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Cold shock proteins and cold acclimation proteins in the psychrotrophic bacterium Pseudomonas putida Q5 and its transconjugant
Andrew W. Gumley and William E. lnniss
Abstract: The production of cold shock proteins (csps) and cold acclimation proteins (caps) was characterized in the psychrotrophic bacterium Pseudomonas putida Q5 and its transconjugant P. putida QST which contains the toluene-degradative TOL (pWWO) plasmid, using two-dimensional gel electrophoresis and computing scanning laser densitometry. Similar growth rates for the psychrotrophic bacterium P. putida Q5 and the transconjugant were found at temperatures ranging from 30 to O°C. Sixteen proteins were quantified and compared in P. putida Q5 and P. putida Q5T following a 25 to 5OC cold shock or constant growth at 5OC. During constant growth at 25OC, a decrease in the synthesis of various proteins occurred in the transconjugant. Following cold shock to 5OC or constant growth at S°C, csps and caps were produced with a greater number occurring in the transconjugant. This may suggest an additional stress response in the transconjugant owing to metabolic load exerted by the TOL plasmid. Growth of P. putida QST with toluate produced seven proteins that appeared to be TOL-plasmid mediated and of which some were also designated as caps.
Key words: cold shock proteins, cold acclimation proteins, TOL pWWO plasmid, psychrotrophic bacterium.
Resume : En utilisant l'electrophorbse en gel bidimensionnelle et la densitomktrie 5 balayage laser informatiske, nous avons caractCrisC la production des protCines consCquentes B un choc par le froid (csps) et les protCines d'acclimatation au froid (caps) chez la bactkrie psychotrophe Pseudomonas putida Q5 et son transconjuguant P. putida Q5T qui possbde le plasmide TOL (pWWO) responsable de la degradation du tolubne. A des temphatures entre 30 et O°C, le P, putida Q5 et le transconjuguant prksentaient les mCmes taux de croissance. Seize protkines ont pu Ctre quantifiCes et comparCes chez P. putida Q5 et P. putida Q5T suite a un choc par le froid de 25 B 5°C ou suite B une tempkrature constante de 5°C. Lors d'une croissance B une tempkature constante de 25OC, une diminution de la production de diverses protkines a Cte notCe chez le transconjuguant mais, suite B une choc par le froid de 5°C ou lors d'une croissance a une tempkrature constante de 5"C, des csps et caps Ctaient produites en plus grande quantitk chez le tarnsconjuguant. Ces rCsultats suggbrent qu'il y a une rCponse supplementaire au stress chez le transconjuguant a cause d'une demande mktabolique exercke par le plasmide TOL. La croissance du P. putida en prCsence de toluate a produit sept protkines qui semblaient contr61Ces par le plasmide TOL et dont quelques-unes ont &tC dCsignCes caps.
Mots cle's : proteines de rCaction B un choc par le froid, protkines d'acclimatation au froid, plasmide TOL pWWO, bactCrie psychrotrophe. [Traduit par la rkdaction]
Introduction expression has been observed in various bacteria. For example, Escherichia coli, when subjected to a 37 to 10°C cold shock,
Various ~h~siological changes have been noted in bacteria induced 13 proteins of some are associated with transla- exposed to low temperature conditions (Gounot 1986. 1991; tional and transcriptional processes and mRNA degradation Russell 1990). One of these changes has been the production of proteins that are believed to be associated with the ability of (Jones et al. 1987). Lelivelt and Kawula (1995) described the
bacteria to survive cold temperatures (Jones and Inouye 1994). induction of a cold shock protein (Hsc66) that exhibits simi-
These proteins that exhibit induced or increased synthesis larities to the heat shock chaperone Hs~70 , and therefore,
during cold shock have been termed cold shock proteins (csps). suggested that some csps may serve to prevent protein misfold- Although the function of these proteins is unknown, their ing at low temperatures. Panoff et al. (1994) described the
production of 10 csps in a Lactococcus lactis subsp. lactis within the first 10 h after a 30 to S0C cold shock. The psychro-
Received January 3 1, 1996. Revision received April 4, 1996. philic bacterium Aquaspirillum arcticum revealed the produc- Accepted April 15, 1996.
