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Systems biology of bacterial persistence, a metabolism-driven strategy for survivalRadzikowski, Jakub

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Chapter 5

Low quorum and/or surface contact abolish

persister formation after a nutrient shift

Jakub Leszek Radzikowski, Sebastiaan P. van Kessel & Matthias Heinemann

Molecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands

Highlights

• The fraction of persisters generated upon a nutrient shift spans over 4 orders of magnitude in a quorum-dependent manner.

• CO2 concentration in the medium could be the quorum signal that influences persister formation.

• Surface contact, or a factor specific to microfluidics, abolishes persister formation upon nutrient shift.

• RcsC could play a role in abolishing persistence in microfluidics, and in CO2-dependent quorum signalling in batch cultures.

Author contributionsJR and MH conceived and designed the study, and wrote the manuscript with input from all authors. JR and SK performed experiments and analyzed the data.

Abstract

Rapid environmental changes, such as nutrient shifts, can induce bacterial persistence. By coincidence, we found that the amount of persister cells formed upon a nutrient shift relies on quorum, sensed through a yet-unknown quorum sensing system. This quorum sensing can change the fraction of generated persisters over 4 orders of magnitude. We also found that persister formation upon the same nutrient shift is abolished in a microfluidic setup where surface contact is involved. Both of these effects could be mediated by the accumulation of CO2 produced by bacteria, and possibly sensed through RcsC, a sensory histidine kinase previously thought to be involved in surface sensing. While more work is necessary to confirm this finding, involvement of CO2 as a quorum signal in persister formation could explain why certain anti-persister therapies work and provide new possibilities for persister eradication.

264 Chapter 5

IntroductionBacteria, such as Escherichia coli, typically reside in constant-

ly changing environments. Upon nutrient shifts, it can happen that

a population of genetically identical cells splits into two phenotypi-

cally different subpopulations through a process called responsive

diversification (Kotte et al., 2014). One of the sub-populations con-

sists of growing cells. The other consists of persister cells. Two pos-

itive feedback mechanisms have been found responsible for this

phenomenon: one that drives the cells to adapt to the new nutrient

(Kotte et al., 2010; Kotte et al., 2014) and one that shuts down cells

to reach the persister state (Radzikowski et al., 2016). The decisive

variable of whether a cell would adopt the normal growth pheno-

type or the persister phenotype is the metabolic flux: both before

and after the nutrient shift (Kotte et al., 2014).

However, as the natural environment is more complex than

the one found in an in vitro batch culture, bacterial persistence

might not only be flux-dependent, but could also depend on other

stimuli (Amato et al., 2014; Kaldalu et al., 2016). For instance, persist-

ers often occur in biofilms, where an extracellular matrix provides

a mechanical stimulus, and changes diffusion constants, as summa-

rized in a recent review (Billings et al., 2015). While persister for-

mation in biofilms can have grounds in metabolic flux perturbations

(Amato and Brynildsen, 2014), it could be other stimuli that trigger

persistence, such as the attachment of the microorganism to a sur-

Low quorum and/or surface contact abolish persister formation after a nutrient shift

265

face or quorum sensing. Moreover, concentration of gasses such as

oxygen, nitric oxide, ammonia or carbon dioxide, might be different

than in a liquid environment due to different diffusion constants and

the properties of the biofilm extracellular matrix.

Concentrations of volatile gasses are known to affect E. coli

metabolism and persistence. Oxygen availability, sensed through

a system of regulatory mechanisms, determines whether E. coli re-

spires or ferments available sugars (Green et al., 2009). Nitric oxide

(NO) gas, which is produced by mammalian cells as a part of the im-

mune response, was found to reduce the amount of persisters when

it was provided to the cells, by inhibiting respiration (Allan et al.,

2016; Orman and Brynildsen, 2016). Moreover, biogenic ammonia

(NH3) was found to enhance the antibiotic tolerance of E. coli (Ber-

nier et al., 2011). Finally, even small changes (from 0 to 0.01%) in

dissolved CO2 concentration can dramatically affect the doubling

time of E. coli, from 75 minutes at the highest concentration to 275

minutes in conditions without CO2 (Repaske and Clayton, 1978).

In this chapter, we present two circumstantial findings we

made during our research of the phenomenon of responsive diver-

sification in E. coli. We found that persister formation depends high-

ly on a yet-to-be-identified quorum sensing system. This quorum

sensing can change the fraction of persisters generated during a

glucose to gluconeogenic carbon source switch over 4 orders of

magnitude. This quorum sensing does not involve autoinducer-2

(AI-2) signalling, nor indole signalling. We also show that persister

266 Chapter 5

formation is abolished in a microfluidic setup where surface con-

tact is involved. The difference in persister cell formation upon nu-

trient shifts, whether at different cell densities, or in microfluidics,

could be mediated by the accumulation of CO2 produced by bacte-

ria. CO2 concentration could be used as a quorum signal, possibly

sensed through RcsC, a sensory histidine kinase previously thought

to be involved in surface sensing. While further work is necessary

to elucidate how this quorum sensing works on the molecular level,

these findings suggest the existence of a quorum sensing system

that can abolish persistence.

Results

Low cell density abolishes persistence

While researching the phenomenon of responsive diversifi-

cation of E. coli, we found that the duration of the lag phase after a

glucose to 2 g L-1 acetate nutrient shift is dependent on the initial

cell density in the acetate culture (Figure 1A). Note, the length of the

lag phase is a good proxy for the fraction of cells assuming the per-

sister state, meaning the longer the lag phase the higher the frac-

tion of the persisters. The identified correlation between the length

of the lag phase and the initial cell density generalized also to shifts

from glucose to 0.75 g L-1 acetate (Figure 1B). At the high inocula-

tion density, these nutrient shifts were previously found to mediate


267

Figure 1 - Lag phase duration after a glucose to acetate shift cor-relates inversely with the initial cell density. (A) Growth curves of cultures shifted from glucose to 2 g L-1 acetate. Empty squares - cells switched at “low” cell density, about 2 x 105 cells mL-1. Grey squares - cells switched at “medium” cell density, about 2 x 106 cells mL-1. Black squares - cells switched at “high” cell density, about 2 x 107 cells mL-1. Lines - smoothing splines fitted to the data. (B) Lag phase duration after a glucose to acetate shift, depending on the initial cell density. See Experimental Procedures for the method of determining the lag phase duration. Empty squares - shift from glu-cose to 2 g L-1 acetate. Empty disks - shift from glucose to 0.75 g L-1 acetate.

