Supplemental Material
Supplementary Methods
The effect of exogenous factors and RpoE on secretion of cargo proteins.
The effect of exogenous factors including polymyxin B sulfate (100 U), iron
chelator 2-(pyridin-2-yl)pyridine (2’2’-dipyridyl, at 100 µM final concentration),
dimethyl sulfoxide (vehicle, DMSO), or bile salts (0.1% final concentration) on the
secretion of cargo proteins was studied in both mid-logarithmic as well as
stationary cultures of three strains of V. cholerae N16961 (wild-type, and wildtype carrying either pMMB67-EH or pRpoE). The bacteria were cultured with
shaking in LB medium at 37°C until it reached OD600 of 0.5. Then, equal amounts
of the cultures were divided into individual test tubes containing the examined
compound. Expression of rpoE was induced with 100 µM IPTG. The
supernatants were separated from bacterial cells by centrifugation at either 30
min-post-exposure (mid-logarithmic phase) or 16 h (late stationary), filtered, and
the
serine
protease
activity
was
determined
as
described
(1).
The
metalloprotease activity, which is associated with a functional T2S system (B. R.
Park and A.E. Sikora, manuscript in preparation), was measured using DQgelatin from pig skin, fluorescein conjugate (EnzCheck, Molecular Probes,
Eugene, OR) following the instructions provided by the manufacturer. The assays
were conducted in a 96-well black microtiter plate format (Greiner BioOne) in the
Synergy HT Multi-Microplate Reader (BioTek). The increase in fluorescence,
which corresponds to the protease activity, was monitored at 37ºC every min
during 10 min after the addition of the substrate and normalized by optical
density of the cultures.
Statistical analyses of eps-lux expression. All analyses were performed in R
version 3.0.2, using the standard packages. Data for this analysis consisted of
eps-lux luminescence time-series measurements for independent experiments of
three strains of Vibrio cholerae (O395, N16961, and C6706), each at two
different
temperatures
(37°C
and
25°C).
For
each
strain-temperature
combination, the Pearson correlation coefficient between the mean eps-lux and
mean OD600 time series was computed. To directly compare the mean eps-lux
time-series from one strain-temperature combination to another, the correlation
matrix for the six different mean eps-lux time series was calculated. The
correlations between the mean time series were subsequently used to construct
a distance for clustering the strain-temperature experiments. The distance
between two different experiments is defined as 1 - Cor(eps-luxi, eps-luxj), where
eps-luxi and eps-luxj are two different strain-temperature time series. This will
result in experiments with mean eps-lux time series that are highly correlated
with each other having small distances, and therefore being placed in the same
cluster.
References:
1.
Sikora AE, Lybarger SR, Sandkvist M. 2007. Compromised outer
membrane integrity in Vibrio cholerae Type II secretion mutants. J.
Bacteriol. 189:8484-8495.
Supplementary Figures
Fig. S1. Correlation between eps-lux expression and bacterial growth (A).
Correlation matrix with a corresponding heat map for the different mean eps-lux
series (B). Hierarchical clustering dendrogram for the strain-temperature
experiments (C).
Fig. S2. Analysis of the timing of the peak eps-lux expression between
different V. cholerae classical (O395) and El Tor (N16961, C6706) strains.
The time series of mean eps-lux luminescence is plotted in black when there is a
statistically significant difference (at level α = 0.05) between the mean eps-lux at
that time point and the peak mean; it is plotted in red when there is no statistically
significant difference between the mean eps-lux and the peak mean.
Fig. S3. Kinetics of eps expression in V. cholerae C6706 wild-type and
ΔhapR strains. Bacterial strains were cultured in LB media at 37°C in 96-well
white, clear bottom microtiter plates. The turbidity of the cultures at OD600 and
eps-lux activity, luminescence, was assessed in technical triplicates and
recorded every 20 min throughout 16 h. Each datum point represents the mean
and SEMs obtained from at least five independent experiments.
Fig. S4. Kinetics of tarp expression in V. cholerae classical O395 strain.
Bacterial strains were cultured in 96-well white, clear bottom microtiter plates in
LB media at either 25°C or 37°C, and under conditions either repressing- or
inducing virulence gene expression, as indicated. The turbidity of the cultures at
OD600 and eps-lux activity, luminescence, was assessed in technical triplicates
and recorded every 20 min throughout 16 h. Each datum point represents the
mean and SEMs (n=4).
Fig. S5. The effect of exogenous factors and overproduction of RpoE on
secretion of cargo proteins. V. cholerae N16961 wild-type, and wild-type
carrying either pMMB67-EH (p) or pRpoE were cultured with shaking in LB
medium at 37°C until it reached OD600 of 0.5. Then, equal amounts of the
cultures were divided into individual test tubes containing the examined
compound including 100 U polymyxin B sulfate, DMSO (vehicle), 100 µM 2’2’dipyridyl, or 0.1% bile salts. Expression of rpoE was induced with 100 µM IPTG.
The supernatants were separated from bacterial cells by centrifugation at either
30 min-post-exposure (mid-logarithmic phase) or 16 h (late stationary), filtered,
and the serine- and metallo-protease activities were determined using specific
fluorogenic substrates as described in Supplementary Methods. The increase in
fluorescence, which corresponds to the protease activity, was monitored at 37ºC
every min during 10 min after the addition of the substrate and normalized by
optical density of the cultures. Each bar represents the mean and SEMs (n=3).