Genome and transcriptome analysis of rock-dissolving Pseudomonas sp. NLX-4 strain

Abstract

Microbial weathering processes can significantly promote soil properties and reduce rock-to-soil ratio. Some soil-inhabiting bacteria exhibit efficient rock-dissolution abilities by releasing organic acids and other chemical elements from the silicate rocks. However, our understanding of the molecular mechanisms involved during bacterial rock-dissolution is still limited. In this study, we performed silicate rock-dissolution experiments on a Pseudomonas sp. NLX-4 strain isolated from an over-exploited mining site. The results revealed that Pseudomonas sp. NLX-4 strain efficiently accelerates the dissolution of silicate rocks by secreting amino acids, exopolysaccharides, and organic acids. Through employing genome and transcriptome sequencing (RNA-seq), we identified the major regulatory genes. Specifically, 15 differentially expressed genes (DEGs) encoding for siderophore transport, EPS and amino acids synthesis, organic acids metabolism, and bacterial resistance to adverse environmental conditions were highly up-regulated in silicate rock cultures of NLX-4 strain. Our study reports a potential bacterial based approach for improving the ecological restoration of over-exploited rock mining sites.

Keywords

Rock mining / Ecological restoration / Silicate rock-dissolution / Pseudomonas sp. NLX-4 strain / Genome sequencing / Transcriptome sequencing

References

[1] Alahari A, Apte SK. A novel potassium deficiency-induced stimulon in Anabaenatorulosa. J Biosci, 2004, 29(2): 153-162.

[2] Aminov RI, . Development, validation, and application of PCR primers for detection of tetracycline efflux genes of Gram-negative bacteria. Appl Environ Microbiol, 2002, 68(4): 1786.

[3] Andrés P, Mateos E. Soil mesofaunal responses to post-mining restoration treatments. Appl Soil Ecol, 2006, 33(1): 67-78.

[4] Arancon NQ, Edwards CA, Atiyeh R, Metzger JD. Effects of vermicomposts produced from food waste on the growth and yields of greenhouse peppers. Biores Technol, 2004, 93(2): 139-144.

[5] Araujo WL, Nunes-Nesi A, Nikoloski Z, Sweetlove LJ, Fernie AR. Metabolic control and regulation of the tricarboxylic acid cycle in photosynthetic and heterotrophic plant tissues. Plant Cell Environ, 2012, 35(1): 1-21.

[6] Audic S, Claverie J-M. The significance of digital gene expression profiles. Genome Res, 1997, 7(10): 986-995.

[7] Balland-Bolou-Bi C, Poszwa A. Effect of calco-magnesian amendment on the mineral weathering abilities of bacterial communities in acidic and silicate-rich soils. Soil Biol Biochem, 2012, 50: 108-117.

[8] Bano N, Musarrat J. Characterization of a new Pseudomonasaeruginosa strain NJ-15 as a potential biocontrol agent. Curr Microbiol, 2003, 46(5): 0324-0328.

[9] Bascos NA. 2008. The requirement of J-domain structural rigidity for cochaperone-mediated allostery in the DnaJ-DnaK molecular chaperone machine. Dissertations and Thesis—Gradworks.

[10] Beckett GJ, Hayes JD. Glutathione S-transferases: biomedical applications. Adv Clin Chem, 1993, 30(1): 281-380.

[11] Boetzer M, Pirovano W. SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information. BMC Bioinformatics, 2014, 15(1): 211.

[12] Braissant O, Cailleau G, Dupraz C, Verrecchia EP. Bacterially induced mineralization of calcium carbonate in terrestrial environments: the role of exopolysaccharides and amino acids. J Sediment Res, 2003, 73(3): 485-490.

[13] Bric JM, Bostock RM, Silverstone SE. Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol, 1991, 57(2): 535-538.

[14] Carrondo MA. Ferritins, iron uptake and storage from the bacterioferritin viewpoint. EMBO J, 2003, 22(9): 1959-1968.

[15] Chen ZH, Schaap P. The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium. Nature, 2012, 488(7413): 680-683.

[16] Chen S, Lian B, Liu C. Effect of Bacillusmucilaginosus on weathering of phosphorite and a preliminary analysis of bacterial proteins. Chin J Geochem, 2008, 27(2): 209-216.

[17] Comino JR, . Quantitative comparison of initial soil erosion processes and runoff generation in Spanish and German vineyards. Sci Total Environ, 2016, 565: 1165-1174.

[18] Crosa JH, Walsh CT. Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev, 2002, 66(2): 223-249.

[19] Deng J, Peng S, Wang L, Bi Y, Yao J, Wang Q. Deng J, Peng S, Wang L, Bi Y, Yao J, Wang Q. Comprehensive utilization of resources. Interpretation of Green Mine Evaluation Index, 2022, Singapore: Springer, 139-180.

[20] Erkossa T, Wudneh A, Desalegn B, Taye G. Linking soil erosion to on-site financial cost: lessons from watersheds in the Blue Nile basin. Solid Earth, 2015, 6(2): 765.

[21] Fersht AR, Matouschek A, Serrano L. The folding of an enzyme: I. Theory of protein engineering analysis of stability and pathway of protein folding. J Mol Biol, 1992, 224(3): 771-782.