B

1

10

0 2 4 6 8 10

no

rma

lize

d c

ell

co

un

t

time [h]

A5 -1

~2x10 cells mL6 -1

~2x10 cells mL7 -1

~2x10 cells mL

0

1

2

3

4

5

6

7

lag

tim

e [

h]

-1initial cell density [mL ]

510 6

107

10 810

low medium high

inoculation density

268 Chapter 5

25.2% ± 3.7% (SD) and 50.5% ± 4.8% (SD) of the total population of

cells to adapt to the new carbon source, for shifts to 0.75 g L-1 and 2

g L-1 acetate, respectively (Kotte et al., 2014).

To determine whether indeed the fraction of persisters would

change after a nutrient-shift in a cell-density dependent manner, we

turned to glucose to 2 g L-1 fumarate shifts, where we had an exper-

imental method to precisely determine the persister fraction. Shifts

to fumarate cause lower alphas, i.e. the fraction of persisters, com-

pared to shifts to acetate, and these lower fractions (alpha values)

are easier to quantify. Also here, we found that alpha was depended

on the initial cell density (Figure 2A). The slope of a linear equation

fitted to the log-transformed data from Figure 2A (see inlay figure)

was significantly different from 0 (p-value < 0.001), which con-

firms the significance of this dependence. Remarkably, we found

that about the same absolute number of cells adapted to the new

carbon source regardless of the initial cell density (Figure 2B), as

indicated by the fact that the slope of a linear regression fitted to

the log-transformed data from Figure 2B (see inlay figure) was not

significantly different from 0 (p-value = 0.536).

These experiments showed that there is a dependence of al-

pha, i.e. a dependence of the formed fraction of persisters, on the

initial cell density after a nutrient shift. As we found this to happen

on both fumarate and acetate, this dependence seems to be carbon

source-independent. The relation of alpha on the initial cell densi-

ty could be mediated by some form of quorum sensing: switching


269

Figure 2 - Fraction of adapting cells (alpha) after a glucose to fumarate shift correlates inversely with the initial cell density. (A) The fraction of adapting cells changes depending on the ini-tial cell density after a glucose to 2 g L-1 fumarate shift. The frac-tion of adapting cells was determined using an algorithm fitting a growth-dependent bimodal Gaussian distribution to the cell count and fluorescence intensity data, as described before (Kotte et al. 2014). Black squares - individual shift experiments. (B) The absolute number of adapting cells seems to be constant regardless of the initial cell density after a glucose to 2 g L-1 fumarate shift. The inlay figures show the same data, but log transformed and with a linear model (black line) with a 95% confidence interval (dashed black line) fit to the data.

0.0001

0.0010

0.0100

0.1000

1.0000

10

100

1000

fract

ion o

f adapting c

ells

(alp

ha)

abso

lute

num

ber

of adapting c

ells

-1 (

in thousa

nds)

[m

L]

A Blow medium high

inoculation density


410

510 610

710 810


4105

10 6107

10 810

4 5 6 7 8-4

-2

0

log(initial cell density)

log

(alp

ha

)

-1

-3

3

4

5

log

(ad

ap

ting

ce

lls)

4 5 6 7 8log(initial cell density)

low medium high

inoculation density

270 Chapter 5

more cells to the new carbon source, i.e. a higher quorum, would

mediate a lower fraction of cells adapting to this new carbon source.

Autoinducer-2 and indole quorum sensing do not affect quorum-dependent persister formation

The quorum-dependent persister formation could be modu-

lated by one of the known quorum sensing systems in E. coli, such

as the indole quorum sensing, shown to be involved in regulation

of persistence, or the system dependent on the Autoinducer-2 (AI-

2) signalling molecule. Thus, we tested different knock-out strains

to test the involvement of these systems. The first three knock-out

strains disabled the AI-2 system: ΔluxS is unable to produce the AI-2

signalling molecule (Surette et al., 1999), ΔlsrK is unable to take up

the AI-2 signalling molecule (Pereira et al., 2012), and in ΔlsrR the

AI-2 system is always active (i.e. never repressed) (Li et al., 2007).

The other two knock-out strains impaired the indole quorum sens-

ing system: Δmtr is impaired in indole transport (Pinero-Fernan-

dez et al., 2011), and ΔtnaA does not produce indole (Li and Young,

2013).

In order to determine whether these strains abolish the rela-

tionship between the duration of the lag phase and initial cell densi-

ty, we switched these strains from glucose to acetate at different cell

densities. We found that lack of the AI-2-mediated quorum sensing

did not abolish the lag phase duration dependence on the initial cell


271

Figure 3 - Known quorum sensing systems are not responsible for the lag phase - initial cell density correlation after a glucose to acetate shift. (A) Knock-outs of genes mediating AI-2 quorum sensing do not affect the dependence of lag phase duration on the initial cell density after a shift from glucose to 0.75 g L-1 acetate. Open disks - wild-type strain. (B) Knock-outs of genes mediating indole quorum sensing do not affect the dependence of lag phase duration on the initial cell density after a shift from glucose to 2 g L-1 acetate. Open squares - wild-type strain.

lag

tim

e [

h]

lag

tim

e [

h]

A B


510 610

710 810


510 610

710 810

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

8low medium high

inoculation density

low medium high

inoculation density

ΔluxSΔlsrRΔlsrK

ΔmtrΔtnaA

272 Chapter 5

density (Figure 3A). Consistent with the fact that indole should not

be produced in absence of exogenous tryptophan (Li and Young,

2013) (which is not present in the M9 minimal medium we use), we

also did not find any effects of the perturbations of the indole-me-

diated quorum sensing: the lag phase duration dependence on the

initial cell density (Figure 3B) was still present. Because of that, we

aimed to look for another quorum sensing molecule.

