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Supplement {-}

\markboth{SUPPLEMENT}{SUPPLEMENT}

Please note: The printed version of this thesis includes a CD with the referenced supplemental files. The .pdf version includes these files as attachments. PDF viewer permitting, they can be opened or saved directly at "available here"-notes. Alternatively, referenced files will also be available at github.com/katrinleinweber/PhD-thesis.

Chapter 2: Biofilm and capsule formation of the diatom Achnanthidium minutissimum are affected by a bacterium {#BF-form-suppl}

\sectionmark{\emph{A.~minutissimum} biofilm formation}

Colorimetric analysis of other carbohydrate standards

Comparison of glucose and glucuronic acid standard curves in Phenol-sulfuric acid assay (N = 1). The largest deviation from both the mix and glucose was found for glucuronic acid (dashes and dotted line). Its linear regression slope is 81 and 88% more shallow respectively, which indicates that this carbohydrate compound either reacts incompletely or yields a products with low absorbance around 488\ nm. In either case, it can only contribute little to the EPS concentrations we report in this. \label{Glu-compare}

Standard curves of several carbohydrates prevalent in A.\ minutissimum (N = 1). In addition to the glucose-based calibration of the phenol-sulfuric acid assay that was used in this study, six most abundant compounds found in A.\ minutissimum previously by @bahulikar_complex_2008 were tested. Dilution series of the standards in natural mixture, as well as individually, yielded the calibration data. Glucose (diamonds and dashed line) exhibits the most pronounced colorimetric reaction. Other sugars have up to 61% more shallow regression slopes. As expected, the natural mix (x with solid line) yielded an average linear regression slope, compared to which the one of glucose is 34% steeper. \label{CHO-STD}

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Growth of Bacteroidetes strain 32 in diatom full medium

Bacteroidetes strain 32 was not able to grow in diatom full medium (BM) without the diatom A.\ minutissimum or the glucose supplement. The bacterium was cultivated in BM medium (N = 2) and in diluted LB (N = 1). Bacteroidetes strain 32 was inoculated in the diatom medium BM and in diluted LB (50% v/v). Therefore, the bacterium was scrapped from an agar plate containing diluted LB, resuspended in 1\ mL BM and 10\ µl were added to 5\ mL of the respective medium in glass reaction tubes. Cultures were incubated at 22°C and 112\ rpm. OD600 was measured with an M107 spectrophotometer (Camspec Ltd, Cambridge, UK). \label{S32-growth-BM}

Quantification of soluble and bound EPS

In a preliminary test we observed the extractability of the capsular material. We therefore observed the cell pellets after treatment with warm water (WW), hot water (HW), hot bicarbonate (HB) and hot alkali (HA) by light microscopy. Extraction of carbohydrates was performed as described in section \nameref{EPS-quant}\ (p.\ \pageref{EPS-quant}) with the only difference that the fraction containing the soluble polymers was filtered using a 0.2\ µm filter previous to evaporation. To highlight bound carbohydrates, the pellets were stained with alcian blue.

\begin{FPfigure} \centering \caption{\textbf{Quantification of carbohydrates and microscopic images of the xenic and axenic \emph{A.~minutissimum} cells in different extracts.} Xenic \emph{A.~minutissimum} cells exhibited capsules even after treatment with warm water and defatting (A and B). Hot water treatment results in a loosening of the capsule structure (C). The capsules were completely dissolved after treatment with hot bicarbonate (D). Treatment with hot alkali solution dissolved all cell structures including the frustules (E). The carbohydrate profile of the xenic culture correlates well with the dissolution of the capsule material as it shows high carbohydrate contents in the HW and HB fractions. \label{CHO-BFM}} \includegraphics{biofilm-formation-figures/S4-CHO-BFM.png} \end{FPfigure}

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Co-cultivation of A.\ minutissimum with different bacterial isolates

Bacteria isolates B-1, B-2, B-4, B-5, B-6, B-7, B-8 and B-10 were isolated from phototrophic, epilithic biofilms from the same sampling site as A.\ minutissimum and Bacteroidetes strain 32 in April 2011. The biofilm was scraped from stone surfaces, diluted in BM and roughly vortexed for 10\ min before the suspension was plated in gradual 1:10 dilutions on agar plates containing diluted LB medium (50% (v/v)). Single colonies were picked and isolated by repeated smear. The bacterial isolates were cultivated as described for strain 32. For co-cultivation, 1\ mL BM was inoculated with 2.8·10^4^ diatom cells/ml and 5\ µl of the bacterial cell suspension (OD600 0.1). The co-cultures and negative controls (axenic diatom and bacteria, respectively) were performed in triplicates, the positive control (co-culture of A.\ minutissimum with strain 32) in duplicates. Biofilm was stained with CV and the absorption of the extracted dye was measured at 580\ nm.

