RESEARCH ARTICLE

E¡ect of temperature and starvation upon survival strategies of

Pseudomonas £uorescens CHA0: comparison with Escherichia coli
Inés Arana, Alicia Muela, Maite Orruño, Carolina Seco, Idoia Garaizabal & Isabel Barcina
Departamento de Inmunologı́a, Microbiologı́a y Parasitologı́a, Facultad de Ciencia y Tecnologı́a, Universidad del Paı́s Vasco, Bilbao, Spain

Correspondence: Isabel Barcina, Abstract

Departamento de Inmunologı́a,
Microbiologı́a y Parasitologı́a, Facultad de
Ciencia y Tecnologı́a, Universidad del Paı́s
Vasco, Apdo. 644, E-48080 Bilbao, Spain.
Tel.: 134 94 601 5090; fax: 134 94 601
3500; e-mail: isabel.barcina@ehu.es
Received 9 July 2010; revised 10 September
2010; accepted 15 September 2010.
Final version published online 18 October 2010.
DOI:10.1111/j.1574-6941.2010.00979.x
Editor: Julian Marchesi
Keywords
Pseudomonas; Escherichia coli; temperature;
starvation; outer membrane.
MICROBIOLOGY ECOLOGY
Microorganisms in aquatic systems are exposed to continuous modifications in
their environmental conditions. In these systems, both autochthonous and
allochthonous bacteria respond to adverse conditions by expressing viable but
nonculturable phenotype. On the basis of this common response, the behaviour of
a few species is extrapolated to others. We compared the survival strategies of
Escherichia coli (allochthonous, mesophile bacterium) and Pseudomonas fluores-
cens CHA0 (ubiquitous, psychrotrophic bacteria) under nonoptimal temperature
and nutrient deprivation. In the absence of nutrients, the effect of temperature on
the loss of culturability did not show a common pattern. Whereas the survival of E.
coli had an inverse relationship with temperature, whereas for P. fluorescens a direct
relationship between temperature and T90 values was only established in the range
5–15 1C, with an inverse relationship at higher temperatures. When the subpro-
teome of the outer membrane of P. fluorescens was comparatively analysed,
starvation was not the main source of change. The most relevant modifications
were due to variations in temperature. OprF, the major surface protein of the genus
Pseudomonas, showed a high expression in nonculturable as well as culturable

populations under all the adverse situations analysed. We therefore propose OprF
as a suitable marker for Pseudomonas detection in the environment.

rates and macromolecular synthesis (Barcina et al., 1997;

Introduction
The microorganisms in natural systems are exposed to
variations in environmental conditions (i.e. variations in
temperature between cold and warm situations) or to
environmental changes (i.e. terrestrial and intestinal bacteria
can reach the aquatic systems after storms and through
wastewater). In both cases, adverse physicochemical condi-
tions induce microbial responses to enable them to persist in
these changing environments.
Bacteria can survive for a long time due to sequential changes
in cell physiology and gradual changes in morphology. A
characteristic response to these adverse situations is to develop
the viable but nonculturable (VBNC) phenotype. The VBNC
state is a strategy of adaptation to adverse environmental
conditions, adopted by some prokaryotes not subjected to
processes of cellular differentiation (Colwell & Gray, 2000).
VBNC cells become nonculturable on media normally used to
culture them, but nonetheless show metabolic activity. More-
over, they exhibit a reduction in nutrient transport, respiration
Colwell & Gray, 2000; Arana & Barcina, 2008; Oliver, 2010).
Previous studies described bacterial species that enter the
VBNC state and the factors inducing this phenotype. Up to
now, 4 60 species adopting this phenotype have been
described and this number is increasing continuously (Oli-
ver, 2010). In aquatic systems, both autochthonous and
allochthonous bacteria respond to adverse environmental
conditions by expressing the VBNC phenotype. On the basis
of this common response, the behaviour of a few species is
frequently extrapolated to that of other species without
considering their origins.
A typical example of autochthonous bacteria in aquatic
systems is Vibrio spp. (psychrophilic, oligotrophic bacteria).
The isolation of Vibrio spp. from aquatic systems is related
to water temperature (Srinivasan et al., 1998; Ohno et al.,
2003; Wang et al., 2006; Julie et al., 2010) and it has been
reported that the VBNC phenotype is generally induced by
low temperatures (4–6 1C) (Smith & Oliver, 2006; Vattaka-
ven et al., 2006; Zhong et al., 2009).