[22] Finlay R, . The role of fungi in biogenic weathering in boreal forest soils. Fungal Biol Rev, 2009, 23(4): 101-106.

[23] Gentry TJ, Fuhrmann JJ, Zuberer DA. Principles and applications of soil microbiology, 2021, Amsterdam: Elsevier.

[24] Gleeson DB, . Characterization of bacterial community structure on a weathered pegmatitic granite. Microb Ecol, 2006, 51(4): 526-534.

[25] Gómez-Lozano M, Marvig RL, Molin S, Long KS. Genome-wide identification of novel small RNAs in Pseudomonas aeruginosa. Environ Microbiol, 2012, 14(8): 2006-2016.

[26] Gupta A, Meyer JM, Goel R. Development of heavy metal-resistant mutants of phosphate solubilizing Pseudomonas sp. NBRI 4014 and their characterization. Curr Microbiol, 2002, 45(5): 323-327.

[27] Hesse E, . Ecological selection of siderophore-producing microbial taxa in response to heavy metal contamination. bioRxiv, 2017

[28] Jiang H, Song W, Li A, Yang X, Sun D. Identification of genes differentially expressed in cauliflower associated with resistance to Xanthomonascampestris pv. campestris. Mol Biol Rep, 2011, 38(1): 621-629.

[29] Kanicky V, Mermet J-M. Selection of internal standards for the determination of major and minor elements in silicate rocks and limestones by laser ablation inductively coupled plasma atomic emission spectrometry. Appl Spectrosc, 1997, 51(3): 332-336.

[30] Kawahara Y, . Simultaneous RNA-seq analysis of a mixed transcriptome of rice and blast fungus interaction. PLoS ONE, 2012, 7(11

[31] Kobayashi K, . Single-cell memory regulates a neural circuit for sensory behavior. Cell Rep, 2016, 14(1): 11-21.

[32] Li R, . SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics, 2009, 25(15): 1966-1967.

[33] Li R, . De novo assembly of human genomes with massively parallel short read sequencing. Genome Res, 2010, 20(2): 265-272.

[34] Lian B, Wang B, Pan M, Liu C, Teng HH. Microbial release of potassium from K-bearing minerals by thermophilic fungus Aspergillusfumigatus. Geochim Cosmochim Acta, 2008, 72(1): 87-98.

[35] Litvinova L, . Nitric oxide and mitochondria in metabolic syndrome. Front Physiol, 2015, 6: 20.

[36] Liu H, Liu X, Li X, Ziyu Fu, Lian B. The molecular regulatory mechanisms of the bacteria involved in serpentine weathering coupled with carbonation. Chem Geol, 2021, 565.

[37] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 2001, 25(4): 402-408.

[38] Maleki F, Khosravi A, Nasser A, Taghinejad H, Azizian M. Bacterial heat shock protein activity. J Clin Diagn Res, 2016, 10(3): BE01.

[39] Maurice PA, Vierkorn MA, Hersman LE, Fulghum JE, Ferryman A. Enhancement of kaolinite dissolution by an aerobic Pseudomonas mendocina bacterium. Geomicrobiol J, 2001, 18(1): 21-35.

[40] Meyer ML, Bloom PR. Lithium metaborate fusion for silicon, calcium, magnesium, and potassium analysis of wild rice. Plant Soil, 1993, 153(2): 281-285.

[41] Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods, 2008, 5(7): 621-628.

[42] Neilands J. Siderophores: structure and function of microbial iron transport compounds. J Biol Chem, 1995, 270(45): 26723-26726.

[43] Neubauer U, Furrer G, Schulin R. Heavy metal sorption on soil minerals affected by the siderophore desferrioxamine B: the role of Fe (III)(hydr) oxides and dissolved Fe (III). Eur J Soil Sci, 2002, 53(1): 45-55.

[44] Ochoa-Cueva P, Fries A, Montesinos P, Rodríguez-Díaz JA, Boll J. Spatial estimation of soil erosion risk by land-cover change in the Andes of Southern Ecuador. Land Degrad Dev, 2015, 26(6): 565-573.

[45] Pan H, Bao W, Xie Z, Zhang J, Li Y. Immobilization of -epoxysuccinate hydrolase activity for d(−)-tartaric acid production. Biotech Lett, 2010, 32(2): 235-241.

[46] Picard L, Turpault MP, Oger PM, Uroz S. Identification of a novel type of glucose dehydrogenase involved in the mineral weathering ability of Collimonaspratensis strain PMB3 (1). FEMS Microbiol Ecol, 2021, 97(1): 232.

[47] Rajkumar M, Nagendran R, Lee KJ, Lee WH. Characterization of a novel Cr6+ reducing Pseudomonas sp. with plant growth–promoting potential. Curr Microbiol, 2005, 50(5): 266-271.

[48] Sahu G, Sindhu S. Disease control and plant growth promotion of green gram by siderophore producing Pseudomonas sp. Res J Microbiol, 2011, 6(10): 735.

