Fluoride Ion Trapping in Bacteria
Under Acidic Environment and
F
Quaternary Structure of CLC -Type
Membrane Transporter
Master's Thesis
Presented to
The Faculty of the Graduate School of Arts and Sciences
Brandeis University
Department of Biochemistry
Christopher Miller, Advisor
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
in
Biochemistry
by
Chunhui Ji
May 2014
Copyright by
Chunhui Ji
© 2014
ABSTRACT
Fluoride Ion Trapping in Bacteria Under Acidic Environment
and Quaternary Structure of CLCF-Type Membrane Transporter
A thesis presented to the Biochemistry Department
Graduate School of Arts and Sciences
Brandeis University
Waltham, Massachusetts
By Chunhui Ji
Microorganisms face environmental threats from fluoride anions (present at 100μM in soil)
that inhibit enolase and pyrophosphatase (Ki~100μM), and it was believed that fluoride toxicity
could be more troublesome under acidic pH level, because membrane permeant HF (pKa ~3.4)
from the acidic extracellular fluid diffuses into the cytoplasm where it dissociates at the neutral
pH, and F- becomes trapped and accumulates. This pH-driven anion accumulation effect is
[!! ]
thought to be governed by the relationship [!! ] !" =
!"#
[!! ]!"#
[!! ]!"
. We sought to directly test the validity
of this thermodynamic principle in E. coli and measure whether this F- ion trapping mechanism
governs the F- load that bacteria endure under acid stress. Using the F--selective LaF3/EuF3
electrode, we continuously monitored external F- concentrations of cultures of E. coli with its
native F- channel knocked out (ΔFluc) during external pH changes and found that under weakly
acidic environment, fluoride anions accumulate in E. coli lacking F- efflux pathways, and the
actual cytoplasmic F- concentrations are in accord with predictions from the weak acid
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accumulation hypothesis [! ! ]!" = [! ! ]!" ×10!"!" !!"!"# . This F- accumulation reaches
equilibrium in ~30 min, has bacteriostatic effects on growth, and prolongs the lag-time before
bacterial growth recovers after removal of acidic stress. The same F- dependent delay on growthrecovery were observed for ΔFluc cells after 2 hours of extreme acid stress (pH 2.5), suggesting
that fluoride anion accumulation in bacteria also holds under extremely acidic environments.
We found representatives from the two families of F− membrane transport proteins—
Fluoride anion channel (Fluc) and CLCF-type F−/H+ antiporter (Pst from Piruella staleyi)–were
both effective at preventing cytoplasmic F- accumulation. Furthermore, amino group crosslinking
by glutaraldehyde treatment of S214K mutant of Pst homologue revealed the homodimeric
structure of the CLCF subclade, in harmony with the dimeric quaternary structure of canonical
CLC transporters.
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Table of Contents
Abstract...........................................................................................................................................iii
Table of Content..............................................................................................................................v
List of Figures and Tables.............................................................................................................vii
List of Abbreviations......................................................................................................................ix
Introduction......................................................................................................................................1
I. Fluoride Anion in the Environment..................................................................................1
I. Fluoride Ion Trapping...................................................................................................... 2
III. Fluoride Efflux Pathways..............................................................................................4
i. F--specific Fluc Channels .....................................................................................6
ii. CLCF-type F−/H+ Antiporters...............................................................................6
IV. Lanthanum Fluoride Ion-Selective Electrode................................................................8
Results and Discussions.................................................................................................................11
I. Fluoride Toxicity vs. Fluoride Resistance......................................................................11
II. Fluoride Ion Trapping in E. coli....................................................................................12
III. Effects of F- Accumulation: Survival vs Growth Lag..................................................16
i. Weakly Acidic Environment: pH 5-6.5..............................................................16
ii. Extreme Acid Environment: pH 1.5-2.5 .........................................................19
IV Structure and Selective Mechanism of F- Transporters ...............................................21
i. CLCF Quaternary Structure: Glutaraldehyde/ cross-linking CLC-pst…………21
Conclusions....................................................................................................................................23
Materials and Methods...................................................................................................................25
I. Reagents..........................................................................................................................25
II. E. coli cell strains and Fluc/CLCF constructs.............................................................. 25
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III. F- Toxicity Rescue. .................................................................................................... 25
IV. F- Accumulation and E. coli Survival in Weakly Acidic Environment...................... 27
V. E. coli Growth Recovery After Weak Acid Fluoride Accumulation...................... ….28
VI. E. coli Survival and Growth Recovery After Acid-Shock.....................................….28
VII. Protein Purification and Liposome Reconstitution. .............................................….28
VIII. Glutaraldehyde Cross-linking..............................................................................….29
References......................................................................................................................................31
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List of Table and Figures
Figure 1. Survey of fluoride levels in the living environment.........................................................1
Figure 2. Weak acid accumulation mechanism of fluoride anion inside membrand bound
organisms.........................................................................................................................................3
Figure 3. Phylogeny of the CLC superfamily..................................................................................5
Figure 4. Two unrelated families of F− membrane transport proteins.............................................5
Figure 5. Structure of the Cl-/H+ antiporter CLC-ec1.......................................................................7
Figure 6. Lanthanum Fluoride ion-selective electrode....................................................................8
Figure 7. Rescue of E. coli from F- toxicity at pH 7......................................................................12
Figure 8. F- toxicity for E. coli as a function of external pH.........................................................13