Carbon dioxide could function as a quorum sensing molecule

For a molecule to act as a quorum sensing molecule, it needs

to be produced by the cells, secreted into the medium and accumu-

lated in the medium. Thus, towards searching for another potential

quorum mediator, we turned to CO2, which is a metabolite that is

always produced and secreted when E. coli grows in the presence of

oxygen. In contrast to other gases, such as O2 or NO, CO2 is special

as once it is dissolved it can be converted into other species and

accumulate. For instance, in M9 medium buffered to pH 7, CO2 accu-

mulates in the form of the non-volatile hydrogen carbonate, HCO3-.

Other species of CO2 that could also be present in the medium in-

clude carbonic acid H2CO3 and carbonate ion CO32-. Here, we will

use the term CO2 to refer to all the species that can be present in

the medium.

In order for CO2 to play a role in the observed relationship

between the fraction of adapting cells and the initial cell density,


273

the concentration of CO2 would need to change in the medium after

a nutrient shift in a cell-density dependent manner. To determine

whether CO2 accumulates after a nutrient shift, we shifted cells

from glucose to fumarate at the low, medium and high cell densi-

ties (cf. Figure 2A). In these experiments, using a prototype CO2

sensor, we obtained some indication that there was an increase in

CO2 concentration after shifts performed at the medium and high

initial cell density (Figure 4A-C), although the sensor worked far

from optimal and we were not able to accurately determine the in-

crease in the dissolved CO2 concentration. However, the observed

CO2 accumulation in the first two hours after a nutrient shift would

be consistent with the fact that there is biomass increase (reductive

division) during that period. Thus, despite the poor quality of the

CO2 measurements due to the prototype nature of the sensor, these

experiments provided an indication that the CO2 indeed accumu-

lates in the medium. As such, CO2 could serve as quorum signal

determining the responsive diversification behaviour of the cells.

In order to determine whether elevated CO2 levels are in-

deed the signal for the altered persister formation, we switched the

cells from glucose to fumarate while bubbling synthetic air (21%

O2, 79% N2) that either contained 200 ppm CO2 or 600 ppm CO2

through the medium. We also bubbled ambient air through the me-

dium. Ambient air in our laboratory contained varying amounts of

CO2, depending on the day and the time of day, but between 400

and 600 ppm. Bubbling gas through the culture medium facilitates

274 Chapter 5

time

aft

er

shift

[h

]

normalized CO2 sensor signal [a.u.] 0

.90

0.9

5

1.0

0

1.0

5

1.1

0

0.0

0.2

0.4

0.6

0.8

A

5-1

ab

ou

t 2

x 1

0 c

ells

mL

time

aft

er

shift

[h

]0

.00

.20

.40

.60

.8tim

e a

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r sh

ift [

h]

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ab

ou

t 2

x 1

0 c

ells

mL

7-1

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x 1

0 c

ells

mL

0.0

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0.0

010

0.0

100

0.1

000

1.0

000

fraction of adapting cells (alpha)

BC

DE

-1in

itial c

ell

densi

ty [m

L]

410

510

610

710

810

gas tank orvacuum pump

~50

mbar

37°C

incu

bato

r

low

me

diu

mh

igh

ino

cula

tion

de

nsi

ty

20

0p

pm

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2

am

bie

nt

air

60

0p

pm

CO

2


275

Fig

ure

4 -

Dis

solv

ed c

arb

on d

ioxi

de

con

cen

trat

ion

cou

ld b

e th

e si

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al m

edia

tin

g t

he

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ha

dep

end

ence

on

th

e in

itia

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ll d

en-

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aft

er a

glu

cose

to

2 g

L-1

fu

mar

ate

shif

t. (

A-C

) A

pro

toty

pe

sens

or in

dic

ates

that

dis

solv

ed C

O2 a

ccum

ulat

es in

the

cult

ure

me-

diu

m a

fter

a s

hift

fro

m g

luco

se t

o 2

g L

-1 f

umar

ate

usin

g “

med

ium

” an

d “

hig

h” c

ell

den

sity

, but

eve

ntua

lly n

ot a

fter

a s

hift

usi

ng “

low

” ce

ll d

ensi

ty. T

he in

crea

se in

CO

2 co

ncen

trat

ion

is a

pp

aren

t in

pan

-el

s B

and

C. D

ata

from

2 (

A),

3 (B

) an

d 2

(C

) re

plic

ates

(in

div

idua

l sh

ift e

xper

imen

ts, i

ndic

ated

by

dif

fere

nt c

olou

rs),

norm

aliz

ed t

o a

read

ing

tak

en j

ust

bef

ore

the

add

itio

n of

cel

ls. T

he d

ata

req

uire

d

norm

aliz

atio

n b

ecau

se o

f lo

ng-t

erm

dri

fts

and

ins

tab

ility

(ov

er t

he

cour

se o

f ho

urs)

of

the

sig

nal

obta

ined

fro

m t

he p

roto

typ

e se

nsor

. In

itia

lly th

e ex

per

imen

ts w

ere

per

form

ed a

t low

sam

plin

g r

ates

, as

visi

ble

for

the

low

cel

l den

sity

dat

a, a

nd la

ter

at h

igh

sam

plin

g r

ates

as

vis

ible

for

the

med

ium

and

hig

h ce

ll d

ensi

ty d

ata.

