1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
Biochemical Society Transactions
Is the cellular and molecular machinery docile in the stationary phase of
Escherichia coli?
Parul Mehtaa,1, Goran Jovanovica, Liming Yingb, Martin Bucka,1.
a
Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom;
National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United
Kingdom
b
1
Corresponding authors, Martin Buck, Department of Life Sciences, Faculty of Natural
Sciences, Imperial College Road, Imperial College, London SW7 2AZ, UK. Tel: +44 (0)207594-5442; E-mail: m.buck@imperial.ac.uk;
Parul Mehta, Division of Cell and Molecular Biology, Department of Life Sciences, Imperial
College London, London SW7 2AZ, United Kingdom Tel: +44 (0)207-594-5366; E-mail:
p.mehta10@imperial.ac.uk.
Keywords: stationary phase, Phage shock protein response (Psp), membrane stress,
cytoplasmic crowding, bacterial enhancer binding protein
Abbreviations: Phage shock protein response (Psp), logarithmic phase (log), innermembrane (IM), Escherichia coli (E. coli), bacterial enhancer binding protein (bEBP)
Abstract
The bacterial cell envelope retains a highly dense cytoplasm. The properties of the
cytoplasm change with the metabolic state of the cell, the logarithmic phase being highly
active and the stationary phase metabolically much slower. Under the differing growth
phases many different types of stress mechanisms are activated in order to maintain cellular
integrity. One such response in enterobacteria is the Phage shock protein (Psp) response
that enables adaptation to inner membrane (IM) stress. The Psp system consists of a
transcriptional activator PspF, negative regulator PspA, signal sensors PspBC and PspA and
PspG acting as effectors. The single molecule imaging of the PspF showed the existence of
dynamic communication between the nucleoid-bound states of PspF and membrane via
negative regulator PspA and PspBC sensors. The movement of proteins in the cytoplasm of
bacterial cells is often by passive diffusion. It is plausible that the dynamics of the
biomolecules differs with the state of the cytoplasm depending on the growth phase.
Therefore, the Psp response proteins might encounter the densely packed glass-like
properties of the cytoplasm in the stationary phase, which can influence their cellular
dynamics and function. By comparing the properties of the log and stationary phases, we
find that the dynamics of PspF are influenced by the growth phase and may be controlled by
the changes in the cytoplasmic fluidity.
Bacterial cells, their different growth phases and the subsequent cellular changes
Bacterial growth involves four different growth phases lag, logarithmic (log), stationary, and
death phase [1]. The bacterial growth dynamics can be followed by plotting cell growth
(absorbance at optical density of 600) versus incubation time and is exponential (log phase)
after leaving lag phase in balanced growth [1, 2]. Each of the bacterial growth phases has its
own characteristic feature, for example the log phase is highly productive phase with active
metabolism, rapid cell division and displays cellular homogeneity [1, 3]. On the other hand
the bacterial cell undergoes a number of structural as well as physiological changes when in
stationary phase, including a decrease in cell volume, cell shape changes, constriction of
nucleoid, alterations in cell wall composition and accumulation of storage materials [3]. The
extracellular environment especially the growth medium of bacterium also changes under
stationary phase, it becomes more basic because of the switch in bacterial lifestyle from
1
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
carbohydrate metabolism to consuming more amino acids [1]. Recently detailed studies on
the properties of the cytoplasm revealed that the bacterial cytoplasm has glass-like
properties, which are specifically perceived by molecular complexes [4]. Such glass-like
properties are dependent on the metabolic states of the cell, and with the late-stationary
phase glass-like properties of the cytoplasm are exaggerated (see figure 1) [4].
Phage Shock response in Escherichia coli
Many enterobacteria reside in harsh environmental conditions such as the human gut and
challenging rhizospheres where they come across different stresses [5, 6]. In order to
combat wide variety of stresses bacteria like E. coli have evolved different types of
responses. One highly specific response to inner membrane (IM) stress is Psp response;
Psp system mounts an adaptation to IM stress, by repairing the IM damage and
consequently conserving the proton motive force and energy production [5]. Induction of Psp
response is unique in the sense that it is dependent on the alternative sigma factor σ54, and
requires a specific activator, bacterial enhancer binding protein (bEBP) [5]. The bacterial 54
systems are of general importance because the transcription mechanism displays
resemblance to eukaryotic transcription using RNA polymerase II and many of the σ54
promoters drive expression of genes involved in biological stress responses such as
pathogenicity, persistence and even biogeochemical cycles [5]. The Psp response is
induced by wide array of external stimuli such as mislocalisation of outer-membrane secretin
such as pIV, iononphores, fatty acids, ethanol, and impaired protein secretion pathways [5,
6].
