International Journal of Food Microbiology 191 (2014) 135–143
Contents lists available at ScienceDirect
International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
Cold stress improves the ability of Lactobacillus plantarum L67 to
survive freezing
Sooyeon Song a, Dong-Won Bae b, Kwangsei Lim c, Mansel W. Griffiths d, Sejong Oh a,⁎
a
Division of Animal Science, Chonnam National University, 77 Yongbong-ro, Gwangju 500-757, Republic of Korea
Central Instrument Facility, Gyeongsang National University, 900 Gajwa-dong, Jinju, Gyeongnam 660-701, Republic of Korea
Dairy Food R&D Center, Maeil Dairies Co., Ltd., 480, Gagok-ri, Jinwi-myun Pyungtaek-si, Republic of Korea
d
Department of Food Science, University of Guelph, Canadian Research Institute for Food Safety, Guelph, ON, Canada
b
c
a r t i c l e
i n f o
Article history:
Received 27 March 2014
Received in revised form 11 August 2014
Accepted 14 September 2014
Available online 18 September 2014
Chemical compounds studied in this article:
Sodium chloride (PubChem CID: 5234)
Phenol (PubChem CID: 996)
Acetone (PubChem CID: 180)
Urea (PubChem CID: 1176)
CHAPS (PubChem CID: 107670)
Dithiothreitol (PubChem CID: 19001)
Isopropanol (PubChem CID: 3776)
Glycerol (PubChem CID: 753)
Bromophenol blue (PubChem CID: 8272)
Ammonium bicarbonate (PubChem CID:
14013)
a b s t r a c t
The stress resistance of bacteria is affected by the physiological status of the bacterial cell and environmental factors such as pH, salts and temperature. In this study, we report on the stress response of Lactobacillus plantarum
L67 after four consecutive freeze–thaw cycles. The cold stress response of the cold-shock protein genes (cspC,
cspL and cspP) and ATPase activities were then evaluated. The cold stress was adjusted to 5 °C when the bacteria
were growing at the mid-exponential phase. A comparative proteomic analysis was performed with twodimensional gel electrophoresis (2D SDS-PAGE) and a matrix assisted laser desorption/ionization-mass
spectrometer. Only 56% of the L. plantarum L67 cells without prior exposure to cold stress survived after four consecutive freeze–thaw cycles. However, 78% of the L. plantarum L67 cells that were treated with cold stress at 5 °C
for 6 h survived after freeze–thaw conditions. After applying cold stress to the culture for 6 h, the cells were then
stored for 60 days at 5 °C, 25 °C and 35 °C separately. The cold-stressed culture of L. plantarum L67 showed an 8%
higher viability than the control culture. After applying cold stress for 6 h, the transcript levels of two genes (cspP
and cspL) were up-regulated 1.4 (cspP) and 1.2 (cspL) times compared to the control. However, cspC was not upregulated. A proteomic analysis showed that the proteins increased after a reduction of the incubation temperature to 5 °C. The importance of the expression of 13 other relevant proteins was also determined through the
study. The exposure of L. plantarum cells to low temperatures aids their ability to survive through subsequent
freeze–thaw processes and lyophilization.
© 2014 Published by Elsevier B.V.
Keywords:
Lactobacillus plantarum L67
Cold stress response
cspC
cspP
cspL
1. Introduction
Lactobacilli and bifidobacteria are well known as the most frequently and safely used starter probiotic bacteria, even for children and immunocompromised individuals (Borriello et al., 2003). In particular,
Lactobacillus plantarum belongs to the facultatively heterofermentative
group of lactobacilli and is a heterogeneous and versatile species that
is encountered in a variety of environmental niches, including dairy,
meat, fish, and many vegetable or plant fermentations. It has a proven
ability to survive gastric transit and colonize the intestinal track of
humans and other mammals (Sartor, 2004; de Vries et al., 2006).
⁎ Corresponding author at: Division of Animal Science Chonnam National University,
77 Yongbong-ro Puk-gu, Gwangju 500-757, Republic of Korea. Tel.: +82 62 530 0822;
fax: +82 62 5302129.
E-mail address: [email protected] (S. Oh).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.017
0168-1605/© 2014 Published by Elsevier B.V.
During the manufacture of lactic acid fermented products, bacterial
cells are exposed to various environmental stresses such as extreme
temperatures, pH, osmotic pressure, oxygen, high pressure and starvation, which may affect the physiological status and the properties of
the cells. However, bacterial cells are naturally equipped with a plethora
of defense mechanisms, such as chaperone proteins (Gro ES/GroEL and
DnaK/ DnaJ/Grp E), proteases, transport systems and proton pumps to
enhance survival in stressful environments (Carcoran et al., 2008).
Moreover, many research studies have reported the positive effects of
the stress response. For example, to improve the viability of the probiotic strain Lactobacillus paracasei NFBC 338 during spray-drying, Desmond
et al. (2004) demonstrated that pre-stressing the culture by exposure to
52 °C for 15 min improved the survival of the strain 700-fold (in
reconstituted skim milk) during heat stress and 18-fold during spraydrying compared to unadapted cells. Exposure to salt stress also
afforded a level of thermo-tolerance. Indeed, following exposure to
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S. Song et al. / International Journal of Food Microbiology 191 (2014) 135–143
0.3 M NaCl, the survival of the strain improved by 16-fold during spraydrying (Desmond et al., 2001).
Freezing is generally used to preserve a starter culture for an extensive amount of time while still maintaining its viability and acidification
activity. After freezing and thawing, many cells are often metabolically
damaged or even killed. The freezing resistance of Lactobacillus is
enhanced by applying cold stress conditions before freezing. In contrast
with Escherichia coli, Bacillus subtilis, and Lactococcus lactis, the function
of the cold response in L. plantarum has not been extensively studied
(Barria et al., 2013). Cold-shock response is classically exhibited when
an exponentially growing culture is shifted from its optimum growth
temperature to a lower temperature. In most bacteria, such as
B. subtilis, a temperature downshift causes a transient cell growth arrest,
during which the general protein synthesis is severely inhibited. However, under these conditions, the synthesis of CIPs is triggered. Eventually, the synthesis of these proteins decreases, the cells become
acclimated to the low temperature, and growth resumes (Jones et al.,
1987). Recently, it has been established that CSPs might regulate the
expression of cold-induced genes such as anti-terminators (Bae et al.,
2000). The regulation of csp genes takes place at several levels. Additionally, the regulation of CspAE expression seemed to occur transcriptionally and post-transcriptionally through protein stability effectors
(Goldenberg et al., 1997). The chromosome of L. lactis contains two
sets of cold-inducible csp genes (cspA/cspB and cspC/cspD) (Wouters
et al., 2000a, 2000b). Small heat shock (shs) genes are also induced by
cold in L. plantarum, and a role for the sHsps in preventing damage by
low temperature has been suggested and L. plantarum strains
overproducing Hsp 18.5, Hsp 18.55 and Hsp 19.3 have improved growth
at low temperature (Spano et al., 2005; Fiocco et al., 2007). L. plantarum
is usually exposed to harsh, stressful environments during handling and
storage. These harsh environments include being either freeze-dried or
just subjected to a regular freeze during the food process through the GI
tract. In the case of L. plantarum, cold stress genes such as cspC, cspL, and
cspP have been identified (Mayo et al., 1997; Derzelle et al., 2000).
