Laboratory for Food Microbiology, Division of Food Science, Wageningen University and Research Centre, Bomenweg 2, 6703 HD Wageningen, The Netherlands1
Microbial Ingredients Section, NIZO food research, Ede, The Netherlands2
Author for correspondence: Tjakko Abee. Tel: +31 317 484981. Fax: +31 317 484893. e-mail: Tjakko.Abee{at}micro.fdsci.wau.nl
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ABSTRACT |
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Keywords: Lactococcus lactis, low-temperature adaptation, cryoprotection, cold-shock proteins
Abbreviations: 2D-EF, two-dimensional gel electrophoresis; CSP, cold-shock protein; LAB, lactic acid bacteria
a Present address: Department of Genetics, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
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INTRODUCTION |
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A remarkable phenomenon is the ability of bacteria to adapt to temperatures that are far below their optimum growth temperature. It has been well established that after a rapid decrease in the temperature of the culture medium (cold shock) a set of proteins is preferentially expressed (see reviews by Jones & Inouye, 1994 ; Graumann & Marahiel, 1996
; Yamanaka et al., 1998
). It has been found in Escherichia coli that about 15 proteins are overproduced after a cold shock from 37 to 10 °C. Among these proteins, CspA (cold-shock protein A, 7 kDa) shows the highest induction (Goldstein et al., 1990
). For Bacillus subtilis a homologous cold-shock protein, termed CspB, has been identified (Willimsky et al., 1992
). A family of nine CspA homologues is present in E. coli of which only CspA, CspB and CspG are cold induced (Lee et al., 1994
; Nakashima et al., 1996
; Yamanaka et al., 1998
). In addition, a family of three cold-induced cold-shock proteins (CSPs) has been observed in B. subtilis (Graumann et al., 1996
). Recently, a family of five csp genes, named cspA, cspB, cspC, cspD and cspE, encoding highly similar CSPs (6585% identity), has been described in the lactic acid bacterium Lactococcus lactis MG1363 (Wouters et al., 1998
). On the L. lactis MG1363 chromosome two tandem groups of csp genes (cspA/cspB and cspC/cspD) were identified, whereas cspE was found as a single gene. Transcription analysis showed that cspE is not cold induced, whereas the other csp genes are induced 10- to 40-fold at different times after cold shock (Wouters et al., 1998
).
Several functions have been shown for CSPs at low as well as at elevated growth temperatures. CSPs may function as RNA chaperones as they are able to bind to mRNA molecules and minimize secondary structure, by which they facilitate the translation process (Graumann et al., 1997 ; Jiang et al., 1997
). CspA of E. coli also appeared to function as a transcriptional activator, as is described for two genes of which the products, H-NS and GyrA, are both involved in DNA supercoiling (LaTeana et al., 1991
; Jones et al., 1992
). Interestingly, it was noted that many organisms develop an increased ability to survive freezing after cold-shock treatment (Goldstein et al., 1990
; Kim & Dunn, 1997
; Panoff et al., 1995
; Thammavongs et al., 1996
; Willimsky et al., 1992
). CspB appeared to be implicated in increased tolerance to freezing, as was shown using a strain in which the gene encoding this protein was disrupted (Willimsky et al., 1992
). However, in previous studies a direct relation of the actual CSP levels with functionality of these proteins was never established.
In this study we provide evidence for an active adaptation response of L. lactis MG1363 to a repetitive freezing challenge after exposure to low temperature. Protein synthesis is required for this adaptation and major differences in the pattern of synthesized proteins are found in the class of 7 kDa CSPs. The specific effect of overproduction of CspD on freeze survival was studied and the correlation between the expression of CSPs and the survival after freezing of L. lactis MG1363 will be discussed.
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METHODS |
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Protein analysis using two-dimensional gel electrophoresis (2D-EF) and one-dimensional SDS-PAGE.
Total cellular proteins were extracted from 10 ml cultures by homogenizing with an MSK cell homogenizer (B. Braun Biotech International) and zirconium beads (0·1 mm, Biospec Products) eight times for 1 min (cooled on ice between treatments). After homogenizing, the zirconium beads were allowed to sediment by gravity, after which the supernatant, containing the cellular proteins, was analysed by 2D-EF or SDS-PAGE. The protein content of the extract was determined using the bicinchoninic acid method as provided by the supplier (Sigma) and equal amounts of protein were applied on the protein gels.