c 2010 Federation of European Microbiological Societies FEMS Microbiol Ecol 74 (2010) 500–509
Published by Blackwell Publishing Ltd. All rights reserved
Pseudomonas fluorescens and E. coli under adverse conditions
For allochthonous bacteria such as Escherichia coli, aqua-
tic systems represent a hostile environment characterized by
a scarcity of nutrients, nonoptimal temperatures and solar
irradiation, etc., all abiotic factors that induce the VBNC
phenotype (Barcina et al., 1997; Arana & Barcina, 2008;
Barcina & Arana, 2009).
Pseudomonas is a genus of truly ubiquitous organisms
because of their simple nutritional requirements, the range
of carbon compounds they use, and their genetic and
metabolic adaptability. Pseudomonas are present in all major
natural environments, including water, soil and rhizosphere,
as well as on the human body (Spiers et al., 2000). Some of
these environments are subject to rapid variations that could
induce the entry into the VBNC state. In the aquatic
systems, environmental factors that may govern the forma-
tion of VBNC cells in Pseudomonas include conditions such
as high NaCl concentrations (Mascher et al., 2000), oxygen
limitations (Binnerup & Sørensen, 1993) and high tempera-
ture (Lowder et al., 2000), but not nutrient starvation (Clegg
et al., 1996; Hase et al., 1999). However, the formation of
VBNC cells under all adverse conditions is controversial and
some authors (Clegg et al., 1996; Hase et al., 1999) have
reported that the decline of several CHA0 derivatives under
starvation conditions did not result in the formation of
VBNC cells. Others (Hase et al., 1999; Mascher et al., 2000)
have suggested that the VBNC state in Pseudomonas fluor-
escens CHA0 does not represent a physiological strategy to
improve survival under adverse conditions.
Despite the large amount of information available, com-
parisons between the dynamics of the transition to the
VBNC state among different bacterial species are limited.
With a few exceptions (Awong et al., 1990; Thomas et al.,
1999), most studies did not use a complete range of
temperatures and the results cannot therefore be extrapo-
lated to other strains or bacterial species due to the different
experimental conditions.
During the last years, the study of the VBNC phenotype
has focused not only on the description and knowledge of
the inducting factors of the process (for a revision, see
Barcina & Arana, 2009), but on the practical aspects
such as the determination of the capacity of reversion of
nonculturable cells to culturable ones and the search for
culture-independent tools that allow the detection of these
bacteria.
Nowadays, the most accepted opinion is that the VBNC
phenotype is made up of live cells with physiological activity
which are infertile, being VBNC cells. Based on this premise,
many studies have been done to establish whether these cells
are able to recover their capacity to generate new cells
(Magariños et al., 1997; Whitesides & Oliver, 1997; Lleó
et al., 2000; Ohtomo & Saito, 2001; Gupte et al., 2003; Wong
et al., 2004). If the hypothesis is true and it is possible to pass
from the VBNC to the culturable state, then the VBNC
FEMS Microbiol Ecol 74 (2010) 500–509
501
phenotype could be considered as part of the life-cycle of
nondifferentiating bacteria.
Researchers working in environmental control consider
that cells entering a VBNC state are a problem due to the
impossibility of detection with classical culture methods.
This problem could be bypassed using molecular techniques.
In this way, the presence of nonculturable Pseudomonas in
aquatic environment has been demonstrated by isolation of
16S rRNA gene with high homology to P. aeruginosa from
deep-sea sediments (Li et al., 1999). Some outer membrane
proteins could be used as diagnostic proteins for Pseudomonas
sensu stricto (Aagot et al., 2001). Kimata et al. (2004) reported
the suitability of outer membrane protein for detection of
Pseudomonas in seawater.
In this work, we did a comparative analysis of the survival
strategies under adverse conditions, specifically nonoptimal
temperature and nutrient deprivation, in two Gram-nega-
tive bacteria: E. coli (allochthonous, mesophile bacterium)
and P. fluorescens CHA0 (Hase et al., 1999) (ubiquitous,
psychrotrophic bacterium). In addition, we studied the
ability of nonculturable P. fluorescens to resuscitate and
become culturable. Finally, we describe a proteomic
approach for P. fluorescens CHA0 to study outer membrane
proteins that could be used as targets for the detection of
culturable and nonculturable cells under adverse conditions.
Materials and methods
Bacterial strains and growth conditions
Two bacterial strains were used in this study. Escherichia coli
strain STCC 416 (Spanish Type Culture Collection) was
maintained on nutrient agar (Oxoid), and P. fluorescens
CHA0 (Hase et al., 1999) on King’s B agar at 4 1C. Strains
were cultured aerobically in Luria–Bertani (LB) broth
(Oxoid) with shaking (120 r.p.m.) at 37 1C (E. coli) or 27 1C
(P. fluorescens). In the preparation of the inocula, E. coli cells
from mid-log phase and P. fluorescens cells from both mid-
log and stationary phase were harvested by centrifugation
(3000 g for 15 min) and washed three times in sterile saline
solution (NaCl 0.9%, w/v). Finally the pellet was suspended
in sterile saline solution to obtain 1010 cells mL–1.
Survival assays
Escherichia coli strain STCC 416 from exponential and
P. fluorescens CHA0 from exponential and stationary growth
phase were incubated under adverse conditions, specifically
nutrient deprivation and nonoptimal temperature. Nutrient
deprivation was implemented by incubating cells in steri-