The purpose of this study was to determine the survival rate of csp
mRNA levels and the inductive proteins associated with L. plantarum
L67 under cold stress.
2. Materials and methods
2.1. Bacteria
L. plantarum L67 from infant feces were selected according to their
colony characteristics, and underwent a gram stain to be tested for catalase activity. These isolates were further screened for their acid and bile
salt tolerance capacities according to Park et al. (2002). For identification of L. plantarum L67, the biochemical properties were first examined
using an API 50CHL kit (BioMerieux, France) and, finally, 16s rDNA
sequencing data, as described by Kim et al. (2004). L. plantarum L67
was grown in MRS broth at 37 °C for 18 h (de Man et al., 1960). The
bacterial cells were separated by centrifugation at 3000 ×g for 15 min
at room temperature. Subsequently, the precipitated cells were washed
twice with sterile saline (0.85% sodium chloride). One milliliter of 10%
skim milk was added to the sediments, and the suspension was stored
at −80 °C until use.
2.2. Growth conditions of L. plantarum L67 and the cold stress treatments
The cells were inoculated in 1% of the overnight culture of the strain
in 10 ml of fresh MRS broth, incubated at 37 °C and harvested when
they reached the mid-exponential phase of growth (OD 600 nm of
0.6). Growth kinetic experiments were performed with MRS broth at
37 °C. For cold shock experiments, the exponentially growing cells
were incubated at 5 °C for 1, 4 or 6 h, and then growth kinetic experiments were performed at 37 °C. All experiments were carried out in
triplicate.
2.3. Freeze–thaw challenge
Two hundred milliliters of MRS broth was inoculated (1%) and incubated at 37 °C. The cells grown to the mid-exponential phase were cold
shocked at 5 °C for 1, 4 and 6 h. Then, the cells were quickly frozen and
stored at − 70 °C. The control group consisted of cells frozen directly
without pre-adaptation for 24 h of freezing. After, the cells were thawed
by placing the tubes in a 37 °C water bath for 5 min. Aliquots of each
sample were taken out and analyzed for viability before they were frozen again at −70 °C. A total of four freeze–thaw cycles were performed.
Each freeze–thaw cycle was performed after 24 h of the previous one.
The thawed cells were serially diluted with 1% peptone water, and the
viable cells were counted.
2.4. Storage stability of freeze-dried L. plantarum L67
L. plantarum L67 was grown in 1 l of MRS broth at 37 °C. When the
growth reached the mid-exponential phase (OD600 of 0.6), the cells
were subjected to 6 h of cold shock treatment at 5 °C in fresh MRS
broth. The cell pellets were collected by centrifugation (5000 ×g at
4 °C) and then re-suspended in skim milk before being frozen at
−70 °C. The frozen cells were freeze-dried for 48 h at 0.2 mbar with a
collecting temperature of −50 °C (IlShin Lab, Yangju, Korea). The cell
suspensions were freeze-dried in duplicates. Duplicate samples of
each treatment were placed in glass vials (40 ml) and purged with
nitrogen gas (10 ml/min) for 10 min. The glass vial lots were stored at
5 °C, 25 °C or 35 °C in the dark for 60 days.
2.5. RNA isolation and RT-qPCR
Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA) with the manufacturer's instructions. The cDNA was prepared from 1 μl of total RNA (1 μg/20 μl) using Maxime RT Premix
Oligo (dT) for the RT-qPCR kit (Intron, Seongnam, Korea). The
primers used in this study were designed by Primer3 software program (www.bioinformatics.nl/primer3plus) based on the genome
sequence of L. plantarum WCFS1 (GenBank accession number NC_
004567). The primers used are shown in Table 1. The expression levels
of the respective genes were measured by real-time PCR using 2× Prime
QMaster Mix (Kapa Biosystems, Boston, USA) and were analyzed using
a CFX96TM Real-Time system (Bio-Rad, California, USA). The reaction
parameters for the real-time PCR analysis were 94 °C for 5 min, followed by 35 cycles of 94 °C for 40 s, 58 °C for 40 s, and 72 °C for 60 s. The
final elongation step was at 72 °C for 5 min. The sample ΔCt (SΔCt)
value was calculated as the difference between the Ct values of the csp
gene before and after applying the cold shock. The ΔCt value of the undifferentiated L. plantarum WFSC1 rpoD (Kleerebezem et al., 2003;
Duary et al., 2012) gene was used as the control ΔCt (CΔCt) value. The
relative gene expression levels between the sample and the control
were determined using the formula: 2−(SΔCt − CΔCt) (Livak and
Schmittgen, 2001).
Table 1
Oligonucleotides used for real-time PCR in this study.
