jobm 201000037

Journal of Basic Microbiology 2010, 50, 507 – 518

507

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Research Paper

Growth temperature associated protein expression
and membrane fatty acid composition profiles
of Salmonella enterica serovar Typhimurium

Sampathkumar Balamurugan

Agriculture and Agri-Food Canada, Lacombe, Alberta, Canada

Total cellular proteins and fatty acid composition profiles of mid-log phase cells of Salmonella
enterica serovar Typhimurium grown at 8, 25, 37 or 42 °C were separated by 2D-PAGE and FAME
analysis. Growth temperature associated protein expression can be grouped into 3 thermal
classes which include proteins whose expression is: I) optimal at 37 °C, meaning their
expression peaked at 37 °C; II) up-regulated with an increase in growth temperature; III) down-
regulated with increase in growth temperature; meaning their expression peaked at 8 °C. At
higher growth temperatures, proteins belonging to the functional groups of amino acid
transport and metabolism, nucleotide metabolism, energy metabolism and post-translation
modifications (chaperones) are present in substantially higher amounts. This increase in
abundance is regulated in a temperature dependent manner. It is important to point out that
proteins involved in energy metabolism observed in higher amounts at higher growth tempe-
ratures all belong to the glycolysis pathway, while at 8 °C they belonged to the TCA cycle.
Increase in growth temperatures results in a decrease in membrane fatty acid unsaturation
and an increase in saturated and cyclic fatty acids. These results provide an insight into the
dynamic molecular and physiological responses of Salmonella Typhimurium during growth at
different temperatures.

Keywords: Salmonella sp. / Growth temperature / Protein expression profile / Membrane fatty acid composition

profile

Received: January 26, 2010; accepted: April 14, 2010

DOI 10.1002/jobm.201000037

Introduction

Temperature has a profound effect on survival and
growth of bacteria. Escherichia coli and Salmonella enterica
serovar Enteritidis will grow over a temperature range
of about 40 °C. For E. coli and Salmonella Enteritidis,
which are typical mesophiles, balanced growth can be
sustained from 10 to almost 49 °C. In the middle of this
temperature range, from approximately 20 to 37 °C, the
rate of growth varies with temperature as though cel-
lular growth, no matter what the medium, were a sim-
ple chemical process [34]. Increasing the temperature
above 42 °C, or decreasing it below 20 °C, leads to pro-

Correspondence: Dr. Sampathkumar Balamurugan, Agriculture and
Agri-Food Canada, Lacombe Research Center, 6000 C&E Trail, La-
combe, Alberta, T4L 1W1, Canada
E-mail: balamurugans@agr.gc.ca
Phone: (403)782-8119
Fax: (403)782-6120

gressively slower growth until finally growth ceases
altogether at 7 or 49 °C [24, 34, 60]. This temperature
range between 7 and 49 °C covers a wide array of envi-
ronments and hosts such as meat surface temperature
in retail meat display cabinets (8 to 10 °C), room tem-
perature (21 to 25 °C), human (37 °C) and avian hosts
(42 °C), that Salmonella routinely encounters.
Several previous studies have probed the effects of
extremes of temperatures, either high or low, on a
variety of cellular processes such as rate of DNA syn-
thesis and repair [15, 72], lipid synthesis and membrane
stability [6, 42], rate of ribosome and protein synthesis
[22, 32] and protein misfolding [31, 35]. In mesophiles, a
significant amount of literature is concentrated on
understanding bacterial adaptations to temperature up-
and down-shifts and the resulting phenotypic re-
sponses. These studies examine changes in gene [54, 57]
and protein expression [5, 75] or membrane fatty acid

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composition [19, 44, 79] changes in cells grown at their
ambient growth temperatures and then shifted to tem-
peratures near the extremes that can sustain growth
for pre-determined periods of time and then determine
their thermotolerance [8, 21] or survival during freez-
ing [8, 10, 45]. These are commonly referred to as heat-
or cold- shock/adaptation responses.
However, relatively little is known about the effect of
growth temperatures on protein expression and mem-
brane fatty acid compositions during steady-state
growth in mesophilic bacteria. This study aims to pro-
vide a snap shot of growth temperature associated pro-
tein expression and membrane fatty acid composition
profiles of mid-log phase cells of Salmonella enterica se-
rovar Typhimurium grown at 8, 25, 37 or 42 °C.

Materials and methods

Bacteria and growth conditions
Salmonella enterica subsp. enterica serovar Typhimurium
ATCC 13311 was obtained from American Type Culture
Collection (Manassas, VA, USA). A loopful of culture
was transferred from a frozen stock and streaked on
tryptic soy agar (TSA, Difco, Becton Dickinson Co.,
Sparks, MD, USA) plates and incubated at 37 °C for
16 h. A single colony from the TSA plate was trans-
ferred to 50 ml tryptic soy broth (TSB, pH 7.4; Difco,
Becton Dickinson Co., Sparks, MD, USA) in 125.0 ml
flasks and incubated aerobically on a rotary shaker (150
rpm) at 37 °C for 24 h. Five hundred microliters of this
culture was transferred to flasks containing 50.0 ml
TSB, pH 7.4 and incubated aerobically at 8, 25, 37 or
42 °C until the cells reached mid-log phase (A600 nm of
0.5 ± 0.05). Cells were harvested by centrifugation at
4,500 × g for 10 min and washed twice with sterile
DNase, RNase free distilled water (Invitrogen Corp.,
Grand Island, N.Y., USA) and stored at –80 °C until
protein and fatty acids extraction. Growth at different
temperatures were repeated 3 separate times and kept
frozen at –80 °C until further analysis.

