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Supporting information Figure S1. A field survey was conducted from Dauphin Island to West Point Island, AL, at five sites in the order indicated. Oil and paired visibly contaminated and uncontaminated sand samples were collected representative of ball and sheet geometries. Location 4 had highest visible oil contamination [1]. 1 Figure S2 Representative chromatograms of field samples collected following the Deepwater Horizon oil spill obtained by GC-MS. a: Chromatogram of sample from an oil sheet, b: Chromatogram of sample from a ball. 2 Figure S3. Comparison of composition of oil samples from sheet and ball geometries determined by GC/MS. Bars are plotted as average and standard deviation of each fraction. a) Comparison 3 of six field-collected sheet and tar ball geometries. The amount of hydrocarbons detected were normalized to the mass of the sample of oil deposit and associated material from the field. No significant difference in oil composition was found between the two geometries according to a Hotelling’s t-squared test. b) GC MS analysis of the oil deposits from the duplicate sand columns after sacrifice. 4 Figure S4. Hierarchical cluster of the sub-samples with depth from blank column core. Depths correspond to those sub-sampled from oiled columns. Red dashed lines indicate that the difference between the clustered samples is not significant. y-axis is indicative of Bray-Curtis similarity at which a cluster is formed. Hierarchical clustering was conducted using ‘Group average’ method [2]. 5 Figure S5. Hierarchical cluster of the samples from the sheet column. Depths correspond to those sub-sampled from oiled columns. Red dashed lines indicate that the difference between the clustered samples is not significant. Y-axis is indicative of Bray-Curtis similarity at which a cluster is formed. Hierarchical clustering was conducted using ‘Group average’ method [2]. 6 Figure S6. Hierarchical cluster of the samples from the duplicate ball columns. Depths correspond to those sub-sampled from oiled columns. Red dashed lines indicate that the difference between the clustered samples is not significant. Y-axis is indicative of Bray-Curtis similarity at which a cluster is formed. Hierarchical clustering was conducted using ‘Group average’ method [2]. 7 Figure S7. 16S rRNA gene copies per gram of sub-sample of column cores with depth. Data for ball columns is average of duplicate columns. Error bars represent the standard deviation of triplicate qPCR measurements of each sample. 8 Table S1. Primer sequences and annealing temperatures employed for PCR and qPCR assays. Target Annealing Reference Primer a Primer (5’-3’) gene Temp. ( °C) 8F AGAGTTTGATCCTGGCTCAG [3] 16S rRNA b 50 1492R GGTTACCTTGTTACGACTT CGCCCGCCGCGCGCGGCGGGCGG I-341 F GC V3 16S GGCGGGGGCACGGGGGGCCTAC [4] 47 rRNA GGGAGGCAGCAG I-533 R TIACCGIIICTICTGGCAC CGGCGTTGCGCATTTYCAYACVV dsrA_290F T [5] dsrA 60 GCCGGACGATGCAGHTCRTCCTG dsrA_660R RWA GGTGGTGTMGGATTCACACARTA mcrA_1035F [5] mcrA 56 YGCWACAGC mcrA_1530R TTCATTGCRTAGTTWGGRTAGTT cat23 F CGACCTGATCTCCATGACCGA [6] cat23 66 cat23 R TCAGGTCAGCACGGTCA 1369F CGACCTGATCTCCATGACCGA [7] 16S rRNA b 60 1492R TCAGGTCAGCACGGTCA a Integrated DNA Technologies Inc (Coralville, IA.) b Primer set 8F and 1492R was used for PCR applications, whereas primer set 1369F and 1492R was used for qPCR. c Touchdown PCR was done for DGGE applications using primer set I-341 F GC and I-533 R. The annealing temperature for first two cycles was 52 °C and was brought down to 47 °C, reducing by 1 °C after every two cycles. 