Gene
Sequence of PCR primers (5′ to 3′)
cspP
Forward: 5′GTGAAGACGGTACCGATGTCTT-3′
Reverse: 5′GTGGTTGAACGTTCGTTGCT-3′
Forward: 5′ATCACTCGCGAAAACGGTAG-3′
Reverse: 5′CCACGATCGCTTTCTTCAAC-3′
Forward: 5′TACTGGTGAAGATGGCAACG-3′
Reverse: 5′GAACAACGTTAGCAGCTTGTGG-3′
Forward: 5′GTGAAGAGGACGATTCACACCT-3′
Reverse: 5′GGTATCAAGGACACCTTCCAG-3′
cspC
cspL
rpoD
S. Song et al. / International Journal of Food Microbiology 191 (2014) 135–143
2.6. Protein extraction of L. plantarum L67
For the proteomic study, 200 ml of the mid-exponential phase
(OD600 of 0.6) cultures was obtained as described above at 37 °C. For
the cold shock experiments, the cells were cultivated at 37 °C at the
mid-exponential phase (OD600 of 0.6) and subjected to 1, 4, or 6 h (respectively) of cold shock treatment at 5 °C. The cell pellets were collected by centrifugation (12,000 ×g, 4 °C). The collected cell pellets were
washed 3 times using 0.85% NaCl. The soluble protein fractions were
extracted twice with phenol (Sigma Chemical Co, St. Louis, USA) (Kim
et al., 2008). Then, 2 ml aliquots of each individual extract were transferred to 15 ml polypropylene tubes. One milliliter of phenol (Sigma
Chemical Co.) was then added. The sample was vortexed after and
was heated at 70 °C for 10 min. The sample was cooled on ice for
5 min, and the phases were separated by centrifugation at 12,000 ×g
for 20 min. The top aqueous phase was discarded and replaced by
1 ml of distilled H2O. The sample was then vortexed and heated at
70 °C for 10 min. The sample was cooled on ice for 5 min, and the phases
were separated again by centrifugation, as before. Then, the top aqueous
phase was discarded. The proteins were precipitated by the addition of
cold acetone at 4 °C for 2 h. After centrifugation, the supernatant was
discarded, and cold acetone was added. The pellet was disrupted by vigorous vortexing, and the precipitated proteins were pelleted by a final
centrifugation of 12,000 ×g for 20 min. The resulting pellet was airdried for 2 h under a hood. The protein concentrations were measured
by a Bradford assay (Bradford, 1976) using the Bio-Rad Protein Assay
(Bio-Rad, California, USA).
137
Mini Bead beater (BioSpec Products, Seoul, Korea). After the addition
of DNase and RNase (10 μg/ml), the solutions were maintained at
room temperature for 45 min. The unbroken cells and debris were subsequently removed by centrifugation at 15,000 ×g for 20 min. The
supernatant was further centrifuged at 20,000 ×g for 20 min, and the
ATPase activity in the supernatant was determined.
2.10. ATPase activity assay
The ATPase activity was estimated from the release of inorganic
phosphate, as measured according to the method of Fiske and
Subbarow (1925). The membrane protein was incubated at 37 °C for
10 min in Tris maleate buffer (pH 7.5, 50 mM) containing 10 mM
MgCl2 and 5 mM ATP (Sigma Chemical Co, St. Louis, USA). The reaction
was stopped by addition of 1 ml of sterile water containing 3.5 N sulfuric
acid, 1 ml of 3.5% ammonium molybdate and 1 ml of 2.1% sodium bisulfite with 0.7% Developer (Kodak Korea, Seoul, Korea) subsequently
added. After 20 min, the absorbance at 660 nm was measured. One
unit of ATPase activity was defined as the amount of enzyme that
1 μmol of inorganic phosphate released in 1 min. The calibration was
performed with a series of Pi standards.
2.11. Statistical analysis
All the experiments were performed in triplicate. The data were
analyzed using Duncan's multiple range test (Duncan, 1955). A P value
of b0.05 in all replicate experiments represented statistical significance.
2.7. Two-dimensional gel electrophoresis
3. Results
Protein loads of 1 mg/ml in 315 μl of rehydration buffer (8 M urea,
2% CHAPS, 50 mM dithiothreitol, 10% isopropanol, 5% glycerol, 0.5%
ampholytes at pH of 3 to 10, and a trace of bromophenol blue) were
used to load the Ready Strip IPG Strips (17 cm, pH 3 to 10, Genomine,
Pohang, Korea) under mineral oil at 20 °C for 15 h. Isoelectric focusing
(IEF) was performed using a PROTEAN IEF cell (Bio-Rad, California,
USA) according to the manufacturer's instructions. The two dimensional
gel electrophoresis (2D SDS-PAGE) was performed at 150 constant volts
using 12.5% SDS-PAGE gels. The separation was conducted using a
PROTEAN II xi system (Bio-Rad, California, USA) with 10 mA per gel
for 1 h and thereafter with 20 mA per gel at 4 °C. The protein spots
were visualized by Coomassie blue staining (Candiano et al., 2004).
The stained gels were scanned with a densitometric scanner (800 by
1600 dpi; UTA 2100XL, UMAX, Techville Inc., Dallas, TX), and the spots
were analyzed with PD Quest software (Bio-Rad, California, USA).
3.1. Effect of temperature on L. plantarum L67 growth
As a first step to understanding the cold stress response in L. plantarum
L67, we constructed a growth curve for L. plantarum L67 at 5 °C, 37 °C and
under cold stress at 5 °C following growth to the mid-exponential phase
at 37 °C. The cell viability at 37 °C after cold shock treatment for 1, 4 or 6 h
was also determined. Fig. 1 shows the growth curve of L. plantarum L67 at
37 °C and 5 °C. The L. plantarum on the MRS broth at 37 °C reached an OD
of 1.2 (OD600) after 6 h, whereas the L. plantarum cells did not grow at
5 °C.
The effect of prior cold stress on the growth kinetics of L. plantarum
L67 at 37 °C is shown in Fig. 2. The cell density reached 1.2 (OD600) at
2.0
2.8. Analysis of matrix assisted laser desorption/ionization-mass
spectrometer (MALDI-MS)
2.9. Crude enzyme extract of L. plantarum L67 for the ATPase activity assay
The L. plantarum L67 cultures at the mid-exponential phase of
growth (OD600 of 0.6) at 37 °C were harvested by centrifugation at
7000 ×g for 10 min. Then, they were washed three times at room temperature in sterile water. The cells were suspended in Tris HCl buffer
(pH 7.5, 75 mM) containing 0.4 mM sucrose and 2 mM MgCl2 and
were disrupted by grinding with 0.1 mm glass beads using the Micro
OD at 600 nm
The protein spots of interest were manually excised. The proteolytic
digestion was performed overnight at 37 °C using trypsin (20 μg/ml;
Promega, WI, USA) in the presence of 25 mM ammonium bicarbonate.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) was employed using the method described
by Kim et al. (2008) for protein identification. The obtained spectra
from the Ettan MALDI-TOF (Amersham Pharmacia Biotech, Uppsala,
Sweden) were subjected to analysis by Mascot software.