In E. coli PspF as an ATPase competent hexamer activates the transcription of physically
distant pspA-E operon and pspG gene under stress inducting conditions (see Figure 2). The
low-order oligomers of PspA bind and negatively regulate PspF activity under non-stress
conditions while upon IM stress functions as a higher-order oligomer to recover IM
functionality [7, 8].
The cellular landscape of the Psp response in E. coli
A detailed knowledge of PspF and PspA localisations and their self-associations is a key to
establishing how the Psp response is controlled and functions. With the help of millisecondresolution single molecule fluorescence microscopy in live E. coli cells we observed the
fluorescently tagged (with Venus fluorescent protein) PspF and PspA proteins under nonstress and IM stress conditions [7,8]. With the imaging studies we could elucidate the
mechanisms of control and activation of the Psp response in intact native cell environment
[7]. The spatial localisations, 2D dynamics and functional stoichiometry of the diffraction
limited, localised spots of fluorescent fusion proteins of PspF and PspA were determined.
The repressive PspF-PspA complex bound to psp promoter(s) on the DNA dynamically and
transiently communicates with the IM under non-stress conditions. Under IM stress
conditions PspA dissociates from the PspF-PspA inhibitory complex and binds the stressed
membrane as a higher order oligomer [7, 8, 9]. Under all experimental conditions the PspF
assembled and localised on the DNA as a single hexameric species, binding and activating
a single psp promoter (pspA or pspG) at a time [7].
The two different functional states of PspF were distinguished by cellular 2D diffusion
coefficients. The PspF actively engaged in transcriptional activation under membrane stress
conditions displayed (apparent diffusion coefficient = 0.018 µm2/s) seven times slower
compared to the PspF within PspF-PspA inhibitory complexes under non-stress conditions
(0.134 µm2/s) [7]. The variations in the dynamic behaviour of PspF were attributed to its
different functional states and interactions with other participating protein complexes,
whereby, PspA (via the PspBC signal sensors) contributes to faster dynamics of PspF such
as movement to the membrane. Clearly, the DNA associated diffusion of PspF changes with
the imposition of IM stress, and other factors influencing the observed cellular dynamics of
PspF such as interactions with other protein complexes, nucleoid organisation and even
cellular crowding needs further exploring [10].
2
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
PspF changes its cellular mobility in stationary phase
The cellular dynamics and localisation of PspF and PspA reported so far have only been
studied in log phase E. coli cells. Here we set out to assess how the cellular landscape of
Psp response changes in early stationary phase under non-stress and stress conditions. The
survival of cells in the stationary phase irrespective of accumulating toxins, energy efficient
lifestyles and elevated pH levels of the extracellular environments requires life-style
adaptions. The cytoplasm crowding is probably exaggerated in the stationary phase with the
reduction in relative cell size and volume. Since the glass-like properties of the cytoplasm
are also pronounced with stationary phase, there is a possibility that the cellular mobility of
the repressive PspF-PspA complex under non-stress conditions and active PspF under IM
stress are reduced. Our direct measurements of apparent 2D diffusion coefficients in early
stationary phase showed that the PspF self assemblies were almost immobile bound to the
DNA (apparent diffusion coefficient = 0.009 µm2/s), there was reduction in the apparent
diffusion coefficient values from the PspF assemblies in the log phase under non-stress
conditions (unpublished work P.Mehta, G.Jovanovic, L.Ying and M.Buck). In addition to the
reduction in PspF mobility, the number of DNA-localised diffraction limited foci increased
from single foci to two foci per chromosome, the cell length was reduced and the relative
protein density increased by 33% in stationary phase as compared to log phase
(unpublished work unpublished work P.Mehta, G.Jovanovic, L.Ying and M.Buck). The
explanation for slower cellular dynamics and more PspF foci/cell include the possibilities that
the binding of PspF to its specific pspA and pspG promoters is more stable and prolonged in
the stationary phase than in log phase. These changes in dynamics and number of foci
could be linked to changes in DNA supercoiling impacting upon transcription factor binding,
including that of PspF, and its target closed promoter complex. As in stationary phase the
nucleoid DNA or plasmid DNA exists in a more relaxed state, which might affect the DNA
binding ability of the proteins [12].