2D-EF was essentially performed as described by OFarrell (1975) using a Pharmacia 2D-EF system. Prior to loading of the samples on the IEF gel, 20 µl protein solution (40 µg protein) was treated with 20 µl lysis solution [9 M urea, 2% 2-mercaptoethanol, 2% IPG buffer 4-7L (Pharmacia Biotech), 2% Triton X-100, 8 mM PMSF] at 37 °C for 5 min, after which 60 µl sample solution (8 M urea, 2% 2-mercaptoethanol, 2% IPG buffer 4-7L, 0·5% Triton X-100, a few grains of bromophenol blue) was added. The total volume (100 µl) was loaded on the acidic end of the first-dimension IEF gel with linear pI ranges from 4 to 7 or from 3 to 10 (Immobiline Dry strips, Pharmacia Biotech). For the second dimension, 15% homogeneous SDS-PAGE gels were used to obtain optimal separation in the low-molecular-mass region. Two molecular mass markers (Pharmacia Biotech) were used, with band sizes of 67, 43, 30, 22·1 and 14·4 kDa and of 16·9, 14·4, 10·7, 8·2, 6·2 and 2·5 kDa, respectively. The gels were silver stained according to Blum et al. (1987)
and were analysed using GEMINI software (Applied Imaging). The intensity of each spot was calculated as a percentage of the total intensity of the spots visualized on a gel, and subsequently, induction factors were calculated. One-dimensional Tricine-SDS-PAGE for the separation of low-molecular-mass proteins was performed as described by Schägger & von Jagow (1987)
.
Freeze survival of L. lactis exposed to different stress conditions.
L. lactis was grown to mid-exponential phase at 30 °C, after which the cells were centrifuged and resuspended in GM17 medium in which they were exposed to the relevant stress conditions. Cells were exposed to either heat stress (10 min 42 °C), salt stress (10 min 0·5 M NaCl), pH stress (10 min at pH 4, adjusted with lactic acid) or stationary-phase stress [4 h after reaching mid-exponential phase (OD600 0·5), which means that cells were at maximum OD600 (approx. 2·4) for 2 h] and subsequently analysed for freeze stability. After stress exposure a 1 ml sample was taken, spun down and resuspended in fresh GM17 medium. The number of c.f.u. was determined before freezing and after each of four repetitive freezethaw cycles.
Northern blot analysis.
For analysis of the csp mRNA levels in L. lactis after exposure to several stress conditions, total RNA was extracted as described by Kuipers et al. (1993) . For Northern blot analysis, 20 µg RNA was glyoxylated and fractionated using a 1% agarose gel as described by van Rooijen & de Vos (1990)
. Equal amounts of RNA were loaded on the gel and RNA was stained using ethidium bromide. A 0·249·5 kb RNA ladder (Gibco-BRL Life Technologies) was used to determine the transcript size. RNA was blotted on a GeneScreen Plus Membrane (Dupont, NEN Research Products). The blot was hybridized using a mix of probes each specific for one of the five csp genes of L. lactis (Wouters et al., 1998
) that were labelled simultaneously. The blots were exposed to X-ray films (X-Omat MS, Kodak).
Overproduction of CspD using a nisin-controlled expression system.