tion of 10 csps from a 10 to O°C cold shock (Roberts and Inniss A.W. Gumley and W.E. 1nniss.l Department of Biology, 1992). Further, the response was found to be temperature University of Waterloo, Waterloo, ON N2L 3G1, Canada. related, where an increase in the production of csps was
Author to whom all correspondence should be addressed dependent on the range and minimum temperature of the (e-mail: [email protected]). cold shock. A similar trend was also observed in Bacillus
Can. J. Microbiol. 42: 798-803 (1996). Printed in Canada / Imprim6 au Canada
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psychrophilus from a 20 to 10,5, and 0°C cold shock with the induction of 5, 7, and 9 csps, respectively (Whyte and Inniss 1992~).
Cold acclimation proteins (caps), which are proteins synthe- sized at a greater level during constant growth at a lower temperature as compared with a higher temperature, were first observed in plants where an increase in production of these proteins occurred during exposure to nonfreezing temperatures (Guy and Haskell 1987). Similar findings have now been reported in bacteria during constant growth at low tempera- tures. In a psychrotrophic Arthrobacter globiformis strain, 13 caps were produced when the bacterium was constantly grown at 10°C as compared with growth at 30°C (Potier et al. 1990). Araki (1991) observed 28 caps in the psychrophile Vibrio sp. ANT-300 during constant growth at 0°C compared with 13°C. Constant growth of B. psychrophilus at 0°C showed an increase in synthesis of 11 caps (Whyte and Inniss 1992~). Aquaspirillum arcticum also showed a similar response with the production of eight caps during constant growth at 0°C (Roberts and Inniss 1992).
Insertion of DNA into bacteria to increase metabolic versa- tility has various biotechnological applications. Such modified microorganisms have been mainly mesophiles, and therefore, their use has been excluded from relatively cold environments. Psychrotrophs, however, which are usually defined as having a minimum growth temperature of 0°C or less and a maximum growth temperature above 20°C (Morita 1975), have the poten- tial to be utilized. Since numerous mesophilic bacteria are metabolically versatile, the metabolic ability of psychrotrophs could be enhanced using DNA from such mesophiles.
The insertion of DNA from mesophilic bacteria does not appear to negatively affect psychrotrophic bacteria based on growth rate. Feller et al. (1989) have shown expression in a psychrotrophic bacterium of a plasmid-encoded antibiotic gene from a mesophile with no apparent effect on the bacterium. This was also observed by Whyte and Inniss (19926) where the insertion and expression of plasmid-encoded chloramphenicol resistance from a mesophile caused no difference in filament formation, growth rate, cold shock protein production, or cold acclimation protein production in the psychrotroph Bacillus psychrophilus. Kolenc et al. (1988) conjugated the psychro- trophic bacterium Pseudomonas putida Q5 with a mesophilic P. putida Pawl containing the TOL (pWWO) plasmid. The resulting psychrotrophic transconjugant P. putida Q5T pos- sessed the TOL plasmid and the ability to degrade toluate at temperatures ranging from 30 to 0°C with no apparent effect on growth.
Understanding the effect of insertion and expression of DNA from a mesophile on the physiology of a psychrotroph could be important for its successful industrial or bioremedial appli- cation. The present report characterizes the effect of the inser- tion of the mesophilic TOL pWWO plasmid into the psychrotrophic bacterium P. putida Q5, on the production of csps and caps, as determined by two-dimensional gel electro- phoresis and computing scanning laser densitometry. In addi- tion, seven proteins were reported that may be encoded on the TOL plasmid and involved in the ability to degrade toluate.
Materials and methods
Growth The microorganisms used in this study were P. putida Q5 isolated from the Bay of Quinte, Lake Ontario (Inniss and Mayfield 1978), and its transconjugant P. putida Q5T containing the mesophilic pWWO TOL plasmid derived by transconjugation of l? putida Q5 with P. putida Pawl (Kolencs et al. 1988). Cultures were grown in 250-mL Erlenmeyer flasks with 50 mL basal salts medium (BSM) broth (4.3 g K2HP04, 3.4 g KH2P04, 2.0 g (NH4)2S04, 0.34 g MgC12. 6H20, 1.0 mg MnClz. 4H20, 0.6 mg FeS04. 7H20, 26.0 mg CaC12. 2H20, 2.0 mg NaMo04. 2H20, 1 L H20, pH 7.0). Salicylate (1 g/L) was used as the sole carbon source for P. putida Q5, whereas P. putida Q5T was grown with either salicylate (1 g/L) or rn-toluate (1 g/L). Growth measurements were conducted spectrophotometrically at 650 nm at 25,10,5, and 0°C on cells incubated in water-bath shakers at 150 rpm.
