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Download (PDF) Supplemental material Cell-density dependent anammox activity of Candidatus Brocadia sinica regulated by N-acyl homoserine lactone-mediated quorum sensing Mamoru Oshiki, Haruna Hiraizumi, Hisashi Satoh, and Satoshi Okabe* Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North-13, West-8, Sapporo, Hokkaido 060-8628, Japan. *Corresponding author: Satoshi OKABE ([email protected]) Division of Environmental Engineering, Faculty of Engineering, Hokkaido University North-13, West-8, Sapporo, Hokkaido 060-8628, Japan. Tel&Fax: (+81)-011-706-6266 This file contains 6 figures, and 1 table. 1 1 Supplementary Figure a) b) Fig. S1 (Oshiki et al.) Fig. S1. Microscopic examination of planktonic (panel a) and dispersed B. sinica (panel b) biomass. a) Planktonic cells were collected from a membrane bioreactor, and hybridized with both TRITC-labeled amx820 (red) and FITC-labeled EUB mix (green) oligonucleotide probes for all the anammox bacteria and most members of the eubacteria, respectively. Scale bar = 25 µm. b) Granular biomass was dispersed into small aggregated biomass using a glass tissue homogenizer, and the cells were stained with 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI). As shown in an imposed image, the small aggregated biomass was mainly composed of the microcolony of B. sinica as examined by fluorescence in-situ hybridization using the amx820 and EUB mix oligonucleotide probes. Scale bar = 100 and 25 µm for the main image and imposed image, respectively. 80 70 60 ) -1 h 50 -1 40 gas production rate 2 N 30 (amol cell 14-15 20 10 Specific 0 106 107 108 109 1010 1011 Cell density (cells mL-1) Fig. S2 (Oshiki et al.) 14-15 Fig. S2. Cell-density dependent activity of N2 gas production examined using a dispersed granular biomass. Granular biomass of B. sinica was dispersed using a glass tissue homogenizer, and serially diluted to be cell density of 106 – 1011 cells mL-1. The culture was anoxically incubated 15 + 14 - 14-15 with addition of 2.5 mM NH4 and NO2 . Production of N2 gas which is specifically produced from the anammox process was examined by gas chromatography mass spectrometry (GC/MS). Error bar represents the range of standard deviation derived from triplicate vials. 100 Without penicillin G 80 With penicillin G ) -1 h -1 60 gas production rate 2 40 N (amol cell 14-15 20 ND ND 0 Specific 8.7 × 106 8.7 × 107 8.7 × 108 Cell density (cells mL-1) Fig. S3 (Oshiki et al.) 14-15 Fig. S3. Influence of penicillin G addition to N2 gas production rate of B. sinica. Planktonic B. sinica cells were serially diluted to be cell density of 8.7 ´ 106 – 108 cells mL-1, and anoxically 15 + 14 - incubated with addition of 2.5 mM NH4 and NO2 . Penicillin G was added at the final concentration of 500 µg mL-1 to inhibit activity of heterotrophic denitrifier. Error bar represents the range of standard deviation derived from triplicate vials. ND; not detected. Desulfacinum infernum DSM 9756 (SHF61659.1) panel a) Desulfacinum infernum (WP 143156457.1) Desulfacinum hydrothermale DSM 13146 (SMC24514.1) Desulfacinum hydrothermale (WP 139796582.1) Syntrophobacter fumaroxidans (WP 