Figure 9. pH-dependent F--uptake by live E. coli cells..................................................................14
Figure 10. Total amount of F--uptake by live E. coli cells as a function of extracellular pH and
total F- concentration......................................................................................................................15
Figure 11. Thermodynamic relationship between E. coli’s internal F- accumulation levels and
extracellular pH..............................................................................................................................16
Figure 12. Effects of F- weak acid accumulation on bacterial survival.........................................18
Figure 13. Effects of F- weak acid accumulation on bacterial growth recovery............................18
Figure 14. Effects of a 2-hour Acid-Shock containing F- on bacterial survival............................20
Figure 15. Effects of a 2-hour Acid-Shock containing F- on bacterial growth recovery...............20
Figure 16. Tertiary homology structure of CLC-pst monomer in cartoon representation showing
amino groups.................................................................................................................................22
Figure 17. Pst architecture. .........................................................................................................22
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Table 1. Summery of E. coli cell strains .......................................................................................25
Figure 18. Fluc-ec2 construct........................................................................................................26
Figure 19. CLC-pst construct. The position of S214K mutation is indicated...............................27
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List of Abbreviations
AR=Acid Resistane
HF=Hydrofluoric acid
F- =fluoride anion
NaF=sodium fluoride
CLC=Cluoride channel
CLCF=Fluoride specific subclade of the CLC superfamily
Fluc=Fluoride channel
ΔFluc=bacterial stains with its endogenous Fluc gene knocked out
CLC-ec1= from E. coli, Synechocystis sp PCC6803
RNAs
Glu=Glutamate
Ser=Serine
Tyr=Tyrosine
LaF3 =lanthanum fluoride
Pst = CLCF-pst homologue from bacterium Pirellula staleyi
WT E. coli = BW21150 strain that contains the endogenous Fluc gene
LB= luria broth
XAR=Extreme Acid Resistance
AS=Acid-Shock
OD=optical density
S214K=Serine to Lysine mutation at residue 214 (of Pst)
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SDS-PAGE= sodium dodecyl sulfate-polyacrylamide gel electrophoresis
EPL= E. coli mixed phospholipids
POPE = 1-palmitoyl, 2-oleoylphosphatidylethanolamine
POPC =1-palmitoyl, 2-oleoylphosphatidylcholine
POPG = 1-palmitoyl, 2-oleoylphosphatidylglycerol
DM = decylmaltoside
MOPS = 3-(N-morpholino)propanesulfonic acid
CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
BOG = n-octyl-beta-D-glucoside
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Introduction
I. Fluoride Anion in the Environment
Fluoride is the smallest and most electronegative anion in the halides family, and while the
biological importance of other halides, such as chloride and iodide, have been well studied [1-5],
that of fluoride are less understood [6]. Fluoride has been widely used as an additive in oral
hygiene and drinking water since 1950s because of its effectiveness at preventing tooth decay.
Fluoride ions present at millimolar levels in bacterial culture media inhibit cell growth [7-9], and
this has been proposed as one of the mechanisms for it efficacy [10-13]. Fluoride is also
ubiquitous in the environment, where it is found in soil and water [14, 15], and it is a highly
abundant element in the earth’s crust (0.32 g/kg) [16-18]. Typically, the environmental
concentrations of fluoride in groundwater, sea, and soil range between 10-100 μM, and
fluoridation of public water supplies adds 50–100 μM to this [14, 19] (Figure 1).
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Figure 1. Survey of fluoride levels in the living environment. Solid samples were diluted
into recording solution and measurements were made using a fluoride ion-selective electrode.
In the meantime, fluoride is toxic to eukaryotic organisms—1% sodium fluoride (about 250
mM) can kill baker's yeast, or Saccharomyces cerevisiae [20]—and inhibits growth of fungi,
including several pathogens [21, 22]. At high concentration, fluoride also has toxic effects on
bacteria and plants, and past efforts to study this antimicrobial effect have been focusing on the
bacteria that cause dental caries [7, 8, 23].
For animals, F- is excreted through the kidneys and does not pose threats at normal levels
[19], but for microorganisms, fluoride is known to be involved in enzyme inhibition and
interactions with important cations in the cell, including as Mg2+ and Ca2+ [24, 25]. Specifically,
it inhibits (Ki = 100 μM) two enzymes essential for glycolytic metabolism and nucleic acid
synthesis [26, 27]: it inhibits enolase through forming a magnesium-fluoride-phosphate complex
[25] and phosphoryl transfer enzymes, including pyrophosphatase, through complexing with
aluminum and beryllium to act as phosphate mimics [24, 28].
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II. Fluoride Ion Trapping
Besides fluoride threats, microorganisms, such as Escherichia coli, also face other
environmental stresses including fluctuations in external pH. However, when grown over a large
range of pH environment from pH 5 to 9, the enteric bacteria E. coli maintains its cytoplasmic
pH within a narrow range around neutral pH [29-31]. When E. coli experiences rapid external
pH drop, the cytoplasmic pH falls, then largely recovers in less than 1 minute [30, 32]. This
regulation of cytoplasmic pH during acid stress depends on catabolic acid consumption and ion
transport [33], and while no single method appears to be essential for E.coli cytoplasmic pH
homeostasis [33-36], mechanisms such as transmembrane K+ flux and osmoprotection by
osmolytes such as NaCl, KCl, proline, and sucrose, were demonstrated to be important [37].
In addition to cooping with weakly acidic environments, food-borne pathogens must also
develop mechanisms to withstand extreme acidic conditions, including the pH 1.5-2.5 gastric
acidity barrier [38], and as expected, the intestinal microorganism E.coli is very effective at
resisting extreme acid stress. It can survive at pH 2 for hours [39, 40] thanks to three acidresistance (AR) systems it posses: the glucose-repressed AR1 requires stationary phase
alternative sigma factor óS (also known as RpoS) and the global regulatory protein CRP (cAMP
receptor protein) to develop acid tolerance [41], and the glutamate-dependent AR2 and argininedependent AR3 each comprises of dedicated pairs of amino acid decarboxylases and antiporters
to export H+ [35]. When external pH drops below pH 2.5, these AR systems help E. coli maintain
an internal pH level between 4.2-4.7 [42].
For either the mildly acidic (pH5) or the extremely acidic (pH2.5) cases, E. coli’s
cytoplasmic pH can be two orders of magnitude higher than its external pH, and this pH gradient
can be the driving force for intracellular F- accumulation (Figure 2). The neutral HF can diffuse
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! 3!!
!
through the lipid bilayer, whereas F- is repelled by the large Born charging energy resulting from
the low dielectric environment of the membrane [43]. Given a pKa of 3.2 and a 1,000,000X
membrane permeability compared to F-, lowered extracellular pH results in uncharged HF
entering the cells and getting trapped as F- [14, 44] (Eq. 1).
