Cold shock proteins and cold acclimation proteins in a psychrotrophic bacterium

LYLE G. WHYTE AND WILLIAM E. INNISS'

Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3Gl

Received March 19, 1992

Revision received July 28, 1992

Accepted July 28, 1992

WHYTE, L. G., and INNISS, W. E. 1992. Cold shock proteins and cold acclimation proteins in a psychrotrophic bacterium. Can. J. Microbiol. 38: 128 1-1285.

The synthesis of proteins in the psychrotrophic bacterium Bacillus psychrophilus in response to both cold shock and continuous growth at low temperatures was examined. Cold shocks of 20 to 0, 5, or 10°C resulted in the induction of nine, seven, and five cold shock proteins, respectively, as determined by 2-dimensional gel electrophoresis and com- puting scanning laser densitometry. Two cold shock proteins, with molecular masses of 61 and 34 kDa, which were induced in B. psychrophilus by cold shocks of 20 to 0 or S°C, were not induced in a cold-sensitive mutant of B. psychrophilus. Analysis of protein profiles of B. psychrophilus during continuous growth at 0, 5, or 10°C revealed the synthesis of 11, 10, and 4 cold acclimation proteins, respectively. Some of these cold acclimation proteins were similar to cold shock proteins. In addition, the relative synthesis of both cold shock proteins and cold acclimation proteins increased with decreasing temperature. Thus, both types of proteins increased both in number and relative synthesis in response to cold shock and continuous growth at low temperature.

Key words: cold shock proteins, cold acclimation proteins, psychrotrophic bacterium.

WHYTE, L. G., et INNISS, W. E. 1992. Cold shock proteins and cold acclimation proteins in a psychrotrophic bacterium. Can. J. Microbiol. 38 : 1281-1285.

Les auteurs ont examine la synthkse des proteines de la bacterie psychrotrophe Bacillus psychrophilus en reponse a un stress par le froid et a une croissance continue a basse temperature. L'electrophorkse en gel a deux dimensions et la densitometrie par balayage au laser assistee par ordinateur ont permis de determiner que des stress par le froid de 20 a 0, 5 ou 10°C provoquent la synthkse respective de neuf, sept et cinq proteines induites par le froid. Deux pro- teines induites par le froid de masses moleculaires de 61 et 34 KDa, qui ont ete induites chez B. psychrophilus lors de stress par le froid de 20 a 0 ou S°C, n'ont pas ete induites chez un mutant de cette espkce sensible au froid. L'analyse des profils proteiques de B. psychrophilus, pendant une croissance continue a 0, 5 ou 10°C, a revele la synthkse respec- tive de 11, 10 et 4 proteines d'acclimatation au froid. Certaines de ces proteines d'acclimatation au froid etaient sem- blables aux proteines induites par le froid. De plus, les synthkses relatives de proteines induites par le froid et de pro- teines d'acclimatation au froid augmente avec des temperatures decroissantes. Ainsi, les deux types de proteines ont augmente en nombre et en synthkse relative en reponse au stress par le froid et a une croissance continue a basse temperature.

Mots cles : proteines induites par le froid, proteines d'acclimatation au froid, bacterie psychrotrophe. [Traduit par la redaction]

Introduction Psychrotrophic and psychrophilic bacteria are capable of

growth at 0°C or less while other microorganisms are not. A molecular determinant of psychrotrophic growth can involve qualitative and quantitative changes in protein synthesis at low temperatures (Inniss and Ingraham 1978; Russell 1990; Gounot 199 1). In the psychrotroph Bacillus psychrophilus, it has been found that the different rates of growth that occur at 5,20, and 30°C corresponded directly with the rates of protein synthesis (Bobier et al. 1972). In addition, microorganisms have been shown to display an increased rate of synthesis of certain proteins in response to cold shock, i.e., a downshift from a higher temperature to a lower temperature. In the eukaryotic mesophilic micro- organism Dictyostelium discoideum, both ubiquitin (Muller- Taubenberger et al. 1988) and a developmentally regulated membrane protein (Maniak and Nellen 1988) were induced by cold shock. In the mesophilic bacterium Escherichia coli, Jones et al. (1987) reported the induction of 13 proteins, which they termed cold shock proteins (csps), in response to a temperature shift of 37 to 10°C. A psychrophilic Vibrio sp. has been found to preferentially synthesize certain pro- teins after a shift down from 13 to 0°C (Araki 1991 b). With