The

bla

ck li

ne

rep

rese

nts

a sm

ooth

ing

sp

line

fit t

o al

l rep

licat

es f

or th

e g

iven

cel

l d

ensi

ty. (

D)

A s

chem

atic

of t

he e

xper

imen

tal s

etup

use

d to

bub

ble

g

as th

roug

h a

bat

ch c

ultu

re. (

E) B

ubb

ling

air

(wit

h 20

0 p

pm

CO

2, 60

0 p

pm

CO

2 or

am

bie

nt a

ir (

bet

wee

n 40

0 –

600

pp

m),

satu

rate

d w

ith

H2O

and

at

abou

t 50

mb

ar p

ress

ure

dif

fere

nce)

, thr

oug

h a

cult

ure

can

incr

ease

the

frac

tion

of a

dap

ting

cel

ls a

t bot

h “l

ow”

and

“hi

gh”

in

itia

l cel

l den

siti

es a

fter

a s

hift

from

glu

cose

to 2

g L

-1 fu

mar

ate.

276 Chapter 5

the gas exchange by increasing the surface to volume ratio and thus

exerts - to some extent - control over the CO2 concentration in the

medium (Figure 4D).

We found that bubbling any of these three kinds of air through

the medium caused less cells to assume the persister phenotype,

both at high and low cell initial inoculation densities (Figure 4E).

Note that in 5 out of 16 experiments bubbling the gas did not me-

diate a change in alpha. This lack of change was probably caused

by inefficient bubbling, as the pressure difference in the setup was

sometimes difficult to keep constant, and the flexible tubing used

for bubbling was sometimes prone to lift out above the surface of

the medium. The fact that we did not observe a difference between

bubbling air with 200 ppm, 600 ppm CO2 or the ambient air (with

400 to 600 ppm) suggests that possibly by bubbling air we remove

another volatile compound from the medium. It could also mean

that at all inoculation densities the dissolved CO2 would accumu-

late to concentrations higher than 600 ppm after a nutrient shift.

While shaking 4 200 mL cultures of E. coli grown on LB medium in

our shaker, we observed the CO2 concentration in the shaker to rise

to values over 1000 ppm, which would suggest that the CO2 con-

centration in the flask could accumulate to higher concentrations

than the ones we have tested. Still, it seems that the dissolved CO2

concentration could be the signal that mediates the quorum-depen-

dent persister formation.


277

A factor specific to a microfluidic device abolishes persistence

Next to the eventually CO2-dependent quorum sensing ef-

fect on the fraction of persisters formed upon a nutrient shift, we

could also dramatically influence the persister formation in a differ-

ent way. While attempting to observe the phenotypic diversification

of E. coli upon a nutrient shift on the single-cell level, using a micro-

fluidic device (Figure 5A-B), we found that almost all cells switched

to the new carbon source during a glucose to fumarate shift (Fig-

ure 5C), meaning that in the microfluidic setup less persisters were

formed. The conditions in the microfluidic device are inherently

different than the conditions in a batch culture. For instance, the

presence of a surface, or the frequent light exposure could have

caused a change in the bacterial behaviour. Moreover, the constant

provision of fresh medium would make the conditions similar to the

conditions in a batch culture with very low cell density and would

prohibit quorum sensing (cf. Figure 2A). Therefore, we set out to

determine which of these factors abolishes persistence.

To exclude the effect of frequent light exposure (i.e. every 10

minutes), we imaged the cells less frequently (i.e. every 2 hours).

Here, we did not find any difference between normal and low ex-

posure conditions (Figure 5C). Thus, frequent light exposure is not

the cause for the altered behaviour in the microfluidic setup. To test

whether quorum sensing could be responsible for the effect ob-

278 Chapter 5

Figure 5 - Performing a nutrient shift in a microfluidic setup abolishes persistence, possibly through RcsC signalling. (A-B) The microfluidic setup used in experiments, with syringe pump (A) or attached batch culture and peristaltic pump (B). (C) In a microflu-idic setup, almost all cells assume the growing phenotype, regard-less of the frequency of light exposure (every 10 minutes or every 2 hours - “low light”) or whether the chip is connected to the flask. The rcsC knock-out strain restores persistence in a microfluidic set-up. Empty disks - measured fractions of adapting cells in individual experiments after a glucose to 2 g L-1 fumarate shift. Full disks - measured fractions of adapting cells in individual experiments after a glucose to 2 g L-1 acetate shift. The experiment with least cells included 70 single colonies, the experiment with most cells includ-ed 297 single colonies. All cells in all experiments were included in the analysis.

A

C

0.0001

0.001

0.01

1

batch culture microfluidic setup

0.1

WTlow

inoc.dens.

WTmedium

inoc.dens.

WThighinoc.dens.

WT WTlowlight

WTwithflask

ΔrcsC ΔrcsCglucose

to acetate

fra

ctio

n o

f a

da

ptin

g c

ells

(a

lph

a)

PDMS

polyacrylamide gel

E.coli cell

cover slip

0

magneticmixer

peristalticpump

batchculturemedium

freshmedium

syringepump

B


279

served in microfluidics, we performed experiments, in which we

fed the cells in the microfluidic chip with medium coming from a

flask containing cells. In this flask, we performed a switch from glu-

cose to fumarate at high cell density. In such a setup setup, if there is

a quorum sensing molecule generated in the batch culture, it would

be provided to the cells in the microfluidic chip. Here, we again did

not find any difference between this and the normal condition (Fig-

ure 5C). Thus, at a first glance, classical quorum sensing utilizing

non-volatile signalling molecules seems to not be responsible for

preventing persister formation in microfluidics, consistent with the

experiments performed in batch cultures inoculated at different

initial densities. Yet, because of the gas permeability of the used

tubing, which is made of PTFE (polytetrafluoroethylene), as well as

of the microfluidic chip made out of PDMS (polydimethylsiloxane),

which could prevent CO2 accumulation in the chip, this result does

not exclude the possibility that a volatile compound, possibly CO2,

mediates a quorum sensing effect in the microfluidic chip.

To determine whether the presence of a surface changes the

fraction of persisters formed in a microfluidic setup, we turned to

test various molecular systems that could be involved in surface

sensing. During previous research during a master project (Radzi-

kowski, 2011), we excluded several mechanisms, with which bacte-

ria could sense the surface and alter the fraction of adapting cells

upon a nutrient shift. Specifically, we tested knock out strains of the

flagellar system, which is involved in mechano-sensing (ΔmotB,

280 Chapter 5

ΔfliL, ΔflhD) (Lele et al., 2013), the Cpx system implicated in sur-

face sensing (ΔcutF) (Otto and Silhavy, 2002), and mechanosensitive

channels (ΔmscL, ΔmscS). These channels do not sense surface di-

rectly, but could be activated upon surface-induced envelope stress

and allow for increased metabolite influx. However, none of these

mutants showed an altered phenotype, compared to the wild-type

strain.