lized saline solution (NaCl 0.9%, w/v). To avoid organic
residue, glass flasks were cleaned with H2SO4 (97%, v/v)
beforehand, rinsed with deionized water, and kept at 250 1C
for 24 h. The assays were carried out in Erlenmeyer flasks
c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
502
containing 2 L sterile saline solution inoculated to reach a
density of 106–107 cells mL–1. The total organic carbon
(TOC), measured with a TOC-5000 Shimadzu carbon
analyser, was o 1 mgC L–1. Incubation temperatures ranged
from 5 to 37 1C.
For each experimental condition, samples were collected
daily to determine the total number of cells (TNC), viable
bacteria and culturable bacteria. Besides, T90 (time requi-
red for 90% reduction in bacterial culturability) was
employed as the representative parameter of culturability
loss. This parameter was determined from plots of E. coli
and P. fluorescens survival.
Bacterial counts
The viable bacteria were estimated as bacteria with intact
cytoplasmic membranes (MEMB1) with the aid of the Live/
Deads BacLightTM kit (Invitrogen) as described by Joux
et al. (1997). The samples stained with the kit were analysed
with a FACSCalibur flow cytometer (Becton Dickinson,
Erembodegem, Belgium). Flow rate was measured at the
beginning and end of each analysis session using BD
TruCOUNTTM tubes (Becton Dickinson). Data were ana-
lysed using CELLQUEST software (V 3.3; Becton Dickinson).
Populations of bacteria were discriminated as two regions of
the log-integrated red fluorescence vs. log-integrated green
fluorescence, and the number of bacteria found within these
regions was used to estimate the number of viable
(MEMB1) and nonviable organisms (cells with a damaged
cytoplasmic membrane; MEMB cells) in the population.
The TNC was estimated by adding the number of MEMB1
cells and MEMB cells.
Culturability, expressed as CFU, was evaluated by the
spread plate method, on tryptone soy agar incubated 24 h at
37 1C for E. coli populations, and on LB agar (Merck) for
24 h at 27 1C for P. fluorescens.
Percentages of each cellular subpopulation were calcu-
lated with respect to TNC counts: culturable cells (C)
directly from CFU, VBNC as MEMB1 CFU, and nonvi-
able cells (NV) as TNC MEMB1 (Arana et al., 2007).
All the results from survival experiments presented below
are the means of at least three experiments; the coefficient of
variation between replicates was o 12%. Differences
between means were assessed by ANOVA. P  0.05 was
considered significant.
Resuscitation procedures
Resuscitation assays were mainly performed as described by
Arana et al. (2007). Suspensions of nonculturable cells
(VBNC and NV cells) of P. fluorescens, obtained from
survival experiments of stationary populations maintained
under nutrient deprivation at 37 1C, were used. Cells were
harvested by centrifugation (4500 g, 10 min) and resus-
c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
I. Arana et al.
pended in sterile saline solution until expected cultur-
ability was o 1 CFU mL 1. These suspensions were diluted
(1 : 100) in flasks containing sterile saline solution, LB
diluted (10%), and LB diluted at 10% and supplemented
with 11 U catalase mL 1. Flasks were incubated with shaking
at 27 1C in the dark for at least 18 days. Daily aliquots were
collected to estimate TNC and CFU counts.
Isolation of P. fluorescens outer membrane
proteins by carbonate extraction
To determine the changes occurring in the outer membrane
protein expression under permanence at nonoptimal tem-
perature, P. fluorescens populations were analysed at the
beginning of experimentation (inocula from stationary
growth phase) and at the end of exposure period.
A total of 2 L of P. fluorescens cultures were harvested by
centrifugation (4500 g, 15 min), washed three times with 50 mM
Tris-HCl (pH 7.3) and resuspended in 5 mL 50 mM Tris-HCl
(pH 7.3) to reach a final density of 109–1010 cells mL 1.
Outer membrane protein preparations were obtained
according to the method described by Molloy et al. (2000)
with minor variations: a 1-mL aliquot of the above-
described cell suspensions was diluted to 6 mL with a buffer
containing 50 mM Tris-HCl (pH 7.3) and 0.7 g DNase I
(Sigma). Cells were disrupted in an SLM Aminco (SLM
Instruments) French press (5.52 MPa). Then the superna-
tant was separated from debris by centrifugation (4500 g,
10 min), diluted in 0.1 M sodium carbonate (pH 11.0) to a
final volume of 60 mL and incubated on ice for 1 h.
Membranes were collected by ultracentrifugation on a Beck-
man centrifuge (rotor 75.13 TFT) at 143 000 g for 1 h at 4 1C.
Finally, the membrane pellet was resuspended in 1 mL
isoelectric-focusing (IEF) buffer.
Interfering substances were eliminated by precipitation of
proteins with 0.15% deoxycholate and 10% tricarboxylic
acid. Pellet was dissolved in 1% sodium dodecyl sulphate
(SDS) and the amount of protein was determined by BCA
protein assay (Pierce), based on the colorimetric method
reported by Lowry et al. (1951) using bovine serum albumin
(Sigma) as a standard.
Two-dimensional (2D) electrophoresis with
immobilized pH gradient (IPG) strips
2D electrophoresis was performed as described by Görg et al.
(2000) using precast IPG strips pH 4–7L, 18 cm length
(Amersham Biosciences), in the first dimension (IEF).
Samples were applied by cup-loading onto previously rehy-
drated strips with 350 mL of rehydration buffer (8 M urea,
0.5% Triton X-100, 0.2% b-mercaptoethanol, 0.6% ampho-
lyte 3–10, and trace amounts of bromophenol blue). Typi-
cally, 300 mg of protein were loaded on each IPG strip and
focusing was carried out in the Multiphor II apparatus
FEMS Microbiol Ecol 74 (2010) 500–509
Pseudomonas fluorescens and E. coli under adverse conditions