Absorption of crystal violet (CV) extracted from biofilms of co-cultures of A.\ minutissimum with different bacterial isolates. Three co-cultures produce a clearly stronger biofilm than the axenic diatom culture, co-cultures with B-1, B-5 and B-6). Only the co-cultures with B-1 and B-5 achieved a biofilm quantity comparable to the co-culture with strain 32. Co-cultures with B-4, B-7 or B-8 showed comparable or even less CV absorptions as the axenic diatom culture. Pure bacteria cultures in BM resulted in low or even no biofilm formation as they presumably did not grow in the used medium without diatom or additional carbon sources. Capsule formation was only visible in the co-culture with strain 32. \label{CV-isolates}

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Colorimetric analysis of defatting fractions during quantification of bound EPS

In order to check for leakage of intracellular carbohydrates into the defatting fractions during the extraction of bound EPS, the procedure described in section\ \nameref{EPS-quant} (p. \pageref{EPS-quant}) was repeated with one stationary axenic and xenic culture each and the EtOH fractions were collected. These defatting fractions were evaporated and taken up in 1\ mL ultrapure water. These samples were analysed in the phenol-sulfuric acid assay as described in this study using the standard curve of the mixture described above (Fig.\ \ref{CHO-STD})

Quantification of carbohydrate release from cells during defatting steps with EtOH (N = 1). Both cultures show similarly high release of presumably intracellular carbohydrates in the first defatting step with EtOH. No carbohydrates could be detected in the following steps which indicates that the pellets did not contain intracellular carbohydrates any more. Furthermore, chl was extracted from the defatted pellet as described in section \nameref{chl-determ}\ (p.\ \pageref{chl-determ}). \label{CHO-defat}

Quantification of chl residue in defatted pellet (N = 1). After defatting of the axenic and xenic pellet, only 4.8 and 1.4% respectively of the cultures' initial chl concentrations were found. A pale, greenish-yellow colouration of the pellet indicated the possibility of small quantities of chl remaining protected by well-developed capsules. However, given these data and the observed green colouration in the first few EtOH fractions, we conclude that the vast majority of chl was extracted by the defatting steps. This in turn means that defatting efficiently breaks open even encapsulated, xenic A.\ minutissimum cells. \label{Chl-defat}

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Chapter 3: A bioassay-guided fractionation of bacterial infochemicals that induce biofilm formation by Achnanthidium minutissimum {#signal-extraction-suppl}

\sectionmark{Bioassay-guided fractionation}

The following good quality 16S ribosomal DNA sequence parts (forward and reverse, respectively1) of Bacteroidetes strain 32 were used for the taxonomic placement as described in section\ \nameref{rDNA-seq} (p.\ \pageref{rDNA-seq}).

\DNA! CTGCAGGCGGCCGCACTAGTGATTAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGGCAGGCTTAATACATGCAAGGCGAGGGGGCAGCAATGTCACCGTCGTACGGGTGCGCAACGCGTATGCAACCTACCTATCACTGGGGGATAGCCCGGGGAAACCCGGATTAATACCGCATAACACAGGGGTCCCGCATGGGTACTATTTGTTAAAGATTTATCGGTGGTAGATGGGCATGCGTTCGATTAGCTAGTTGGTATAGGTAACGGCTTACCAAGGCTACGATCGATAGGGGAGCTGAGAGGTTGATCCCCCACACGGGCACTGAGATACGGGCCCGACTCCTACGGGAGGCAGCAGTAGGGAATATTGGGCAATGGATGCAAGTCTGACCCAGCCATGCCGCGTGCCGGATGAAGGCCCTCAGGGTTGTAAACGGCTTTTATTCGGGAAGAAGAGCAGGGATGCGTCCTTGTGTGACGGTACCGAATGAATAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTGTCCGGATTTATTGGGTTTAAAGGGTGCGTAGGTGGCTTGTTAAGTCAGTGGTGAAATACAGCCGCTCAACGGTTGAGGTGCCATTGATACTGACAAGCTTGAAACAAGTGGAGGCTGCCGGAATGGATGGTGTAGCGGTGAAATGCATAGATATCATCCAGAACACCGATTGCGAAGGCAGGTGGCTACGTTTGATTTGACACTGAGGCACGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGGACTCGCTGTTGGCCTGTCAAGGGTCAGCGGCTTAGGGAAACCGTTAAGTCCTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGTCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCTGGGCTAAATCACACTAGACGCATTCAGAAATGGGTGTTCCAGCAATGGCTGGTGTGAAGGTGCTGCATGGCTGTCGTCAGCTCGTGT!