Figure 2. Weak acid accumulation mechanism of fluoride anion inside membrand bound
organisms. pH-driven accumulation of F- in bacteria is a afunctin of the high pKa of HF (3.2),
membrande permeaability of HF, and higher pH inside than outide.
[!! ]!"
[!! ]
!"#
=
[!! ]!"#
Eq. 1
[!! ]!"
[! ! ]!" = [! ! ]!" ×10!"!" !!"!"#
Eq. 2
In human, it has been shown that F- inhibition of ameloblast cell functions is pH-dependent; a
lower pH environment renders it more susceptible to F− toxicity, and this has been proposed as
the basis of fluorosis [44]. The same inferences can be made about microorganisms: an E.coli
cell with neutral cytoplasmic pH experiencing the pH 5 environment of the small intestine,
which contains 100 μM F-, will accumulate 10 mM F- in its cytoplasmic (Eq. 2), unless its
membrane possesses pathways to dissipate the accumulated F-. However, the validity of this
thermodynamic relationship governing weak acid driven fluoride accumulation in
microorganisms has not been tested; the physiological effects of fluoride anion trapping have not
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been characterized, and the mechanisms bacteria employ, if any, to minimize or cope with such
threat are not know.
III. Fluoride Efflux Pathways
Given the potential existence of such a steep F- concentration gradient across the cell
membranes, it is reasonable to expect that either an active F- transporter or a passive membrane
channel would effectively guard the cell against extracellular fluoride threats. Accordingly,
unicellular microorganisms have evolved such F--specific membrane transport proteins. To date,
two phylogenetically unrelated families of F− transport proteins are known: CLCF-type
F−/H+ antiporters, a subclass of the widespread CLC anion-transporter superfamily, and a group
of small membrane proteins known as the “crcB” or “Fluc” family (Figure 3). These F- exporter
proteins are widespread among unicellular organisms and green plants, potentially to minimize
the toxic effects of this anion’s by keeping its concentration in the cytoplasm low [16, 19, 45].
Figure 3. Phylogeny of the CLC superfamily. The clade that comprises F- riboswitch-controlled
CLCs is highlighted in red. Two other clades with available proteins functional or structural data
are: antiporters CLC-ec1 from E. coli, Synechocystis sp PCC6803, and Salmonella enterica in
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light blue and eukaryotic Cl- channels CLC-0, -1, and -2, and the Cl-/H+ antiporter from the
thermophilic alga Cyanidioschyzon merolae in dark blue [19].
Figure 4. Two unrelated families of F− membrane transport proteins. “Fluc”, also known as the
“crcB” family (A), is a groups of small membrane proteins widespread among unicellular
organisms; CLCF-type F−/H+ antiporters (B) are a subclass of the widespread CLC aniontransporter superfamily [45]
i. F--specific Fluc Channels
The discovery of the upstream regulatory RNA motif for this F--specific channel in 2012
shed lights on the mechanism by which cells respond to toxic levels of fluoride anion. These
fluoride-sensing regulatory RNAs, or Riboswitches, bind to F- and regulate the expression of
downstream genes in response to this anion in eubacteria and archaea [6, 46] (Figure 4A). Fluc
is one of these genes originally identified in E. coli, and it encodes a small protein (127 amino
acids) with four transmembrane domains [6].
It is proposed that Fluc functions as a dimer of identical or homologous membraneembedded domains arranged in an inverted-topology fashion, which is similar to several small
multidrug transporters [45]. Deletion of the single Fluc gene in E. coli (ΔFluc) produces
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hypersensitivity to F−, and this growth-phenotype may be rescued by transformation with
F− exporter genes from a variety of bacterial species [16, 19]. Recently, it was found that, as
compared to neutral pH, yeast cells lacking these F- transporter genes experienced greater toxic
effects from environmental F- levels (100 μM) when they were grown in low pH conditions (pH
6.5 or below) [6]. Again, high cytoplasmic F- level due to the pH difference across the lipid
membrane has been proposed to be the culprit. However, we would like to gather direct evidence
of such fluoride anion accumulation in vivo to explain the subsequent cell responses.
ii. CLCF-type F−/H+ Antiporters
The F- hypersensitivity of these ΔFluc E. coli can also be rescued by insertion of a different
family of F- exporters: the CLCF-type proton-coupled antiporters (purified proteins catalyzed Ftransport in liposomes) [19]. Character members of the widespread CLC family are doublebarreled Cl- specific ion channels, but the family contains two mechanistically disparate
subtypes: Cl- anion channels and Cl-/H+ antiporters (including the bacterial Cl-/H+ antiporter
CLC-ec1, the only CLC protein for which a crystal stricture has been solved for) (Figure 5) [4753]. These transporter proteins participate in diverse biological tasks requiring transmembrane
anion conductance, such as acidification of lysosomes, control of skeletal muscle excitability,
renal regulation of blood pressure, and extreme acid resistance in enteric bacteria [54]. Although
functionally diverse, most CLCs are structurally similar and use Cl- as their substrate, but the
riboswitch-controlled CLCF antiporters (Figure 4B) were recently found to have unusual
characteristics: they lack conserved residues (central serine and tyrosine) that form the anion
binding site in canonical CLCs; they exhibit selectivity for F- over Cl-; they bear a channel-like
valine rather than a transporter-like glutamate at a residue thought to distinguish CLC channels
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and transporters; and they bear 1:1 stoichiometry for the F-/H+ exchange rather than the usual
value of 2:1 [19].
Figure 5. Structure of the Cl-/H+ antiporter CLC-ec1—a character member of the double-barreled
Cl- specific ion channels. Ribbon diagram of CLC-ec1 is shown with extracellular side up, Cl−
ions as green spheres. (Left) Rendered in minimally cutaway view to visualize more easily side
chains Gluex at the outer gate and Tyrc and Serc at the inner gate, as indicated [55].