1.5
1.0
0.5
0.0
0
3
5
6
9
12
24
Incubation time (h)
Fig. 1. Growth curve of L. plantarum L67 at 37 °C (□), 5 °C (*) and cold stress at 5 °C (△)
after growth at 37 °C until reaching an OD600 of 0.6. The arrow (→) indicates a temperature change shift to 5 °C. The cells were inoculated in 1% of 10 ml of overnight culture in
10 ml of fresh MRS broth and incubated at the appropriate temperature (37 °C and
5 °C). For the cold stress experiments, when the cultures reached 0.6 (OD600) at 37 °C,
they were transferred to incubation at 5 °C. The optical density (OD) was measured
with a Synergy HT multimode plate reader (BioTek, Winooski, VT). The results are the
means of triplicated trials, and the error bars indicate the standard errors.
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2.0
1.8
1.6
OD at 600 nm
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
3
6
9
12
Incubation time (h)
15
18
24
Fig. 2. Effect of cold stress on the growth kinetics of L. plantarum L67. The cells were cultured in MRS broth at 37 °C prior to cold stress; control ( ); 5 °C (□); cold stress at 5 °C for 1 h (▲),
4 h (×), and 6 h (●). The cells were inoculated in 1% of 10 ml of overnight culture in 10 ml of fresh MRS broth and were incubated at 37 °C. For the cold stress, the inoculated culture was
incubated at 5 °C for 1, 4 or 6 h, and then growth kinetic experiments were performed at 37 °C. For the control, the growth kinetic experiments were performed without cold stress. The OD
was measured with a Synergy HT multimode plate reader (BioTek, Winooski, VT). The results are the means of triplicated trials, and the error bars indicate the standard errors.
37 °C in 6 h, whereas it reached only 0.6 after prior exposure to 5 °C for
1 h. However, after 9 h of pre-exposure at the lowest temperature, the
cells grew at a similar rate to those of the control. The fastest resumption
of growth occurred for the cells that had been subjected to cold stress
for 6 h.
3.2. Freeze–thaw challenge of L. plantarum L67
To measure the freezing tolerance, the freeze and thaw cycle was repeated for 4 cycles. The survival ratio of the cells after the freeze–thaw
was then determined and is shown in Fig. 3. Only 56% of the control cells
without cold shock survived after 4 consecutive freeze–thaw cycles.
However, the cells adapted for 4 and 6 h at 5 °C exhibited increased survival (up to 69 and 78%, respectively). The cells exposed to cold stress
for 6 h exhibited an almost constant level of survival after freeze–
thaw following an initial reduction in count. This result indicates that
cryotolerance is improved by subjecting the cells to prior cold stress.
3.3. Storage stability of dried L. plantarum L67
The viabilities of the L. plantarum L67 cultures were initially 3.8 ×
1011 CFU/g and 2.21 × 1011 CFU/g for the cold-stressed cultures and
the controls, respectively. At 5 °C, the cell population of the coldstressed L. plantarum L67 remained stable at 1.05 × 1010 CFU/g, while
the cell numbers in the control samples declined (1.82 × 109 CFU/g).
L. plantarum L67 was stored for 60 days.
At 25 and 35 °C storage, dramatic losses in cell count were observed
in the freeze dried cultures of the probiotic, with levels of L. plantarum
L67 cells that were subjected to prior cold-stress decreasing to 4.6 ×
108 CFU/g (at 25 °C) and 5.4 × 108 CFU/g (at 35 °C), whereas the
100
Survival (%)
90
80
70
60
50
40
0
1
2
3
4
Freeze-Thaw Cycle (Times)
Fig. 3. Survival ratio of the L. plantarum L67 cells after freeze–thaw cycles prior to cold stress. Control (□); cold stress at 5 °C for 1 h ( ), 4 h ( ), and 6 h (■). Two hundred milliliters of MRS
broth was inoculated (1%) and incubated at 37 °C. The cells were grown to the mid-exponential phase and were treated with cold stress at 5 °C for 1, 4 or 6 h; then the cells were frozen and
stored at −70 °C. The control cells were frozen directly without the cold stress. After 24 h of freezing, the cells were thawed by placing the tubes in a 37 °C water bath for 5 min. A total of
four freeze–thaw cycles were carried out. The 4 cycles of the freeze–thaw cycle was performed every 24 h. The thawed cells were serially diluted with 1% peptone water, and the viable
cells were counted. The results are the means of triplicated trials, and the error bars indicate the standard errors.
S. Song et al. / International Journal of Food Microbiology 191 (2014) 135–143
139
cspP
Transcription fold level
(normalized by rpo D)
1.5
*
*
*
1.0
0.5
0.0
1h
4h
cspL
Transcription fold level
(normalized by rpo D)
1.5
6h
*
1.0
0.5
0.0
1h
Transcription fold level
(normalized by rpo D)
cell counts in the control sample fell to 8.7 × 107 CFU/g (at 25 °C) and
9.8 × 107 CFU/g (at 35 °C) following 60 days of storage (Fig. 4).
6h
cspC
1.5
Fig. 4. Survival of the freeze-dried L. plantarum L67 during storage for 60 days. L. plantarum
L67 was grown in 1 l of MRS broth at 37 °C. The white bar indicates the powders prepared
with the control cultures, while the black bar indicates the powders prepared with the
cold-stress cultures. When the growth reached the mid-exponential phase (OD600 of
0.6), the cells were subjected to 6 h of cold shock treatment at 5 °C. The cell pellets were
collected by centrifugation (5000 ×g at 4 °C) and were then re-suspended in skim milk before being frozen at −70 °C. The frozen cells were freeze-dried. Duplicate samples of each
treatment were stored at 5 °C, 25 °C or 35 °C in the dark for 60 days. The cells were diluted
with 1% peptone water, and the viable cells were counted after 15 days. The results are the
means of triplicate drying trials, and the error bars indicate the standard errors.
4h
1.0
0.5
0.0
3.4. mRNA expression and ATPase activity under cold stress
RT-qPCR was performed to determine the expression of the coldshock genes in L. plantarum L67 (Fig. 5). After applying the cold stress,
two genes (cspP and cspL) were significantly expressed, but cspC was
not expressed at 6 h compared to the control.
1h
4h
6h
Fig. 5. Relative mRNA expression of the cold shock protein genes of L. plantarum L67. The
mRNA levels (transcription fold level (normalized by rpoD)) were calculated relative to
the transcription level detected in the control. Total RNA from mid-exponential phase cultures (OD600 of 0.6) prior to cold stress. * indicates a significant difference between the
control and the treatment of cold stress, P b 0.05.
