Defining molecular details of the chemistry of biofilm formation by Raman microspectroscopy.

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1 5 April 2017 Supplementary Information Defining molecular details of the chemistry of biofilm formation by Raman microspectroscopy. Paul R. Carey*, Blake R. Gibson, Jordan F. Gibson, Michael E. Greenberg, Hossein Heidari- Torkabadi 1, Marianne Pusztai-Carey, Sean T. Weaver, Grant R. Whitmer Experimental Details Bacteria E. Coli BW or Staphylococcus epidermidis RP62A, were grown in Mueller Hinton broth at 37ºC to an OD 600nm, of 0.6 to mls of the cells were rapidly filtered on a vacuum filter with a 22 micron Millipore filter, and washed with 10 ml aqeous isotonic NaCl. The filter was placed in a 50 ml Falcon tube and frozen in liquid N 2 (the time from filtration to freezing is seconds). The sample was then freeze dried for hours. The freeze dried cells, identified as planktonic cells, were then placed in an indentation in aluminium foil (below) and examined under the Raman microscope. It is notable that the Raman spectra of freeze dried planktonic cells are essentially identical to the spectra from planktonic cells dried at room temperature or dried at 50 ºC (unpublished work this laboratory). Biofilm formation. OD cells were concentrated by centrifugation, 50 mls of cells were spun down at 8000 rpms for six minutes. The supernatant was poured off and the cells were suspended in 1mL of 160 mm NaCl. The cells were spun down again at 8000rpms for four minutes and the supernatant discarded. The cells were resuspended in another 1 ml of 160 mm sodium chloride and 50 microliters were placed in each aluminum indentation.. The samples were placed in a sealed incubator under saturated water vapor pressure, for 24 to 48 hours. The samples were dried at 50 ºC and washed to remove planktonic cells and other debris and dried again. Raman microscopy Raman spectra were recorded using a Raman microscope as detailed in reference 1S. The instrument is a simple dispersive spectrometer with cw nm laser excitation milli-watts of slightly off focus power was employed (to ensure photodecomposition did not occur). The focal volume was approximately 50 microns (width) and microns (depth). Each data set was recorded in1x100 seconds. The spectra were calibrated for intensity and wavelength using a standard neon lamp and HOLOGRAMS software. The only data manipulation was background correction using GRAMS/AI software. However, some samples showed a modest luminescent background. This was reduced substantially by exposing the sample to 35 mw nm excitation for a few minutes prior to recording the Raman data. 1

2 Raman sample holders. The cell holders were devices constructed from readily available laboratory materials. A 10 x 7 cm. sheet of Al kitchen foil was glued to the lid of a 96 well plate. Indentations were made over the depressions in the lid. Each indentation was approximately 7 mm in diameter and 1-2 mm deep. Following sterilization the samples were placed in the depressions. The 96 well plates were placed on the microscope stage for visual inspection and spectroscopic data collection. Freeze-dried planktonic cells or biofilm samples gave high quality Raman spectra with minimal background interference. Photography Fresh-dried cells and dried biofilm were present in aluminum indentations as described in the protocol when photographs were taken. All photographs were taken using reflectance microscopy at a total magnification of 100x. Photographs of E. coli and S. epidermidis biofilms are compared with those of their planktonic cells in Figure S1. Fig S1. Photographs (X100 magnification) of dried planktonic E.coli (top left), E. coli biofilm (bottom left): dried planktonic S. epidermidis (top right), S.epidermidis biofilm (bottom right). See reference (S2) for an E. coli biofilm showing a similar morphology. 2

3 Staining with methyl violet E.coli and S.epidermidis at OD were plated onto aluminum foil indented into circles of inverted lids of a 96-well plate. For planktonic samples the cells were dried immediately at 50ºC. for 1hr. Biofilm samples were made as described above. The dried cells were stained with 125μl 0.06% crystal violet for 10 minutes at RT, washed exhaustively with water and air dried. The relative amounts were quantified by dissolving the dried crystal violet stain in 30% acetic acid and spectrophotometric absorption measured at OD 595 in a Victor Multilabel counter (Perkin Elmer) plate reader. The results are shown in the main text Figure 1. Test for the reproducibility of the Raman data Figure S2 compares the Raman spectra of two independent preparations of biofilm from S. epidermidis cells. The cells were grown at a 17 day interval and the cells were standing in aluminum indentations, in a saturated water vapor atmosphere, for 24 hours. Each sample was dried at 50ºC prior to recording its Raman spectrum. Fig. S2 illustrates that the key features discussed in the main text are reproducible. 3

4 Fig. S2. Comparison of the Raman spectra of S. epidermidis biofilm from two independent cell samples (grown two weeks apart). Carbohydrate samples. To provide initial comparison with standard carbohydrate Raman spectra, the spectra of peptidoglycan and the capsular polysaccharide A from bacteroides fragilis are shown in Figures S3 and S4, respectively 4

5 Fig. S3. Raman spectrum of the cell wall (peptidoglycan) from E. coli DH10B Fig. S4. Raman spectrum of capsular polysaccharide A from Bacteroides fragilis. 5

6 Wavenumber (cm -1 ) -1 ) Assignment 1661 amide I 1618 tyrosine ring 1606 phenylalanine ring 1575 guanine ring (major contributor), adenine ring (minor) 1475 (shoulder) guanine ring (major contributor), adenine ring (minor) 1449 CH 2 deformations unresolved intensity due to carbohydrates 1337 adenine ring mode and CH2 deformation modes (non-aromatic residues), α-helices 1316 CH2 deformations (non-aromatic residues) amide III (disordered protein 2 structure) 1174 guanine ring 1142 thymine ring 1125 triphosphates from NTPs, non-aromatic C-C stretch phospholipids, carbohydrates, and PO - 2 in nucleic acids backbone 1031 phenylalanine ring 1003 phenylalanine ring 963 unassigned (possibly α-helices and/or carbohydrates) 852 tyrosine ring 826 tyrosine ring 810 RNA phosphodiester backbone (major contributor), DNA phosphodiester backbone (minor) DNA phosphodiester backbone, RNA phosphodiester backbone (minor contributor), cytosine ring 783 (minor contributor) 756 unassigned 747 thymine ring 725 adenine ring 669 guanine ring 644 tyrosine ring 621 phenylalanine ring 577 guanine ring (broad) unassigned Table S1. Raman peak assignments for Figure 2 in main text. Supplementary Reference (S1) Carey, P. R. Ann. Rev. Phys. Chem. 2006, 57, (S2) Serra, D. O.; Richter, A. M., Hengge, R. J. Bacteriol. 2013, 195,