[49] Sambrook J, Russell DW. Standard ethanol precipitation of DNA in microcentrifuge tubes. Cold Spring Harb Protoc, 2006, 2006(1): pdb. prot4456.

[50] Slater GSC, Birney E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics, 2005, 6(1): 31.

[51] Sohpal VK, Dey A, Singh A. MEGA biocentric software for sequence and phylogenetic analysis: a review. Int J Bioinform Res Appl, 2010, 6(3): 230-240.

[52] Spaepen S, Vanderleyden J, Remans R. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev, 2007, 31(4): 425-448.

[53] Sun LL, . Differences in the gene expressive quantities of carbonic anhydrase and cysteine synthase in the weathering of potassium-bearing minerals by Aspergillusniger. Sci China Earth Sci, 2013, 56(12): 2135-2140.

[54] Sun W, Ji B, Khoso SA, Tang H, Liu R, Wang L, Hu Y. An extensive review on restoration technologies for mining tailings. Environ Sci Pollut Res, 2018, 25(34): 33911-33925.

[55] Tourney J, Ngwenya BT. The role of bacterial extracellular polymeric substances in geomicrobiology. Chem Geol, 2014, 386: 115-132.

[56] Tribelli PM, . Novel essential role of ethanol oxidation genes at low temperature revealed by transcriptome analysis in the Antarctic bacterium Pseudomonasextremaustralis. PLoS ONE, 2015, 10(12

[57] Vandevivere P, Welch S, Ullman W, Kirchman D. Enhanced dissolution of silicate minerals by bacteria at near-neutral pH. Microb Ecol, 1994, 27(3): 241-251.

[58] Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet, 2009, 10(1): 57-63.

[59] Wang W, Lian B, Pan L. An RNA-sequencing study of the genes and metabolic pathways involved in Aspergillusniger weathering of potassium feldspar. Geomicrobiol J, 2015, 32(8): 689-700.

[60] Wang Y-L, Sun L-J, Xian C-M, Kou F-L, Zhu Y, He L-Y, Sheng X-F. Interactions between biotite and the mineral-weathering bacterium Pseudomonasazotoformans F77. Appl Environ Microbiol, 2020, 86(7): e02568-e2619.

[61] Wang YL, Dong W, Xiang KX, Wang Q, He LY, Sheng XF. A combination of genomics, transcriptomics, and genetics provides insights into the mineral weathering phenotype of Pseudomonasazotoformans F77. Appl Environ Microbiol, 2021, 87(24): e01552-e1621.

[62] Wang P, Niu Y, Sun P, Wang X, Guo P, Gong H, Duan M, Shen F, Shi Y, Xue S, Chen Y. Iron isotope compositions of coexisting sulfide and silicate minerals in sudbury-type ores from the Jinchuan Ni-Cu sulfide deposit: a perspective on possible core-mantle iron isotope fractionation. Minerals, 2021, 11(5): 464.

[63] Welch SA, Vandevivere P. Effect of microbial and other naturally occurring polymers on mineral dissolution. Geomicrobiol J, 1994, 12(4): 227-238.

[64] Wu Y-W, Zhang J-C, Wang L-J, Wang Y-X. A rock-weathering bacteria isolated from rock surface and its role in ecological restoration on exposed carbonate rocks. Ecol Eng, 2017, 101: 162-169.

[65] Wu Y, Zhang J, Guo X. An indigenous soil bacterium facilitates the mitigation of rocky desertification in carbonate mining areas. Land Degrad Develop, 2017

[66] Wu Y, Zhang J, Guo X, Wang Y, Wang Q. Isolation and characterisation of a rock solubilising fungus for application in mine-spoil reclamation. Eur J Soil Biol, 2017, 81: 76-82.

[67] Wu B, . The performance of biochar-microbe multiple biochemical material on bioremediation and soil micro-ecology in the cadmium aged soil. Sci Total Environ, 2019, 686: 719-728.

[68] Wyman SK, Jansen RK, Boore JL. Automatic annotation of organellar genomes with DOGMA. Bioinformatics, 2004, 20(17): 3252-3255.

[69] Xiao B, Lian B, Shao W. Do bacterial secreted proteins play a role in the weathering of potassium-bearing rock powder?. Geomicrobiol J, 2012, 29(6): 497-505.

[70] Xiao B, Lian B, Sun L, Shao W. Gene transcription response to weathering of K-bearing minerals by Aspergillusfumigatus. Chem Geol, 2012, 306–307(1): 1-9.

[71] Xiao B, Lian B, Sun L, Shao W. Gene transcription response to weathering of K-bearing minerals by Aspergillusfumigatus. Chem Geol, 2012, 306: 1-9.

[72] Zhang XQ, . Genome-wide analysis of DNA methylation in rat lungs with lipopolysaccharide-induced acute lung injury. Mol Med Rep, 2013, 7(5): 1417-1424.

[73] Zhou J, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. Appl Environ Microbiol, 1996, 62(2): 316-322.

Funding

National Special Fund for Forestry Scientific Research in the Public Interest of China(201504406)

Jiangsu Agriculture Science and Technology Innovation Fund(CX (17) 1004)

Canada Ontario Research Chair Funding

Ontario Trillium Foundation