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Exploring the Interactions and the Metabolome of the Soil

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1 Supplementary Material 2 Do size and shape matter? Exploring the interactions and the 3 metabolome of the soil isolate Hylemonella gracilis 4 5 Olaf Tyc1,2*, Purva Kulkarni1,3, Adam Ossowicki1, Vittorio Tracanna4, Marnix H. 6 Medema4, Peter van Baarlen5, W.F.J. van Ijcken6, Koen J. F. Verhoeven1 and Paolina 7 Garbeva1, 7 8 9 1Netherlands Institute of Ecology (NIOO-KNAW), Department of Microbial Ecology, PO 10 BOX 50, 6700 AB Wageningen, Netherlands 11 12 2Goethe University, Department of Internal Medicine I, University Hospital Frankfurt, 13 Theodor-Stern-Kai 7, 60590 Frankfurt, Germany 14 15 3Translational Metabolic Laboratory, Radboud University Medical Center, Geert Grooteplein 16 zuid 10, 6525 GA Nijmegen, Netherlands 17 18 4Wageningen University, Department of Plant Sciences, Bioinformatics Group, 19 Droevendaalsesteeg 1, 6708 PB Wageningen, Netherlands 20 21 5Wageningen University, Department of Animal Sciences, Host-Microbe Interactomics, 22 Postbus 338, 6700 WD Wageningen, Netherlands 23 24 6Center for Biomics, Erasmus University Medical Center, Building Ee, Room Ee6.79b 25 Wytemaweg 80, 3015 CN Rotterdam Rotterdam, Netherlands 26 27 7Department of Plant and Environmental Sciences, Faculty of Natural and Life Sciences, 28 University of Copenhagen, Denmark 29 30 Correspondence: 31 Goethe University, Department of Internal Medicine I, University Hospital Frankfurt, 32 Theodor-Stern-Kai 7, 60590 Frankfurt, Germany, E-Mail: [email protected] 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Supplementary Figure 1: Schematic overview of the isolation method used to isolate H. 50 gracilis from soil. 51 52 53 Supplementary Figure 2: Box plots for the significantly differentially abundant metabolites 54 and their corresponding ion intensity maps found using Mass spectrometry imaging (MSI) 55 during the interaction of H. gracilis with Paenibacillus and Serratia. 56 57 58 59 Supplementary Figure 3: Volcano plots and Venn diagram to demonstrate metabolite 60 concentration differences and unique/shared metabolites of the LAESI-MSI data. (a) 61 Volcano plot for H. gracilis monoculture (HM) vs. Paenibacillus sp. AD87 - H. gracilis 62 coculture (PH). (b) Volcano plot for Paenibacillus sp. AD87 monoculture (PM) vs. 63 Paenibacillus sp. AD87 - H. gracilis coculture (PH). (c) Volcano plot for H. gracilis 64 monoculture (HM) vs. S. plymuthica PRI-2C - H. gracilis coculture (SH). (d) Volcano plot for 65 S. plymuthica PRI-2C monoculture (SM) vs. S. plymuthica PRI-2C - H. gracilis coculture (SH). 66 Each point in the volcano plot represents one metabolite. Significantly differentially abundant 67 metabolites were calculated with a fold change (FC) threshold of 2 on the x-axis and a t-tests 68 threshold of 0.1 on the y-axis. The red and the green dots indicate statistically significant 69 metabolites. The vertical FC threshold lines indicate an increase or decrease in concentration 70 of metabolites. Negative log2 (FC) values indicated in red represent lower concentrations in 71 native than in range expanding species; positive values indicated in green represent higher 72 concentrations of metabolites in native than in range expanding species. (e) Venn diagram for 73 HM-PM-PH. (f) Venn diagram for HM-SM-SH. To construct the Venn diagram, a single mass 74 feature was considered even if it was present in only one replicate for a specific sample species. 