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APTase activity
(pi μmol/min/mg protein)
10.0
*
8.0
*
6.0
4.0
Standard
1h
4h
6h
Fig. 6. ATPase activity of L. plantarum L67 under cold stress. ATPase activity was estimated
from the release of inorganic phosphate as measured according to the method of Fiske and
Subbarow (1925). * indicates a significant difference between the control and the treatment of cold stress, P b 0.05.
The ATPase activity of L. plantarum L67 subjected to cold stress is
shown in Fig. 6. The ATPase activity of L. plantarum L67 grown at 37 °C
was 8.5 unit enzymes (UE). The ATPase activity in the cells that were
cold stressed for 1 h at 5 °C was 7.2 UE, while following cold stress for
4 h, the enzyme activity reached the maximum observed level of
9.2 UE. The degree of ATPase activity (8.7 UE) after cold stress for 6 h
was slightly lower compared to the levels in cells that underwent cold
stress for 4 h. In the early stages of cold stress, ATPase activity was decreased, but after 4 h, the ATPase activity of the cells returned to normal
levels.
3.5. Inductive protein by cold stress in L. plantarum L67
Fig. 7 displays the 2D SDS-PAGE gels of the CBB stained proteins extracted from L. plantarum L67 following growth at 37 °C (OD600 of 0.6)
and subjected to cold stress for 6 h, which resulted in the highest survival
rate of cells after the freeze–thaw cycles. Under cold stress, 24 spots were
significantly overexpressed compared with the control. The proteins
identified by MALDI-TOF/MS were classified according to their functions
(Table 2). The thirteen identified proteins were classified into six groups:
cell growth (31%), unknown function (23%), energy (15%), stress response (15%), signal transduction (8%) and transcription (8%) (Fig. 8).
4. Discussion
L. plantarum is important within the food industry as a starter strain
for the fermentation of food. When foods are processed, bacteria are exposed to low temperature stresses. Hence, it is important to understand
the cold shock response and cold adaptation phenomenon of bacteria
such as L. plantarum L67. As a first step to understanding the cold stress
response in L. plantarum L67, we determined the L. plantarum L67 viability after cold shock treatment for 1, 4 and 6 h before incubation again at
the optimal temperature by measuring the growth kinetics. The results
of the study assumed that the cells might have a tolerance against freezing upon preadaptation to cold stress due to the expression of stress
proteins such as CSPP and CSPL (Fig. 3). The relevance of cold shock
and cryotolerance was first reported in B. subtilis, where it was determined that the removal of the cold shock protein B resulted in increased
sensitivity to freezing (Willimsky et al., 1992). In many bacteria, survival
to cold shock prior to freezing increases survival because cold adaptation likely results in a greater proportion of unsaturated fatty acids in
the plasma membrane of the affected cells compared to untreated cultures. However, the exact function of cold shock proteins in relation to
cryotolerance is still unknown (El kest and Marth, 1992).
In a recent report (Derzelle et al., 2000), it was shown that the
overproduction of each CSP causes distinct phenotypic effects in
L. plantarum. It is speculated that the different CSP proteins may antiterminate the transcription of the three csp genes of L. plantarum, thus
allowing the cell to adequately respond to stressful environmental challenges. In this study, RT-qPCR was performed to measure the expression
of cold-shock genes. After cold stress, two genes (cspP and cspL) were
significantly expressed, but cspC was not significantly expressed at 6 h
compared to the control. Recent reports have shown that cold inducible
CSP proteins may also be expressed at specific stages of the normal
growth cycle (Weber et al., 2001; Graumann and Marahiel, 1999). The
highest mRNA level of cspC is expressed during early exponentially
growing cells at the optimal temperature, while the cspC mRNA level
declines in the stationary phase (Derzelle et al., 2000). Derzelle et al.
(2003) have suggested that cspC overproduction improves growth resumption at the optimal temperature. Although cspC is not primarily
implicated in cold shock adaptation, it may play a positive role during
the acclimation phase of growth at cold temperatures. To better understand the mechanisms involved in this cold response, the role of the
cspC gene of L. plantarum will be further studied in the future. This result
is similar to that described by Derzelle et al. (2003). Derzelle et al.
(2002) also demonstrate that cold shock has induced the cspL gene.
This is a key result of the present study because it demonstrates that
csp transcripts may be more stable and more efficiently expressed
than other cellular messengers even if their absolute level does not significantly increase during cold exposure. It has been reported that the
overproduction of cspL after cold shock transiently augments growth,
while the overproduction of cspP and cspC has no effect on growth compared to the control (Derzelle et al., 2003). Additionally in this study, as
the mRNA levels of cspP increased, the survival ratio of the cells after
freeze–thaw also increased. However, we could not observe the
CSP protein in the proteomic analysis. While the csp gene was
overexpressed, the CSP protein was not expressed. It may be because
Fig. 7. Comparative two-dimensional gel electrophoresis patterns of the intracellular proteins of L. plantarum L67 growth at 37 °C (A) and under cold stress at 5 °C for 6 h (B). The proteins
were subjected to isoelectric focusing (pH 3–10) and resolved in the second dimension by 12.5% SDS-PAGE; the proteins were visualized with CBB staining.
S. Song et al. / International Journal of Food Microbiology 191 (2014) 135–143
141
Table 2
Differentially expressed proteins in L. plantarum L67 with cold shock for 6 h at 5 °C. The proteins were identified by MALTI-TOF/MS.
Spot no. Accession
number
Homology
% coverage Matched Mascot score Mr value Species
peptides
2
3
4
5
6
7
11
15
18
19
21
F4XMM6
A8S6V7
B3X7G0
C3KZR4
gi|167622455
F7MRE9
C1FJ41
UPI0001E29AA2
D3HC60
Q334H4
A8TV77
20
17
12
19
10
13
21
21
54
99
46
11
10
13
12
7
4
8
9
9
5
10
59
59
61
59
60
61
59
59
54
52
55
23509
68556
38641
41066
34112
5412
15350
10913
31481
4285
28634
Lyngbya majuscula
Faecalibacterium prausnitzii
Shigella dysenteriae
Clostridium botulinum
Shewanella halifaxensis HAW-EB4
Clostridium botulinum C str. Stockholm
Micromonas sp. RCC299
Pseudomonas syringae pv. syringae
Streptococcus gallolyticus subsp. gallolyticus
Triticum monococcum
Alpha proteobacterium
22
24
F0S3W5
F3KX52
Transposase
Putative uncharacterized protein
Pantothenate kinase
Putative YqaJ
hypothetical protein Shal_0515
Arsenate reductase-like protein
Predicted protein
phage integrase family protein
Pantothenate synthetase
Heat shock protein 101
Universal stress protein UspA and related
nucleotide-binding protein
Multi-sensor signal transduction histidine kinase
Regulatory protein, LysR:LysR, substrate-binding
29
38
11
10
57
56
74423
36076
Desulfurobacterium thermolithotrophum DSM
Hylemonella gracilis ATCC 19624
each CSP protein has a different induction time; for example, the csp
gene induction time was different. Although the protein was not
expressed under cold stress for 6 h, we observed other stress response
proteins such as Hsp and the universal stress protein UspA.