Finally, we turned to investigate the Rcs system, which has

been found to respond to solid surfaces. The Rcs system relies on

RcsC, a sensory histidine kinase that is required for biofilm forma-

tion and which responds to an unknown surface-related stimulus

(Ferrieres and Clarke, 2003). RcsC phosphorylates RcsB via another

protein, RcsD (Takeda et al., 2001). RcsB is a transcription factor that

is activated by phosphorylation, and in tandem with RcsA, regulates

the transcription of various genes for instance involved in cell di-

vision, motility and biofilm formation. The role of the Rcs system

in Enterobacteria has been summarized in a review (Huang et al.,

2006).

To test the involvement of the Rcs system in the regulation of

persistence in response to surface, we switched the ΔrcsC mutant

strain from glucose to fumarate in the microfluidic chip. We found

that none of the observed cells assumed the growing phenotype

upon a glucose to fumarate shift (Figure 5C). Moreover, while in a

glucose to 2 g L-1 acetate shift performed in a flask the fraction of

adapting cells was found to be 0.5 ± 0.048 (SD) (Kotte et al., 2014),


281

we found that in microfluidics during a similar shift in 3 out of 5

replicates none, or almost none of the cells adapted. In the other

replicates, we observed growth of almost all cells (Figure 5C, Sup-

plementary Figure 1), which we attribute to eventual presence of

a contamination of the microfluidic setup with undesired nutrients.

These nutrients would cause the switch resemble a gradual, diauxic

shift rather than a sharp nutrient shift. Gradual diauxic shifts gener-

ate less persister cells (Amato et al., 2013) than rapid nutrient shifts

(Kotte et al., 2014).

Thus, RcsC-mediated surface sensing could be responsible

for the suppression of persistence in microfluidics. However, while

it was shown that RcsC responds to solid surface, the actual signals

to which this protein responds are not known (Ferrieres and Clarke,

2003). In fact, it could also be a gaseous quorum signal. Although

through the above-described experiments, in which we fed the

chip with medium from a batch culture, we excluded a AI-2 or in-

dole quorum signal, these experiments did not exclude a quorum

signal mediated by a highly volatile compound. The reason for this

is that in the utilized microfluidic setup, accumulation of volatile

compounds is unlikely, due to a high surface-to-volume ratio of the

chip and the tubing, and the gas permeability of the used materi-

als. It could be that the RcsC system actually responds to such a

volatile compound, and not necessarily to surface contact as such.

If RcsC responds to a gaseous compound, rather than surface, the

rcsC knock out should also have an effect in batch cultures.

282 Chapter 5

RcsC could sense CO2 and could be responsible for the alpha dependence on quorum

To determine whether a rcsC knock-out would change the re-

lation between alpha and the initial cell density in batch cultures,

we switched the rcsC knock-out strain from glucose to fumarate at

different cell densities. Here, we found that the knock-out strain had

indeed an altered behaviour, and the inverse correlation between

alpha and the initial cell count was abolished (Figure 6A). In fact,

now a positive correlation was found. Instead of more cells adapting

at lower inoculation densities, less cells adapted at lower inocula-

tion density. At the low inoculation density, no growth was observed

for 48 hours, and thus we determined the alpha to be equal to 0.

Moreover, the lag phase duration was elongated for each inocula-

tion density (Figure 6B).

Because we found that in batch cultures where surfaces are

absent the rscC deletion strain has a different phenotype than the

wildtype, we concluded that RscC might be involved in sensing a

signal not specific to surface. It seems thus that the signal for the

activation of RcsC is present also in batch cultures, and possibly, it is

a quorum-dependent signal.


283

Figure 6 - Sensory histidine kinase rcsC knock-out abolishes the inverse correlation between alpha and the initial cell den-sity after a glucose to 2 g L-1 fumarate shift. (A) Alpha values es-timated for the rcsC knock-out strain. For the lowest initial density, no growth was observed for 48 hours and thus the alpha was deter-mined to be equal to 0. (B) Additional data containing growth curves of cultures switched at low, medium and high inoculation density for which alpha could not be estimated due to technical problems with the flow cytometer. Black points - wild type strain, grey and white points - rcsC knock-out strain. Different shades of grey indicate rep-licate experiments. Cell count normalized to first time point in each experiment.

0.0001

0.0010

0.0100

0.1000

1.0000

0fra

ctio

n o

f a

da

ptin

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ells

(a

lph

a)

0.1

1

10

0.1

1

10

0 20 40 60 80

time [h]

0.1

1

10


410

510 610

710 810

A B

no

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nt in

ocu

latio

n d

en

sity

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me

diu

mh

igh

low medium high

inoculation density

ΔrcsC

284 Chapter 5

DiscussionIn summary, we found that the fraction of persister cells that

are generated upon a nutrient shift can span over 4 orders of magni-

tude depending on quorum, but not any of the known quorum sens-

ing systems. We have found indications that CO2 concentration in

the medium could be the quorum signal that influences persister

formation. Moreover, we have found that surface contact, or a factor

specific to microfluidics, abolishes persister formation upon nutri-

ent shifts. A sensory histidine kinase, RcsC could play a role in abol-

ishing persistence in microfluidics, and in CO2-dependent quorum

signalling in batch cultures.

Still, more evidence is needed for these claims to be con-

firmed. For instance, more replicate experiments with bubbling gas

through the medium at high cell densities need to be performed.

Moreover, experiments with synthetic air with an increased CO2

concentration should be done. Increased CO2 concentration should

result in lower alphas, and such a result would provide further ev-

idence for the role of this metabolite in modulating persistence.