75 76 77 Supplementary Table 1: Bacterial organisms used in this study. Strain / isolate / organism Phylum/class Genbank Reference Hylemonella gracilis isolate NS1 beta-proteobacteria CP0310395 this study Paenibacillus sp. AD87 Firmicutes KJ685299 Tyc et al. 2014 Serratia plymuthica PRI-2C gamma-proteobacteria CP015613 Schmidt et al. 2017 78 79 80 81 Supplementary Table 2: Overview of the transcriptome analysis, number (n) of significantly 82 differentially expressed genes of H. gracilis responding to Serratia plymuthica PRI-2C or to 83 Paenibacillus sp. AD87, Serratia plymuthica PRI-2C responding to H. gracilis and 84 Paenibacillus sp. AD87 responding to H. gracilis at day 5 and day 10. Significantly time differentially expressed Organism Interacting organism point genes Total (n) (d) up- down- regulated regulated 5 25 36 61 Serratia plymuthica PRI-2C 10 10 0 10 Hylemonella gracilis 5 8 0 8 Paenibacillus sp. AD87 10 0 0 0 5 1 0 1 Serratia plymuthica PRI-2C 10 129 53 182 Hylemonella gracilis 5 0 0 0 Paenibacillus sp. AD87 10 4 11 15 Total (n) 177 100 277 85 86 87 88 89 Supplementary Table 3: Significantly differentially expressed genes of Paenibacillus sp. 90 AD87, responding to H. gracilis at day 10. Gene logFC PValue FDRFunction gpAD87_RS06700 -2.9328905 8.66E-15 6.48E-11 rpoE; RNA polymerase sigma-70 factor, ECF subfamily gpAD87_RS13150 -1.8451399 7.78E-05 0.04477567 opuBD; osmoprotectant transport system permease protein gpAD87_RS13150 -1.8451399 7.78E-05 0.04477567 opuC; osmoprotectant transport system substrate-binding protein gpAD87_RS30390 -1.6913798 2.06E-06 0.0030772 tatD; TatD DNase family protein [EC:3.1.21.-] gpAD87_RS19945 -1.2794042 1.17E-05 0.01253836 UXS1; UDP-glucuronate decarboxylase [EC:4.1.1.35] gpAD87_RS00275 -1.2612217 1.63E-05 0.01628367 E2.2.1.2; transaldolase [EC:2.2.1.2] gpAD87_RS19920 -1.2608789 1.78E-05 0.0166361 pimB; phosphatidyl-myo-inositol dimannoside synthase [EC:2.4.1.346] gpAD87_RS00270 -1.2512648 5.28E-05 0.03593459 PGD; 6-phosphogluconate dehydrogenase [EC:1.1.1.44 1.1.1.343] gpAD87_RS19895 -1.1972691 4.98E-06 0.00620873 UGDH; UDPglucose 6-dehydrogenase [EC:1.1.1.22] gpAD87_RS21205 -1.1167672 2.65E-06 0.00360466 N/A gpAD87_RS26695 -0.9979737 3.18E-05 0.02642266 gerKA; spore germination protein KA gpAD87_RS00725 1.0213585 9.92E-06 0.01142133 E3.1.3.15B; histidinol-phosphatase (PHP family) [EC:3.1.3.15] gpAD87_RS17335 1.43673471 9.05E-05 0.0483872 abrB; transcriptional pleiotropic regulator of transition state genes gpAD87_RS10500 1.48682555 6.63E-05 0.04132522 deoC; deoxyribose-phosphate aldolase [EC:4.1.2.4] 91 gpAD87_RS11290 1.69953743 1.47E-06 0.00243757 folD; methylenetetrahydrofolate dehydrogenase (NADP+) 92 93 94 Supplementary Table 4: Significantly differentially expressed genes of Serratia plymuthica 95 PRI-2C responding to H. gracilis at day 5. Gene logFC PValue FDR Function 96 Q5A_025180 1.484670421 4.37298312 1.16E-05 rph; ribonuclease PH [EC:2.7.7.56] 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 Supplementary Table 5: Significantly differentially expressed genes of Serratia plymuthica 119 PRI-2C responding to H. gracilis at day 10. Gene logFC PValue FDR Function Q5A_020525 -3.539216745 3.79E-13 2.98E-10 hcp; type VI secretion system secreted protein Hcp Q5A_020555 -2.840557251 6.03E-06 0.0011281 fimA; major type 1 subunit fimbrin (pilin) Q5A_023300 -2.169738036 6.89E-08 2.10E-05 malK; multiple sugar transport system ATP-binding protein [EC:3.6.3.