincher GB. Heterogeneity in the chemistry, structure and function of plant cell walls. Nat Chem Biol. 2010;6:724–732. doi: 10.1038/nchembio.439. PubMed DOI

Cellulase-Hemicellulase Activities and Bacterial Community Composition of Different Soils from Algerian Ecosystems

Bacteria from the endosphere and rhizosphere of Quercus spp. use mainly cell wall-associated enzymes to decompose organic matter

Forest Soil Bacteria: Diversity, Involvement in Ecosystem Processes, and Response to Global Change