Additional experiments in a microfluidic setup with a controlled

atmosphere with increased CO2 concentration could show that it is

the lack of CO2 accumulation, and not solid surface, that abolishes

persistence in microfluidics. Finally, more replicate experiments

confirming the ability of the rcsC knock out to abolish the depen-

dence of persistence on quorum are needed.


285

The mechanism in which rcsC would modulate persistence

can be revealed by studies of strains with the Rcs system always

active or disabled. These strains have been constructed, and test-

ing them also in combination with experiments with modified atmo-

sphere would be the key to understanding this novel mechanism of

quorum sensing. The sensory histidine kinase RcsC has homology

to a kinase from Rhodobacter spp., which is involved in CO2 sens-

ing and regulates alternative CO2 fixation pathways (Mosley et al.,

1994). As such, it could be possible that RscC is part of a CO2 sens-

ing mechanism in E. coli that affects gene expression and prevents

persistence.

Overall, we envision that the dependence of fraction of per-

sisters generated upon a nutrient shift on quorum or the use of a

microfluidic device could be explained as described in Figure 7.

Low quorum, or efficient gas exchange (as in the microfluidic de-

vice) prevents CO2 accumulation. When CO2 accumulates, it could

promote bacterial persistence. CO2 was found to regulate bacteri-

al processes, as well as bacterial growth via various mechanisms.

These mechanisms could involve CO2 interfering with membrane

functioning, acidification of the cytoplasm by dissolved CO2, or di-

rect interactions of CO2 with enzymes, which has been summarized

in a review (Stretton and Goodman, 1998). One of such enzymes

could be the RcsC sensory histidine kinase. Moreover, CO2 accumu-

lation could directly regulate the metabolic flux in two ways: (i) by

changing the reaction rates of enzymes that produce or utilize CO2,

286 Chapter 5

such as the phosphoenolpyruvate carboxylase, or (ii) by changing

the pH in the cytoplasm, which would affect enzymatic activity. Fi-

nally, as suggested in the previous chapter of this thesis, the acidifi-

cation of cytoplasm could directly prevent ppGpp hydrolysis, lead-

ing to enhanced persister formation at higher cell densities when

CO2 accumulates (Figure 7).

If our findings are confirmed, it would mean that CO2 could

be a universal, interspecies quorum signal mediating growth or

persistence. Existence of such a mechanism would have multiple

implications. First, bacteria in presence of other organisms produc-

ing CO2 would decide to enter persistence and survive until they

can outcompete the other organisms. Second, with the CO2 concen-

tration in the atmosphere having risen to 400 ppm due to the action

of humans (Ed Dlugokencky and Pieter Tans, NOAA/ESRL (www.

esrl.noaa.gov/gmd/ccgg/trends/)), the increase in atmospheric

CO2 could have a direct effect on the frequency of occurrence of

persister cells and re-occurrence of infections. Third, the ability to

prevent persistence with low CO2 would also shed some light on

why some therapies are successful in treating persister infections.

Patients with tuberculosis are best treated at high altitude, which

has been attributed to lower oxygen pressure (Murray, 2014), but

could also be attributed to lower concentration of carbon dioxide

in non-urbanized areas (George et al., 2007). Moreover, hyperbaric

oxygen therapy, in which infections can be treated in hyperbaric

chambers in presence of 100% oxygen atmosphere without CO2,


287

seems to enhance the killing of bacteria by antibiotics (Cimsit et

al., 2009). While the effectiveness of these therapies could not be

fully attributed to specific mechanisms, carbon dioxide sensing and

its effect on bacterial persistence would provide an explanation

that could be further used in sensitizing persister cells to antibiotic

treatment.

Figure 7 - Potential regulation of persistence by CO2 accumu-lation. Low quorum, or efficient gas exchange can prevent CO2 ac-cumulation. CO2 accumulation could cause bacterial persistence in several ways. First, CO2 could be sensed by RcsC, or possibly another sensing system that through downstream regulation would prevent persister formation. Second, CO2, could affect metabolic flux either by affecting enzymes that perform metabolic reactions involving CO2. Finally, CO2 could acidify the cytoplasm and as such, could regulate ppGpp hydrolysis, as suggested in a previous chap-ter in this thesis.

CO2

accumulation

RcsC

low quorum

efficientgas exchange(as in microfluidics)

persistence

intracellularpH drop

metabolicflux

288 Chapter 5

ExpErimEntal procEdurES

Bacterial strains used in this study

Strain Source

BW25113 Obtained from (Baba et al., 2006)

BW25113 ΔrcsC Obtained from (Baba et al., 2006)

BW25113 ΔluxS P1 phage transduced into BW25113 from a strain from (Baba et al., 2006), verified with PCR

BW25113 ΔlsrR P1 phage transduced into BW25113 from a strain from (Baba et al., 2006), verified with PCR

BW25113 ΔlsrK P1 phage transduced into BW25113 from a strain from (Baba et al., 2006), verified with PCR

BW25113 Δmtr Obtained from (Baba et al., 2006)

BW25113 ΔtnaA Obtained from (Baba et al., 2006)

Nucleotide sequences for primers used for verification of the mutant strains>luxS-forwardTGACTAGATGTGCAGTTCCTGC>luxS-reverseTTACCGGAGGTGGCTAAATG>lrsK-forwardACTATAACCCAGGCGCTTTCC>lrsK-reverseGCCGAGGATAATCTAATGG>lrsR-forwardCTACGTAAAATCGCCGCTGCTG>lrsR-reverseTATAAACCGAGCGGGCGCAAAG

Media and cultivation

Escherichia coli K12 strain BW25113 was used for all exper-

iments. All experiments were performed using M9 minimal medi-

um, which was prepared as previously described (Kotte et al., 2014)

by mixing all the components of the medium, adjusting the vol-


289

ume with water and filtering through a 0.2 μm polyethersulphone

(PES) bottle-top filter. M9 medium was supplemented with a carbon

source to a final concentration of 5 g L-1 glucose or 2 g L-1 fumarate,

unless indicated otherwise. The carbon source stock solutions were

made by dissolving the carbon source in demineralized water, ad-

justing the pH to 7 with NaOH or HCl, and filtering through a 0.2 μm

PES filter. Cultivations were done in 50 mL of M9 medium in a 500

mL Erlenmeyer flask closed with a 38 mm silicone sponge closure

(Bellco Glass) at 37 °C, 300 rpm, and 5 cm shaking diameter. On the

following day, cells were diluted into a new culture prepared in the

same way as the overnight culture, incubated and further diluted as

needed in order to keep the cells in mid-exponential phase.