IV. Lanthanum Fluoride Ion-Selective Electrode
The lanthanum fluoride electrode permits continuous monitoring of the fluoride anion
concentration in an aqueous solution, and it has proven to be a very useful technique in our
studies of the F--specific transport proteins. The construction of a lanthanum fluoride electrode
(Figure 6) consists of a fluoride ion-sensitive membrane sealed over the end of an inert plastic
tube, which contains an internal electrode and filling solution: the membrane consists of a single
crystal of lanthanum fluoride LaF3, doped with a divalent ion such as Eu2+ to create lattice
vacancies; the internal electrode would be a silver-silver chloride electrode immersed in a sealed
internal filling solution containing both chloride and fluoride ions. As transference of charge
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through the crystal is almost exclusively due to fluoride, the electrode is highly specific to
fluoride.
Figure 6. Lanthanum Fluoride ion-selective electrode. The internal electrode is a silver-silver
chloride electrode immersed in a sealed internal filling solution containing both chloride and
fluoride ions, and the lanthanum fluoride ion-sensitive membrane consists of a single crystal of
lanthanum fluoride LaF3, doped with a divalent ion such as Eu2+ to create lattice vacancies where
fluoride anions can fit, therefore, the electrode is highly specific to fluoride because transference
of charge through the crystal is almost exclusively due to fluoride.
While the fluoride electrode measures cell potential in mV of an aqueous solution, these
measurements can be directly translated into fluoride anion concentrations in the reaction
chamber through the Nernst equation (Eq. 3) where E is the measured cell potential in mV, E0 is
the standard cell potential, R is the ideal gas constant, T is the temperature in kelvins, F is the
Faraday constant (9.65×104 C·mol−1), and aF− is the activity of the fluoride ion [56].
! = !! −
!"
!
!!"!!!!
Eq. 3
For the purpose of our experiments, a more useful form of the Nernst equation (Eq. 4)
directly associates change in voltage measured in mV (ΔV) and change in F- concentration in the
reaction solution measured in mM (ΔCF-), and CF-0 represent the initial F- concentration in the
reaction solution in mM.
∆! =
!
!"
!
∆!!!
!"!(1 + !
! 9!!
!!
!
)
Eq. 4
!
Since the discovery of these families of F--specific transporters, efforts to characterize their
structure and functions have mainly been focusing on neutral pH, and less is known about the
potentially grievous effects of cytoplasmic F- accumulation when E. coli experiences pH
fluctuations. In addition, study of their importance to bacteria has been based on the assumption
that the weak acid accumulation mechanism of F- depicted in Fig. 2 and Eq. 1 holds under
cellular conditions. We seek to directly test this thermodynamic principle in E. coli and find out
whether the proposed equilibrium relationship suggesting F- ion trapping (Eq. 1) actually
governs the F- load that bacteria endure under acid stress. Relatedly, we want to characterize the
timescale over which F- breaches the cell membrane, the associated physiological impacts, and
the cell’s coping strategies, if any, for this genuine threat. Moreover, it is also important to
investigate if the F- transporters function differently, or as effectively, under acidic conditions
than under neutral pH.
Beyond characterizing their physiological functions under acid stress, it is only reasonable
to continue to seek structural and mechanistic explanations of the functions of these novel
transporters. Specifically, we referenced the CLC-eca homology model to analyze the quaternary
structure of the CLCF-pst homologue (from bacterium Pirellula staleyi, and it shares all other
characteristic traits of the F--specific superfamily, although it is the only CLCF homologue
known to be free of riboswitch-control [19]) as well as examining specific amino acids know to
be crucial in the canonical CLC Cl- pathway, in order to understand of the F--selective pathways
in the CLCF clade.
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Results and Discussion
I. Fluoride Toxicity vs. Fluoride Resistance
It was previously tested that E. coli containing the WT Fluc gene (BW21150 strain) could
withstand up to 5 mM F- in LB supplemented with NaF at pH 7 (Figure 7A) whereas this Fconcentration threshold before E. coli started to experience growth arrest is 50 μM for the ΔFluc
strain (Figure 7B) [19]. Transformation of ΔFluc E. coli with arabinose-induced rescue vector
bearing synthetic CLC-pst gene restored growth under elevated (500 μM) F- conditions,
confirming that the F--transport activity of the CLCF homologue is sufficient to protect bacteria
against F-toxicity (Figure 7C). For Fluc, while purified proteins allowed F- efflux in
proteoliposomes [45], previous attempts to rescue ΔFluc E.coli growth arrest against F- toxicity
with synthetic Fluc homologues were unsuccessful. However, when the C-terminal hexahistidine
tag that comes with commercially available gene construct for the Fluc-Ec2 homologue (from E.
coli virulence plasmid [45]) was eliminated, Ec2 produced successful rescue (Figure 7D). While
this phenomenon confirmed that Fluc alone is sufficient at protecting bacteria against fluoride
toxicity, it also served as evidence that Fluc’s inverted-dimer membrane construction is crucial
for the channel’s F--transporting activity in vivo, and the orientation bias brought upon by the
addition of the C-his tag renders this natural construction.
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Figure 7. Rescue of E. coli from F- toxicity at pH 7. Representative growth curves for WT E.
coli (A), ΔFluc strain transformed with an empty vector (B) with CLC-pst (C) or with Fluc-ec2
(D) in pH 7 LB supplemented with the indicated NaF.
II. Fluoride Ion Trapping in E. coli
To characterize the pH-dependent F- accumulation in bacteria and its physiological
consequences, we first examined the threshold growth-inhibitory F- concentration under different
acidity. Similar to previous observations in yeast cells [6], we found that E. coli lacking the Fluc
gene exhibited increased hypersensitivity to F- toxicity as extracellular pH decreased (Figure 8).
At pH7, ΔFluc E. coli started to experience growth arrest when external F- concentrations
exceeded 50 μM, and this threshold F- concentration lowered to 5 μM at pH 5, while WT cells
showed no growth-inhibition at all conditions tested. ΔFluc E. coli’s elevated hypersensitivity to
F- at low external pH hints pH-driven cytoplasmic fluoride anion trapping, and internal Fconcentration can reach the inhibition threshold (Ki=100 μM) even when external Fconcentration is low.