Separation of total cellular proteins
by two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE)
Sample preparation: All chemicals for sample prepara-
tion and 2D-PAGE were obtained from GE Healthcare if
otherwise indicated. Total cellular protein was ex-
tracted by sonicating cell pellets in 1.0 ml lysis buffer
[8.0 M urea, 4.0% (w/v) CHAPS, 40 mM DTT and 2.0%
(v/v) pH 3–10 IPG buffer] thrice for 15 s each at 40 Ω
amplitude on ice with 1 min interval between pulses.

The extract was then treated with 0.1 volume buffer
containing 50 mM MgCl

, 1 mg DNase I ml

–1

and

0.25 mg RNase A ml

–1

for 15 min in ice. The reaction

was stopped by mixing it with 3 volumes ice-cold ace-
tone. Proteins were then precipitated for 2 h at –20 °C.
The precipitate was collected by centrifugation at
4,500 × g for 15 min, followed by decanting super-
natant and drying to remove residual acetone. The
protein precipitate was then resuspended in IPG rehy-
dration solution [8.0 M urea, 2.0% (w/v) CHAPS, 40 mM
DTT, 2.0% (v/v) pH 3–10 IPG buffer]. The dye-binding
assay of Bradford [11] was then performed to quantify
the protein concentration.
Isoelectric focusing: Three gels were run per sample
replicate resulting in a total of 9 gels per treatment.
Thirteen-centimetre Immobiline DryStrip gels (pH 4–7)
were rehydrated with 250 μl IPG gel rehydration buffer
[composition as above except that IPG buffer pH 4–7
was used and also contained 0.01% (w/v) bromophenol
blue] containing 400 μg of total protein for 16 h at
room temperature. Isoelectric focusing was achieved
when the total run time yielded 72 kVh at 20 °C follow-
ing the step and hold setting described by Gorg et al.
[29]. Isoelectric focusing was performed using a Mul-
tiphor II (GE Healthcare) electrophoresis system. Fol-
lowing isoelectric focusing, the gels were equilibrated
twice for 15 min each in 10 ml isoelectric focusing gel
equilibration buffer [50 mM Tris/HCL (pH 8.8), 6.0 M
urea, 30.0% (v/v) glycerol, 2.0% (w/v) SDS and 0.002%
bromophenol blue] containing 100 mg DTT for the first
equilibration and 250 mg iodoacetamide for the second
equilibration.
SDS-PAGE: Equilibrated isoelectric focused strips and
a 0.5 cm

filter paper pad impregnated with 10 μl of

prestained molecular mass ladder (Bio-Rad) were placed
on top of a uniform 14.0% SDS-polyacrylamide gel
(20 cm tall) and sealed with low melting point overlay
agarose (Bio-Rad) for second-dimension electrophoresis
using a Protean II xi electrophoresis system (Bio-Rad).
Second-dimension separation was carried out at 20 mA
per gel constant current for 30 min (for proteins/dye to
migrate from IPG strip to SDS-polyacrylamide gel) fol-
lowed by 25 mA constant current at room temperature
for 270 min (enough for the bromophenol blue dye
front to exit the gel). Proteins in each gel were fixed
using 300 ml of fixing solution (40% methanol and 10%
glacial acetic acid) for 30 min and stained for 18 h with
250 ml of Bio-Safe Colloidal G-250 (Bio-Rad) at room
temperature under gentle agitation on a shaker.
Stained gels were washed 3 times with 400 ml of deion-
ised H

O for 1 h each before imaging using an Epson

Expression 1680c pro desktop scanner as 300 dpi 16-bit

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Protein expression and membrane fatty acid profiles of Salmonella Typhimurium

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grayscale TIFF format images using the transmissive
mode.
Analysis of protein spots on 2D-PAGE gels: Protein
spots separated by 2D-PAGE were analysed for differen-
tial expression using ImageMaster™ 2D Platinum soft-
ware Version 5.0 (GE Healthcare). Statistical analysis
using trimmed means and their mean squared devia-
tions were calculated for spots within groups of gels.
This allows one to analyze variations in protein expres-
sion among treatments. ImageMaster provides the most
commonly used descriptive statistics of central tend-
ency (trimmed mean) and dispersion (mean squared
deviation) to analyze variations in protein expression
among treatments. A twofold difference in expression
was set as a threshold during image analysis to detect
proteins that were differentially expressed.