(Amersham Biosciences) in two steps. The first step, for
sample introduction, was 500 V until 1500 Vh were reached,
and then 3500 V until total 75 000 Vh. After IEF separation,
the strips were equilibrated 2  10 min with 50 mM Tris-
HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS and a trace of
bromophenol blue. The first equilibration solution con-
tained 2% dithiothreitol, and 4% iodoacetamide was added
in the second equilibration step. The strips were loaded onto
EXCELGEL XL SDS 12–14% gels (Amersham Biosciences) as
the second dimension. Electrophoresis was carried out at a
constant current (20 mA) for 45 min and at 40 mA for
160 min. Voltage was constrained to 1000 V, wattage to
40 W and temperature to 10 1C. The pH gradient was
determined with an IEF calibration kit (broad pI kit, pH
3–10, from Pharmacia). Molecular masses were estimated
on the basis of comigrating broad-range standards (broad
MW kit, 250–10 kDa, BioRad) in the second dimension.
After electrophoresis, gels were stained with an MS-
compatible silver nitrate procedure (Yan et al., 2000) and
scanned using a computer-assisted densitometer (Amer-
sham Biosciences). Spot detection, gel alignment and spot
quantification were performed using Image Master 2-D ELITE
software, version 4.01 (Amersham Biosciences). To correct
for variability resulting from silver staining, spot intensity
was normalized. Each spot was expressed as percent of
P
volume (%V), where %V = spot volume/ volumes of all
spots resolved in the gel.
To analyse the outer membrane subproteome, three gels
were analysed for each experiment and the software (Image
Master 2-D ELITE software, version 4.01) created averaged
gels for each experimental situation. These are artificial gels
created from a combination of the normal gels. The spot
values in the averaged gel are calculated from the mean
values of the spots in the gels that were used to construct
them. Protein spots were considered when present in two of
the three gels. Differences between the means were detected
using one-way ANOVAs. P  0.05 was considered significant.
Protein identification
After new electrophoresis, gels were stained with the Coo-
massie-blue procedure (Meyer & Lambert, 1965). Selected
protein spots were excised manually from the gel and
subjected to in-gel digestion with trypsin (Roche, Basel,
Switzerland) according to Shevchenko et al. (1996) with
minor modifications. The gel pieces were swollen in diges-
tion buffer containing 50 mM ammonium bicarbonate and
12.5 ng mL 1 proteomics grade trypsin and the digestion
proceeded at 37 1C overnight. The supernatant was recov-
ered and peptides were extracted twice: first with 25 mM
ammonium bicarbonate and acetonitrile, and then with
0.1% trifluoroacetic acid and acetonitrile. The recovered
supernatants and extracted peptides were pooled, dried in a
FEMS Microbiol Ecol 74 (2010) 500–509
503
SpeedVac (Thermo Electron, Waltham, MA), redissolved in
10 mL of 0.1% formic acid (FA) and sonicated for 5 min. LC-
MS/MS spectra were acquired using a MALDI SYNAPT
HDMS mass spectrometer (Waters, Milford, MA) interfaced
with a nanoAcquity UPLC System (Waters). A 7-mL aliquot
of each sample was loaded onto a Symmetry 300 C18,
180 mm  20 mm precolumn (Waters) and washed with
0.1% FA for 3 min at a flow rate of 5 mL min 1. The
precolumn was connected to an XBridge BEH130 C18,
75 mm  200 mm, 1.7 mm (Waters) equilibrated in 1% acet-
onitrile and 0.1% FA. Peptides were eluted with a 30-min
linear gradient of 3–60% acetonitrile directly onto a NA-
noEase Emitter (Waters). Obtained spectra were processed
using PROTEINLYNX GLOBAL SERVER 2.3 (Waters) and searched
against the UniprotKB/Swiss-Prot database using MASCOT
(Matrixscience). The following parameters were adopted
for protein identification: carbamidomethylation of
cysteines as fixed modification, oxidation of methionines as
variable modification, to p.p.m. of peptide mass tolerance,
0.1 Da fragment mass tolerance and one missed cleavage.
Results
The effects of temperature upon culturability, viability and
integrity of E. coli or P. fluorescens populations under
nutrient starvation (saline solution) are shown in Figs 1
and 2. Figure 3 represents T90 values calculated for each
bacteria and survival condition.
In all cases, we observed that both bacterial strains
maintained their integrity and TNC that did not change
throughout the experimentation time (Figs 1 and 2). In the
case of E. coli (inocula from exponential phase), culturabil-
ity decrease was more quickly affected by the increasing
temperature (Figs 1 and 3). In the experiments carried out at
20–37 1C, about 0.1% of remaining population was cultur-
able at the end of incubation period. The optimum survival
temperature was 5 1C. Reductions in MEMB1 counts
were detected only for those populations maintained at
5 and 20 1C. This meant that the NV fraction was pre-
dominant (Fig. 1a and b). Above 25 1C, the behaviour of
E. coli populations varied, and the VBNC cells represented
an important fraction of the final populations (Fig. 1c
and d).
Figure 2 shows the effect of temperature conditions and
absence of nutrients on P. fluorescens CHA0 counts and
on the percentages of the different subpopulations
formed. With respect to E. coli dynamics, some differences
were observed. For exponential populations (Fig. 2a–d),
decreases in culturability were not related to the tempera-
ture increment (see T90 values in Fig. 3). Thus, optimum
survival temperature for P. fluorescens was 15 1C (Figs 2 and
3). The largest differences between both bacteria were
observed at 5 1C (Figs 1a and 2a), not only in the dynamics
c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
504 I. Arana et al.