011698453.1) Syntrophobacteraceae bacterium (HAA02343.1) Desulfobacteraceae bacterium (PNV87514.1) Deltaproteobacteria bacterium (RPJ09486.1) Deltaproteobacteria bacterium SM23 61 (KPK91470.1) Deltaproteobacteria bacterium (RPJ42197.1) Deltaproteobacteria bacterium (RLB82582.1) Desulfobacterales bacterium S7086C20 (OEU44390.1) Syntrophobacterales bacterium (TFG43892.1) Desulfatitalea sp BRH c12 (KJS33672.1) Desulfobacteraceae bacterium 4484 190.3 (OPX40766.1) Deltaproteobacteria bacterium HGW-Deltaproteobacteria-12 (PKN69762.1) Deltaproteobacteria bacterium HGW-Deltaproteobacteria-9 (PKN05184.1) Desulfuromonas sp (PLY07421.1) Desulfuromonas sp (PLX75823.1) Desulfuromonas sp DDH964 (WP 066724894.1) Desulfuromonas sp (PLX86277.1) Desulfuromonas sp (PLX81189.1) Deltaproteobacteria bacterium (RMF47584.1) Bacterium 311FMe.002 (TLN02637.1) Geobacter sp DSM 2909 (WP 108100805.1) Geobacter sp (RII28541.1) Geobacter daltonii (WP 012648861.1) Geobacter sp. (WP 135868902.1) Geobacter sp (TSK07283.1) Geobacter sp S62 (WP 136524105.1) Deltaproteobacteria bacterium (PXF54342.1) Dissulfuribacter thermophilus (WP 067615611.1) Desulfobulbaceae bacterium (TBV81431.1) Deltaproteobacteria bacterium RIFOXYD12 FULL 56 24 (OGQ86836.1) Deltaproteobacteria bacterium RIFOXYD12 FULL 53 23 (OGR03181.1) Deltaproteobacteria bacterium (RLA81943.1) Deltaproteobacteria bacterium GWB2 55 19 (OGP25471.1) Deltaproteobacteria bacterium GWA2 54 12 (OGP14868.1) Deltaproteobacteria bacterium GWA2 55 10 (OGP15796.1) Deltaproteobacteria bacterium GWA2 55 82 (OGP16026.1) Deltaproteobacteria bacterium (HBG47212.1) Nitrospinae bacterium RIFCSPLOWO2 12 39 16 (OGW07665.1) Nitrospinae bacterium (HAP67921.1) Nitrospinae bacterium RIFCSPHIGHO2 02 39 11 (OGV97554.1) Nitrospina sp (TDJ58734.1) Nitrospinae bacterium RIFCSPLOWO2 12 FULL 47 7 (OGW26394.1) Nitrospina sp (HCG72090.1) Nitrospinae bacterium CG22 combo CG10-13 8 21 14 all 47 10 (PIP74194.1) Nitrospina sp (HCK68969.1) candidate division WOR-1 bacterium RIFOXYA2 FULL 37 7 (OGC16123.1) candidate division WOR-1 bacterium RIFOXYA2 FULL 36 21 (OGC06874.1) candidate division WOR-1 bacterium RIFCSPHIGHO2 01 FULL 53 15 (OGB89293.1) Candidatus Saganbacteria bacterium CG08 land 8 20 14 0 20 45 16 (PIS30258.1) candidate division WOR-1 bacterium RIFCSPHIGHO2 02 FULL 45 12 (OGB88681.1) Geovibrio sp L21-Ace-BES (WP 022849983.1) Candidatus Margulisbacteria bacterium GWD2 39 127 (OGH95216.1) Rickettsiales bacterium (MAH80481.1) Planctomycetes bacterium RIFCSPHIGHO2 02 FULL 40 12 (OHB90678.1) Planctomycetes bacterium GWA2 40 7 (OHB34627.1) Candiadtus Scalindua japonica (WP 096895452.1) Candidatus Brocadiaceae bacterium S225 (WP 034401907.1) Candidatus Scalindua rubra (ODS33399.1) Candidatus Scalindua sp SCAELEC01 (RZV90178.1) Planctomycetes bacterium GWA2 50 13 (OHB37071.1) Planctomycetes bacterium RIFCSPHIGHO2 02 FULL 52 58 (OHB88847.1) Candidatus Jettenia caeni (WP 007223357.1) Candidatus Jettenia ecosi (TLD40608.1) Planctomycetes bacterium RIFCSPHIGHO2 12 39 6 (OHC03869.1) Planctomycetes bacterium RIFCSPHIGHO2 02 FULL 38 41 (OHB83252.1) Planctomycetes bacterium GWA2 39 15 (OHB35085.1) Candidatus Kuenenia stuttgartiensis (WP 099325574.1) Candidatus Kuenenia stuttgartiensis (CAJ71509.1) Planctomycetes bacterium RBG 16 41 13 (OHB74022.1) Candidatus Kuenenia stuttgartiensis (TVM02158.1) Candidatus Brocadia sinica the BROSI A2810 gene Planctomycetes bacterium GWB2 41 19 (OHB40458.1) Planctomycetes bacterium RIFOXYB12 FULL 42 10 (OHC17127.1) Planctomycetes bacterium GWB2 41 19 (OHB42320.1) Candidatus Brocadia caroliniensis (OOP57939.1) Candidatus Brocadia sp WS118 (TVM03687.1) Candidatus Brocadia fulgida (KKO18383.1) Candidatus Brocadia sp UTAMX2 (OQZ01679.1) Candidatus Brocadia sapporoensis (WP 070066996.1) Candidatus Brocadia sp BL1 (TVL95132.1) Candidatus Brocadia sp UTAMX1 (OQZ02920.1) Candidatus Brocadiaceae bacterium B188 (TWU52472.1) 0.20 Candidatus Brocadiaceae bacterium B188 (WP 146326139.1) Fig. S4a (Oshiki et al.) Streptomyces sp NRRL F-525 (WP 033278240.1) panel b) Streptomyces xylophagus (WP 043668500.1) Achromobacter sp SLBN-14 (WP 142149975.1) Streptomyces sp NL15-2K (WP 124444230.1) Streptomyces sp DI166 (WP 093719214.1) Streptomyces aureus (WP 037616639.1) Streptomyces mangrovisoli (WP 046587529.1) Streptomyces (WP 095856002.1) Streptomyces olivochromogenes (WP 067378728.1) Streptomyces (WP 054228974.1) Streptomyces (WP 099940105.1) Streptomyces sp OK885 (WP 101407840.1) Streptomyces sp 94 (WP 099924060.1) Streptomyces mirabilis (WP 075025340.1) Streptomyces sp F001 (WP 129796613.1) Streptomyces avermitilis (WP 010988506.1) Streptomyces sp NRRL S-475 (WP 030835407.1) Streptomyces sp Ag82 G6-1 (WP 097215324.1) Streptomyces sp NRRL S-146 (WP 031107896.1) Streptomyces sp SLBN-192 (WP 142165253.1) Streptomyces (WP 031137082.1) Streptomyces (WP 104781978.1) Streptomyces sp A244 (WP 107457329.1) Streptomyces sp XY006 (WP 094054303.1) Streptomyces iakyrus (WP 033307244.1) Streptomyces (WP 045562358.1) Streptomyces sp WAC 01325 (WP 126903024.1) Streptomyces chartreusis (WP 107912054.1) Streptomyces sp KS 5 (WP 093867157.1) Streptomyces (WP 030957722.1) Streptomyces sp CNH189 (WP 024884920.1) Streptomyces phaeochromogenes (WP 055617089.1) Streptomyces sp IB201691-2A2 (WP 143642970.1) Streptomyces sp NRRL F-2580 (WP 030711540.1) Streptomyces (WP 031121837.1) Streptomyces vinaceus (WP 043247476.1) Streptomyces (WP 030118858.1) Streptomyces sp M3 (WP 129260977.1) Streptomyces sp ADI93-02 (WP 124278322.1) Streptomyces argenteolus (WP 145811575.1) Streptomyces sp AcH 505 (WP 042000251.1) Streptomyces klenkii (WP 120757240.1) Streptomyces sp MBT76 (WP 079110012.1) Streptomyces roseoverticillatus (WP 078659897.1) Streptosporangium nondiastaticum (WP 106681860.1) Streptomyces sp NRRL F-5755 (KOT95015.1) Streptomyces (WP 030598265.1) Streptomyces rimosus (WP 030652610.1) Streptomyces sp NRRL WC-3701 (KOT32086.1) Streptomyces sp WAC 06725 (WP 125533898.1) Streptomyces rimosus (WP 030371557.1) Streptomyces rimosus (WP 033029997.1) Streptomyces rimosus subsp rimosus (KOT56898.1) Frankia (WP 044887245.1) Frankia sp ACN1ag (WP 055408987.1) Frankia sp CpI1-P (KQM03826.1) Gammaproteobacteria bacterium (TLZ13723.1) Allonocardiopsis opalescens (WP 106246274.1) Thermoanaerobacter ethanolicus (WP 003870115.1) Thermoanaerobacter wiegelii (WP 052877885.1) bacterium 42 11 (KUK14005.1)
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