Cell staining

1.5 x 109 cells from an exponentially growing culture were

harvested by centrifugation (5 min, 4000 g, 4 °C) in 15 mL conical

tubes. The supernatant was removed by aspiration with a 10-mL

serological pipette. The cells were resuspended in 0.5 mL of dilu-

ent C (Sigma-Aldrich) by pipetting. A freshly prepared mixture of

0.5 mL diluent C and 10 μL of the PKH67 dye (Sigma-Aldrich) was

added to the cells, vortexed for 3 seconds, and incubated at room

temperature for 3 minutes. Afterwards, 4 mL of ice-cold 1% solution

(w/v) of bovine serum albumin in M9 medium was added and the

tube was vortexed again. The cells were then centrifuged (5 min,

4000 g, 4 °C), supernatant was aspirated with a 10-mL serological

290 Chapter 5

pipette, ant the pellet was then re-suspended in 5 mL of ice-cold

M9 medium. This procedure was repeated twice more, except that

the cells were finally re-suspended in 1 mL of ice-cold M9 medium,

after which they were used for further experiments.

Flow cytometric analyses

Cell counts, fluorescence intensity and forward scatter val-

ues were determined with an Accuri C6 flow cytometer. Cells were

diluted to an appropriate density with M9 medium without a car-

bon source directly prior to analysis. The flow cytometer was set to

measure 20 μL volume, with the fluidics setting set to ‘medium’. The

SSC-H and FSC-H thresholds were set to 500 and 8000, respectively,

in order to cut off most of the electronic noise. The Accuri CFlow

Plus software was used for data analysis.

Lag phase duration determination

To determine the lag-phase we fitted an exponential regres-

sion to cell count measurements in the exponential phase of the

growth curve. The lag-phase was determined by calculating the

intersection of the exponential regression and the cell density at

the moment after the reductive division, usually 2 hours after the

nutrient shift.


291

Measurements of dissolved CO2 concentration

Measurements of dissolved CO2 concentration were per-

formed using a prototype CO2 sensor (Presens). Single-use self-ad-

hesive fluorescent spots were placed on the side of an Erlenmeyer

flask. Then, with a special harness, an optical fibre was attached to

the flask. Through this fibre, the light exciting the spots and the fluo-

rescent emission of the spots was transferred between the flask and

the measuring device. Once measurements were started, the flask,

filled with 50 mL of M9 medium supplemented with 2 g L-1 fumarate,

and with attached sensor was then incubated in a shaker (200 rpm,

3 cm diameter) for 24 hours in order to equilibrate the sensor. Then,

1 mL of inoculum was added. The data was then normalized to a

measurement taken about 1 minute before the inoculation.

Experiments with bubbling air

Experiments with bubbling air were performed in special Er-

lenmeyer flasks, equipped with a GL45 thread on the top of the bot-

tle, and an additional port on the side equipped with a GL25 thread.

A cap with two hose connectors was placed on the top thread, with

a 5 mm silicone hose reaching down to the bottom of the flask. The

side port was closed with a blank cap. Such equipped flask was then

autoclaved. The air was provided through a PVC hose from a gas

tank equipped with a precision valve, at approximately 50 mbar dif-

292 Chapter 5

ference of pressures, or from the outlet of a vacuum pump. The air

provided was first bubbled through 400 mL of water, with the aid of

an aquarium air stone. Then, the cultivation of bacteria and switch

experiments for alpha determination were performed as described

above.

Polyacrylamide gel pads and PDMS flow channel

For the polyacrylamide gel pads, acrylamide solution (3.33

mL 30% acrylamide/bis solution (Bio-rad), 100 μL APS (Bio-rad), 4

μL TEMED (Bio-rad), H2O to 10 mL) was casted into a SDS-PAGE gel

casting system (1.5 mm thickness, Bio-rad). After polymerization,

the gel was cut into squares roughly 10 x 10 mm in size and washed

in 50 mL of M9 medium supplemented with 2g L-1 fumarate, at 4°C

for at least 24 hours. For the PDMS flow channel, 45 g of silicone

elastomer mixed with 4.5 mL of curing agent (Sylgard 184 Silicone

elastomer, Dow-Corning) was poured into a 12 cm glass petri dish

with channel molds made out of 5 layers of centrifuge tube labels

(Tough-Tags, Diversified Biotech) cut into 10 x 1 x 0.2 mm strips.

The petri dish with the polymer was then desiccated in a vacuum

chamber and left to polymerize over the weekend at room tempera-

ture. PDMS was cut into squares, each containing one channel, and

removed from the petri dish. At both ends of each channel holes

were made using a hypodermic needle (18G x 1”, Thin Wall Nee-

dle, Terumo) which tip was flattened and then sharpened with P800


293

water sandpaper.

Microfluidic device

The microfluidic device was constructed in a stainless-steel

bracket which fitted into the microscope stage. All device parts were

washed with acetone, ethanol and demineralized water. The device

was based on a 24 x 24mm glass cover slip placed in the bracket. 3

μL of cell suspension at OD between 0.01 and 0.1 was placed on the

cover slip and covered with a precisely trimmed polyacrylamide

gel pad. The pad was surrounded with a seal made from a thin layer

of cured PDMS and then covered with a PDMS flow channel (Figure

5A). The whole device was then squeezed and tightened to the met-

al bracket using a plexiglas frame and bolts. The device was fitted

with an inlet and outlet tube (PTFE microbore tubing, 0.3 x 0.76 mm,

Cole Parmer). The outlet tube was directed into a waste container.