\DNA! GTCGCCGATTTTACCCTAACAGTGTCTTTAACCTACTGCTTCAGGTCTCCCCGACTCCCATGGCTTGACGGGCGGTGTGTACAAGGTCCGGGAACGTATTCACCGCGCCATAGCTGATGCGCGATTACTAGCGATTCCAGCTTCATAGAGTCGAGTTGCAGACTCCAATCCGAACTGAGAACGGCTTTTTGGGATTGGCATCTCATCGCTGAGTAGCTACCCTCTGTACCGCCCATTGTAGCACGTGTGTTGCCCTGGACGTAAGGGCCATGATGACTTGACGTCGTCCCCTCCTTCCTCTCTGTTTGCACAGGCAGTCTGATTAGAGTCCCCACCATTACGTGCTGGCAACTAACCATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTTCACACCAGCCATTGCTGGAACACCCATTTCTGAATGCGTCTAGTGTGATTTAGCCCAGGTAAGGTTCCTCGCGTATCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGACCCCCGTCAATTCCTTTGAGTTTCACCGTTGCCGGCGTACTCCCCAGGTGGAGGACTTAACGGTTTCCCTAAGCCGCTGACCCTTGACAGGCCAACAGCGAGTCCTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCATGCTTTCGTGCCTCAGTGTCAAATCAAACGTAGCCACCTGCCTTCGCAATCGGTGTTCTGGATGATATCTATGCATTTCACCGCTACACCATCCATTCCGGCAGCCTCCACTTGTTTCAAGCTTGTCAGTATCAATGGCACCTCAACCGTTGAGCGGCTGTATTTCACCACTGACTTAACAAGCCACCTACGCACCCTTTAAACCCAATAAATCCGGACAAC!

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The following 16S ribosomal DNA consensus sequence2\ of Bacteroidetes strain 32 was used for the BLASTN analysis which is summarised in table\ \ref{dyado-hits}.

\DNA! GCCCGGGGAAACcCGGATTAATACCGCATAACACAGGGGTCCCGCATGGGTACTATTTGTTAAAGATTTATCGGTGGTAGATGGGCATGCGTTCGATTAGCTAGTTGGTATAGGTAACGGCTTACCAAGGCTACGATCGATAGGGGAGCTGAGAGGTTGATCCCCCACACGGGCACTGAGATACGGGCCCGACTCCTACGGGAGGCAGCAGTAGGGAATATTGGGCAATGGATGCAAGTCTGACCCAGCCATGCCGCGTGCCGGATGAAGGCCCTCAGGGTTGTAAACGGCTTTTATTCGGGAAGAAGAGCAGGGATGCGTCCTTGTGTGACGGTACCGAATGAATAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTGTCCGGATTTATTGGGTTTAAAGGGTGCGTAGGTGGCTTGTTAAGTCAGTGGTGAAATACAGCCGCTCAACGGTTGAGGTGCCATTGATACTGACAAGCTTGAAACAAGTGGAGGCTGCCGGAATGGATGGTGTAGCGGTGAAATGCATAGATATCATCCAGAACACCGATTGCGAAGGCAGGTGGCTACGTTTGATTTGACACTGAGGCACGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGGACTCGCTGTTGGCCTGTCAAGGGTCAGCGGCTTAGGGAAACCGTTAAGTCCTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGTCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCTGGGCTAAATCACCACaGGAATCATTCAGAAATGGGTGATCCAGCAATGGCTTGTTTGAAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATGGTTAGTTGCCAGCACGTAATGGTGGGGGACTCTAATCAGACTGCCTGTGCAcACAAGAgAGGAAGGAGGGGACGACGTCAAGTCATCATGGGCCcTTTACGTCCAGGGcCAACACAACGTGCTTACAaTGGGCGGGTACAgAAGGGTTAGCtACCTCCAcCGATGAGAATGCCAATCCCAAAAAGCCGTTcTCCCAgTTCCGAaTTGGAATCCTgCACCTCGACTCCTATGGAAGaCTGGGAATCCCTTAgATAATCCCCCCACCCCttATGGGggCaGGTGaAAAaa!