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Figure 8. F- toxicity for E. coli as a function of external pH. Growth curves for WT E. coli and
ΔFluc strain in LB buffered to the indicated pH and supplemented with the indicated NaF.
To establish the direct relationship between growth arrest and high cytoplasmic F- levels,
we continuously monitored fluoride anion concentration in live bacteria culture at changing
external pH using the lanthanum fluoride electrode, and directly observed the pH-driven Fintake process by ΔFluc cells. Concentrated (10X) ΔFluc E. coli overnight cell culture with 0.4
mM baseline NaF was treated with a 0.1 mM F- calibration shot followed by addition of
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concentrated hydrochloric acid to lower the extracellular pH from 7 to 5.3 (Figure 9). After the
initial drop in extracellular F- concentration due more F- existing in its conjugate acid form (HF)
at lower pH, Fluc-mediated F− efflux immediately equilibrated intracellular and extracellular Flevels for WT cells. In contrast, the ΔFluc culture showed apparent fluoride-uptake as the
external F- concentration decreased and reached equilibrium in 30 minutes.
Figure 9. pH-dependent F- uptake by live E. coli cells. Raw-data traces of extracellular Fconcentration in suspensions of WT (middle) or ΔFluc (bottom) E. coli in response to acid
exposure, and top trace shows buffer only without cells. Data collected with F- electrode. A 0.1
mM calibration pulse of NaF (time zero) and addition of HCl to drop the pH from 7 to 5.5
(arrow) are shown.
We quantified ΔFluc E. coli’s internal F- concentrations at various pH to verify the merits of
the governing equilibrium relationship (Eq. 1). The final cytoplasmic F- level after fluorideuptake can be calculated by factor in the measured change in F- concentration in the 2 mL
reaction solution and the total cell volume (approximately 1% of reaction chamber volume: 0.1
nL/ cell [57] multiplied by 2.7 × 109 viable E. coli cells). Varying the extracellular condition
demonstrated that the magnitude of E. coli fluoride-uptake increased as the magnitude of
external pH-drop from pH7 increased (Figure 10A) and as the total available fluoride level in
the reaction chamber increased (Figure 10B). Knowing that E. coli maintains a neutral internal
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pH when external pH fluctuates within the range of pH 5 to pH 9 [29-31], the ratio of
intracellular to extracellular F- concentrations varied almost exactly in 1:1 proportions with the
ratio of extracellular to intracellular H+ concentrations (Figure 11A), and is independent of the
total fluoride level (Figure 11B). These results fit predictions from the equation
[F-]in = [F-]out×10pHin-pHout, and cytoplasmic did indeed reach 15 mM at pH 5.2 when the
environment only contains 500 μM F- (Figure 11A, inset).
A
B
Figure 10. Total amount of F--uptake by live E. coli cells as a function of extracellular pH and
total F- concentration. Change in extracellular F- concentration in ΔFluc E. coli suspensions in
response to acid exposure. Raw data collected with F- electrode and a 2 mL reaction volume was
used to calculate the changes in F- concentration. (A) Amount of F--uptake by E. coli cells after
addition of HCl (time zero) to drop the extracellular pH from 7 to indicate values, when the total
F- level in the reaction chamber was kept constant at 0.5 mM. (B) Amount of F--uptake by E. coli
cells after addition of HCl (time zero) to drop the extracellular pH from 7 to 5.5, when the total
F- levels in the reaction chamber varied as indicated.
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Figure 11. Thermodynamic relationship between E. coli’s internal F- accumulation levels and
extracellular pH. Internal![F5!]in!and!external![F5!]o!F5!concentrations!of!a!single!bacterium!
were!calculated!from!measuring!total!amount!of!F55uptake!by!all!E.#coli!cells!in!a!2!mL!
reaction!chamber!containing!a!total!of!0.5!mM!(A),!or!indicated!levels!of!(B),!NaF.!
Individual!cell!volume!of!0.1!nL/!cell![57]!and!a!total!of!2.7!×!109!viable!E.#coli!cells!in!the!
reaction!chamber!were!used!in!the!calculations.!The!ratios!of!external!to!internal!H+!
concentrations!([H+!]o!/![H+!]in!in!A)!were!calculated!from!measured!extracellular!pH!values!
and!a!cytoplasmic!pH!of!7![29531].!The!predicted![F5!]in!/![F5!]o!!values!according!to!Eq.!1!
were!plotted!as!two!dashed!lines,!and!the!two!subplots!show!the!actual!internal!F5!
concentrations!bacteria!endure!at!a!given!external!condition.!(A)!The!ratio!of!intracellular!
to!extracellular!F5!concentrations!([F5!]in!/![F5!]o!)!varied!almost!exactly!in!1:1!proportions!
with!the!ratio!of!extracellular!to!intracellular!H+!concentrations,!and!(B)!this!ratio!is!
independent!of!the!total!F5!concentrations!available.!
III. Effects of F- Accumulation: Survival vs Growth Lag
i. Weakly Acidic Environment: pH 5-6.5
Since the predicament of toxic fluoride anion-buildup to above 10 mM when bacteria
lacking F- efflux pathways encounter weakly acidic environment of naturally prevailing Fconcentrations is factual and imminent, we seek to better understand the physiological
consequences and bacteria’s response to this problem. We incubated stationary-phase WT and
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ΔFluc E. coli cultures in pH 5.5 minimal media containing 500 μM NaF for up to 26 hours, and
viable counts at various time points indicated that an extended period of high cytoplasmic Fconcentration did not affect E. coli survival (Figure 12); on the other hand, it did affect E. coli’s
ability to recover growth after removal of the acid and fluoride stresses (Figure 13). Growthphase (pH 7) ΔFluc E. coli culture was treated with the addition of 500 μM NaF (with or without
adding concentrated hydrochloric acid to the lower the extracellular pH from 7 to 5.5) for 2
hours followed by subsequent wash with pH 7 LB to remove excess fluoride; we found that
while E. coli all exhibited growth arrest during fluoride treatment, when excess F- and H+ were
removed form the growth media, E. coli experienced a F- treatment at pH 5.5 showed an almost
1-hour lag before they started exponential growth again, whereas the same ΔFluc cells treated
with F- at pH 7 started exponential growth immediately (Figure 13). Since ΔFluc E. coli lacks a
F- efflux pathway, the lag indicates the time needed for the high concentration (more than 10X)
of F- trapped in the cytoplasm during acid-treatment to leak out in the form of membranepermeable HF. These results suggest that in weakly acidic environments, F-accumulation inside
bacteria cytoplasm over an extended period of time is not bactericidal but bacteriostatic.