9 Table S2. PCR reaction matrix a Reagent Amount or concentration in 25 µµµl master mix 10 ××× PCR buffer d 2.5 µl 5××× PCR buffer d 5 µl dNTPs e 0.2 mM Forward and reverse primer a 0.25 µM Taq DNA polymerase f 1.75 U Formamide g 0.25 µl DNA template 1 µl Mg 2+ d 2mM a Integrated DNA Technologies Inc (Coralville, IA.) d MasterTaq kit (Eppendorf, Westbury, NY) was used for PCR assays. e (Promega Corporation, Madison, WI) f TAQ DNA POL (5U/ µl) (MP Biomedicals, Solon, OH) g Formamide Mol Bio Grade (Fisher Scientific Company LLC, Suwanee, GA.) 10 Table S3. qPCR reaction matrix Reagent Concentration in 20 µµµl master mix Forward and reverse primer a 0.3 µM SsoFast EvaGreen Supermix 1× iTaq h 0.1 µl a Integrated DNA Technologies Inc (Coralville, IA.) h (Bio-Rad, Hercules, CA); iTaq was only used for amplifying gene specific to alpha subunit dissimilatory sulfite reductase using dsrA_ 290F and dsrA_ 660R primer set. a All standard curves of qPCR were constructed from serial dilutions of cloned positive controls ranging from 10 8 to 10 2 gene copies per µL. Potential effects of inhibitors were assessed by serially diluting select samples and comparing PCR efficiencies with that of standards. It was found that a dilution of 1:50 effectively minimized inhibitory effects and was applied across samples. Samples were analyzed in triplicate with a standard curve and negative control included in each run. 11 Tabel S4. Clonal sequences of the qPCR product. mcr A Highest Match (GenBank Closest cultured relative (GenBank accession accession number) number) Uncultured Methanococcus Methanococcus maripaludis X1 (CP002913) sp. (AY454745) Methanococcus Methanococcus maripaludis strain S2 (BX950229) maripaludis (AF298223) Uncultured methanogenic Methanothermococcus okinawensis strain DSM archaeon clone 14208 (AY354033) (HQ635384) Methanobacterium Methanobacterium aarhusense methyl-coenzyme M aarhusense (AY386125) reductase alpha subunit ( mcr A) gene, partial cds.(AY386125) Uncultured methanogenic Methanothermococcus thermolithotrophicus mcrA archaeon clone gene for methyl CoM reductase subunit alpha, (AY354026) partial cds. (AB353226) Uncultured archaeon clone Methanobrevibacter smithii ATCC (GQ120965) 35061(CP000678) Uncultured methanogenic Methanosphaera stadtmanae DSM 3091 archaeon clone (CP000102) (DQ260943) Uncultured archaeon Methanothermococcus thermolithotrophicus mcrA (FN650317) gene for methyl CoM reductase subunit alpha, partial cds. (AB353226) Uncultured archaeon clone Methanothermococcus okinawensis IH1 (CP002792) ( EU495427) 12 Methanosarcina mazei Methanosarcina mazei strain Goe1 (AE008384) strain Goe1 (AE008384) Uncultured archaeon clone Methanothermobacter thermautotrophicus str. Delta (GU357471) H (AE000666) dsr A Highest Match (GenBank Closest cultured relative (GenBank accession accession number) number) Uncultured bacterium clone Desulfovibrio burkinensis DSM 6830 dissimilatory (EU192621) sulfite reductase alpha subunit ( dsr A) and dissimilatory sulfite reductase beta subunit ( dsr B) genes, partial cds. (AF418186) Uncultured bacterium clone Desulfovibrio burkinensis DSM 6830 dissimilatory 13_43 dissimilatory sulfite sulfite reductase alpha subunit ( dsr A) and reductase ( dsr A) gene, dissimilatory sulfite reductase beta subunit ( dsr B) partial cds. (EU192417) genes, partial cds. (AF418186) Uncultured sulfate- Desulfovibrio burkinensis dsr A, dsr B genes for reducing bacterium sulfite reductase alpha and beta subunits, partial cds. (AM494487) (B061536) Uncultured bacterium clone Olavius ilvae Delta 1 endosymbiont clone OIOsp2- (EU192588) 1-7 dissimilatory (bi)sulfite reductase alpha ( dsr A) and dissimilatory (bi)sulfite reductase beta ( dsr B) genes, partial cds. (DQ058671) Uncultured bacterium clone Desulfovibrio burkinensis DSM 6830 dissimilatory 12_23 dissimilatory sulfite sulfite reductase alpha subunit ( dsr A) and reductase ( dsr A) gene, dissimilatory sulfite reductase beta subunit ( dsr B) partial cds. (EU192450) genes, partial cds. (AF418186) Uncultured bacterium clone Desulfosalsimonas