Mass spectrometry of proteins
Mass spectrometry and protein identification was per-
formed by the Institute of Biomolecular Design, Uni-
versity of Alberta, Edmonton, Canada. The solvents
used (water, acetonitrile, formic acid) are HPLC grade.
All other reagents were ultra pure grade. Stained spots
of interest were excised and an automated in-gel tryptic
digestion was performed on a Mass Prep Station (Mi-
cromass, UK). The gel pieces were de-stained, reduced
(DTT), alkylated (Iodoacetamide), digested with trypsin
(Promega Sequencing Grade Modified) and the resulting
peptides extracted from the gel.
In-gel tryptic digests were analyzed using a Bruker
Ultraflex TOF/TOF mass spectrometer. Sample was ap-
plied to an AnchorChip plate (Bruker) as previously
described by Zhang and coworkers [81]. Essentially 1 μl
of sample was applied to the plate and allowed to dry.
Then 0.8 μl of a 0.5 mg/ml CHCA (α-cyano-4-hydoxycin-
namic acid) in 70% acetonitrile and 0.1% TFA was ap-
plied on top of the sample. This was allowed to dry and
the spot was washed with 0.5% TFA. Mass spectra and
tandem mass spectra were then obtained using a
Bruker Ultraflex TOF/TOF in an automated fashion
using the AutoXecute function. Typically 100 shots
were accumulated to generate a peptide mass finger
print. The 5 most intense peaks were then selected to
have ms/ms performed where 400 shots on average
were used to generate ms/ms profile. The spectra were
then processed using Flexanalysis and mgf files pro-
duced using BioTools and finally searched using Mas-
cot.

Fatty acid methyl esters (FAME) analysis
Bacterial FAME analysis was performed at the Lipid
Biochemistry Lab, Agriculture & Agri-Food Canada,

Lacombe, Canada. Bacterial FAME were prepared from
40 to 50 mg (wet weight) cell pellet according to MIDI
Technical Note #101 [58]. Fatty acid methyl esters were
analyzed using a Varian (Varian Inc., Walnut Creek,
CA, USA) 3800 gas chromatograph (GC) equipped with a
Varian 8200 Autosampler, Varian 1079 injector, and a
flame ionization detector. The column used was a Su-
pelco (Supelco, Bellefonte, PA, USA) SPB-5 fused silica
capillary column 30 m length × 0.25 mm I.D. × 0.25 μm
film thickness. Gas chromatograph conditions were as
follows: column oven temperature initial hold of 4 min
at 150 °C then to 250 °C at 4 °C min

–1

, injector tempera-

ture held at 250 °C, detector temperature held at
280 °C, gas flow was held at a constant pressure of
15 psi, 1.0 μl of sample was injected and split 10:1.
Fatty acid methyl esters were identified compared to a
standard (Supelco Bacterial Acid Methyl Ester CP Mix)
and presented as percentage mean of 3 separate replica-
tions. The t-test for two samples assuming unequal
variance was performed using Microsoft Excel, to de-
termine statistical significance.

Results

At 8 °C, Salmonella Typhimurium reaches mid-log phase
(A600 nm of 0.5 ± 0.05) in ~66 h (3960 min). As the
growth temperature is increased, the time required for
Salmonella Typhimurium to reach A600 nm of 0.5 ± 0.05
decreased to 255, 145 and 135 min at 25, 37 and 42 °C,
respectively.

Growth temperature associated changes in protein
expression profiles
Growth temperature associated changes in protein
expression observed in this study can be grouped into 3
thermal classes (Table 1). These thermal classes include
proteins whose expression is: I) optimal at 37 °C, mean-
ing their expression peaked at 37 °C; II) up-regulated
with an increase in growth temperature; III) down-
regulated with increase in growth temperature; mean-
ing their expression peaked at 8 °C. Table 1 lists the
number of quantitative changes and associated cellular
function of proteins within the 3 thermal classes. Ta-
ble 2 lists the various differentially expressed proteins
within these 3 thermal classes.
At higher growth temperatures, proteins belonging
to the functional groups of amino acid transport and
metabolism, nucleotide metabolism, energy metabo-
lism and post-translation modifications (chaperones)
are present in substantially higher amounts (Tables 1
and 2; Fig. 1). This increase in abundance is regulated in

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Table 1. Number of quantitative changes, thermal classes* and
associated cellular functions identified through proteomic ana-
lysis.

Cellular function

Number of

proteins

I. Optimal expression at 37 °C

Amino acid transport and metabolism

Cell envelope (surface structure)

Nucleotide metabolism

II. Up-regulated with increase in growth temperature

Energy metabolism

Post-translation modifications (chaperone)

Protein synthesis

III. Down-regulated with increase in growth temperature

Biosynthesis of cofactors, prosthetic groups

and carriers

Cell envelope

Cellular processes

Energy metabolism

Protein synthesis

Post-translation modifications (chaperone)

Regulatory function

Signal transduction

Transport and binding

* In italics

a temperature dependent manner. However, the ex-
pression of a subset of these proteins belonging to the
functional groups, amino acid transport and metabo-
lism, nucleotide metabolism and flagellar structural
protein are optimal at 37 °C (Class I; Table 1 and 2),
meaning they were present in substantially higher
amounts at 37 °C and then decreased at 42 °C. In con-
trast, at the lower (8 °C) growth temperature, proteins
belonging to the functional groups of cellular envelope
and processes, energy metabolism, protein synthesis,
biosynthesis of cofactors, regulatory functions, signal
transduction, and transport are present in substantially
higher amounts (Tables 1 and 2; Fig. 1). Unlike growth
at higher temperature, only one chaperone protein
(HscA) was present in higher amounts in cells grown at
low temperatures (Tables 1 and 2; Fig. 1), although
other stress related proteins were present in higher
amounts in cells grown at low temperature. It is impor-
tant to point out that proteins involved in energy me-
tabolism observed in higher amounts at higher growth
temperatures all belong to the glycolysis pathway,
while at 8 °C they belonged exclusively to the tricar-
boxylic acid (TCA) cycle, except one that belonged to
the Pentose Phosphate pathway (PPP) (Table 2).