However, the residual effect in the form of lag-time in growth creates disadvantages for bacteria
whose survival depends on their ability to quickly colonize the host environment, such as the
human small intestine.
!
! 17!
!
!
Figure 12. Effects of F- weak acid accumulation on bacterial survival. Viable counts of WT and
ΔFluc E. coli cells at various times during a 26-hour incubation of the indicated cells in minimal
media containing 0.5 mM NaF at indicated pH.
Figure 13. Effects of F- weak acid accumulation on bacterial growth recovery. Growth-phase
(OD 1) ΔFluc E. coli culture in pH 7 LB was treated with the addition of 500 μM NaF (at hour
3) and incubated in either pH 7 (top) or pH 5.5 (bottom, by adding HCl) for two hours. The cell
culture was then centrifuged and pellets were re-suspended in fresh LB where growths were
monitored again. E. coli that experienced pH-driven F- accumulation (bottom) showed a 1-hour
lag before they started to growth again.
!
! 18!
!
!
ii. Extreme Acid Environment: pH 1.5-2.5
In addition to coping with the weak acid effects in the duodenum, food-borne bacteria are
also confronted with the harsh pH 2 milieu of the stomach for hours during their passage through
the human digestive tract [35]. It is well-known that E. coli activate the XAR systems to survive
the pH 1.5-2.5 environment, however, there is still a 2-unit pH difference between the
extracellular pH and the pH 4.5 cytoplasmic pH bacteria actively maintain, which serve as the
driving force for F- buildup. It is reasonable to ask if E. coli respond to the combined stress of Fand acid the same way under these extreme conditions. Acid-adapted over-night WT, ΔFluc, and
Pst E. coli cultures were subjected to a 2-hour treatment in pH 2.5 growth media containing
various levels of NaF—fluoride Acid-Shock (AS), after which their survival and growth in
neutral LB media free of F- were monitored. For all 3 strains, the AS challenge alone killed over
95% of the cells, and 100% of the cells died during AS when F- level in AS media reached
10mM. Interestingly, as compared to ΔFluc cells, protection from F- membrane transporters (Pst
and WT) did not improve survival rates when F- concentrations during AS were at intermediate
levels (100 μM and 1 mM) (Figure 14). Again, the strain difference manifested itself in terms of
lag time before growth recovery after the E. coli cells were removed from stressful conditions.
When AS contained 100 μM NaF, ΔFluc E. coli lagged 4-8 hours behind WT and Pst E. coli in
growth-recovery after AS (Figure 15). Although having physiological F- level during a 2-hour
AS does not worsen survival of E. coli lacking F- efflux pathways, the 10-hour lag time before
growth is restored can be detrimental for an enteric bacterium who needs to start dividing as soon
as it moves out of the stomach to colonize the host intestine.
!
! 19!
!
!
%
150"
100"
WT"
ΔFluc"
50"
CLC/pst"
0"
0"
0.1"
1"
10"
[F$]&in&AS&media,&mM
Figure 14. Effects of a 2-hour Acid-Shock containing F- on bacterial survival. Survival counts of
WT, ΔFluc, and Pst E. coli after a 2-hour incubation in Acid-Shock media containing indicated
F- concentrations were standardized against the survival rates of the same cell strain treated with
Acid-Shock that did not contain fluoride.
Figure 15. Effects of a 2-hour Acid-Shock containing F- on bacterial growth recovery. The lagtime before growth recovery after 2-hour incubations of WT, ΔFluc, and Pst E. coli cells in
Acid-Shock media containing indicated F- concentrations. The lag-time was measured by the
additional time it took the E. coli culture to reach OD 1 as compared to E. coli suspended in pH
5.5 minimal media during the two hours.
!
! 20!
!
!
IV Structure and Selective Mechanism of F- Transporters
i. CLCF Quaternary Structure: Glutaraldehyde/ cross-linking CLC-pst
Given the validity of the thermodynamic principles governing the pH-driven cytoplasmic Faccumulation and the consequences of this accumulation on bacteria growth, it is essential for
enteric bacteria that need to quickly colonize the alimentary tract to employ F- efflux pathways.
This demands structural and mechanistic understandings of the two types of F- transporters. So
far, it is know that unlike the double-barreled Cl--specific canonical CLC channels, Friboswitch-controled CLCF homologues lack key Cl--binding residues, select F- over Cl--, bares a
channel-like valine as an antiporter, and exhibit unconventional 1:1 F-/H+ exchange
stoichiometry [19]. Lacking the clarification from a crystal structure, we would like to get a
better sense of the structural features that give rise to these strangest characteristics.
To verify the dimeric structure of CLC-pst, as suggested by size-exclusion chromatography
(Randy 2012), we performed amino-group cross-linking of Pst subunit with glutaraldehyde in
liposomes loaded with 300 mM NaF. Pst contains five amino groups in its native form and
homology model (Figure 16) suggested two solvent-accessible lysine residues were located near
the TM interface (K204 and K373 on the top interface loops), so glutaraldehyde would be
expected to cross-link a parallel dimer. While native Pst showed partial cross-linking after
glutaraldehyde treatment (Figure 17A), the Pst S214K mutant with an additional lysine
substituted on the inter-face top loop lead to almost 100% dimerization, indicated by the intense
80 kD dimer band on the PAGE gel (Figure 17B). With a parallel homodimer structure, the two
newly added lysine would mirror each other at the same position on the TM interface when a
dimer is formed. This suggested the validity of the homology model from Ec1 and served as
proof that CLCF-Pst adopts a parallel-topology homodimer structure.