The activity of ATPase in the cells exposed to cold stress for 1 h
showed a significantly reduced activity compared to the control cells.
However, the cells exposed for 4 h showed significantly higher activities
compared to the control cells. The results indicated that when the cells
were exposed to low temperatures, ATPase activity decreased, whereas
the ATPase activity of the adapted cells increased. We suggest that
ATPase activity is related to the maintenance of membrane fluidity.
When the growth temperature decreases, there is an increase of
Unknown funtion
23%
unsaturated fatty acids and methyl branching. Thus, the decrease of average chain length improves the membrane fluidity (Casanueva et al.,
2010).
For the proteomic analysis, we performed 2D SDS-PAGE (Fig. 7).
Under cold stress, twenty four spots were overexpressed and thirteen
spots were identified. The proteins were classified into six groups: energy metabolism, cell growth, unknown function, signal transduction,
stress response and transcription (Fig. 8).
Thirty-one percent of the overexpressed proteins were related to
cell growth. We reported that spot 4 and spot 18 were related to pantothenate kinase and synthetase. Pantothenic acid is used in the synthesis
of coenzyme A (CoA). CoA is important to energy metabolism for
Energy
metabolism
15%
Signal
transduction
8%
Stress response
15%
Cell growth
31%
Transcription
8%
Fig. 8. Functional classification of the identified proteins from L. plantarum L67 analyzed by MALDI-TOF/MS using Map Man ontology as described by Beven et al. (1998).
142
S. Song et al. / International Journal of Food Microbiology 191 (2014) 135–143
pyruvate to enter the tricarboxylic acid cycle (TCA cycle) as acetyl-CoA
and for α-ketoglutarate to be transformed to succinyl-CoA in the TCA
cycle (Gropper et al., 2008). CoA is also important for the biosynthesis
of many important compounds such as fatty acids, cholesterol, and acetylcholine (Gropper et al., 2008). Spot 2 had homology with a
transposase protein, which is an enzyme that binds to the ends of a
transposon and catalyzes the movement of the transposon to another
part of the genome by a cut and paste mechanism or through a replicative transposition mechanism (Jones et al., 2011). Spot 5 had homology
with a putative protein, YqaJ. The function of this protein domain is to
digest DNA for recombination. This recombination is crucial to viral replication (Vellani and Myers, 2003). DNA exonucleases play roles in DNA
metabolism, e.g., in replication, repair, and recombination. Spot 15 was
associated with the homology phage integrase family protein. Spots 2, 5
and 15 were related to chromosome structuring.
The proteins related to energy metabolism were identified as spots 3
and 7. Spot 3 was recognized as a protein involved in electron transport
(Sudarsanam et al., 2007). Spot 7 was related to arsenate reductases
such as oxidoreductases.
We observed an up-regulation of the proteins involved in the stress
response, which included spots 19 and 21. Spot 19 was identified as a
heat shock protein (Hsp), which is a stress response protein. Wu et al.
(2009) have reported that heat shock proteins respond to low pH stress;
the proteins have the function of preventing the accumulation of unfolded protein intermediates under stress. Additionally, Fiocco et al.
(2007) have reported that sHsp may respond to specific environmental
stresses. L. plantarum transformed strains were able to tolerate high and
low temperature stresses. Taken together, these observations suggest
that signal transduction machinery and transcription factors that mediate the genetic response to stressful conditions appear to be shared by
different stress-regulative networks (Capozzi et al., 2011). The universal
stress protein UspA-related nucleotide-binding protein was induced
(spot 21). The levels of UspA have been evaluated in the response to
the cell status of E. coli under stressful conditions (Gustavsson et al.,
2002; Kvint et al., 2003). The UspA protein of E. coli has a role in resisting
damage to DNA. Based on our conclusions, the induced expression of
the nucleotide-binding protein UspA may be associated with protecting
the DNA of cells during cold stress. However, the exact physiological
role of UspA in L. plantarum L67 requires further study.
Spot 22 had homology with multi-sensor signal transduction histidine kinase. Histidine kinases (HK) are multifunctional and typically
transmembrane; they are proteins of the transferase class that plays a
role in signal transduction across the cellular membrane (Wolanin
et al., 2002). Spot 24 was a regulatory protein involved in regulated
transcription (Chen et al., 2011).
Among the identified proteins, spots 6 and 11 have yet to be identified according to their function (Worden et al., 2009).
Many L. plantarum L67 proteins were overexpressed in the cells exposed to low temperatures, and these proteins may interact with each
other, resulting in cryotolerance.
Cold stress induced proteins play roles in a variety of cellular processes such as fatty acid metabolism, chromosome structuring, transcription, translation, general metabolism, energy metabolism, and
stress response (Graumann et al., 1997; Wouters et al., 2000a, 2000b;
Yamanaka et al., 1998). Additionally, the induced proteins in this
study were related to energy metabolism, cell growth, signal transduction, stress response and transcription.
In conclusion, we suggest that L. plantarum L67 adapts to low temperatures by a constitutive expression of the potentially cryoprotective
cspP gene and proteins related to metabolism and stress response. In a
bio-preservation strategy, the protective strains are generally cultivated at their optimum temperatures, eventually freeze-dried, and then
directly inoculated into products that are stored at chilled temperatures. Cold acclimation thus constitutes a demonstrable advantage in
bacterial competition against spoilage and pathogenic psychrotrophic
bacteria in terms of food preservation. This property would enable
cells to multiply at optimal temperatures and to be successfully inoculated into chilled foodstuffs without delaying the food preservative
effect.
Acknowledgments
This research was supported by iPET (Korea Institute of Planning and
Evaluation for Technology in Food, Agriculture, Forestry and Fisheries),
Ministry for Food, Agriculture, Forestry and Fisheries, Republic of
Korea (112121-2).