CspD was overexpressed using the nisin-controlled expression system as described by Kuipers et al. (1995) and de Ruyter et al. (1996). Using the oligonucleotides OECspDFor (5'-GCTGCCATGGCAAATGGAACAGTAAAATGG-3') and OECspDRev (5'-CACGAAGCTTTTTCCTCTTGCTGGCTAAGT-3'), containing a NcoI site and a HindIII site (underlined), respectively, the cspD gene could be amplified using PCR. The obtained fragment was digested with NcoI and HindIII and subsequently cloned in vector pNZ8032 (De Ruyter et al., 1996
), thereby replacing the gusA gene that was originally present in pNZ8032. In this way, a translational fusion was obtained of the nisA promoter to the ATG start codon of the cspD gene. The plasmid generated (pNZOECspD) was transformed into L. lactis NZ3900, a derivative of L. lactis MG1363 in which the two-component regulatory nisR/nisK pathway is integrated into the chromosome. Upon addition of different concentrations of the inducer M17W nisin (0, 0·1, 0·2 and 0·5 ng ml-1; Kuipers et al., 1995
) CspD could be overproduced in high quantities as was analysed using SDS-PAGE. To study the freeze survival of L. lactis NZ3900(pNZOECspD), this strain was cultured at 30 °C to OD600 0·3, after which different concentrations of nisin were added. After 90 min of incubation (final OD600 approx. 1·5) protein samples and samples to analyse the freeze survival (performed in triplicate) were taken. As a control, a freeze-challenge was performed with L. lactis NZ3900(pNZ8020); pNZ8020 carries the nisA promoter without any fused gene (de Ruyter et al., 1996
).
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RESULTS |
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Survival after freezing of L. lactis following different temperature shocks
The mRNA levels of the csp genes of L. lactis were analysed at different times after cold shock from 30 to 20 °C and from 30 to 4 °C (Fig. 4a). It appeared that the total mRNA level for the csp genes was only slightly increased at 30 min after cold shock to 20 °C. After longer incubation at this temperature, no csp mRNA could be detected. After a temperature downshock from 30 to 4 °C, clear induction of csp genes was observed at 1 h after cold shock. This induction was approximately 10-fold lower than observed at 4 h after cold shock to 10 °C (Fig. 4a
, lane 9). At 2 h after cold shock to 4 °C csp mRNA levels were decreased (Fig. 4a
). 2D-EF analysis revealed that only CspB and CspD were slightly induced at 4 h after cold shock to 4 °C and slight induction of CspD was observed at 4 h after cold shock to 20 °C (Fig. 4b
). Furthermore, no induction of CspA and CspC was observed after cold shock from 30 °C to 20 or 4 °C (data not shown). These results indicate that the lactococcal CSPs are tightly temperature-regulated and that the expression is highest after cold shock to 10 °C. After cold shock to 20 °C and 4 °C for 4 h, 20 and 10 other induced proteins, respectively, are observed apart from the five 7 kDa CSPs (data not shown).
Remarkably, after exposure of the L. lactis cells to 4 °C for 2 and 4 h, significant cryoprotection was also observed. The survival after four repetitive freezethaw cycles was approximately 130-fold higher than the survival of mid-exponential-phase cells. When L. lactis was shifted to 20 °C an adaptive response to freezing was also found, which led to an approximately 30-fold increased survival after four freezethaw cycles compared to mid-exponential-phase cells grown at 30 °C. This adaptive response was already observed at 30 min after cold shock (Fig. 4c). These data show that an increased freeze survival coincides with increased CSP expression and in more detail coincides with CspD expression, for which induction is observed under all cold-shock conditions tested. However, the percentage of surviving cells after freezing is quantitatively not directly correlated with the expression level of the CSPs or one specific CSP.
Survival after freezing of L. lactis following different stress exposures
Following cold shock from 30 to 10 °C a high csp mRNA induction was observed (Fig. 5a, lane 6). However, after exposures to either heat, pH, acid or stationary-phase stress no significant increase of the csp mRNA levels was observed (Fig. 5a
). Also at the protein level no up- or down-regulation of the CSPs was found after the different stress exposures. Only a slight induction of CspD was observed under salt-stress conditions. CspE could be detected under all conditions tested and appears to be constitutively expressed (Fig. 5b
). The survival after freezing following exposure to heat, salt and acid stress was similar to the survival of mid-exponential-phase cells cultured at 30 °C. Surprisingly, stationary-phase cells showed a 20-fold higher survival after four repetitive freezethaw cycles than the control cells (30 °C mid-exponential phase; Fig. 5c
). For stationary-phase cells no increased CSP levels were observed, and apparently other factors are involved in the increased freeze survival. 2D-EF gels revealed induction of 19 proteins (at least twofold induction) at stationary phase (data not shown). Three of these proteins were also induced at 4 h after cold shock to 20, 10 and 4 °C (spots 6, 12 and 15; Table 2
).