-] Q5A_011700 -1.997102593 1.42E-05 0.0021876 ynfM; MFS transporter, YNFM family, putative membrane transport protein Q5A_003445 -1.972950006 1.13E-06 0.00024804 garD; galactarate dehydratase [EC:4.2.1.42] Q5A_018430 -1.744985992 9.02E-05 0.00993022 N/A Q5A_009750 -1.734981082 4.74E-05 0.00622171 ftnA; ferritin [EC:1.16.3.2] Q5A_020535 -1.729517104 0.000274679 0.02461418 impB; type VI secretion system protein ImpB Q5A_023295 -1.625377127 2.34E-10 1.17E-07 lamB; maltoporin Q5A_003465 -1.528329707 2.21E-05 0.00327673 garL; 2-dehydro-3-deoxyglucarate aldolase [EC:4.1.2.20] Q5A_003455 -1.527498481 8.21E-05 0.00952465 gudX; glucarate dehydratase-related protein Q5A_003450 -1.467669658 3.67E-06 0.00074239 gudP; MFS transporter, ACS family, glucarate transporter Q5A_010395 -1.445444456 9.84E-06 0.00165079 puuD; gamma-glutamyl-gamma-aminobutyrate hydrolase [EC:3.5.1.94] Q5A_020530 -1.425017256 5.82E-05 0.00719325 impC; type VI secretion system protein ImpC Q5A_002075 -1.344637355 0.000219471 0.02052737 ABC-2.P; ABC-2 type transport system permease protein Q5A_015990 -1.328859825 1.07E-07 3.15E-05 paaC; ring-1,2-phenylacetyl-CoA epoxidase subunit PaaC [EC:1.14.13.149] Q5A_010390 -1.305376856 1.67E-07 4.46E-05 puuR; HTH-type transcriptional regulator, repressor for puuD Q5A_023310 -1.296519929 5.59E-09 1.99E-06 malE; maltose/maltodextrin transport system substrate-binding protein Q5A_005185 -1.29424411 1.11E-07 3.19E-05 cybB; cytochrome b561 Q5A_003460 -1.287052853 0.000331618 0.02833289 gudD; glucarate dehydratase [EC:4.2.1.40] Q5A_015985 -1.157880882 1.10E-05 0.00177766 paaB; ring-1,2-phenylacetyl-CoA epoxidase subunit PaaB Q5A_009735 -1.129234286 2.54E-08 8.63E-06 dksA; DnaK suppressor protein Q5A_016000 -1.118109907 1.34E-05 0.0021073 paaE; ring-1,2-phenylacetyl-CoA epoxidase subunit PaaE Q5A_015980 -1.11728173 4.91E-06 0.00094221 paaA; ring-1,2-phenylacetyl-CoA epoxidase subunit PaaA [EC:1.14.13.149] Q5A_017330 -1.103510732 4.63E-06 0.00091123 katE; catalase [EC:1.11.1.6] Q5A_015995 -1.101590323 7.03E-06 0.00123738 paaD; ring-1,2-phenylacetyl-CoA epoxidase subunit PaaD Q5A_015975 -1.093213554 3.94E-06 0.00078613 paaZ; oxepin-CoA hydrolase / 3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase Q5A_016010 -1.074845013 1.36E-05 0.00212107 paaG; 2-(1,2-epoxy-1,2-dihydrophenyl)acetyl-CoA isomerase [EC:5.3.3.18] Q5A_013825 -1.003817338 0.000406824 0.03308758 osmB; osmotically inducible lipoprotein OsmB Q5A_008760 -0.988582472 0.000254571 0.02322956 hspQ; heat shock protein HspQ Q5A_009380 -0.977583963 0.000538345 0.04089512 bssS; biofilm regulator BssS Q5A_006035 -0.974655768 1.64E-05 0.00249851 acpD; FMN-dependent NADH-azoreductase [EC:1.7.-.-] Q5A_003705 -0.961603947
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    Supplemental Table 7. Every Significant Association

