Supplemental Information
Flow cytometry analysis was used to quantify fluorescence produced by autoinducer-2
(AI-2) induced samples.
Fig. S1 demonstrates the details for E. coli CT104
(W3110∆lsrFG∆luxS) induced with 8 μM AI-2 (II) compared to an uninduced control (I), each
measured at a 5h timepoint. Forward and side scatter were optimized to define the sample
population (Fig. S1a). PMT settings for both the FITC and PE channels were monitored to
detect cell fluorescence (Fig. S1b); red fluorescence was observed by a shift in the population
along the PE, but not the FITC, axis. The red fluorescence profile of each sample was further
analyzed by plotting PE channel data as a histogram (Fig. S1c). The fluorescence profile of the
non-fluorescing control was gated as P1. The complement of P1, NOT(P1), was used to define
the DsRed-expressing subpopulation (shown in inset histogram). In Fig. S1d, NOT(P1) data
were reported as a raw count and a percentage of the total population (circled).
Figure S1. Flow cytometry analysis of E. coli CT104 strain (W3110∆lsrFG∆luxS) demonstrating the
induction of DsRed in response to exogenous AI-2. 10,000 events were collected per sample. (a) Light
scatter plots of E. coli CT104 strain (W3110∆lsrFG∆luxS) 5 hours post exposure to 0 µM AI-2 (I) and
8 µM AI-2 (II), respectively. (b) Fluorescence scatter plots comparing green and red fluorescence
intensity of the same samples shown in a, using FITC and PE settings, respectively. (c) Histograms of
red fluorescence intensity from b used to gate cells against autofluorescence and quantify the
fluorescent portion of the population (shown by inset histogram). (d) Tables showing number of
fluorescent and non-fluorescent events and their proportions of the entire population.
Motility and Gene Expression Sensitivity and Response Times
70
QS @ 7hrs
60
mo lity @ 1hr
40
10
30
20
Mo lity (%)
mo lity @ 2 hrs
50
QS DsRed (%)
15
QS @ 2hrs
5
10
0
0
0.05
0.5
5
50
AI-2 concentra on (mM)
Fig. S2. Data from the current study superimposed onto motility data from Wu et al., 2013.
The data from Wu et al., [1] Supplemental section were collated and are displayed in Fig.
S2 here jointly with data from Fig. 2. E. coli CT104 cells were incubated with varied levels of
AI-2. QS activity corresponds to DsRed expression and is presented as a percent of the total cell
population expressing DsRed. Data are depicted for both 2 and 7 hr samples. As indicated in Fig.
2, the quantized quorums are established only after an incubation period (e.g, 7 hrs here). The
motility experiments from Wu et al., were carried out identically, but sample data are included
from 1 and 2 hr samples. The motility data are resolved displaying quantized units by 2 hrs
suggesting a quicker response. These are consistent with the chemotaxis data also depicted in
Wu et al., 2013. Finally, these data also point to the apparent enhanced sensitivity to AI-2 for
motility as opposed to gene expression. The molecular bases for these activities are different
owing to chemoreceptor dynamics for the former.
Coincident Expression of Native Genes and T7 Polymerase Amplification
Fig. S3A (below) shows qPCR measurements of E. coli MDAI2 pCT6 pET200 that were
grown to mid log phase and then incubated with 0 μM AI-2, 4 μM AI-2 and 40 μM AI-2,
respectively. At each time point and concentration, the pET transcript (amplified by T7
polymerase) is normalized to the T7 RNA polymerase transcript that is controlled via the native
lsr promoter. Note that at 0 hrs and 0 μM AI-2, the pET transcript is expressed approximately 82
fold higher than the T7 RNA polymerase transcript. This amplification is fairly consistent
throughout. In Fig. S3B, we illustrate that the addition of 40 μM of AI-2 up-regulates the
expression of the T7 polymerase transcript in a manner that closely follows the up-regulation of
the QS-dependent genes lsrR and lsrK. The data are normalized to 16sRNA. We note also that
this data shows that the addition of 4 μM of AI-2 results in a smaller up-regulation of the T7RNA
polymerase, lsrK and lsrR genes compared to 40 μM of AI-2, as one might have expected.