Growth temperature associated changes
in membrane fatty acid composition
Table 3 shows the changes in membrane fatty acid
composition of Salmonella Typhimurium cells grown to

mid-log phase at different temperatures. Major fatty
acids identified were n-dodecanoic (lauric, 12:0), n-tetra-
decanoic (myristic, 14:0), n-hexadecanoic (palmitic,
16:0), cis-9-hexadeceonic (palmitoleic, 16:1–9c), cis-11-
octadecenoic (cis-vaccenic, 18:1–11c), cyclo-heptadeca-
noic (17:0 cyclo) and cyclo-nonadecanoic (19:0 cyclo)
acid. The main change observed in membrane fatty acid
composition is the decrease in degree of fatty acid un-
saturation as the growth temperature is increased. This
occurs due to the decrease in the cis-vaccenic and
palmitoleic acid proportion and increase in palmitic,
cyclo-heptadecanoic and cyclo-nonadecanoic acid in a
temperature dependent manner (Table 3).

Discussion

Expression of chaperone and stress response
proteins
When cells are exposed to temperatures at the mini-
mum or maximum that can sustain growth, pressure is
exerted on these cells to maintain cellular functions.
These extreme temperatures severely affect protein
synthesis resulting in the accumulation of unfolded or
improperly folded proteins which are not functional.
Under these conditions, for cells to sustain growth,
these proteins would either have to be degraded or
folded to their correct confirmation to restore activity.
Molecular chaperone are a diverse group of proteins
that oversee the correct intracellular folding and as-
sembly of polypeptides without being components of
the final structure. Many chaperones are heat shock
proteins (HSP), that is, proteins expressed in response to
elevated temperatures or other cellular stresses [20].
Increase in the temperature of growth induces the heat
shock response. In the present study heat shock pro-
teins belonging to HSP60 (GroEL, GroES), HSP90 (HtpG)
and HSP100 (Clp) family (Fig. 1; Spots 3, 17, 2, 1; Table 2)
are up-regulated. While these HSPs are either undetect-
able or in very low concentrations in cells grown at
8 °C, their expression is up-regulated in a temperature
dependent manner (Fig. 1). These results are in agree-
ment that HSPs are constitutively expressed through-
out the growth cycle of mesophiles, but are dramati-
cally up-regulated at elevated temperatures [16, 31].
In contrast at 8 °C, only one chaperone protein, HscA
(Hsc66), was induced during grow (Fig. 1A; Spot F). This
protein was not detected at elevated temperatures. It
has been previously reported that chaperone protein
HscA, a HSP70 homolog, is induced approximately 11-
fold, 3 h after a shift from 37 to 10 °C and the expres-
sion of hscA was induced by cold shock and not upon

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Table 2. Differentially expressed proteins under different thermal classes in mid-log phase cells of Salmonella enterica serovar
Typhimurium ATCC 13311 grown at different temperatures.

Function Spot

Gene

Description

I. Optimal expression at 37 °C

Amino acid transport and metabolism

aspA

Aspartate ammonia-lyase

glyA

Serine hydroxymethyltransferase

carA

Carbamoyl-phosphate synthetase

cysK

Cysteine synthase A

artJ

Probable arginine-binding periplasmic protein

Cell envelope (surface structure)

fljB

Phase-2 flagellin structural protein

Nucleotide metabolism

purH

Phosphoribosylaminoimidazolecarboxamide formyltransferase

purC

Phosphoribosylaminoimidazole-succinocarboxamide synthase

II. Up-regulated with increase in growth temperature

Energy metabolism

pmgl

Phosphoglyceromutase

pfkA

6-phosphofructokinase

11,

gap

Glyceraldehyde-3-phosphate dehydrogenase

Post-translation modifications (chaperone)

clpB

Heat shock protein f84.1

htpG

Chaperon Hsp90, heat shock protein C62.5

groEL

60 kDa chaperonin, GroEL, Hsp 60 class

groES

Co-chaperone GroES, 10 kDa chaperonin

Protein synthesis

rpsB

30 S ribosomal protein subunit S2

III. Down-regulated with increase in growth temperature

Biosynthesis of cofactors, prosthetic groups

and carriers

ribB

3,4-dihydroxy-2-butanone 4-phosphate synthase

iscU

NifU homologue

Cell envelope

yaeT

Outer membrane protein precursor

Cellular processes

pnp

Polynucleotide phosphorylase

osmY

Hyperosmotically inducible periplasmic protein

tpx

Thiol peroxidase

Energy metabolism

pta

Phosphate acetyltransferase

sfcA

NAD-linked malic enzyme; malate oxidoreductase

aceF

Dihydrolipoamide acetyltransferase

lpd

Lipoamide dehydrogenase (NADH)