' ~ u t h o r to whom all correspondence should be addressed. Printed in Canada / lmprime au Canada

regard specifically to psychrotrophic microorganisms, induc- tion of csps has been shown in the psychrotrophic yeast Trichosporon pullulans and was found to be influenced by the range of the cold shock (Julseth and Inniss 1990). The function(s) of csps has (have) yet to be determined. Some evidence suggests that these proteins may be involved in the reinitiation of protein synthesis upon exposure to colder tem- peratures (Szer 1970; Broeze et al. 1978; Jones et al. 1987). In psychrotrophic and psychrophilic microorganisms, the production of csps may constitute an adaptive response to allow for survival during the stress of a cold shock and also, perhaps, to prepare for growth at continuously cold temperatures.

In the present study, the induction of csps in a psychro- trophic bacterium is described and compared with that of a cold-sensitive (CS) mutant produced from it. The synthesis of cold acclimation proteins (caps), i.e., those proteins syn- thesized at a greater level during continuous growth at lower temperatures as compared with a higher temperature, was also examined.

Materials and methods Cells and media

The microorganisms used in this study were the psychrotrophic bacterium Bacillus psychrophilus (ATCC 23304), and a CS mutant

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(CS-2, hereafter referred to as CS mutant) derived from it by mutagenization and penicillin counterselection, using procedures as previously described by Murray and Inniss (1980). The micro- organisms were grown in a basal broth developed specifically for psychrotrophic bacilli by Adam and Stokes (1968) except that glucose was omitted. A modification of the glucose-salts medium of McCallum et al. (1986), with 0.1% ammonium citrate replac- ing 0.1% glucose, was used for the radiolabelling of cellular proteins.

Growth The growth of B. psychrophilus and the CS mutant was

monitored at 0, 5, 10, and 20°C by optical density measurements at 650 nm. All growth measurements were conducted on cells in 250-mL Erlenmeyer flasks containing 50 mL of the citrate basal broth and incubated in water-bath shakers at 150 rpm for the indicated times and temperatures.

Cold shock protein radiolabelling Cells, which were grown .at 20°C in 250-mL Erlenmeyer flasks

to an OD,,, of 0.5 (midlogarithmic growth), were subjected to the appropriate cold shock temperatures and incubation times in water- bath shakers. Cells (200-pL amounts) of the wild type or CS mutant were transferred into 1.5-mL microcentrifuge tubes preincubated at the appropriate cold shock temperature, centrifuged at 12 000 x g for 1 min, and resuspended in radiolabelling medium preincubated at the appropriate temperature. Radiolabelling of cellular proteins was initiated with the addition of 150 pCi . mL - ' (1 Ci = 37 GBq) of L-[35~]methionine (New England Nuclear, Montreal, Que.). For the 20 to 5 and 10°C cold shocks, cells were radioactively labelled for the last hour of the cold shock periods (2, 12, and 24 h). To obtain sufficient incorporation of the radio- label suitable for 2-dimensional gel electrophoresis for the 20 to 0°C cold shock, B. psychrophilus cells and CS mutant cells were radiolabelled for the final 2 and 4 h, respectively, of the cold shock treatment. Cells were then reisolated by centrifugation at 12 000 x g for 1 min and the radiolabelling medium was removed. The cells were either processed for electrophoresis immediately or stored at - 20°C. To the cell pellet, 10 pL of 1% lysozyme in 0.25 M Tris-HC1 (pH 8.0), and 10 pL of nuclease solution (20 mM CaCl,, 50 mM MgCl,, 1.0 mg . mL ' RNase A, 1.0 mg mL - ' DNase 1, in 0.5 M Tris-HC1, pH 7.0) were added, vortexed, and incubated for 5 min. To complete cell lysis, 30 pL of the lysis buf- fer of O'Farrell(1975) (9.5 M urea, 2% (v/v) Nonident-P-40,2% 3-10 ampholytes (Bio-Rad Laboratories, Mississauga, Ont.), and 5% 2-mercaptoethanol) was added, vortexed, and incubated for 20 min followed by freezing on dry ice (30 s). To maintain the con- centration of 9.5 M urea in the samples, 11.4 mg of urea was added to each microcentrifuge tube, followed by centrifugation at 12 000 x g for 10 s. Trichloroacetic acid (TCA) insoluble radio- activity of the supernatant fraction was determined by liquid scin- tillation spectrometry (Berg et al. 1987).