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Table: \label{dyado-hits} BLASTN hits of 16S ribosomal DNA consensus sequence from Bacteroidetes strain 32. Abbreviated genus name D. stands for Dyadobacter, and P. for Persicitalea. Query and hits are partial sequences with lengths around 1.25 kb, E values of 0.0, Query covers of 97%. Max and Total scores were equal, and are thus summarily listed as Score here. Row with dashes (--) indicates the gap of 480 score points and 7 percentage points in identity that separate Dyadobacter hits from the next closest genus. Repeating the BLASTN analysis with the 16S sequences of these hits again yielded Dyadobacter clusters with the same ranges of query covers and sequence identities, as well as a >6%-point gap to the next closest genus.

Species (strain) Score Identity Accession (NR_)
D. koreensis (NBRC 101116) 1873 95% 113977.1
D. koreensis (KCTC 12537) 1873 95% 044041.1
D. psychrophilus (BZ26) 1868 95% 117212.1
D. ginsengisoli (Gsoil 04) 1838 94% 041372.1
D. hamtensis (HHS 11) 1838 94% 042226.1
D. jejuensis (AM1R11) 1825 94% 109488.1
D. fermentans (NS114) 1794 94% 027533.1
D. alkalitolerans (12116) 1790 93% 044476.1
D. soli (MJ20) 1788 93% 117263.1
D. tibetensis (Y620-1) 1777 94% 109648.1
D. arcticus (R-S7-29) 1757 93% 109479.1
D. crusticola (CP183-8) 1744 93% 042335.1
D. beijingensis (A54) 1705 92% 043725.1
-- -- -- --
P. jodogahamensis (NBRC 103568) 1225 85% 114246.1

Method of pyrolysis-field ionisation mass spectrometry (Py-FIMS) {#Py-FIMS-suppl}

\sectionmark{Py-FIMS}

Duplicates of 5\ µL of MeOH-dissolved samples were injected into annealed quartz crucibles and dried overnight in a desiccator. Pyrolysis was carried out directly in the ion source of a double-focusing Finnigan MAT95 (emitter: 4.7\ kV, counter electrode -5.5\ kV). The samples were heated in a vacuum of 10^-4^\ Pa from 50 to 700°C in steps of 10°C over a timespan of 15\ min. 60 spectra of the mass range 15-900\ m/z were recorded at 10\ millimasses accuracy. Results were interpreted with the help of marker signals (m/z) of relevant substance groups [@hempfling_chemical_1988; @schnitzer_analysis_1992; @schulten_characterization_1996; @van_bochove_pyrolysis-field_1996;@leinweber_analytical_2009; @leinweber_advances_2013].

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Chapter 4: A semi-automated, KNIME-based workflow for biofilm assays {#assay-opt-suppl}

\sectionmark{Semi-automated biofilm assay}

Detailed instructions for the manual preparation of the Achnanthidium\ minutissimum bioassays, based on the protocol by @windler_biofilm_2015. See figure \ref{BF-assay-wf}, p. \pageref{BF-assay-wf} for method overview. \label{BF-assay-wf-suppl}

Please note: \texttt{.tar} files need to be unpacked (e.g. with \href{http://7-zip.org/}{7-Zip}) before import into the respective programs.