tal

Transaldolase

sucD

Succinyl-CoA synthetase alpha subunit

Protein synthesis

tsf

Elegation factor EF-2

proS

Prolyl-tRNA synthetase

rpsF

30S ribosomal protein subunit S6

Post-translation modifications (chaperone)

hscA

Chaperone protein HscA

Regulatory function

fur

Ferric uptake regulator

Signal transduction

bipA

GTP-binding proteins

Transport and binding

ybiT

ABC transporter ATP-binding protein

heat shock [41]. HscA is a molecular chaperone which
along with HscB a co-chaperone plays an important role
in iron-sulphur (FeS) cluster formation, which are im-
portant cofactors for numerous proteins involved in
electron transfer, in redox and non-redox catalysis, in
gene regulation, and as sensors of oxygen and iron [14].
However, the significance of the increased expression
of HscA, IscU and Fur at low temperatures in not ap-
parent.
In Gram negative bacteria,

-barrel proteins are a

major class of outer membrane proteins. In E. coli these
proteins are synthesized in the cytoplasm, translocated
across the inner membrane and assembled in the outer
membrane through an unknown mechanism that re-
quires the outer membrane YaeT complex [71, 74] and
the periplasmic chaperones SurA, DegP, and Skp [61,

70]. The presence of misfolded proteins up-regulates
the expression of these chaperones. Cells exposed to
extreme temperatures normally have an abundance of
misfolded proteins. Therefore YaeT in cells growing at
extreme temperatures could be expected to be at high
levels. However, in the present study, YaeT levels de-
creased as the growth temperature of cells was in-
creased (Fig. 1A; Spot A). No apparent reason could be
attributed for this observation at this time.
In

E. coli a downshift in temperature causes a tran-

sient inhibition of most protein synthesis, resulting in a
growth lag called the acclimation phase. It is during
this acclimation phase, cold shock proteins (CSP) are
dramatically induced [37]. These CSPs are loosely cate-
gorized into 2 classes [67]. Class I proteins are induced
to very high levels (>10-fold) during the acclimation

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Figure 1. Protein expression profiles of mid-log phase cells of Salmonella enterica serovar Typhimurium grown at 8 (A), 25 (B), 37 (C), and
42

°C (D). Identities and expression of labelled spots are indicated in Table 2.

phase and their levels fall back to non CSPs levels when
cold adapted. Some of the Class I proteins include CspA
family, CsdA, RbfA, NusA and PNP proteins. Class II
proteins are induced a few fold (<10-fold) during the
acclimation phase and their levels fall back to non CSPs
level when cold adapted. The cells examined in this
study were past the acclimation phase and were in a
mid-log phase/steady state of growth, which explains
why CSPs were not detected in cells grown at 8 °C. The
higher abundance of proteins like elongation factor,

t-RNA synthetase and ribosomal subunit proteins are
indicative of the protein synthesis and growth. Elonga-
tion factors are a set of proteins that facilitate the
events of translational elongation, the steps in protein
synthesis from the formation of the first peptide bond
to the formation of the last one. Thus during the ex-
tended cold acclimation period, synthesis of most cellu-
lar proteins is repressed while expression of CSPs are
induced and cells are cold adapted, following which
growth resumes at a new much reduced growth rate.

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Table 3. Fatty acid composition profiles of mid-log phase cells of Salmonella enterica serovar Typhimurium ATCC 13311 grown at
different temperatures.

Composition (% ± SD)*

Fatty acid

8 °C

25 °C

37 °C

42 °C

SFA

C12:0

1.11 ± 0.02

3.07 ± 0.11

4.22 ± 0.03

3.77 ± 0.07

C14:0

6.4 ± 0.15

6.12 ± 0.09

6.39 ± 0.05

6.19 ± 0.08

C15:0

0.08 ± 0.02

0.11 ± 0.01

0.05 ± 0.01

C16:0

23.423 ± 0.19

27.53 ± 0.54

32.55 ± 0.41

37.03 ± 0.86

C17:0

0.06 ± 0.02

0.09 ± 0.03

0.06 ± 0.00

C18:0

0.35 ± 0.08

0.68 ± 0.06

0.59 ± 0.15

0.79 ± 0.03

C19:0

0.11 ± 0.02

0.07 ± 0.02

0.05 ± 0.01

Total SFA

31.29 ± 0.31a

37.65 ± 0.49b

44.01 ± 0.55c

47.95 ± 1.02d

USFA

C16:1-9c

33.08 ± 0.43

25.45 ± 0.37

21.45 ± 0.07

19.97 ± 0.23

C18:1-9c

0.56 ± 0.12

0.13 ± 0.08

0.35 ± 0.01

C18:1-11c

21.39 ± 0.39

22.79 ± 0.52

19.38 ± 0.21

16.73 ± 0.17

Total USFA

54.48 ± 0.25a

48.80 ± 0.75b

40.96 ± 0.26c

37.06 ± 0.37d

CFA

C17:0 cyclo

0.84 ± 0.03

1.13 ± 0.04

2.49 ± 0.04

3.75 ± 0.08

C19:0 cyclo

0.28 ± 0.01

0.73 ± 0.04

0.63 ± 0.04

0.90 ± 0.01

Total CFA

1.12 ± 0.04a

1.87 ± 0.07b

3.13 ± 0.07c

4.65 ± 0.08d

OH-FA

C14:0 2-OH

0.13 ± 0.05

0.20 ± 0.01

0.13 ± 0.05

0.17 ± 0.01

C14:0 3-OH

9.71 ± 0.19

9.16 ± 1.17

8.83 ± 0.76

8.08 ± 1.28

Total OH-FA

9.85 ± 0.18a

9.36 ± 1.18a

8.96 ± 0.74a

8.25 ± 1.28a

Other FA

3.26 ± 0.10a

2.32 ± 0.10bd

2.94 ± 0.09c

2.09 ± 0.17d

Total FAME

100.00

* Values in the same row with different alphabets are significantly different (P < 0.05).