of loss of culturability, but also in the formation of sub- When cultures from stationary phase of growth were
populations (VBNC or NV). Moreover, P. fluorescens popu- submitted to the same adverse conditions, all the tempera-
lations lost viability at 25 and 37 1C (decreases in MEMB1 tures tested resulted in loss of culturability (Figs 2e–h and
counts) during the last weeks of experimentation (Fig. 2c 3). The main difference between cultures from stationary
and d). and exponential phases (Fig. 2) was the magnitude of this
loss of viability during exposure at 25 and 37 1C (Fig. 2c–d
(a) and g–h).
100 8
When forward light-scattering signal (FSC-Height) was
7 used for analysing changes in the cell size of P. fluorescens
Log no. cells mL–1

80
6 during experiments comparing the results with cells col-
Percentage

60 5 lected at the beginning and after 80 days of incubation, no

4 statistically significant decrease was detected (data not
40 3 shown).
2 The resuscitation ability of Pseudomonas populations
20
1 exposed to absence of nutrients and nonoptimum tempera-
0 0 ture (37 1C) was tested in the presence of nutrients (10%
0 20 40 60 80 100
Time (days) LB), absence of oxidative stress (10% LB supplemented with
catalase) and incubation at optimum growth temperature
(b) (27 1C). Increases in cellular density (TNC) were observed
100 8
when at least one culturable cell was inoculated in diluted
7
Log no. cells mL–1

80 LB. These increases corresponded to the growth of remain-

6
ing culturable cells and the calculated instantaneous growth
Percentagee

60 5
rates (from CFU counts) were similar to that obtained from
4
laboratory growth curves (data not shown). In the absence
40 3
of nutrients, no change in cellular counts was detected (data
2
20 not shown); neither resuscitation nor growth were observed.
1 To determine the effect of the absence of nutrients and
0 0
0 20 40 60 80 100
nonoptimum temperature on outer membrane protein
Time (days) expression, P. fluorescens populations were analysed at the
beginning and end of the exposure period. A total of 177
(c) spots were considered in the inocula from the stationary
100 8
phase of growth. At the end of the exposure time, the
7
80 number of spots detected diminished for the three tempera-
Log no. cells mL–1