References
Bae, W., Xia, B., Inouye, M., Severinov, K., 2000. Escherichia coli cspA family RNA chaperones are transcription terminators. Proc. Natl. Acad. Sci. 97, 7784–7789.
Barria, C., Malecki, M., Arraiano, C.M., 2013. Bacterial adaptation to cold. Microbiology
159, 2437–2443.
Beven, M., et al., 1998. Analysis of 1.9 Mb contiguous sequence from chromosome 4 of
Arabidopsis thaliana. Nature 391, 485–493.
Borriello, S.P., Hammoles, W.P., Holzapfel, W., Marteau, P., Schrezenmeir, J., Vaara, M.,
Valtenen, V., 2003. Safety of probiotics that contain lactobacilli or bifidobacteria.
Clin. Infect. Dis. 36, 775–780.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72, 248–254.
Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G.M., Carnemolla, B., Orecchia,
P., Zardi, L., Righetti, P.G., 2004. Blue silver: a very sensitive colloidal Coomassie G-250
staining for proteome analysis. Electrophoresis 25, 1327–1333.
Capozzi, V., Weidmann, S., Fiocco, D., Rieu, A., Hols, P., Guzzo, J., Spano, G., et al., 2011. Cold
stress response in lactic acid bacteria. Stress Responses of Lactic Acid BacteriaFood
Microbiology and Food Safety. Springer Science-Business Media, LLC 2011, New
York, pp. 96–110 (Chapter 5).
Carcoran, B.M., Stanton, C., Fitsgerald, G., Ross, R.P., 2008. Life under stress: the probiotic
stress response and how it may be manipulated. Curr. Pharm. Des. 14, 1382–1399.
Casanueva, A., Tuffin, M., Cary, C., Cowan, D.A., 2010. Molecular adaptations to
psychrophily: the impact of ‘omic’ technologies. Trends Microbiol. 18, 374–381.
Chen, S., Beeby, M., Murphy, G.E., Leadbetter, J.R., Hendrixson, D.R., Briegel, A., Li, Z., Shi, J.,
Tocheva, E.I., Muller, A., Dobro, M.J., Jensen, G.J., 2011. Structural diversity of bacterial
flagellar motors. EMBO J. 30, 2972–2981.
de Man, J.C., Rogosa, M., Elisabeth, S., 1960. A medium for the cultivation of lactobacilli. J.
Appl. Microbiol. 23, 130–135.
de Vries, M.C., Vaughan, E.E., Kleerebezem, M., de Vos, W.M., 2006. Lactobacillus plantarum
survival, functional and potential probiotic properties in the human intestinal tract.
Int. Dairy J. 16, 1018–1028.
Derzelle, S., Hallet, B., Francis, K.P., Ferain, T., Delcour, J., Hols, P., 2000. Changes in cspL,
cspP, and cspC mRNA abundance as a function of cold shock and growth phase in
Lactobacillus plantarum. J. Bacteriol. 182, 5105–5113.
Derzelle, S., Hallet, B., Francis, K.P., Ferain, T., Delcour, J., Hols, P., 2002. Cold shock induction of the cspL gene in Lactobacillus plantarum involves transcriptional regulation. J.
Bacteriol. 184, 5518–5523.
Derzelle, S., Hallet, B., Francis, K.P., Ferain, T., Delcour, J., Hols, P., 2003. Improved adaptation to cold-shock, stationary phase, and freezing stresses in Lactobacillus plantarum
overproducing cold-shock proteins. Appl. Environ. Microbiol. 69, 4285–4290.
Desmond, C., Stanton, C., Fitzgerald, G.F., Collins, K., Ross, R.P., 2001. Environmental adaptation of probiotic lactobacilli towards improvement of performance during spray
drying. Int. Dairy J. 11, 801–808.
Desmond, C., Fitzgerald, G.F., Stanton, C., Ross, R.P., 2004. Improved stress tolerance of
GroESL-overproducing Lactococcus lactis and probiotic Lactobacillus paracasei NFBC
338. Appl. Environ. Microbiol. 70, 5929–5936.
Duary, R.K., Batish, V.K., Grover, S., 2012. Relative gene expression of bile salt hydrolase
and surface proteins in two putative indigenous Lactobacillus plantarum strains
under in vitro gut conditions. Mol. Biol. Rep. 39, 2541–2552.
Duncan, D.B., 1955. Multiple range and multiple F tests. Biometrics 11, 1–42.
El kest, S.E., Marth, E.H., 1992. Freezing of Listeria monocytogenes and other microorganisms: a review. J. Food Prot. 55, 639–648.
Fiocco, D., Capozzi, V., Goffin, P., Hols, P., Spano, G., 2007. Improved adaptation to heat,
cold, and solvent tolerance in Lactobacillus plantarum. Appl. Microbiol. Biotechnol.
77, 909–915.
Fiske, C.H., Subbarow, Y., 1925. The colorimetric determination of phosphorus. J. Biol.
Chem. 66, 375–400.
Goldenberg, D., Azar, I., Oppenheim, A.B., Brandi, A., Gualerzi, C.O., Pon, C.L., 1997. Role of
Escherichia coli cspA promoter sequences and adaptation of translational apparatus in
the cold shock response. Mol. Gen. Genomics. 256, 282–290.
Graumann, P.L., Marahiel, M.A., 1999. Cold shock proteins CspB and CspC are major
stationary-phase-induced proteins in B. subtilis. Arch. Microbiol. 171, 135–138.
Graumann, P., Wendrich, T.M., Michael, H.W., Weber, Katja, S., Mohamed, A.M., 1997. A
family of cold shock proteins in Bacillus subtilis is essential for cellular growth and
for efficient protein synthesis at optimal and low temperatures. Mol. Microbiol. 25,
741–756.
Gropper, S.S., Smith, J.L., Groff, J.L., 2008. Advanced Nutrition and Human Metabolism, 5th
ed. Cengage learning, Belmont, CA.
S. Song et al. / International Journal of Food Microbiology 191 (2014) 135–143
Gustavsson, N., Diez, A., Nystro¨m, T., 2002. The universal stress protein paralogues of
Escherichia coli are coordinately regulated and cooperate in the defense against
DNA damage. Mol. Microbiol. 43, 107–117.
Jones, P.G., Van Bogelen, R.A., Neidhardt, F.C., 1987. Induction of proteins in response to
low temperature in Escherichia coli. J. Bacteriol. 169, 2092–2095.