Radiolabelling of cold shock proteins Cells, which were grown to an OD650 of 0.25 (midlogarithmic growth) at 25"C, were subjected to a cold shock at 5OC in a water-bath shaker. Samples (200 pL) of P. putida Q5 grown with salicylate or l? putida Q5T grown with either salicylate or toluate were removed from either the control cells or cold shock cells and transferred to a 1.5-mL Eppendorf tube preincubated at 25 or 5OC. For comparison of the cold shock response in P. putida Q5 and its transconjugant, the cold shock time period, where the maximum csps were produced based on one-dimensional gel electrophoresis analysis (data not shown), was used. Both l? putida Q5 and P. putida Q5T grown with salicylate were cold shocked for 2 h, whereas P. putida Q5T grown with toluate was cold shocked for 4 h. Radiolabelling of the cellular proteins was initiated by the addition of 150 pCi/mL of L-[35S]methionine (New England Nuclear, MontrCal, P.Q.) to each sample for the last hour for the cold shocked cells and for 1 h for the control cells. Radiolabelling was stopped by centrifuging at 11 600 x g for 2 min at 4°C and removal of the supernatant. The pellet was either stored at -20°C or processed immediately for electrophoresis. To the pellet, 10 pl of 0.1% lysozyme in 0.25 M Tris-HCl (pH 8.0) and 10 pL of nuclease solution (50 mM MgC12, 20 mM CaC12, 1.0 mg RNase A, and 1.0 mg DNase I in 0.5 M Tris-HC1, pH 7.0) were added, vortexed, and incubated for 5 min. After incubation, 30 pL of lysis buffer (O'Farrell 1975) consisting of 9.5 M urea, 2% vlv Nonidet P-40,2% 3-10 ampholytes (BioRad laboratories, Mississauga, Ont.), and 5% v/v 2-mercaptoethanol was added. The samples were vortexed, incubated for 20 min, and subjected to two freeze-thaw cycles in dry ice each for 30 s. The urea concentration of 9.5 M was maintained by the addition of 11.4 mg of urea to each tube, followed by centrifugation at 11 600 x g for 10 s. Trichloroacetic acid (TCA) insoluble radioactivity of the Supernatants was determined by liquid scintillation spectrometry (Berg et al. 1987).
Radiolabelling of cold acclimation proteins Identification of caps from constant growth of P.putida Q5 with salicylate and its transconjugant P. putida Q5T grown with either salicylate or toluate was performed. Cells were grown at 5OC for 10 generations. At midlogarithmic growth = 0.25), cells were radiolabelled for 1 h as previously described.
Radiolabelling of suspected TOL-encoded proteins To determine whether the additional proteins observed in l? putida Q5T when grown with toluate could be induced in the parent, l? putida Q5 and P. putida Q5T were grown at 25OC in BSM with both salicylate and toluate. Cells were grown to midlogarithmic growth = 0.25) and radiolabelled as previously described. Cells were radio- labelled for 1 h at 25OC.
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Table 1. Comparison of generation times of P. putida Q5 and its transconjugant P. putida Q5T at various temperatures.
Temperature Generation time (h)
("(3 @-so Q5T-Sb QST-T"
"Pseudomonas putida Q5 grown with salicylate. bPseudomonas putida QST grown with salicylate. CPseudomonas putida QST grown with toluate.
Fig. 1. Relative synthesis of seven of the selected 16 proteins in P. putida Q5 (0) and its transconjugant (M) during constant growth at 25OC with salicylate as the sole carbon source. Total percent protein for 16 selected proteins was compared using two-dimensional gel electrophoresis and computing scanning laser densitometry.