Fatty acid compositions are average of three separate replications.

Although only one chaperone protein was detected
in high levels in mid-log phase cells grown at 8 °C,
other proteins connected to bacterial stress response
were detected in these cells. Proteins connected to
stress response are either proteases, ribonucleases, or
involved in transport of newly synthesised proteins.
Polynucleotide phosphorylase (PNPase), GTP-binding
protein (BipA), hyperosmotically inducible periplasmic
protein (OsmY), and thiol peroxidase (Tpx) are some of
the stress response proteins present in high levels in
mid-log phase cells grown at 8 °C.
As the accumulation of stress response proteins ap-
pear to be deleterious to cells, the overproduction is
prevented by ribonucleases and proteases, which are
induced by the same stress. In the case of heat shock
response, σ

which plays the major role in regulating

heat shock response [80] is degraded by a specific prote-
ase, FtsH, which is also induced by heat shock [33, 68].
In the case of cold shock and adaptation, mRNA stabil-
ity is the major factor for regulation of CSP expression
[12, 28]. Polynucleotide phosphorylase (PNPase) is a cold
shock-induced exoribonuclease and a component of

RNA degradosomes involved in mRNA degradation [4, 7,
76]. Yamanaka and Inouye [77] reported that the syn-
thesis of CSPs was not repressed in the E. coli pnp mu-
tant, but was extremely sensitive to low temperature
and was unable to form colonies below 25 °C. The cold-
sensitive growth phenotype of the pnp mutant was fully
complemented by transforming cells with plasmid
carrying the pnp+ gene [78], indicating that PNPase
function is essential for growth at low temperature. A
similar observation was reported in a PNPase-deficient
mutant of Yersinia enterocolitica that was unable to de-
grade CSP mRNA properly after cold shock [48]. This is
in agreement with the higher abundance of PNPase
(Fig. 1A; Spot B) in cells grown at 8 °C in the present
study.
In E. coli BipA has been shown to be required for
growth at low temperatures [50]. This is in agreement
with the higher abundance of this protein in cells
grown at 8 °C in the present study (Fig. 1A, Spot E).
BipA is a highly conserved prokaryotic GTPase that func-
tions to regulate numerous actions in bacteria such as
virulence [23, 59], host defence [53], and starvation [26].

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Journal of Basic Microbiology 2010, 50, 507 – 518

www.jbm-journal.com

OsmY is a periplasmic protein induced by hyperos-
motic stress. The function of this protein has not been
determined, but it is expected to regulate the transport
of osmolytes. The role of accumulating osmolytes as a
cryoprotectant and enabling growth at low tempera-
tures in Listeria has been studied in detail [64, 69]. How-
ever, the role of accumulating osmolytes as cryoprotec-
tants in Salmonella and E. coli is not clear. Thus, the
abundance of OsmY protein in mid-log phase cells
grown at 8 °C suggests that there could be a common
factor that regulates the response to cold and osmotic
stress. The answer could be in the role RpoS plays in
stress response regulation. At low growth temperature
RpoS expression has been observed in log-phase cells
[62]. Cold shock proteins have been proposed to act as
an RNA chaperone that facilitates translation at low
temperature by blocking the formation of secondary
structures [36]. Among the Csp proteins, CspC and CspE
are unique, as they seem to function at both physio-
logical temperature and during cold shock [51]. Several
diverse functions, including, for example, chromosome
condensation and regulation of the PNPase activity,
have been assigned to CspE [25]. CspC and CspE have
been shown to upregulate the expression of the gene
encoding a global stress response regulator RpoS [51].
The upregulation is caused by the rpoS RNA stabiliza-
tion. RpoS is a regulatory element of OsmY [51]. Thus
the high levels of OsmY in mid-log phase cells grown at
8 °C could be indirectly regulated by Csp proteins that
are constitutively expressed in cells at both physiologi-
cal temperature and cold shock.
Extended exposure of E. coli to temperatures above
and below their growth optimum leads to significant
increase in intracellular H

concentration and oxi-

dized glutathione level against their low levels at opti-
mum growth temperature [63], meaning that at ex-
treme temperatures cells could be experiencing oxi-
dative stress, which would explain why thiol peroxidase
(Tpx) was present in high levels in mid-log phase cells
grown at 8 °C. However, high levels of thiol peroxidase
and other antioxidants were not observed in cells
grown at high temperatures.
The gene product of ribB, 3,4-dihydroxy-2-butanone
4-phosphate synthase, catalyzes the conversion of D-
ribulose 5-phosphate to formate and 3,4-dihydroxy-2-
butanone 4-phosphate, which is an important step in
riboflavin biosynthesis. The abundance of ribB is the
highest at 25 °C and is reduced in cells grown at higher
temperatures (Fig. 1A; Spot N). This finding is in
agreement with prior report that ribB is expressed at all
temperatures, but accumulation of transcripts decline
with raising temperature and its expression is re-

pressed by heat shock [56]. However, it’s role in Salmo-
nella growing at low temperatures in unclear.