Jones, A.C., Monroe, E.A., Podell, S., Hess, W.R., Klages, S., Esquenazi, E., Niessen, S., Hoover,
H., Rothmann, M., Lasken, R.S., Yates, J.R., Reinhardt, R., Kube, M., Burkart, M.D., Allen,
E.E., Dorrestein, P.C., Gerwick, W.H., Gerwick, L., 2011. Genomic insights into the
physiology and ecology of the marine filamentous cyanobacterium Lyngbya
majuscula. Proc. Natl. Acad. Sci. U. S. A. 108, 8815–8820.
Kim, S.J., Kim, J.H., Park, J.Y., Kim, H.T., Ha, Y.L., Yun, H.D., Kim, J.H., 2004. Cold adaptation
of Lactobacillus paraplantarum C7 isolated from kimchi. J. Microbiol. Biotechnol. 14,
474–482.
Kim, Y., Oh, S., Park, S., Seo, J., Kim, S.H., 2008. Lactobacillus acidophilus reduces expression
of enterohemorrhagic Escherichia coli O157:H7 virulence factors by inhibition
autoinducer-2-like activity. Food Control 19, 1042e50.
Kleerebezem, M., Boekhorst, J., van Kranenburg, R., Molenaar, D., Kuipers, O.P., Leer, R.,
Tarchini, R., Peters, S.A., Sandbrink, H.M., Fiers, M.W., Stiekema, W., Lankhorst, R.M.,
Bron, P.A., Hoffer, S.M., Groot, M.N., Kerkhoven, R., de Vries, M., Ursing, B., de Vos,
W.M., Siezen, R.J., 2003. Complete genome sequence of Lactobacillus plantarum
WCFS1. Proc. Natl. Acad. Sci. 100, 1990–1995.
Kvint, K., Nachin, L., Diez, A., Nystro¨m, T., 2003. The bacterial universal stress protein:
function and regulation. Curr. Opin. Microbiol. 6, 140–145.
Livak, k.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time
quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408.
Mayo, B., Derzelle, S., Fernandez, M., Leonard, C., Ferain, T., Hols, P., Suarez, J.E., Delcour, J.,
1997. Cloning and characterization of cspL and cspP, two cold-inducible genes from
Lactobacillus plantarum. J. Bacteriology 179, 3039–3042.
Park, Y.S., Lee, J.Y., Kim, Y.S., Shin, D.H., 2002. Isolation and characterization of lactic acid
bacteria from feces of newborn baby and from Dongchimi. J. Agric. Food Chem. 50,
2531–2536.
Sartor, R.B., 2004. Therapeutic manipulation of the enteric microflora in inflammatory
bowel diseases: antibiotics, probiotics, and prebiotics. Gastroenterology 126,
1620–1633.
Spano, G., Beneduce, L., Perrotta, C., Massa, S., 2005. Cloning and characterization of the
hsp 18.55 gene, a new member of the small heat shock genes family isolated from
wine Lactobacillus plantarum. Res. Microbiol. 156, 219–224.
143
Sudarsanam, P., Ley, R., Guruge, J., Turnbaugh, P.J., Mahowald, M., Liep, D., Gordon, J., 2007.
Draft Genome Sequence of Faecalibacterium prausnitzii M21/2. EMBL/GenBank/DDBJ
databases.
Vellani, T.S., Myers, R.S., 2003. Bacteriophage SPP1 Chu is an alkaline exonuclease in the
SynExo family of viral two-component recombinases. J. Bacteriol. 185, 2465–2474.
Weber, M.H., Beckering, C.L., Marahiel, M.A., 2001. Complementation of cold shock proteins by translation initiation factor IF1 in vivo. J. Bacteriol. 183, 7381–7386.
Willimsky, G., Bang, H., Fischer, G., Marahiel, M.A., 1992. Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures. J.
Bacteriol. 174, 6326–6335.
Wolanin, P.W., Thomason, P.A., Stock, J.B., 2002. Histidine protein kinases: key signal
transducers outside the animal kingdom. Genome Biol. 3, 3013.1–3013.8.
Worden, A.Z., Lee, J.H., Mock, T., Rouzé, P., Simmons, M.P., Aerts, A.L., Allen, A.E., Cuvelier,
M.L., Derelle, E., Everett, M.V., Foulon, E., Grimwood, J., Gundlach, H., Henrissat, B.,
Napoli, C., McDonald, S.M., Parker, M.S., Rombauts, S., Salamov, A., Von Dassow, P.,
Badger, J.H., Coutinho, P.M., Demir, E., Dubchak, I., Gentemann, C., Eikrem, W.,
Gready, J.E., John, U., Lanier, W., Lindquist, E.A., Lucas, S., Mayer, K.F., Moreau, H.,
Not, F., Otillar, R., Panaud, O., Pangilinan, J., Paulsen, I., Piegu, B., Poliakov, A.,
Robbens, S., Schmutz, J., Toulza, E., Wyss, T., Zelensky, A., Zhou, K., Armbrust, E.V.,
Bhattacharya, D., Goodenough, U.W., Van de Peer, Y., Grigoriev, I.V., 2009. Green evolution and dynamic adaptations revealed by genomes of the marine picoeukaryotes
Micromonas. Science 324, 268–272.
Wouters, J.A., Mailhes, M., Rombouts, F.M., de Vos, W.M., Kuipers, O.P., Abee, T., 2000a.
Physiological and regulatory effects of controlled overproduction of five cold-shock
proteins of Lactococcus lactis MG1363. Appl. Environ. Microbiol. 66, 3756–3763.
Wouters, J.A., Rombouts, F.M., Kuipers, O.P., de Vos, W.M., Abee, T., 2000b. The role of
cold-shock proteins in low-temperature adaptation of food related bacteria. Syst.
Appl. Microbiol. 23, 165–173.
Wu, R., Wang, W., Yu, D., Zhang, W., Li, Y., Sun, Z., Wu, J., Meng, H., Zhang, H., 2009. Proteomics analysis of Lactobacillus casei Zhang, a new probiotic bacterium isolated from
traditional home-made koumiss in inner Mongolia of China. Mol. Cell. Proteome 8,
2321–2338.
Yamanaka, K., Fang, L., Inouye, M., 1998. The cspA family in Escherichia coli: multiple gene
duplication for stress adaptation. Mol. Microbiol. 27, 247–255.