Molecular Mass ( m a )
Electrophoretic conditions and analysis For determination of the time period following cold shock where maximum csps were induced, one-dimensional gel electrophoresis was conducted as outlined by Berg et al. (1987). Protein profiles were analyzed every hour for 12 h after cold shock. Two-dimensional gel electrophoresis was conducted according to the principles of O'Farrell (1975) and as outlined by Julseth and Inniss (1990) and Whyte and Inniss (1992a). All experiments were repeated at least six times. Sixteen proteins were chosen for comparison between t? putida Q5 grown with salicylate and its transconjugant grown with either salicy- late or toluate. These 16 proteins also included all csps and caps obtained from cold shock or cold acclimation. Quantification of
Can. J. Microbial. Vol. 42, 1996
Fig. 2. Relative production of csps after a 25 to 5OC cold shock in P. putida Q5 grown with salicylate (u), P. putida Q5T grown with salicylate (U), and P. putida Q5T grown with toluate (M) as determined by two-dimensional gel electrophoresis and computing scanning laser densitometry. Relative increase represents production of proteins compared with those levels during constant growth at 25OC.
15 20 28 29 30a 42 74 78 81b
Molecular Mass ( m a )
proteins was performed on fluorograms prepared as described by Berg et al. (1987), using a computing scanning laser densitometer (Molecular Dynamics, Sunnyvale, Calif.) and ImageQuant 3.0 com- puter software (Molecular Dynamics). Volume integration was used to determine the individual densities of the proteins. Those proteins whose relative synthesis (based on six replicated experiments), com- pared with the 25°C control, increased a minimum of two fold (i.e., above the maximum variability observ~d for identical samples) at the low temperature were designated either csps or caps, depending on their production after cold shock or cold acclimation, respectively.
Results
Effect of TOL plasmid insertion in f! putida Q5 Growth rates of P. putida Q5 and its transconjugant P. putida Q5T were compared at various temperatures. Similar rates of growth, based on four replicate experiments, were observed between P.putida Q5 grown with salicylate and its trans- conjugant P. putida Q5T grown with either salicylate or toluate (Table 1). These values, which are in general agreement with Kolencs et al. (1988), suggest that the insertion of the meso- philic TOL plasmid does not negatively affect the growth rate of the psychrotrophic bacterium.
Examination of protein synthesis at 25OC by two- dimensional gel electrophoresis showed that neither the synthe- sis of new proteins nor the absence of proteins occurred in the
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Fig. 3. Relative production of caps in P. putida Q5 grown with salicylate (o), l? putida Q5T grown with salicylate (N), and P. putida Q5T grown with toluate (m) during constant growth at S°C as determined by two-dimensional gel electrophoresis and computing scanning laser densitometry. Relative increase represents production of proteins compared to those levels during constant growth at 2S°C.
20 28 29 40 42 52 72 74 78 81b
Molecular Mass (kDa)
transconjugant compared with the parent P. putida Q5 when grown with salicylate as the sole carbon source. Of the 16 rep- resentative proteins analyzed, 7 proteins with molecular masses of 15, 28, 30b, 56, 76, 78, and 81b kDa showed a significant decrease in protein synthesis levels in P. putida Q5T (Fig. 1). This may suggest that, although generation time is not affected, the insertion of the TOL plasmid might have an adverse effect on protein production.
Cold shock proteins in P. putida Q5 and its transconjugant
One-dimensional gel electrophoresis was used to determine the time period after cold shock where maximum induction of csps occurred. Both P putida Q5 and its transconjugant, when grown with BSM with salicylate, showed a maximum number of csps at 2 h after cold shock. The transconjugant, when grown with toluate, produced the maximum number of csps at 4 h after cold shock. Using these time periods, two-dimensional gel electrophoresis, followed by computing scanning laser densi- tometry, was used to quantify the relative protein synthesis of the representative 16 proteins. After the cold shock from 25 to 5"C, P. putida Q5 showed an increased synthesis of four csps with molecular masses of 20, 29, 42, and 74 kDa. The trans- conjugant P. putida Q5T, grown with salicylate, produced seven csps with molecular masses of 15,20,28,29,42,74, and
Fig. 4. Induction of TOL encoded proteins in P. putida Q5T with toluate and salicylate at 25OC. Cellular proteins were radiolabelled with 150 pCi/mL of L-[35S]methionine for 1 h of constant growth at 2S°C. Equivalent amounts of TCA- insoluble cell extracts were subjected to two-dimensional gel electrophoresis. Location of TOL encoded proteins are identified by their molecular mass (kDa). (A) Pseudomonas putida Q5 with salicylate and toluate; (B) P. putida Q5T with salicylate and toluate.