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DISCUSSION |
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Since many starter LAB are stored frozen prior to use in fermentations there is much interest in the survival of these strains after freezing. The survival after freezing of L. lactis MG1363 increased approximately 100-fold when this bacterium was exposed to 10 °C for 4 h (Fig. 2); the addition of chloramphenicol to the growth medium during a cold-shock treatment blocked the cryprotection process. These results indicate that within a few hours of incubation at low temperature significant cryoprotection is obtained for which protein synthesis is required. Since 2D-EF analysis revealed that the members of the 7 kDa CSP family of L. lactis are the most stongly induced proteins after cold shock, it is tempting to speculate that these proteins are directly involved in the protection against freezing. Willimsky et al. (1992)
showed that deletion of the gene encoding CspB of B. subtilis resulted in a decreased freeze survival and the authors suggested a role as anti-freeze proteins for CSPs, because of their low molecular mass and their abundant presence. Our study shows that the csp mRNA and CSP expression levels in L. lactis increased upon cold shock to 20 and 10 °C as well as to 4 °C. However, the induction of CSPs was highest after cold shock to 10 °C. Upon cold shock to 20 °C induction of CspD was observed and upon cold shock to 4 °C the level of both CspB and CspD increased. However, these cold-shock conditions did not result in induction levels comparable to those seen after exposure to 10 °C. In contrast, the survival after freezing was similarly increased after exposure to 20 and 4 °C compared to exposure to 10 °C, indicating that neither the csp mRNA level nor the CSP level quantitatively correlates with improved cryoprotection. Since CspD was induced under all cold-shock conditions, the specific impact of CspD on the freeze survival was monitored using controlled overproduction. This revealed a slight increase in freeze survival at high overproduction levels and no protective effect at CspD levels comparable to cold-shock conditions. In conclusion, the 7 kDa cold-shock protein CspD may enhance the survival capacity after freezing but this protein is evidently not the only factor determining cryoprotection. Probably other proteins are needed for this protective effect and a concerted action of several proteins can not be excluded. Overproduction of CspD results in increased levels of several proteins; however, it does not induce the expression of the proteins induced by both low temperature as well as stationary-phase conditions (Table 2
, data not shown).
In contrast to observations for CspA, CspB and CspG of E. coli (Etchegaray & Inouye, 1999 ), no induction of the lactococcal CSPs was observed in the presence of chloramphenicol 100 µg ml-1) at 10 °C. Our investigations show that growth is blocked, no new proteins are synthesized and no increase in survival after freezing is observed upon addition of chloramphenicol. We speculate that the translational machinery in L. lactis is not intact and that during this condition also csp genes cannot be translated.
Since the actual CSP levels do not directly correlate with increased freeze survival, it might be speculated that one (or more) member(s) of the CSP family regulate(s) the adaptive response to freezing by regulating the expression of other proteins. It has been reported that CspA of E. coli regulates the expression of genes belonging to the cold-shock stimulon (LaTeana et al., 1991 ; Jones et al., 1992
). In L. lactis MG1363 various other proteins than the 7 kDa CSPs were found to be induced following cold shock, ranging from 16, 20 and 10 induced proteins after shock to 10, 20 and 4 °C, respectively. Strikingly, the levels of three proteins (spots 6, 12 and 15; Table 2
) were increased after all cold-shock treatments and under stationary-phase conditions, but not under any of the other stress conditions. For all these conditions increased survival after freezing was observed and it is tempting to speculate that these unidentified proteins may play a role in cryoprotection.