Concluding remarks
Single molecule fluorescence microscopy of chromosomally encoded fluorescently tagged
PspF and PspA fusion proteins offers new mechanistic insights into the control of the Psp
response at the cellular level. The mobility and localisation of the PspF activator and the
PspA negative regulator/effector proteins and their complexes in log phase live E. coli cells
revealed the surveillance mode of the PspF-PspA complex for IM damage [7]. In the
stationary phase, the localisation and dynamics of PspF changes suggesting some longer
lived DNA interactions. These have the potential to impact upon the kinetics of stress
response induction, and the degree of cell to cell variability expected from inherently
stochastic events which occur during transcription such as bursts in gene expression and
phenotypic variation between genetically identical cells. Each of these aspects of gene
expression and phenotype can now be explored. The application of advanced data analyses
and mathematical modelling has enabled to characterise complex phenomenon (such as
redefining of cytoplasmic properties) [4, 11]. Being able to analyse changes in protein
behaviour in the different growth phases of bacteria at the single molecule level will not only
expand our understanding of bacterial cell biology. It will also enable to understand the
survival mechanisms developed by bacteria in order to flourish in harsh growth-limiting
conditions [13].
Acknowledgements
This work was supported by project funding from the Leverhulme Trust.
References
1) Farrell, M.J. & Finkel, S.E. (2003) The growth advantage in stationary phase phenotype
conferred by rpoS mutations is dependent on the pH and nutrient environment. J.
Bacteriol. 185, 7044-7052.
3
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
2) Whitman, W.B. (2009) The Modern Concept of the prokaryote. Journal of Bacteriology.
191, 2000-2005.
3) Makinoshima, H., Aizawa, S., Hayashi, H., Takeyoshi., M. et al. (2003) Growth phasecoupled alterations in cell structure and function of Escherichia coli. Journal of
Bacteriology. 185, 1338-1345.
4) Parry, B.R., Surovtsev, I.V., Cabeen, M.T., O’Hern, C.S., et al. (2014) The bacterial
cytoplasm has glass-like properties and is fluidized by metabolic activity. Cell. 156, 183194.
5) Joly, N., Engl, C., Jovanovic, G., Huvet, M., et al. (2010) Managing membrane stress:
the phage shock protein (Psp) response, from molecular mechanisms to physiology.
FEMS Microbiology Reviews. 34, 797-827.
6) Darwin, A.J. (2005) The phage-shock-protein response. Molecular Microbiology. 57, 621628.
7) Mehta, P., Jovanovic, G., Lenn, T., Bruckbauer, A., et al. (2013) Dynamics and
stoichiometry of a regulated enhancer-binding protein in live Escherichia coli cells.
Nature Communications. 4:1997, 1-9.
8) Jovanovic, G., Mehta, P., McDonald, C et al. (2014) The N-terminal amphipathic helices
determine regulatory and effector functions of phage shock protein A (PspA) in
Escherichia Coli. J. Mol. Biol. 426, 1498-511.
9) Jovanovic, G., Mehta, P., Ying, L., Buck, M (2014). Anionic lipids and the cytoskeletal
proteins MreB and RodZ define the spatio-temporal distribution and function of
membrane stress controller PspA in Escherichia coli. Microbiology. In press
10) Mika, J.T. & Poolman, B. (2011) Macromolecule diffusion and confinement in prokaryotic
cells. Current Opinion in Biotechnology. 22, 117-126.
11) Golding, I. & Cox, E. (2006) Physical Nature of Bacterial Cytoplasm. Physical Review
Letters. 96, 098102 1-4.
12) Reyes-Dominguez, Y., Contreras-Ferrat, G et al. (2003). Plasmid DNA supercoiling and
Gyrase activity in Escherichia coli wild-type and rpoS stationary-phase cells. J. of
Bacteriol. 185, 1097-1100.
13) Finkel, S.E. (2006). Long-term survival during stationary phase: evolution and the GASP
phenotype. Nature Rev. Microbiol. 4, 113-120.
Figure legends
4
195
196
197
198
199
200
201
Figure 1 Schematic of dense bacterial cytoplasm: The figure illustrates the possible constraints in the cellular
mobility of the phage shock protein F (PspF) assembly when communicating with other proteins like phage shock
protein A (PspA), RNApolymerase holoenzyme with σ54, enhancer DNA sites or with membrane in the latestationary phase with denser cytoplasm and more stored materials. The main mode of movement in the cell for
protein complexes is by passive diffusion [4, 10].
5
202
203
204
205
206
207
Figure 2 Schematic of Phage Shock Protein (Psp) Response The schematic represents the possible cellular
landscape of the Psp response showing the different spatial localisation and respective function of Psp proteins.
PspF functions as a bacterial enhancer binding protein activating σ54 dependent transcription from either the pspA
promoter or pspG promoter. PspA is a dual function protein, acting as a repressor of PspF during non-stress
conditions and as an effector to help maintain membrane integrity under stress conditions [7].
6