Radiolabelling of cold acclimation proteins To determine if B. psychrophilus produced certain proteins at

an increased rate during continuous growth at low temperature, cells were grown continuously at 0, 5, and 10°C and radiolabelled during midlogarithmic growth (OD,,, = 0.5) essentially as previ- ously described for cold shock radiolabelling. Cells were radiolabelled for 1 h at 5 and 10°C, and 2 h at 0°C.

Electrophoretic conditions Two-dimensional gel electrophoresis was conducted according

to the principles of O'Farrell (1975) using a Mini-Protean I1 2-D module (Bio-Rad Laboratories). Equal amounts (200 000 cpm; 1 cpm = 0.0167 Bq) of TCA-precipitable radioactivity, suspended in equal volumes of lysis buffer, were placed onto isoelectric focus- ing gels prepared from 5.5 g of urea, 1.33 mL of 30% acrylamide stock solution (28 g of acrylamide and 1.62 g of piperazine diacrylamide in 100 mL of distilled water), 2.0 mL of 10% Nonident-P-40, 0.25 mL of 3-10 ampholytes, 0.25 mL of 5-7

Hours FIG. 1. Growth of B. psychrophilus and the CS mutant at 5°C.

After 140 h, the CS mutant was shifted to incubation at 20°C ( A , B. psychrophilus; m, CS mutant).

ampholytes, 1.97 mL of distilled water, 10 pL of fresh 10% ammonium persulfate, and 10 pL of N, N, N', N1-tetramethyl- ethylenediamine. Samples were overlayed with 25 pL of sample overlay buffer (3.0 g of urea, 2.0 mL of Nonident-P-40,0.25 mL each of 3-10 and 5-7 ampholytes, and sterile distilled water to 10 mL). The electrophoresis was performed as described by Julseth and Inniss (1990) and all experiments were carried out in, at least, triplicate. Fluorograms, which were prepared as described by Berg et al. (1987), were then examined by a computing scanning laser densitometer using ImageQuant 3.0 software (Molecular Dynamics, Sunnyvale, Calif.). The individual densities of the proteins were determined by volume integration. The proteins, whose relative syntheses were found to be increased by a minimum of 2-fold (on the basis of replicate experimentation) at the low temperature as compared with the level found at 20°C, were designated to be either csps or caps, depending on the conditions that produced them.

Results Growth

The growth patterns of B. psychrophilus and the CS mutant at different temperatures were compared. The paren- tal psychrotroph possessed specific growth rates of 0.012, 0.033, 0.070, and 0.177 h - ' at 0, 5, 10, and 20°C, respec- tively. The CS mutant had specific growth rates of 0.007 and 0.153 h - ' at 10 and 20°C, respectively, but was unable to grow at 0 and 5°C. However, after transferring the CS mutant incubated at 5°C for 140 h, for example, to the per-

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FIG. 2. Induction of csps in B. psychrophilus after a 20 to 0°C cold shock for 24 h. Cellular proteins were radiolabelled with 150 pCi of L-[35~]methionine for the last 2 h of the cold shock. Equivalent amounts of TCA-insoluble radioactive counts were sub- jected to 2-dimensional gel electrophoresis (PI range 5.2-6.4). The location and the molecular mass (in kDa) of the csps are as indicated on the fluorogram.

missive temperature (20°C), it grew at the same rate as the wild type, indicating that the cold sensitivity was reversible (Fig. 1).

Cold shock proteins of B . psychrophilus and the CS mutant When B. psychrophilus was subjected to cold shock from

20 to 0, 5, or 10°C and the protein profiles obtained after 2, 12, and 24 h were examined by 2-dimensional gel elec- trophoresis, the induction of various proteins was observed, for example, as shown for a representative gel (Fig. 2). Then, the synthesis of these csps was directly quantified, using computing scanning laser densitometry. A 20 to 0°C cold shock induced nine csps with molecular masses of 72, 61, 54, 52, 36, 34, 33,27, and 21 kDa (Fig. 3). A 20 to 5°C cold shock induced seven csps (72, 61, 54, 52, 36, 34, and 27 kDa), while the least severe cold shock, from 20 to 10°C, induced five csps (72, 61, 54, 52, and 36 kDa). Thus, the number of csps appeared to be correlated with the severity of the cold shock. In addition, comparison of the relative synthesis levels suggested that the greater the cold shock, the higher the synthesis level of the csps. For example, seven out of the nine csps (72, 61, 36, 34, 33, 27, and 21 kDa) had a greater level of relative synthesis after the cold shock from 20 to 0°C as compared with the cold shock from 20 to 10°C (Fig. 3). In addition, five out of the nine csps (72, 61, 34, 33, and 21 kDa) demonstrated a greater level of relative synthesis after the cold shock from 20 to 0°C as compared to the cold shock from 20 to 5°C.