Expression of proteins involved
in energy metabolism
Extracts from mid-log phase cells grown at 8 °C con-
tained increased levels of proteins involved in the TCA
cycle, while glycolytic proteins were present in high
levels in extracts from mid-log phase cells grown at
higher temperatures (Table 2). This observation is con-
sistent with previous reports [27]. The increased levels
of proteins that are required for energy generation [17]
in cells with reduced capacities for protein synthesis
indicate that the cells grown at 8 °C are stressed by
energy limitation. Noticeable of the TCA cycle proteins
are proteins involved in pyruvate metabolism. Dihy-
drolipoamide acetyltransferase (Fig. 1A; Spot I) and lipo-
amide dehydrogenase (Fig. 1A; Spot K) along with dihy-
drolipoamide reductase are subunits of pyruvate
dehydrogenase complex which is the first enzyme in
the TCA cycle catalyzing the breakdown of pyruvate to
acetyl-CoA, while Phosphate acetyltransferase (PTA;
Fig. 1A; Spot D) and succino-CoA synthetase (Fig. 1A;
Spot N) catalyze the breakdown of acetyl-CoA and suc-
cinyl-CoA, respectively, to release CoA, which is fed
back into pyruvate metabolism for the formation of
acetyl-CoA. Acetyl-CoA is an important substrate for
TCA cycle as well as fatty acid synthesis. Another TCA
cycle protein detected in higher levels, malate oxidore-
ductase (Fig. 1A; Spot G) catalyzes the conversion of
malate to pyruvate and NADH. In addition to TCA cycle
proteins, transaldolase (Fig. 1A; Spot L), one of the 2
pentose phosphate pathway (PPP) proteins is present in
higher amount in low temperature grown cell extracts.
Transaldolase along with transketolase converts glu-
cose 6-phosphate to fructose 6-phosphate and glyceral-
dehyde 3-phosphate. The fructose 6-phosphate can be
changed back to glucose 6-phosphate while glyceralde-
hyde 3-phosphate is converted to pyruvate by glycolytic
enzymes. The detection of TCA cycle and PPP proteins
at higher levels in extracts of mid-log phase cells grow-
ing at low temperature suggests the presence of high
concentration of pyruvate in the cold adapted cells
which could serve as the primary substrate for energy
production when cold adapted cells resume growth at
low temperatures. However, this would require glyco-
lytic break down of glucose to pyruvate or acetate dur-
ing cold acclimation. Wouters et al. [73] examined gly-
colytic activity of mid-exponential phase cells of Lacto-
coccus lactis exposed to 10 °C for several hours. They
reported maximal glycolytic activity after 4 to 5 h at
10 °C. After longer exposure (20 h), they saw a signifi-

Journal of Basic Microbiology 2010, 50, 507 – 518

Protein expression and membrane fatty acid profiles of Salmonella Typhimurium

515

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cant drop in glycolysis. This suggests that during the
extended lag/cold acclimation phase, glycolytic activity
could have been induced resulting in the breakdown of
glucose to pyruvate and release of limited amount of
ATP which could be utilized for the synthesis of CSPs. It
has to be remembered that cells examined in the pre-
sent study were past their lag/cold acclimation stage
and were in steady state growth which could be why
glycolytic enzymes were not detected in higher
amounts. In contrast, in cells growing at higher tem-
peratures there is either a very short or no noticeable
lag phase. When cells are exposed to ambient growth
temperatures in a nutrient rich medium, cells enter a
rapid state of growth resulting in increase in cell num-
ber until nutrients are depleted or when exposed to
adverse environmental conditions. As a consequence of
aerobic growth and utilization of glucose, glucose is
converted to pyruvate by glycolysis, followed by break
down of pyruvate to acetyl-CoA by the pyruvate dehy-
drogenase complex. Acetyl-CoA enters the TCA cycle
and is oxidized to yield energy. The pyruvate level in
the cells reaches a maximum when cells have utilized
all the glucose and reach early stationary phase [30].
This could be why increased levels of glycolytic proteins
were observed in mid-log phase cells growing at higher
temperatures. However this does not mean that TCA
cycle proteins are not expressed. It is evident from
Fig. 1 that both glycolytic and TCA cycle proteins are
being expressed under all growth temperatures but
glycolytic proteins are present in increased levels in
mid-log phase cells growing at ambient or higher tem-
perature. The products of TCA cycle could be diverted
towards the synthesis of amino acids, proteins, fatty
acids, nucleotides and flagella biosynthesis leading to
increase in cell numbers.