6
Percentage

tures studied, 5, 15 and 37 1C. In fact, almost 50% of the

60 5
initial proteins were lost during survival experiments. The
4
40 number of proteins detected at the end of experimentation
3
increased as the temperature increased (70, 107 and 114
2
20 spots for 5, 15 and 37 1C, respectively).
1
Only 21 proteins present at the beginning of survival
0 0
0 20 40 60 80 100 experiments remained in all experiments (approximately
Time (days) 12% of initial outer membrane proteins). The presence of a
set of proteins with a molecular weight of approximately
(d) 35 kDa was notable, and represented some of the most
100 8
7
Log no. cells mL–1

80
6
Percentage

60 5
4
Fig. 1. Changes in bacterial counts and percentages of subpopulations
40 3 during the permanence of Escherichia coli cells from the exponential
2 phase of growth in sterile saline solution incubated at 5 1C (a), 20 1C (b),
20
1 25 1C (c) or 37 1C (d). , Total number of bacteria; ’, number of bacteria
0 0 with intact cytoplasmic membranes; ., number of culturable bacteria.
0 20 40 60 80 100 Percentages of VBNC (striped bars) and nonviable cells (open bars). Data
Time (days) shown are averages of three experiments.

c 2010 Federation of European Microbiological Societies FEMS Microbiol Ecol 74 (2010) 500–509
Published by Blackwell Publishing Ltd. All rights reserved
Pseudomonas fluorescens and E. coli under adverse conditions 505

(a) (e)

Log no. cells mL–1

Log no. cells mL–1

100 8 100 8
7 7
80 80

Percentage

Percentage
6 6
60 5 60 5
4 4
40 3 40 3
20 2 20 2
1 1
0 0 0 0
0 20 40 60 80 100 0 20 40 60 80 100
Time (days) Time (days)

(b) (f) 100

Log no. cells mL–1

Log no. cells mL–1

100 8 8
7 7

Percentage
80 80

Percentage
6 6
60 5 60 5
4 4
40 3 40 3
20 2 20 2
1 1
0 0 0 0
0 20 40 60 80 100 0 20 40 60 80 100
Log no. cells mL–1 Time (days) Time (days)

(c) 100 (g) 100

Log no. cells mL–1

8 8
7 7
80 80

Percentage
Percentage

6 6
60 5 60 5
4 4
40 3 40 3
20 2 20 2
Fig. 2. Changes in bacterial counts and 1 1
0 0 0 0
percentages of subpopulations during the 0 20 40 60 80 100 0 20 40 60 80 100
permanence of Pseudomonas fluorescens CHA0 Time (days) Time (days)
cells from the exponential phase of growth (a–d)
and from the stationary phase of growth (e, f) in (h) 100
Log no. cells mL–1

Log no. cells mL–1

(d) 100 8 8
sterile saline solution. Incubation temperatures: 7 7
80 80
Percentage
Percentage

5 1C (a, e), 15 1C (b, f), 25 1C (c, g) and 37 1C (d, 6 6

60 5 60 5
h). , Total number of bacteria; ’, number of 4 4
bacteria with intact cytoplasmic membranes; . 40 3 40 3
number of culturable bacteria. Percentages of 20 2 20 2
1 1
VBNC (striped bars) and nonviable cells 0 0 0 0
(open bars). Data shown are averages of three 0 20 40 60 80 100 0 20 40 60 80 100
experiments. Time (days) Time (days)

120 with the inoculum (72%). However, most proteins (64%)

100 from the 37 1C gel were exclusive to this temperature. A
great number of these exclusive proteins (about 40% of
Time (days)

80
proteins) had a molecular weight between 15 and 30 kDa.
60
Additionally, at this temperature it was observed that 12
40
proteins present in the other three gels disappeared, the
20 most significant being a set of proteins of about 30 kDa,
0 which were some of the predominant proteins in the other
0 10 20 30 40
conditions.
Temperature (°C)
In addition, there were two proteins (about 24 kDa),
Fig. 3. Mean T90 values obtained from survival experiments for Escher- common to all the survival experiments, which was not
ichia coli () and Pseudomonas fluorescens CHA0 (&, ’) in sterile saline present in the initial situation.
solution. Inocula from the exponential (open symbol) or stationary (solid
symbol) phase of growth.

highly expressed proteins. These proteins could be identified

Discussion
by LC-MS/MS and corresponded to isoforms of OprF. Variation in the environmental temperature is probably the
The outer membrane subproteome for the optimum most common stress factor in the aquatic systems affecting
survival temperature (15 1C) showed the greatest similarities the survival of the bacterial populations. The results