propionicica strain PropA Chap_PS2_18 dissimilatory dissimilatory sulfite reductase alpha subunit ( dsr A) sulfite reductase alpha and dissimilatory sulfite reductase beta subunit subunit ( dsr A) gene, partial (dsr B) genes, partial cds. (DQ386237) cds. (HM562943) 13 Uncultured bacterium clone NA CF3 dissimilatory sulfite reductase (AF442719) Desulfohalobium utahense Desulfohalobium utahen se strain EtOH3 strain EtOH3 dissimilatory dissimilatory sulfite reductase alpha subunit (dsrA) sulfite reductase alpha and dissimilatory sulfite reductase beta subunit subunit ( dsr A) and (dsr B) genes, partial cds. (DQ386236) dissimilatory sulfite reductase beta subunit (dsr B) genes, partial cds. (DQ386236) Uncultured sulfate- Olavius ilvae Delta 1 endosymbiont clone OIOsp2- reducing bacterium dsr A, 1-7 dissimilatory (bi)sulfite reductase alpha ( dsr A) dsr B genes for and dissimilatory (bi)sulfite reductase beta ( dsr B) dissimilatory sulfite genes, partial cds. (DQ058671) reductase alpha subunit, dissimilatory sulfite reductase beta subunit, partial cds (AB263151) Uncultured prokaryote Desulfosalsimonas propionicica strain PropA clone GSL_27_55 dissimilatory sulfite reductase alpha subunit ( dsr A) dissimilatory sulfite and dissimilatory sulfite reductase beta subunit reductase alpha subunit (dsr B) genes, partial cds (DQ386237) (dsr A) gene, partial cds. (DQ386256) cat23 Highest Match (GenBank Closest cultured relative (GenBank accession accession number) number) Uncultured bacterium clone Pseudomonas sp. IC catechol 2,3-dioxygenase C23O-2 catechol-2,3- (bph E) gene, complete cds, and 2-hydroxymuconic dioxygenase gene, semialdehyde dehydrogenase ( bph G) gene, partial complete cds. (DQ408374) cds. (U01825) Pseudomonas sp. KB35B Pseudomonas sp. KB35B catechol-degrading gene catechol-degrading gene cluster, complete sequence. (DQ265742) 14 cluster, complete sequence. (DQ265742) Pseudomonas sp. KB35B Pseudomonas sp. 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    Flavobacterium Gliding Motility: from Protein Secretion to Cell Surface Adhesin Movements

    University of Wisconsin Milwaukee UWM Digital Commons Theses and Dissertations August 2019 Flavobacterium Gliding Motility: From Protein Secretion to Cell Surface Adhesin Movements Joseph Johnston University of Wisconsin-Milwaukee Follow this and additional works at: https://dc.uwm.edu/etd Part of the Biology Commons, Microbiology Commons, and the Molecular Biology Commons Recommended Citation Johnston, Joseph, "Flavobacterium Gliding Motility: From Protein Secretion to Cell Surface Adhesin Movements" (2019). Theses and Dissertations. 2202. https://dc.uwm.edu/etd/2202 This Dissertation is brought to you for free and open access by UWM Digital Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UWM Digital Commons. For more information, please contact [email protected]. FLAVOBACTERIUM GLIDING MOTILITY: FROM PROTEIN SECRETION TO CELL SURFACE ADHESIN MOVEMENTS by Joseph J. Johnston A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Sciences at The University of Wisconsin-Milwaukee August 2019 ABSTRACT FLAVOBACTERIUM GLIDING MOTILITY: FROM PROTEIN SECRETION TO CELL SURFACE ADHESIN MOVEMENTS by Joseph J. Johnston The University of Wisconsin-Milwaukee, 2019 Under the Supervision of Dr. Mark J. McBride Flavobacterium johnsoniae exhibits rapid gliding motility over surfaces. At least twenty genes are involved in this process. Seven of these, gldK, gldL, gldM, gldN, sprA, sprE, and sprT encode proteins of the type IX protein secretion system (T9SS). The T9SS is required for surface localization of the motility adhesins SprB and RemA, and for secretion of the soluble chitinase ChiA. This thesis demonstrates that the gliding motility proteins GldA, GldB, GldD, GldF, GldH, GldI and GldJ are also essential for secretion.