    Supplemental Table 7. Every significant association between an individual covariate and functional group (assigned to the KO level) as determined by CPGLM regression analysis. Variable Unit RelationshipLabel See also CBCL Aggressive Behavior K05914 + CBCL Emotionally Reactive K05914 + CBCL Externalizing Behavior K05914 + K15665 K15658 CBCL Total K05914 + K15660 K16130 KO: E1.13.12.7; photinus-luciferin 4-monooxygenase (ATP-hydrolysing) [EC:1.13.12.7] :: PFAMS: AMP-binding enzyme; CBQ Inhibitory Control K05914 - K12239 K16120 Condensation domain; Methyltransferase domain; Thioesterase domain; AMP-binding enzyme C-terminal domain LEC Family Separation/Social Services K05914 + K16129 K16416 LEC Poverty Related Events K05914 + K16124 LEC Total K05914 + LEC Turmoil K05914 + CBCL Aggressive Behavior K15665 + CBCL Anxious Depressed K15665 + CBCL Emotionally Reactive K15665 + K05914 K15658 CBCL Externalizing Behavior K15665 + K15660 K16130 KO: K15665, ppsB, fenD; fengycin family lipopeptide synthetase B :: PFAMS: Condensation domain; AMP-binding enzyme; CBCL Total K15665 + K12239 K16120 Phosphopantetheine attachment site; AMP-binding enzyme C-terminal domain; Transferase family CBQ Inhibitory Control K15665 - K16129 K16416 LEC Poverty Related Events K15665 + K16124 LEC Total K15665 + LEC Turmoil K15665 + CBCL Aggressive Behavior K11903 + CBCL Anxiety Problems K11903 + CBCL Anxious Depressed K11903 + CBCL Depressive Problems K11903 + LEC Turmoil K11903 + MODS: Type VI secretion system K01220 K01058 CBCL Anxiety Problems K11906 + CBCL Depressive
  • Functional and Physiological Discovery in the Mannonate Dehydratase Subgroup of the Enolase Superfamily

    Functional and Physiological Discovery in the Mannonate Dehydratase Subgroup of the Enolase Superfamily

    FUNCTIONAL AND PHYSIOLOGICAL DISCOVERY IN THE MANNONATE DEHYDRATASE SUBGROUP OF THE ENOLASE SUPERFAMILY BY DANIEL JOSEPH WICHELECKI DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2014 Urbana, Illinois Doctoral Committee: Professor John Gerlt, Chair Professor John Cronan Professor Scott Silverman Professor Wilfred van der Donk ABSTRACT In the current post-genomic world, the exponential amassing of protein sequences is overwhelming the scientific community’s ability to experimentally assign each protein’s function. The use of automated, homology-based annotations has allowed a reprieve from this efflux of data, but has led to widespread misannotation and nonannotation in protein sequence databases. This dissertation details the functional and physiological characterization of the mannonate dehydratase subgroup (ManD) of the enolase superfamily (ENS). The outcome affirms the dangers of homology-based annotations while discovering novel metabolic pathways. Furthermore, the experimental verification of these pathways ( in vitro and in vivo ) has provided a platform to test the general strategies for improved functional and metabolic characterization being developed by the Enzyme Function Initiative (EFI). Prior to this study, one member of the ManD subgroup had been characterized and was shown to dehydrate D-mannonate to 2-keto-3-deoxy-D-gluconate. Forty-two additional members of the ManD, selected from across the sequence space of the subgroup, were screened for activity and kinetic constants were determined. The members of the once isofunctional subgroup were found to differ in both catalytic efficiency and substrate specificity: 1) high 3 4 -1 -1 efficiency (k cat /K M = 10 to 10 M s ) dehydration of D-mannonate, 2) low efficiency (k cat /K M = 10 1 to 10 2 M-1s-1) dehydration of D-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested).
