Microsoft Word - Sullivan ACS 2011 supplementary info.docx

Supplemental Materials:

METHODS:

Bacterial strains and culture conditions

The bacterial strains used in this study are

listed in Supplementary Table 1. Lysogeny Broth (LB) medium was used for all imaging

experiments. All cultures for imaging and phenaz

ine extraction were inoculated from

early stationary phase cultures at an OD

500

of 0.001 in 50

ml of LB and incubated

shaking at 30

°

C overnight in 250

ml baffled flasks. When the large cultures reached

early stationary phase (OD

500

between 2.1 and 2.5), both imaging samples and samples

for extraction were collected. For imaging, 1

ml of the culture was pelleted by micro

centrifugati

on, resuspended in 50

ul of 1x PBS, and imaged as described below. For

phenazine extraction, 30

ml of culture was pelleted by centrifugation at 5000xg for 10

min, the supernatant was decanted and the pellet and supernatant were frozen

separately at

–

°

Frozen samples were processed at a later time as described

below. Because preliminary experiments suggested that only reduced phenazines were

associated with the cells, care was taken to disrupt the cells as little as possible to avoid

altering the redox

state of the phenazines and therefore the phenazine concentration

measured by extraction or imaging.

Phenazine extraction and quantification.

For phenazine extractions, the frozen pellets

were thawed, resuspended in 3

ml of 1x PBS, and sonicated on ice

. The cell lysate

sample was divided and 1.5

ml samples were extracted for pyocyanin or PCA.

Additionally, 1.5

ml of the culture supernatant was also extracted for each phenazine.

For pyocyanin, the sample was first extracted with chloroform and subsequ

ently

extracted by 0.01

N HCl. The sample for PCA extraction was first acidified by addition of

ul of 6

N HCl and then extracted with ethyl acetate followed by 1

M NaOH. For both

phenazines, standard curves were extracted in parallel and all extractio

n steps were well

vortexed and incubated while shaking for at least 30

min to maximize extraction

efficiency.

The phenazine concentrations in the cell extract and the supernatant were measured

with respect to the extracted standard curve using a Biotek S

ynergy plate reader. Acidic

PYO was measured at 520 nm and basic PCA was measured at 367 nm and both were

compared to the baseline reading at 700 nm.

Two

Photon Microscope Set

up.

Two

photon imaging took place in a custom

made

system. Samples were exci

ted by focusing a pulsed laser beam generated by a MIRA

900F titanium

sapphire laser pumped by a Verdi 10 laser (Coherent Inc.) using a 40x C

Apochromat 1.2

NA water immersion objective lens (Carl Zeiss Microimaging). A 3D

image of the specimen was acquire

d by raster scanning the focus point inside the

specimen using a computer

controlled pair of scanner mirrors (6350 Cambridge

Technology) and a piezoelectric objective actuator (P720, Physik Instrumente). The

emitted light is collected via the epi

luminesce

nce path in a de

scanned configuration.

The signal is separated from the laser beam via a dichroic mirror (675DCSPXR, Chroma

Technology), filtered via a short

pass filter (E625SP, Chroma Technology), spectrally

resolved in a spectrograph (MS125 Newport Cor

p.) equipped with a ruled grating (77414

Newport Corp.), and detected by a 16

channel photomultiplier tube (R5900U

01,

Hamamatsu Photonics). The PMT output is processed (amplification, discrimination,

single photon counting) and transmitted to a control PC

by custom

made electronics

(

)

. For bacteria imaging, the laser beam wavelength was 780

nm, the laser po

wer

delivered to the sample was 7

mW, and the pixel sampling time was 125

sec. For pure

compound imaging, the laser beam wavelength was tuned between 740 and 890

nm,

the laser power delivered to the sample was 1

mW, and the pixel sampling time was 1

msec. All images digitized a 25

m field of view into 256

256 pixels, 16 cha

nnels per

pixel. Each PMT channel counted photons within a 13

wide range of the spectrum,

so that the 16 channels of the PMT cover the range [390, 600] nm.

Supplementary Table 1:

Strains used in this work

Strain name

Genotype

Source (Reference)

N263

PA14 (wildtype)

(

)

DKN330

PA14

Δ

phz

G1,

Δ

phz

(

)

LD375

PA14

Δ

phz

L. Dietrich, Columbia University

DKN619

PA14

∆

pvdA,

∆

pchE

(

)

DKN620

PA14

Δ

phz

G1,

Δ

phz

G2,

∆

pvdA,

∆

pchE

(

)

Supplementary Figure 1:

Comparison of the emission spectra of PYO

red

, PCA

red

1OHP

red

phenazines and F8820 fluorescent microsphere

s (Invitrogen) after single

photon (top) and two

photon excitation (bottom). Although the emission spectra of

PYO

red

, 1OHP

red

and F8820 do not depend on the mode of fluorescent excitation (single

photon or two

photon), the emission spectrum of PCA

red

is bl

shifted approximately 20

nm after two

photon excitation compared to single

photon excitation. The emission

peaks of PCA

red

and F8820 are approximately the same after single

photon excitation,

however they differ significantly after two

photon excitation

. The x

axis in the single

photon measurement corresponds to the exact wavelength of the measurement. The x

axis in the two

photon measurement shows the center wavelength of each one of the 16

sensor channels (each channels detects photons in a 13 nm range

of the spectrum).

Supplementary Figure 2:

Example of a raw image (sum of the 16

channel raw data)

and its segmentation into bacteria and medium pixels. Segmentation was based on the

known noise properties of the sensor and a halo surrounding the bacteria

was excluded

from both groups of pixels.

References:

21.

Buehler, C., Kim, K. H., Greuter, U., Schlumpf, N., and So, P. T. (2005) Single

photon counting multicolor multiphoton fluorescence microscope.

J Fluoresc

–

51.

22.

Rahme, L. G., Stevens, E. J

., Wolfort, S. F., Shao, J., Tompkins, R. G., and

Ausubel, F. M. (1995) Common virulence factors for bacterial pathogenicity in

plants and animals.

Science

268

, 1899

–

902.

23.

Wang, Y., Wilks, J.C., Danhorn, T., Ramos, I., Croal, L., and Newman, D.K.

(2011)

Phenazine

carboxylic acid promotes bacterial biofilm development via

ferrous iron aquisition,

J. Bacteriol.

, in press