Expression of proteins
at ambient (37

°C) growth temperature

Cells growing at the ambient growth temperature of
37 °C had proteins involved in amino acid and nucleo-
tide metabolism in abundance (Fig. 1C). These proteins
are essential for cellular growth. Our observation is
consistent with Gadgil et al. [27] who reported a signifi-
cant drop in expression of genes involved in amino acid
biosynthesis and transport and nucleotide metabolism
during a temperature downshift from 37 °C. Such de-
crease in expression probably reflects the reduced
growth rate and the accompanying reduction in energy
and amino acid demand at lower temperatures. Al-
though the growth rate continues to increase at 42 °C,
the abundance of these proteins goes down at the ele-
vated temperature (Fig. 1D, Table 2). Another protein,

flagellin phase-2 structural protein (Fig. 1C; Spot 4) is
present in higher levels in cells grown at 37 °C and
their abundance goes down at both higher and lower
temperatures (Fig. 1, Table 2). Up-regulation of the flag-
ellin protein is an indication of flagellation. This is in
agreement with other findings that transfer of Salmo-
nella Typhimurium to temperature above (44 °C) and
below (20 °C) optimum (37 °C) prevented flagellation
[39, 43, 55]. Similar effects of elevated temperatures on
flagellation have been observed in Proteus vulgaris [9],
E. coli [46, 47], and Pasteurella pseudotuberculosis [52].
Andrade et al. [3] observed flagella regardless of the
temperature of growth, although growth at 37 °C ap-
peared to be the most favourable for flagellum produc-
tion in E. coli. These reports are consistent with the
observations in the present study.

Temperature dependent changes
in membrane fatty acid profiles
The decrease in the degree of fatty acid unsaturation is
the most commonly reported response of Gram-nega-
tive bacteria to an increase in growth temperature.
However, the relative proportion of cis-vaccenic to
palmitoleic acid during the decrease in degree of fatty
acid unsaturation in not very clear. It is thought that
the decrease in palmitoleic acid seems to occur espe-
cially within the low-growth temperature range of 0 to
20 °C, while the decrease in cis-vaccenic acid seems to
occur within a higher growth temperature range of 20
to 40 °C [65]. Such a preference in the reduction of an
unsaturated fatty acid over the temperature range ex-
amined in this study was not observed. Another impor-
tant observation is the degree of fatty acid cyclization
which appears to increase with an increase in the
growth temperature (Table 3), and has been reported in
a great variety of Gram-negative bacteria [13, 18]. How-
ever, fatty acid cyclization does not seem to be an abso-
lute requirement for adaptation of Gram-negative bac-
teria to variations in the environmental temperatures
because in several studies no changes occur in fatty
acid cyclization [1, 49].
Bacterial cytoplasmic membrane which consists
mainly of lipids is the boundary between the cytoplasm
and the external environment which regulates the flow
of nutrients and metabolic products in and out of the
cell, thereby permitting homeostasis of the cytoplasmic
environments [38]. When bacteria are exposed to ad-
verse environmental conditions they change the com-
position of the membrane lipids. These changes in the
composition of membrane lipid affect mainly the fluid-
ity of the cellular membrane and are thought to occur
in order to maintain both membrane integrity and

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functionality in the face of the external conditions [66].
For the most part, the ratio of saturated fatty acids
(SFA) to unsaturated fatty acids (USFA) affects mem-
brane fluidity. An increase in the ratio suggests de-
creased membrane fluidity. In the present study, with
an increase in the temperature of growth, the ratio of
saturated to unsaturated fatty acids increases from 0.57
at 8 °C to 0.77 at 25 °C to 1.07 at 37 °C to 1.29 at 42 °C.
Previous studies have reported that as the temperature
of growth increases, fatty acids with higher melting
point (palmitic acid, T

: 41 °C) are incorporated into the

lipid bilayer, which increases the phospholipids gel to
liquid-crystalline-phase transition temperature of the
membrane, thereby maintaining fluidity and compen-
sates for the increase in temperature [40, 66]. Simulta-
neously, there is a decrease in the composition of low
melting point fatty acids (palmitoleic acid T

: –36.0 °C;

cis-vacennic acid T

: –19.0 °C) which are converted to

their corresponding cyclic fatty acids (CFA; cyclo-hepta-
decanoic, 17:0 cyclo; and cyclo-nonadecanoic, 19:0
cyclo, respectively). Similar observations of increase in
CFA and decrease in USFA at high growth temperatures
have been reported [2] and are in agreement with the
findings in this study.

Conclusions

As the temperature of growth is increased the func-
tional groups with the largest number of proteins dif-
ferentially expressed are energy metabolism, amino
acid transport and metabolism and finally chaperone
proteins at 42 °C. Although growth rate is higher at
temperatures above optimum (37 °C), cells shift to a
stress combating mode which is evidenced by the in-
crease in chaperone proteins in cells grown at 42 °C.
Another important functional group; energy metabo-
lism, is unique to the growth temperature. Glycolytic
proteins are in abundance with an increase in growth
temperature, while TCA cycle proteins are abundant in
cells grown at low temperatures (8 °C). Increase in
growth temperatures results in a decrease in mem-
brane fatty acid unsaturation and an increase in satu-
rated and cyclic fatty acids. These results provide an
insight into the dynamic molecular and physiological
responses of Salmonella Typhimurium during growth at
different temperatures.

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