  • Novel Non-Phosphorylative Pathway of Pentose Metabolism from Bacteria

    Novel Non-Phosphorylative Pathway of Pentose Metabolism from Bacteria

    www.nature.com/scientificreports OPEN Novel non-phosphorylative pathway of pentose metabolism from bacteria Received: 22 March 2018 Seiya Watanabe1,2,3, Fumiyasu Fukumori4, Hisashi Nishiwaki1,2, Yasuhiro Sakurai5, Accepted: 30 September 2018 Kunihiko Tajima5 & Yasuo Watanabe1,2 Published: xx xx xxxx Pentoses, including D-xylose, L-arabinose, and D-arabinose, are generally phosphorylated to D-xylulose 5-phosphate in bacteria and fungi. However, in non-phosphorylative pathways analogous to the Entner-Dodorof pathway in bacteria and archaea, such pentoses can be converted to pyruvate and glycolaldehyde (Route I) or α-ketoglutarate (Route II) via a 2-keto-3-deoxypentonate (KDP) intermediate. Putative gene clusters related to these metabolic pathways were identifed on the genome of Herbaspirillum huttiense IAM 15032 using a bioinformatic analysis. The biochemical characterization of C785_RS13685, one of the components encoded to D-arabinonate dehydratase, difered from the known acid-sugar dehydratases. The biochemical characterization of the remaining components and a genetic expression analysis revealed that D- and L-KDP were converted not only to α-ketoglutarate, but also pyruvate and glycolate through the participation of dehydrogenase and hydrolase (Route III). Further analyses revealed that the Route II pathway of D-arabinose metabolism was not evolutionally related to the analogous pathway from archaea. Te breakdown of D-glucose is central for energy and biosynthetic metabolism throughout all domains of life. Te most common glycolytic routes in bacteria are the Embden-Meyerhof-Parnas, the Entner-Doudorof (ED), and the oxidative pentose phosphate pathways. Te distinguishing diference between the two former glyco- lytic pathways lies in the nature of the 6-carbon metabolic intermediates that serve as substrates for aldol cleav- age.
  • Index of Recommended Enzyme Names

    Index of Recommended Enzyme Names

    Index of Recommended Enzyme Names EC-No. Recommended Name Page 1.2.1.10 acetaldehyde dehydrogenase (acetylating) 115 1.2.1.38 N-acetyl-y-glutamyl-phosphate reductase 289 1.2.1.3 aldehyde dehydrogenase (NAD+) 32 1.2.1.4 aldehyde dehydrogenase (NADP+) 63 1.2.99.3 aldehyde dehydrogenase (pyrroloquinoline-quinone) 578 1.2.1.5 aldehyde dehydrogenase [NAD(P)+] 72 1.2.3.1 aldehyde oxidase 425 1.2.1.31 L-aminoadipate-semialdehyde dehydrogenase 262 1.2.1.19 aminobutyraldehyde dehydrogenase 195 1.2.1.32 aminomuconate-semialdehyde dehydrogenase 271 1.2.1.29 aryl-aldehyde dehydrogenase 255 1.2.1.30 aryl-aldehyde dehydrogenase (NADP+) 257 1.2.3.9 aryl-aldehyde oxidase 471 1.2.1.11 aspartate-semialdehyde dehydrogenase 125 1.2.1.6 benzaldehyde dehydrogenase (deleted) 88 1.2.1.28 benzaldehyde dehydrogenase (NAD+) 246 1.2.1.7 benzaldehyde dehydrogenase (NADP+) 89 1.2.1.8 betaine-aldehyde dehydrogenase 94 1.2.1.57 butanal dehydrogenase 372 1.2.99.2 carbon-monoxide dehydrogenase 564 1.2.3.10 carbon-monoxide oxidase 475 1.2.2.4 carbon-monoxide oxygenase (cytochrome b-561) 422 1.2.1.45 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase .... 323 1.2.99.6 carboxylate reductase 598 1.2.1.60 5-carboxymethyl-2-hydroxymuconic-semialdehyde dehydrogenase . 383 1.2.1.44 cinnamoyl-CoA reductase 316 1.2.1.68 coniferyl-aldehyde dehydrogenase 405 1.2.1.33 (R)-dehydropantoate dehydrogenase 278 1.2.1.26 2,5-dioxovalerate dehydrogenase 239 1.2.1.69 fluoroacetaldehyde dehydrogenase 408 1.2.1.46 formaldehyde dehydrogenase 328 1.2.1.1 formaldehyde dehydrogenase (glutathione)