Upon exposure of L. lactis to stresses other than cold stress (heat, salt, acid and stationary-phase stress) the csp mRNA levels and the expression of CSPs were not affected. For CspD of E. coli (Yamanaka & Inouye, 1997 ) and CspB and CspC of B. subtilis (Graumann et al., 1997
) stationary-phase induction has been observed. L. lactis cells that were in stationary phase for more than 2 h exhibited increased survival after freezing as compared to mid-exponential cells, whereas other stress exposures did not result in protection against freezing. These observations indicate that during stationary phase, factors other than CSPs are important as cryoprotectants. Generally, many organisms show increased resistance to stress conditions during stationary phase (Kolter et al., 1993
). Starved L. lactis IL1403 showed enhanced resistance to heat, ethanol, acid, osmotic and oxidative stress (Hartke et al., 1994
) and our data show also enhanced resistance to freezing stress. For L. lactis MG1363 we observed increased levels of 19 proteins under stationary-phase conditions. A central position for mediating the stress responses might be assigned to alternative sigma factors, but thus far only a vegetative factor has been described for L. lactis (Gansel et al., 1993
). In related Gram-positive organisms, like B. subtilis and Listeria monocytogenes, alternative sigma factors have been shown to coordinate responses to a variety of signals such as temperature, pH, osmolarity and stationary phase (Hecker et al., 1996
; Becker et al., 1998
). To study cross-protection under different stress conditions it is of great interest to elucidate the presence of such general stress proteins and to verify whether the stress response in L. lactis involves similar sigma factors or is co-ordinated via alternative pathways.
In this study, the actual expression levels of CSPs have for the first time been correlated with a physiological function in low-temperature adaptation, in this case freeze survival of the industrially important bacterium L. lactis. Exposure to 4, 10 and 20 °C for several hours leads to increased freeze survival and this coincides with increased CSP expression. However, the observed level of freeze protection does not quantitatively correlate with the csp gene expression levels. In addition, L. lactis cells specifically overproducing CspD at 30 °C show a 210-fold increased survival after freezing compared to control cells. This indicates that the 7 kDa cold-shock protein CspD may enhance the survival capacity after freezing but that this protein is probably not the only factor determining cryoprotection. The exact functioning of the members of the CSP family in L. lactis in relation to freeze adaptation is not yet known. In order to gain more insight into this aspect, csp disruption mutants of L. lactis will be constructed.
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REFERENCES |
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Blum, H., Beier, H. & Gross, H. J. (1987). Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93-99.
El-kest, S. E. & Marth, E. H. (1992). Freezing of Listeria monocytogenes and other microorganisms: a review. J Food Protein 55, 639-648.
Etchegaray, J.-P. & Inouye, M. (1999). CspA, CspB, and CspG, major cold shock proteins of Escherichia coli, are induced at low temperature under conditions that completely block protein synthesis. J Bacteriol 181, 1827-1830.
Franks, F. (1995). Protein destabilization at low temperatures. Adv Protein Chem 46, 105-139.[Medline]
Gansel, X., Hartke, A., Boutibonnes, P. & Auffray, Y. (1993). Nucleotide sequence of the Lactococcus lactis NCDO763 (ML3) rpoD gene. Biochim Biophys Acta 1216, 115-118.[Medline]
Gasson, M. J. (1983). Plasmid complements of Streptococcus lactis NCDO712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154, 1-9.[Medline]
Goldstein, J., Politt, N. S. & Inouye, M. (1990). Major cold shock protein of Escherichia coli. Proc Natl Acad Sci USA 87, 283-287.[Abstract]
Graumann, P. & Marahiel, M. A. (1996). Some like it cold: response of microorganisms to cold shock. Arch Microbiol 166, 293-300.[Medline]
Graumann, P., Schröder, K., Schmid, R. & Marahiel, M. A. (1996). Cold shock stress-induced proteins in Bacillus subtilis. J Bacteriol 178, 4611-4619.[Abstract]
Graumann, P., Wendrich, T. M., Weber, M. H. W., Schröder, K. & Marahiel, M. A. (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.[Medline]
Hartke, A., Bouche, S., Gansel, X., Boutibonnes, P. & Auffray, Y. (1994). Starvation-induced stress resistance in Lactococcus lactis subsp. lactis IL1403. Appl Environ Microbiol 60, 3474-3478.[Abstract]
Hecker, M., Schumann, W. & Völker, U. (1996). Heat-shock and general stress response in Bacillus subtilis. Mol Microbiol 19, 417-428.[Medline]
Jiang, W., Hou, Y. & Inouye, M. (1997). CspA, the major cold-shock protein of Escherichia coli, is an mRNA chaperone. J Biol Chem 272, 196-202.