Fig. S3: Strains of E. coli MDAI2 pCT6 pET200 were grown to mid-exponential phase (OD ~ 0.4),
and then incubated with 0 μM AI-2, 4 μM AI-2 and 40 μM AI-2, respectively, for 6 hours. RNA was
extracted prior to and after incubation with the RNAqueous kit. RNA was reverse transcribed to
cDNA and amplified using the BIO-73005 SensiFast SYBR Hi-Rox One Step Kit. For the selected
candidate genes, primers were designed using Primerquest. 16s rRNA was used as a control
housekeeping gene, and qPCR was performed using a 7900HT real time PCR System (Applied
Biosystems) with thermal conditions of 10 min at 45°, 2 min at 95°, and 40 cycles of 5 sec at 95°and
20 sec at 60°. In A) the relative gene expression level of each target gene was normalized at each data
point to 16s rRNA, and then at each condition and timepoint to the T7RNA polymerase transcript. B)
After 6 hours of incubation, the relative gene expression level of each target gene was normalized at
each data point to the 16s rRNA. The control for each dataset was chosen to be the 0 μM AI-2 case.
Fold change and error bars were calculated using the ΔΔCT relative comparative method.
AI-2 Mediated Cell Recruiting
Shown in Fig. 4a, we designed cell “recruits” that express multiple fluorescent proteins
in order to monitor the process of AI-2-based chemotaxis; constitutively expressed red
fluorescence provides continuous traceability while AI-2-induced expression adds AI-2
dependent green fluorescence.
“Recruiter” cells were encapsulated within a biocompatible
matrix in order to centralize the source of AI-2 generation. Recruiter genotype was chosen such
that the AI-2 synthase gene, luxS, was either included for AI-2 production or absent for negative
control cells.
Red recruits were separated from recruiter capsules via a transwell membrane
with 1 μm porosity to significantly reduce passive transmigration.
The two sections of the transwell apparatus were sampled at multiple timepoints and
imaged by fluorescence microscopy to probe for recruitable red cells. In Fig. 4b(i), the bar graph
shows, at two timepoints, the number of red cells counted in the microscope field of view (200X)
from the recruited fraction (average of four image counts). The green cell count is also plotted.
Representative images from each sample area shown in Fig. 4b (ii), using both red and green
filters. The gray value profiles for both red and green were plotted for the highlighted portions
of the images from luxS+ treated red recruits. Colocalization is observed where strong red and
green peaks are overlaid.
The data shows multiple AI-2-dependent phenotypic responses by a single cell
population over different time scales. Within the 16 h timeframe, AI-2 recruiting led to 10-fold
increase in cell migration across the membrane toward the recruiter cells (Fig. 4b(i)). Over 40 h,
the passively diffusing luxS- treated red recruits never surpassed the recruited cell count of luxS+
treated cells. Additionally, by 40 h, a significant subpopulation of the luxS+ treated cells
(compared to none of the luxS- treated cells) fluoresced green (Fig. 4b(ii)).
Plotting the
fluorescence profiles quantitatively reveals the amplitude increase (10-fold in some cases) of
green fluorescence over time, and also the colocalization of both red and green fluorescence
where strong peaks are overlaid.
Methods
The capsule development was noted in the main article. Three capsules were added to the
bottom well per transwell system and were confined to the center of each.
A transwell
membrane with a 1μm pore size (BD Biosciences) was inserted. LB was diluted 10-fold in PBS
(10 mM), supplemented with calcium chloride (0.5 mM, to maintain capsule integrity), and used
as the incubation medium; three volumes were added to the bottom well per volume in the top of
the transwell insert. Finally, CT104 + pCT6 + pT5RT7G, grown at room temperature to OD 0.1,
was diluted 1000-fold into the top of the transwell apparatus. The system was sealed with
Parafilm M® and incubated for 16 hr or longer at 30 °C followed by 24 h at room temperature.
Initial Conditions for Study:
We counted 8x107 cells per mL per 0.1OD. We used 8x104 cells per mL in the partitioned
fraction when diluted 1/1000. We used 2uL sample for imaging = 160 cells per coverslip. The
data are presented as number of cells in microscopic fields of view (0.57 mm2 field of view with
20X objective). We have 113mm2 area of coverslip, 6mm radius, so that the field of view is
1/200 of coverslip area, leading to 1-2 cells, on average, in view for the initial conditions.
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