The inlet tube was connected to a 50 mL syringe (TERUMO) filled

with M9 medium (optionally supplemented with carbon source) by

a larger tube (PTFE microbore tubing, 0.76 x 2.29 mm, Cole Parm-

er), a gauge luer stub (Instech Solomon) and a 0.2 μm PES syringe

filter. The syringe filter was first used to filter 50 mL of M9 medium

without a carbon source in order to flush out any impurities. The

syringe was then placed in a syringe pump (11 Elite, Harvard appa-

ratus). The flow rate was set to 2 mL h-1 and the device was placed on

the microscope optical table. When external cell culture was used, a

peristaltic pump set at the same flow rate was used to draw the cell

294 Chapter 5

culture medium, with cells, from a 50 mL Erlenmeyer flask closed

with a silicone sponge closure (Bellco glass), and stirred with a

magnetic bar at 300 rpm. The whole setup and the microscope were

incubated at 37 °C.

Microscopy

All microscopy experiments were done using a Nikon Eclipse

Ti microscope (Nikon Ti-E inverted microscopes with either an An-

dor 897 Ultra EX2 EM-CCD camera or Andror LucaR EM-CCD cam-

era, Nikon PFS dynamic focusing system, objective: MRD01901 CFI

Plan Apochromat VC 100XH N.A.1.40 oil, W.D. 0.13 mm, Spring-load-

ed) controlled by NIS Elements (Nikon). For each experiment, ap-

proximately 20 positions were selected. A picture was taken every

10 minutes at each position in Brightfield DIC.


295

AcknowledgementsWe thank the Single Molecule Biophysics and Molecular Bio-

physics groups at the University of Groningen for allowing us to use

their equipment.

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Kotte, O., Volkmer, B., Radzikowski, J.L., and Heinemann, M. (2014). Phenotypic bistability in Escherichia coli’s central carbon metabolism. Molecular Systems Biology 10, 736.Kotte, O., Zaugg, J.B., and Heinemann, M. (2010). Bacterial adaptation through distributed sensing of metabolic fluxes. Molecular Systems Biology 6, 355.Lele, P.P., Hosu, B.G., and Berg, H.C. (2013). Dynamics of mechanosensing in the bacterial flagellar motor. Proc. Natl. Acad. Sci. U. S. A. 110, 11839-11844.Li, G., and Young, K.D. (2013). Indole production by the tryptophanase TnaA in Escherichia coli is determined by the amount of exogenous tryptophan. Microbiology-Sgm 159, 402-410.Li, J., Attila, C., Wang, L., Wood, T.K., Valdes, J.J., and Bentley, W.E. (2007). Quorum sensing in Escherichia coli is signaled by AI-2/LsrR: Effects on small RNA and Biofilm architecture. J. Bacteriol. 189, 6011-6020.Mosley, C., Suzuki, J., and Bauer, C. (1994). Identification and Molecular-Genetic Characterization of a Sensor Kinase Responsible for Coordinately Regulating Light-Harvesting and Reaction-Center Gene-Expression in Response to Anaerobiosis. J. Bacteriol. 176, 7566-7573.Murray, J.F. (2014). Tuberculosis and High Altitude Worth a Try in Extensively Drug-Resistant Tuberculosis? American Journal of Respiratory and Critical Care Medicine 189, 390-393.Orman, M.A., and Brynildsen, M.P. (2016). Persister formation in Escherichia coli can be inhibited by treatment with nitric oxide. Free Radical Biology and Medicine 93, 145-154.Otto, K., and Silhavy, T. (2002). Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc. Natl. Acad. Sci. U. S. A. 99, 2287-2292.Pereira, C.S., Santos, A.J.M., Bejerano-Sagie, M., Correia, P.B., Marques, J.C., and Xavier, K.B. (2012). Phosphoenolpyruvate phosphotransferase system regulates detection and processing of the quorum sensing signal autoinducer-2. Mol. Microbiol. 84, 93-104.Pinero-Fernandez, S., Chimerel, C., Keyser, U.F., and Summers, D.K. (2011). Indole Transport across Escherichia coli Membranes. J. Bacteriol. 193, 1793-1798.Radzikowski, J.L. (2011). Investigating the effect of solid surface on Escherichia coli metabolism. Master Thesis, Molecular System Biology group at the University of Groningen.Radzikowski, J.L., Vedelaar, S., Siegel, D., Ortega, ÁD., Schmidt, A., and Heinemann, M. (2016). Bacterial persistence is an active σS stress response to metabolic flux limitation. Mol Syst Biol 12, Repaske, R., and Clayton, M. (1978). Control of Escherichia-Coli Growth by CO2. J. Bacteriol. 135, 1162-1164.Stretton, S., and Goodman, A. (1998). Carbon dioxide as a regulator of gene expression in microorganisms. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology 73, 79-85.Surette, M., Miller, M., and Bassler, B. (1999). Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: A new family of genes responsible for autoinducer production. Proc. Natl. Acad. Sci. U. S. A. 96, 1639-1644.Takeda, S., Fujisawa, Y., Matsubara, M., Aiba, H., and Mizuno, T. (2001). A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC -> YojN -> RcsB signalling pathway implicated in capsular synthesis and swarming behaviour. Mol. Microbiol. 40, 440-450.


297

Supplementary figures

Supplementary Figure 1 - Growth curves of rcsC knock-out strain cells switched from glucose to acetate in a microfluidic setup.

Areas of cell colonies were measured and normalized to t=0. At least 12 cells measured for each replicate. Black dots - individual cell colony areas. Black line - a smoothing spline fit to the data.

no

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lize

d c

ell

colo

ny

are

a [

a.u

.]

0.5

1

5

10

time [h]0 4 8 12

time [h]0 4 8 12

time [h]0 4 8 12

time [h]0 4 8 12

time [h]0 4 8 12

ɑ = 0.006 ɑ = 0 ɑ = 0.898 ɑ = 0 ɑ = 0.788

university of groningen systems biology of bacterial ... · the log-transformed data from figure 2b...

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