Jones, P. G. & Inouye, M. (1994). The cold-shock response a hot topic. Mol Microbiol 11, 811-818.[Medline]
Jones, P. G., Krah, R., Tafuri, S. R. & Wolffe, A. P. (1992). DNA gyrase, CS7·4, and the cold shock response in Escherichia coli. J Bacteriol 174, 5798-5802.[Abstract]
Kim, W. S. & Dunn, N. W. (1997). Identification of a cold shock gene in lactic acid bacteria and the effect of cold shock on cryotolerance. Curr Microbiol 35, 59-63.[Medline]
Kolter, R., Stiegele, D. A. & Tormo, A. (1993). The stationary phase of the bacterial life-cycle. Annu Rev Microbiol 47, 855-874.[Medline]
Kuipers, O. P., Beerthuyzen, M. M., Siezen, R. J. & de Vos, W. M. (1993). Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis: requirement of expression of the nisA and nisI genes for development of immunity. Eur J Biochem 216, 281-291.[Abstract]
Kuipers, O. P., Beerthuyzen, M. M., de Ruyter, P. G. G. A., Luesink, E. J. & de Vos, W. M. (1995). Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J Biol Chem 270, 27299-27304.
LaTeana, A., Brandi, A., Falconi, M., Spurio, R., Pon, C. L. & Gualerzi, C. O. (1991). Identification of a cold shock transcriptional enhancer of the Escherichia coli major cold shock gene encoding nucleoid protein H-NS. Proc Natl Acad Sci USA 88, 10907-10911.[Abstract]
Lee, S. J., Xie, A., Jiang, W., Etchegaray, J., Jones, P. G. & Inouye, M. (1994). Family of the major cold-shock protein, CspA (CS7.4), of Escherichia coli, whose members show a high sequence similarity with the eukaryotic Y-box binding proteins. Mol Microbiol 11, 833-839.[Medline]
Nakashima, K., Kanamaru, K., Mizuno, T. & Horikoshi, K. (1996). A novel member of the cspA family of genes that is induced by cold shock in Escherichia coli. J Bacteriol 178, 2994-2997.[Abstract]
OFarrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007-4021.[Abstract]
Panoff, J.-M., Thammavongs, B., Laplace, J.-M., Hartke, A., Boutibonnes, P. & Auffray, Y. (1995). Cryotolerance and cold adaptation in Lactococcus lactis subsp. lactis IL1403. Cryobiology 32, 516-520.
Rallu, F., Gruss, A. & Maguin, E. (1996). Lactococcus lactis and stress. Antonie Leeuwenhoek 70, 243-251.
van Rooijen, R. J. & de Vos, W. M. (1990). Molecular cloning, transcriptional analysis, and nucleotide sequence of lacR, a gene encoding the repressor of the lactose phosphotransferase system of Lactococcus lactis. J Biol Chem 265, 18499-18503.
de Ruyter, P. G. G. A., Kuipers, O. P. & de Vos, W. M. (1996). Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62, 3662-3667.
Schägger, H. & von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368-379.[Medline]
Thammavongs, B., Corroler, D., Panoff, J.-M., Auffray, Y. & Boutibonnes, P. (1996). Physiological response of Enterococcus faecalis JH2-2 to cold shock: growth at low temperatures and freezing/thawing challenge. Lett Appl Microbiol 23, 398-402.[Medline]
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.[Abstract]
Wouters, J. A., Sanders, J.-W., Kok, J., de Vos, W. M., Kuipers, O. P. & Abee, T. (1998). Clustered organization and transcriptional analysis of a family of five csp genes of Lactococcus lactis MG1363. Microbiology 144, 2885-2893.[Abstract]
Yamanaka, K. & Inouye, M. (1997). Growth-phase-dependent expression of cspD, encoding a member of the CspA family in Escherichia coli. J Bacteriol 179, 5126-5130.[Abstract]
Yamanaka, K., Fang, L. & Inouye, M. (1998). The CspA family in Escherichia coli: multiple gene duplication for stress adaptation. Mol Microbiol 27, 247-255.[Medline]
Received 20 April 1999;
revised 6 August 1999;
accepted 9 August 1999.