FEMS Microbiol Ecol 74 (2010) 500–509

c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
506
obtained in this work and supported by previous ones
(Arana et al., 2003, 2007) confirmed the influence of
temperature on the survival of E. coli and P. fluorescens.
Moreover, these results also show that, in the absence of
nutrients, the effect of temperature on the loss of cultur-
ability is not a common pattern (Fig. 3).
The survival of E. coli follows an inverse relation with
temperature. Escherichia coli, an allochthonous mesophilic
bacterium, responds to the changes of temperature accord-
ing to a program in which low temperature causes a slowing
down of the metabolism and, as consequence, a delay in
cellular damage (Fig. 3). This behaviour has been previously
described for this bacterium and other pathogens in aquatic
systems such as Campylobacter jejuni and Legionella pneu-
mophila (Thomas et al., 1999; Ohno et al., 2003; Craig et al.,
2004). It has been speculated that, at low temperatures, E.
coli is ‘waiting for better days’ (Koch, 1971).
However, for P. fluorescens, a psychrotrophic bacterium
common in aquatic systems, a direct relation between
temperature and T90 values was only established in the range
5–15 1C and this relation was inverse for higher tempera-
tures. Guillou & Guespin-Michel (1996) and Hemery et al.
(2007) considered the temperature of 17 1C critical because
it corresponds to the junction of two separate physiological
growth domains–cold (0–17 1C) and suboptimal (17–28 1C).
This behaviour was similar to that described for Vibrio spp.
(psychrophilic bacterium). Some authors (Oliver et al., 1995;
Day & Oliver, 2004; Randa et al., 2004; Hernroth et al., 2010)
have stated that low water temperatures have a negative effect
on isolation of Vibrio and that a temperature outside the
range of 13–22 1C reduces the time of survival in sterilized
seawater (Kaspar & Tamplin, 1993).
The survival patterns of P. fluorescens and E. coli can be
related to the temperature range over which they can grow
(psychrotroph 0–30 1C or mesophiles 10–50 1C). When
comparing the survival behaviour of allochthonous bacteria,
among them the pathogenic bacteria (mostly mesophiles),
with that of other bacterial species, it is necessary to consider
this fact. Comparing data from the literature (Kaspar &
Tamplin, 1993; Oliver et al., 1995; Ohno et al., 2003; Randa
et al., 2004; Hernroth et al., 2010) as well as those obtained
in this work for the two microorganisms studied, there was a
common point – the optimum survival temperature was
below the optimum growth temperature (Fig. 3).
When the subproteome of the outer membrane of
P. fluorescens was comparatively analysed, we observed that
starvation was not the main source of change. As tempera-
ture modifications were critical, nutrient deprivation was
responsible for the expression of only two outer membrane
proteins. Guillou & Guespin-Michel (1996) demonstrated,
when growing P. fluorescens MF0, a biphasic variation of
total protein concentration with respect to temperature,
with a maximum within 17–20 1C. In our work, a direct
c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
I. Arana et al.
relationship was detected between temperature and outer
membrane proteins, with a maximum spot detection at
37 1C (114 spots). Most of the new proteins originated at
37 1C, the most stressful temperature for P. fluorescens, and
had a low molecular weight, which could be related to an
increased proteolysis and/or a protein turnover. Similar
results had been previously reported for E. coli cells main-
tained in seawater under illumination (Muela et al., 2008). A
large number of protein species were induced by exposure to
seawater, and most of these proteins exhibited a molecular
weight o 29 kDa. This was mainly attributed to protein
turnover carried out before the loss of culturability as a
response to adverse environmental conditions.
We observed that the physiological state of P. fluorescens
cells did not determine the survival pattern, which was
similar for cells from the exponential or stationary growth
phase. Greater survival was observed at 15 1C for cells from
the exponential phase. Hengge-Aronis (1993) described a set
of stress proteins synthesized just before entering the
stationary phase of growth which helps cells to resist adverse
conditions and delays their loss of cultivability. Several
authors (Munro et al., 1995; Dantur & Pizarro, 2004) found
that E. coli cells in the exponential phase are less resistant to
stress and lose cultivability more quickly than cells in the
stationary phase in which these proteins are present. In this
study, a remarkably enhanced resistance of P. fluorescens
populations was not observed in culture at stationary phase
submitted to nutrient deprivation. However, the survival
process in stationary phase as a reaction to temperature
seems to be mitigated in comparison with the behaviour in
exponential phase (Fig. 3).
There was a loss of culturability under stress conditions
and the adoption of VBNC phenotype in several bacteria