Induction of csps in the CS mutant was also examined and compared with induction in the parental psychrotroph. When the CS mutant was shocked from 20 to 5"C, some csps (72, 54, 52, 36, and 27 kDa) were induced in both the wild type and the CS mutant (Fig. 4). However, two csps with molecular masses of 61 and 34 kDa, respectively,

72 61 54 52 36 34 33 27 21

MOLECULAR MASS (kDa)

FIG. 3. Synthesis of csps in B. psychrophilus after cold shocks from 20 to 0°C (m), 5°C (a), or 10°C (a). The fold increase represents the relative synthesis of csps, as determined by comput- ing scanning laser densitometry, compared with 20°C before cold shock. The fold increase levels shown were found maximally after either 2, 12, or 24 h of cold shock.

appeared to be not induced in the CS mutant. After the 20 to 0°C cold shock, similar results were obtained, in that the 61- and 34-kDa csps were again not induced in the CS mutant after 4 h at 0°C.

Cold acclimation proteins of B . psychrophilus Two-dimensional gel electrophoresis of the proteins pro-

duced by B. psychrophilus during continuous growth at 0, 5, and 10°C revealed increased levels of a number of proteins that were designated as caps; this term is similar to that used to describe proteins produced in certain plants during growth at low, nonfreezing temperatures (Guy and Haskell 1987). The increased synthesis of these caps was quantified by com- puting scanning laser densitometry as previously described and compared with the level of synthesis on 20°C control gels. At O°C, 11 caps were found with molecular masses ranging from 72 to 21 kDa (Fig. 5). Eight of these proteins (72, 61, 54, 52, 36, 34, 27, and 21 kDa) were similar to B. psychrophilus csps. However, measurement of the relative synthesis levels of these proteins suggested that most of them were produced at relatively lower levels during con- tinuous growth at cold temperatures when compared with the maximum relative synthesis level caused by a cold shock to the same temperature. Production of two caps (67 and 28 kDa) was only observed during continuous growth at low temperatures and was not induced by a cold shock. At 5 and 10°C, the number of caps was 10 (72, 67, 61, 54, 52, 37, 34, 28, 27, and 21 kDa) and 4 (72, 67, 52, and 28 kDa), respectively. Similar to the trend observed with csp induc- tion, the greatest level of production of caps was found at the lowest temperature. At O°C, 7 of the 11 caps (61, 54, 37, 36, 34, 27, and 21 kDa) were produced at relatively higher levels than in cells grown at 10°C, and 4 of the 11 caps (67, 54, 37, and 36) were also produced at a greater level than in cells grown at 5°C. As would be expected, com- parison of the synthesis of caps in the CS mutant was not possible because of insufficient incorporation of the

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72 61 54 52 36 34 27

MOLECULAR MASS (kDa)

FIG. 4. Synthesis of csps in B. psychrophilus (m) and the CS 72 67 61 54 52 37 36 34 28 27 21

mutant (El) after a cold shock from 20 to 5°C. The fold increase MOLECULAR MASS (kDA)

represents the relative synthesis of csps, as determined by com- FIG. 5. Caps produced in B. psychrophilus during continuous puting scanning laser densitometry, compared with 2O0C before growth at ooc (.), 5 0 ~ (a), and 1 0 0 ~ (a). The fold increase cold shock. The fold increase levels shown were found maximally represents the relative synthesis of caps, as determined by ,-om- after either 2, 12, or 24 h of cold shock. puting scanning laser densitometry, compared with 20°C.

radiolabel at 0, 5, and 10°C owing to the lack of significant Goldstein et al. (1990) reported that a major csp (CS7.4) growth at these temperatures, which are restrictive for the of E. coli possessed hydrophilic characteristics and proposed CS mutant. that it may act similarly to fish antifreeze proteins (AFPs),

protecting E. coli from freezing injury. The amino acid Discussion sequence of CS7.4 has been reported to be very similar to