!
! 21!
!
!
Figure 16. Tertiary homology structure of CLC-pst monomer in cartoon representation showing
amino groups. Extracellular side up in side-view (left) and top-view (right) showing the dimeric
interface helix and loops (yellow) of the Pst monomer. The five naturally occurring lysine
residues (blue) are shown and the Serine in the S214K mutant is shown in pink. This figure was
made using pymol version 1.3. (PDB 4JL6, Cho, Y.-J.).
Figure 17. Pst architecture. 12% SDS-PAGE of WT (A) or S214K (B) Pst in liposomes treated
with indicated levels (0.125-0.5%) of glutaraldehyde for the indicated times. Monomer and
dimer bands are as indicated.
!
! 22!
!
!
Conclusion
The idea of pH-driven accumulation of fluoride anion inside lipid-membrane bound
organisms tested in this thesis study is fundamental to the study of the recently identified
fluoride-specific membrane transport proteins. Previous studies of either the CLCF membrane F/H+ antiporters or the Fluc membrane F- channels have assumed the significance of these proteins
comes from their ability to protect bacteria lacking other F- efflux pathways from the
cytoplasmic build-up of toxic fluoride anions in response to the pH gradient across the lipid
membrane. However, whether the thermodynamic principle is effective at predicting the actual
F- load inside a living, metabolizing microorganism when it encounters shifts in external pH was
not established
We’ve proved the validity of the proposed weak acid accumulation mechanism of fluoride
anion—membrane!permeant!HF!(pKa!~3.4)!from!the!acidic!extracellular!fluid!diffuses!into!
the!cytoplasm!where!it!dissociates!at!the!neutral!pH,!and!F5!becomes!trapped!and!
accumulates—and!our!results!show!almost!exact!15to51!relationships!in!the!equation!
[! ! ]!"
[! ! ]!"#
=
[! ! ]!"#
[! ! ]!"
.!We!continuously!monitored!external!F5!concentrations!of!cultures!of!E.#coli!
with!its!native!F5!channel!knocked!out!(ΔFluc)!during!external!pH!changes!and!found!that!
while!the!magnitude!of!pH!gradient!across!bacterial!membrane!and!the!total!level!of!F5!
available!in!the!environment!can!both!drive!up!cytoplasmic!F5!concentrations!for!E.#coli;!
However,!the!ratio!of!internal!to!external!F5!concentrations!only!depends!to!on!the!internal!
and!external!pH!differences,!and!is!independent!of!the!total!F5!concentrations!(this!ratio!
!
! 23!
!
!
remained!at!~60!when!total!F5!level!varied!between!0.1!mM!and!1.5!mM).!When!fluoride!
anions!accumulates!in!E.#coli!lacking!F5!efflux!pathways,!and!the!actual!cytoplasmic!F5!
concentrations!are!in!accord!with!predictions!from!the!weak!acid!accumulation!hypothesis!
[! ! ]!" = [! ! ]!" ×10!"!" !!"!"# .!This!F5!accumulation!reaches!equilibrium!in!~30!min,!has!
bacteriostatic!effects!on!growth,!and!prolongs!the!lag5time!before!bacterial!growth!
recovers!after!removal!of!acidic!stress.!The!same!F5!dependent!delay!on!growth5recovery!
were!observed!for!ΔFluc!cells!after!2!hours!of!extreme!acid!stress!(pH!2.5),!suggesting!that!
fluoride!anion!accumulation!in!bacteria!also!holds!under!extremely!acidic!environments.
The fact that fluoride level can accumulate to over 10 mM just under environmental Flevels indicates the necessity for bacteria to possess F- specific transporters.!We!found!
representatives!from!the!two!families!of!F−!membrane!transport!proteins—Fluoride!anion!
channel!(Fluc)!and!CLCF5type!F−/H+!antiporter!(Pst!from!Piruella#staleyi)–were!both!
effective!at!preventing!cytoplasmic!F5!accumulation.!Furthermore,!amino!group!
crosslinking!by!glutaraldehyde!treatment!of!S214K!mutant!of!Pst!homologue!revealed!the!
homodimeric!structure!of!the!CLCF!subclade,!in!harmony!with!the!dimeric!quaternary!
structure!of!canonical!CLC!transporters.!!Although!the confirmation that CLC-pst exists in a
dimeric topology does not come as a surprise, this does not begin to explain the unusual
functionalities of this F- -transporting subclade, and!given!the!unconventional!discrepancies!
between!characteristic!traits!of!CLCF!!and!that!of! canonical CLC’s, a better understanding of
the fluoride selective and transport mechanism is demanded.
!
! 24!
!
!
Materials and Methods
I. Reagents
Chemicals from Sigma-Aldrich (St. Louis, MO) were of highest grade obtainable.
Detergents were obtained from Anatrace, 1-palmitoyl, 2-oleoylphosphatidylcholine (POPC), and
1-palmitoyl, 2-oleoylphosphatidylglycerol (POPG) from Avanti Polar Lipids, and fluorophores
from Invitrogen or GE-Biosciences.
II. E. coli cell strains and Fluc/CLCF constructs
Constructs used in this study are summarized in Table 1. Synthetic gene construct for
Fluc-Ec2 was inserted into a pASK vector with a C-terminal LysC recognition site and
hexahistidine tag (TRKAASLVPRGSGGHHHHHH). Site-directed mutagenesis was performed
using standard PCR techniques.
Table 1. Summery of E. coli cell strains
Homologue
Source organism
nickname
WT
E. coli BW21150
ΔFluc
E. coli BW21150 with endogenous
Fluc gene knocked-out
WT CLC-pst Pirellula staleyi
S214K Pst
Ec2
E. coli virulence plasmid
NCBI reference sequence
Protein yield, mg/L
0.1
0.5
YP_003370005.1
1.0
1.0
YP_001481330.1
III. F- Toxicity Rescue.
Synthetic gene constructs coding for CLC-pst homologue or Fluc-ec2 were purchased
from Genscript and inserted into the pBAD18 expression vector using XbaI/HindIII restriction
!