(Arana & Barcina, 2008; Barcina & Arana, 2009; Oliver,
2010). Although both E. coli and P. fluorescens enter into the
VBNC state (Figs 1 and 2), differences in the behaviour and
importance of VBNC fractions were found. Arana et al.
(2007) previously stated that the VBNC subpopulation is
not the dominant subpopulation during the survival process
of E. coli in aquatic ecosystems. In P. fluorescens, as the
temperature was raised, the formation of VBNC cells
decreased, but at low temperatures the VBNC fraction was
clearly predominant. Clegg et al. (1996) and Hase et al.
(1999) reported that the decline of several CHA0 derivatives
under starvation conditions did not result in the formation
of VBNC cells. These results were obtained in experiments
carried out for 7 days at 27 1C. In our work, 13 days were
necessary for VBNC detection at 25 1C (Fig. 2).
Whitesides & Oliver (1997) and Wong et al. (2004) have
described that the VBNC state in Vibrio is typically resusci-
tated by temperature upshift treatment; and Magariños et al.
(1997) have obtained similar results in Pasteurella pisticida
when nutrient conditions are favourable. The removal of
FEMS Microbiol Ecol 74 (2010) 500–509
Pseudomonas fluorescens and E. coli under adverse conditions
environmental stress led to the resuscitation of Salmonella,
Enterococcus, E. coli and Aeromonas (Lleó et al., 2000;
Ohtomo & Saito, 2001; Gupte et al., 2003; Maalej et al.,
2004). Nevertheless, resuscitation is a very controversial
topic and other studies have reported that nonculturable
bacteria cannot be resuscitated (Kolling & Matthews, 2001;
Ziprin et al., 2003; Arana et al., 2007). Our results agree with
last authors; there was no indication that nonculturable P.
fluorescens CHA0 could be resuscitated by the removal of
environmental stress.
Irrespective of the contradictions concerning the capacity
for resuscitation of some bacteria, it is of fundamental
importance to consider the role that populations of bacteria
in the VBNC state play in nature. These VBNC populations
maintain activity and therefore, participate in the functioning
of ecosystems in the carbon cycle and in energy production.
However, for environmental control laboratories, these VBNC
cells are a problem because of the impossibility of detection
with classical culture methods. Molecular techniques could
resolve this problem. Thus, the presence of nonculturable
Pseudomonas in aquatic environments has been demonstrated
by isolation of 16S rRNA genes closely related to P. aeruginosa
from deep-sea sediments (Li et al., 1999). However, Rezzonico
et al. (2003) have reported that QC-PCR gives effective results
in vitro only when applied to samples in which most P.
fluorescens CHA0 cells were culturable.
The use of immunological methods for the detection of
specific microorganisms is a rapid and simple technique
(Rompré et al., 2002), although it should be noted that these
methods cannot be used for viability studies. Some outer
membrane proteins might be used as diagnostic proteins for
Pseudomonas sensu stricto (de Mot et al., 1994; Aagot et al.,
2001). Kimata et al. (2004) investigated the presence and
distribution of P. aeruginosa in seawater using OprP detec-
tion, a protein synthesized specifically under phosphate-
deficient conditions.
Unlike other Gram-negative bacteria such as E. coli,
Pseudomonas sp. and other closely related organisms use
almost exclusively specific porins for uptake through the
outer membrane (Tamber et al., 2006). OprF is the major
surface protein of the genus Pseudomonas and found only in
this genus (Bodilis et al., 2006). This protein is an OmpA
homologue, which plays a role as nonspecific porin and
membrane structural protein, required for the maintenance
of the cell shape (Hancock & Brinkman, 2002; Bodilis et al.,
2006). In our study, we found a protein with a molecular
weight of about 35 kDa, which was identified as OprF. It is
the first time that the presence of an integral outer mem-
brane protein of Pseudomonas spp. showing a high expres-
sion in nonculturable as well as culturable populations is
characterized in all the adverse situations analysed. So, this
OprF protein could be suitable for Pseudomonas detection in
environment.
FEMS Microbiol Ecol 74 (2010) 500–509
507
We can conclude that E. coli (mesophile bacteria) and
P. fluorescens (psychrotrophic microorganism) present
different survival patterns under temperature stressing
conditions. Moreover, after the analysis of outer membrane
subproteoma of Pseudomonas, we can conclude that tem-
perature, not nutrient deprivation, is principally responsible
for changes in the outer membrane protein composition.
Our results also show that OprF could be a target to detect
Pseudomonas in environment.
Acknowledgements
The Spanish Government (CTM2006-09532/TECNO) has
financially supported this work. MS analysis was performed
in the Proteomics Unit at the University of the Basque
Country (SGIker, member of ProteoRed). Technical and
human support provided by SGIker (UPV/EHU, MICINN,
GV/EJ, ESF) is gratefully acknowledged.
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