Determination of protein profiles for the psychrotroph the conserved region of several eukaryotic DNA binding pro- B. psychrophilus in response to cold shock or during con- teins (Wistow 1990). Interestingly, certain antibiotics that tinuous growth at low temperatures revealed increased syn- target the prokaryotic ribosome are reported to induce csps thesis of both csps and caps. Both the number and relative synthesis levels of these designated types of proteins increased with lowered temperature. Similar relationships have been reported with regard to the heat shock response in two psychrotrophic yeasts, T. pullulans (Julseth and Inniss 1990) and Sporobolomyes salmonicolor (Berg et al. 1987), in which the higher heat shock temperature resulted in an increase in both the number and relative synthesis levels

and hsps in E. coli, implicating the ribosome as a cellular sensor of temperature (VanBogelen and Neidhardt 1990). Previously, Szer (1970) had described a ribosomal protein from a psychrophilic Pseudomonas that allowed E. coli washed ribosomes to function at colder temperatures in vitro.

Analysis for production of caps in B. psychrophilus revealed the presence of 11 caps, of which 8 were also

of heat shock proteins (hsps). In addition, in B. psychrophilus designated as csps. Therefore, these eight proteins may be and the arctic psychrophilic bacterium Aquaspirillum of some particular importance during both cold shock stress arcticum (Butler et al. 1989) the number of hsps increased and continuous low temperature growth. Little information in response to higher heat shock temperatures (McCallum exists concerning the production of specific proteins by et al. 1986). In the present study, the correlation between microorganisms during continuous low temperature growth. the increase in numbers and relative synthesis levels of the Herendeen et al. (1979) have described in E. coli a small csps and caps with lower cold shock or growth temperatures supports the consideration that the production of such pro- teins may constitute an adaptative response by the bacterium to allow or facilitate growth at lower temperatures.

Comparison of the synthesis of csps by B. psychrophilus with the CS mutant disclosed the apparent lack of induc- tion of two csps (61 and 34 kDa) in the CS mutant. This suggests that these two proteins may possibly be involved in the inability of the CS mutant to grow at lower tempera- tures and thus, perhaps, may be important for the capability of the parental psychrotroph for growth at low temperature.

There is a dearth of information concerning the induction of csps and their function in prokaryotes, particularly in psychrophilic and psychrotrophic microorganisms. In the mesophile E. coli, several of the 13 csps identified on 2-dimensional gels were suggested to be involved in tran- scription and translation (Jones et al. 1987). In addition,

number of proteins having a marked increase in synthesis during steady-state growth at 13.5"C as compared with 37°C. In a psychrotrophic Arthrobacter globiformis strain, the production of 13 temperature-specific proteins during exponential growth at 10°C as compared with 32°C (the maximum growth temperature) was reported by Potier et al. (1990). These proteins were exclusive to, or present in, increased amounts, only at 10°C. In the psychrophile Vibrio sp. ANT-300, 28 proteins from a soluble fraction involved in protein synthesis displayed higher levels during growth at 0°C than during growth at 13°C (Araki 1991a). It was suggested that changes in the levels of these proteins may account for the temperature-dependent differences observed in protein synthesis. In plants, the synthesis of novel pro- teins during the cold acclimation period has been docu- mented to a much greater extent (Guy 1990). In some cases, the production of these caps has been linked with subsequent

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freezing tolerance. Perras and Sarhan (1990) found that four proteins were induced during the cold acclimation period in a freezing-tolerant wheat strain but not in a freezing- sensitive strain. More recently, frost-tolerant winter rye leaves have been found to possess both antifreeze activity and ice nucleation activity attributable to AFPs and ice- nucleating proteins (Griffith and Marentes 1992). Interest- ingly, Kurkela and Franck (1990) discovered a cold-induced gene, kinl, in Arabidopsis whose 6.5-kDa protein product had similarity to fish AFPs. Certain overwintering insects produce thermal hysteresis proteins, which like AFPs act in a noncolligative manner, lowering the freezing point of intracellular water and thereby protecting the organism from freezing iujury (Cannon and Block 1988).

In conclusion, increasing evidence suggests that organisms produce specific proteins for low temperature adaptation. The present study, showing the synthesis of both csps (at least two of which appear not to be induced in a CS mutant that cannot grow at low temperatures) and caps by the psychrotroph B. psychrophilus, supports this hypothe- sis. Moreover, both the number and relative synthesis levels of both the csps and caps were found to increase in response to lower cold shock and growth temperatures. Continuing research will be directed toward determining the actual involvement of these proteins in psychrotrophy.

Acknowledgements This research was supported by a Natural Sciences and

Engineering Research Council (NSERC) grant to W .E. I. In addition, the support of an NSERC equipment grant (EQP0090276) is gratefully acknowledged.

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