  • plate-layout-template.xlsx \textattachfile[color=blue]{supplement/plate-layout-template.xlsx}{(available here)}
    Plate layout template for recording sample placement in multi-well plates and merging metadata with measurements in KNIME. See Fig.\ \ref{BF-assay-wf}, p.\ \pageref{BF-assay-wf} for illustration.
  • Viaflo-scripts.tar \textattachfile[color=blue]{supplement/Viaflo-scripts.tar}{(available here)}
    Viaflo electronic pipetting scripts to successively remove cells, crystal violet staining solution and wash water after steps 1, 3 and 5 (Table\ \ref{Viaflo-scripts}). See Fig.\ \ref{BF-assay-wf}, p.\ \pageref{BF-assay-wf} for experimental context and \href{http://www.integra-biosciences.com/sites/vialink.html}{Vialink}'s built-in help for importing instructions.
  • Magellan-readout.mth \textattachfile[color=blue]{supplement/Magellan-readout.mth}{(available here)}
    Plate-reading method for Tecan's Magellan software. See section \emph{\nameref{BF-quant-method}}, p.\ \pageref{BF-quant-method} for details.
  • KNIME-workflow.tar \textattachfile[color=blue]{supplement/KNIME-workflow.tar}{(available here)}
    Importable workflow for the KNIME Analytics Platform to demonstrate the merging of sample metadata (\texttt{plate-layout-template.xlsx}) and Magellan-measured absorbance data (\texttt{.asc} files). See Fig.\ \ref{KNIME-wf} for illustration. Please note that importing will return an error initially, because the file paths can not match, and have to be corrected as described in section \emph{\nameref{knime-workflow}}. In case of the \texttt{FileReader} nodes, this correction should be conducted with the option \texttt{Preserve user settings for new location} activated. If forgotten, and if the data preview shows a column filled with question marks, please right-click on that column and activate the option \texttt{DON'T include column in output table}. Traffic light symbols below the nodes will indicate whether corrections are still necessary (red), or whether the nodes can be executed (yellow).
    Upon execution of this workflow, data files are read in and the \texttt{Expand    WellPosition} nodes ensure equal formatting of the sample metadata and measurement results according to the well coordinates (defined in the \texttt{.xlsx} file and present in the \texttt{.asc} files). \texttt{Joiner} combines these tables per row, discarding incongruencies between plate layouts and measurement data. \texttt{Concatenate} progressively merges two plates' data tables into one. \texttt{PlateHeatmapViewer} provides a visual comparison of the data processing result with the visual impression of a plate. In particular, the expected locations of biofilm-negative and -positive controls are easily discernible. In the concatenated table, \texttt{PlateRowConverter} and \texttt{ColumnCombiner} regenerate the alphanumeric well coordinates so that the data and visual impression of individual wells can be compared. \texttt{ColumnFilter} and \texttt{ColumnResorter} exclude obsolete coordinate metadata and pre-format the remaining table for export by \texttt{CSV~Writer}.
  • plot-KNIME-output.R \textattachfile[color=blue]{supplement/plot-KNIME-output.R}{(available here)}
    R code to demonstrate the plotting of KNIME-processed data. Please note that due to a randomisation function in the plate layout \texttt{.xlsx} file, editing the latter and running the KNIME workflow and this script again may produce a plot with different assignments of data points to the levels \texttt{X}, \texttt{Y} and \texttt{Z}.
  • plot-Tecan-figures.tar \textattachfile[color=blue]{supplement/plot-Tecan-figures.tar}{(available here)}
    R code and data (\texttt{.csv} format) used to produce the plots in this article.

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Chapter 5: Capsules of the diatom Achnanthidium minutissimum arise from fibrillar precursors and foster attachment of bacteria {#capsule-microstructure-suppl}

\sectionmark{Capsule microstructure}

Identification of A.\ minutissimum cell clusters in axenic culture by subsequent observation by both bright-field (A) and scanning electron (B) microscopy (scale bars: 5\ µm). Demonstration of the same technique used to identify the appearance of xenic biofilms and dehydrated capsule material in SEM (Fig.\ \ref{CLEM},\ p.\ \pageref{CLEM}) in axenic cultures after 31\ days of incubation with much fewer adherent cells and no capsules. \label{CLEM-ax}

\begin{FPfigure} \centering \includegraphics[width=1\textwidth]{capsule-microstructure-figures/ax-fibrils.png} \caption{\textbf{Scanning electron micrographs of fibril-covered \emph{A.minutissimum} frustules from axenic culture.} Samples were prepared for SEM after 20days of incubation. \textbf{A} (scale bar: 1µm) & \textbf{B} (scale bar: 200m): Frustules with few, short fibrils, which were not found in xenic biofilms. \textbf{C} (scale bar: 1µm) & \textbf{D} (scale bar: 200nm): Frustule with medium-dense fibrillar mesh, as also seen in xenic biofilm (main Fig.\ref{stages}A). \textbf{E} (scale bar: 200nm): Fibrils are not only flatly attached to the frustule but also stick out into space and make contact with other cells (arrows), as also seen in xenic cultures (main Fig.~\ref{stages}B).} \label{ax-fibrils} \end{FPfigure}

Footnotes

  1. Complete sequences and RDP classification results are in Supplemental File S32-16S-rpd-classified-hierarchy.txt \textattachfile[color=blue]{supplement/S32-16S-rpd-classified-hierarchy.txt}{(available here)} ↩

  2. S32-16S-consensus.fas \textattachfile[color=blue]{supplement/S32-16S-consensus.fas}{(available here)} ↩