! 25!
!
!
sites. Standard site-directed mutagenesis was performed to change two amino acids before the Cterminal hexahistidine tag into stop codons. E. coli (BW2113 background strain or ΔFluc) was
transformed with pBAD containing the Pst (Pst strain) or Ec2 (Ec2 strain) insert. Overnight
cultures from single colonies were diluted 100-fold in LB and grown for approximately 2
doubling times (35 min) before inducing protein expression with 0.2% arabinose. After an hour,
NaF was added to the desired concentration, whereupon growth was monitored by optical
density (OD).
Figure 18. Fluc-ec2 construct. The C-terminal hexahistidine tag was cut off by mutating the first
two amino acids of the linker sequence into stop codons (as indicated).
!
! 26!
!
!
Figure 19. CLC-pst construct. The position of S214K mutation is indicated.
IV. F- Accumulation and E. coli Survival in Weakly Acidic Environment
Overnight E. coli cultures (WT, ΔFluc, and Pst strains) from single colonies were
pelleted and re-suspended in 1/10 volume of pH 7 recording media (150 mM NaCl, 10 mM
KPO4, 0.4 mM NaF). Fluoride anion appearance in the extracellular solution in the 2 mL reaction
cuvette was continuously monitored using the F--selective Cole-Parmer LaF3/EuF3 electrode,
during which an initial calibration dose of 0.1 mM F- (from 50 mM NaF stock solution) was
added, followed by addition of concentrated hydrofluoric acid (from 1N stock solution) to
decrease the reaction media pH to desired levels. The F- concentration was continuously
monitored for another 30 minutes, and then the final pH of the reaction media was determined.
For the case where pH decrease was 1.5 pH units, the reaction media containing E. coli cells was
!
! 27!
!
!
subsequently incubated for a total of 26 hours during which viable cell counts were taken
periodically.
V. E. coli Growth Recovery After Weak Acid Fluoride Accumulation
Overnight ΔFluc E. coli culture from single colony was diluted 50-fold into fresh LB
media at pH 7, and cell growth was monitored with OD. At OD 1, 500 μM NaF (from 50 mM
stock solution) was added to the growth-phase cell culture, which was then incubated at pH 7 or
pH 5.5 (by addition of 1N stock of concentrated hydrofluoric acid) for 2 hours. Excess fluoride
in the media was washed away by centrifuging the culture and re-suspending the cell pellet in
equal volume of fresh pH 7 LB. the growth of the cell culture was then monitored with OD.
VI. E. coli Survival and Growth Recovery After Acid-Shock
E. coli (WT, ΔFluc, and Pst strain) cell cultures from single colonies were grown
overnight in LB media buffered to pH5.5 with 100mM 2-(N-morpholino)ethanesulfonic acid
(MES). Overnight cell cultures were centrifuged and cell pellets were re-suspended and
incubated at 37oC in equal volume pH5.5 minimal media (40 mM KCl, 80 mM KH2PO4, 33 mM
H3PO4, pH5.5) or in pH 2.5 Acid-Shock media (40 mM KCl, 80 mM KH2PO4, 33 mM H3PO4,
1.7 mM Na3C6H5O7, pH 2.5) containing desired concentrations of NaF for 2 hours. Then, a viable
cell-count was obtained through 108 dilutions onto LB-agar plates, and growth of cells—pelleted
and re-suspended in 500-time volume fresh pH 7 LB media—was monitored with OD.
VII. Protein Purification and Liposome Reconstitution.
!
! 28!
!
!
For overexpression and biochemical applications, the coding sequences were inserted
into a pASK vector with an C-terminal hexahistidine tag. E. coli (BL21-DE3) was induced with
anhydrotetracycline at an optical density of 1.0, and protein was expressed for 3 h or until optical
density fell below 0.8. Cells were lysed by sonication and extracted with 40 mM decylmaltoside
(DM) for 2 h at room temperature. After pelleting the cell debris, the clarified extract was passed
over cobalt affinity beads (1 mL/L culture), washed with 100 mM NaCl, 20 mM imidazole,
5 mM DM, and eluted with solution containing 400 mM imidazole. Protein was further purified
on a Superdex200 gel-filtration column in 100 mM NaF, 50 mM 3-(Nmorpholino)propanesulfonic acid (MOPS), pH 7, and 5 mM DM. Proteins eluting as
monodisperse peaks at positions expected for a CLC homodimer were carried forward for
functional analysis.
Proteoliposomes of low protein density (1μg/mg lipid) were reconstituted by dialysis of the
mixture of Pst protein, 7.5 mg/mL 1-palmitoyl, 2-oleoyl phosphatidylcholine + 2.5 mg/mL 1palmitoyl, 2-oleoyl phosphatidylglycerol (POPC/POPG), 35 mM 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) against 300 mM NaF, 25
mM MOPS, pH7 at room temperature for 36 h. Liposomes were stored in aliquots at -80 °C until
the day of use. Prior to functional assays, liposomes were extruded 21 times through a 400 nm
membrane filter.
VIII. Glutaraldehyde Cross-linking
To determine the quaternary structure of Pst. proteoliposomes (POPC/POPG, 10 μg/mL)
loaded with 300 mM in 300 mM NaF, 25 mM MOPS, pH7 was incubated with 0.125%–0.5%
glutaraldehyde (∼500–2000-fold molar excess) for 2–60 min. The reaction was quenched with a
!
! 29!
!
!
10-fold molar excess of 2-caboxyethylr (Tris)-HCl (TCEP; Toronto Research Chemicals,
Toronto, Canada), pH 7.5 before liposomes were disrupted with 30 μM detergent n-octyl-beta-Dglucoside (BOG), and samples were run on a 12% SDS-PAGE gel. Visualization was made with
silver stain due to the low protein concentrations in the reaction mixture (~1ug/lane).
!
! 30!
!
!
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