78 kDa. Additional production of csps was observed in the transconjugant grown with toluate with a total of nine csps (15, 20, 28, 29, 30a, 42, 74, 78, and 81b kDa) being synthesized (Fig. 2). All of the csps (20, 29, 42, and 74 kDa) in P. putida Q5 were also found as csps in the transconjugant. Three csps (15, 28, and 78 kDa) were produced in the transconjugant regardless of the sole carbon source (salicylate or toluate), whereas the 30a- and 8 lb-kDa csps were produced only in the transconjugant with toluate as the carbon source. Thus, the insertion of the TOL plasmid appears to result in the increased production of csps as compared with the parent P. putida Q5, with a further increase when the transconjugant is grown with toluate. Comparison of the relative increases of the csps showed generally higher values for the four csps common to the transconjugant and the parent, with the highest synthesis levels occurring when grown with toluate.
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Cold acclimation proteins in f! putida Q5 and its transconjugant
Pseudomonas putida Q5 and its transconjugant were grown at 5°C for 10 generations in BSM with salicylate (P. putida Q5) or with either salicylate or toluate (P. putida QST). Two- dimensional gel electrophoresis and computing scanning laser densitometry were used to quantify the representative 16 pro- teins synthesized in P. putida Q5 and its transconjugant. Pseudomonasputida Q5 produced only one cap with a molecu- lar mass of 72 kDa (Fig. 3). The transconjugant, when grown with salicylate, produced five caps (28,52,72,74, and 78 kDa) whereas, when grown with toluate, it produced nine caps (20, 29, 40, 42, 52, 72, 74, 78, and 81b kDa). Three caps (52,74, and 78 kDa) were common in the transconjugant, regardless of the carbon source, and the 72-kDa cap was common in both the parent and the transconjugant.
TOL-encoded proteins When protein synthesis of the transconjugant grown in BSM with toluate was examined, seven proteins were produced (22, 24,26,31,35,36, and 53b kDa) that were not produced in the parent P putida Q5 or in the transconjugant grown with salicy- late (Fig. 4). Since expression of these proteins only occurred when the transconjugant was grown with toluate, it was con- sidered that these proteins may be induced by toluate and are involved in its degradation. Thus, protein synthesis was com- pared in P. putida Q5 and P. putida Q5T when grown in the presence of both toluate and salicylate at 25°C. The seven proteins again were only expressed in the transconjugant and not in the parent, suggesting that they may be encoded by the TOL plasmid and induced in the presence of toluate (Fig. 4). In addition, of the 16 proteins examined, 3 of the possible TOL encoded proteins (24,26, and 3 1 kDa) were also induced in the transconjugant grown constantly at 5°C with toluate and thus were also designated as caps.
Discussion
The presence of the TOL plasmid from the mesophile in the psychrotrophic bacterium P. putida Q5 showed no effect on growth, based on generation times, at various temperatures. The insertion of foreign DNA into bacteria can cause a deple- tion of energy used for the maintenance and expression of cell plasmids, resulting in a metabolic burden on the bacterium (Glick 1995). Such an impairment has been observed in bacte- ria as a decrease in growth rate and cell size (Cheah et al. 1987; Hong et al. 1991a, 1991b). In the present study, quantification of selected proteins in P. putida Q5 and its transconjugant showed a decrease in the synthesis of seven proteins in the transconjugant. This may suggest that protein synthesis can be a more sensitive indicator of metabolic load than growth rate. Hong et al. (1995) has suggested that siderophore production may be a more sensitive indicator of metabolic load than generation time.
Following cold shock, a marked increase in numerous pro- tein levels (csps) was observed in P. putida Q5 and its trans- conjugant. A similar response has been shown in various bacteria (Jones et al. 1987; Lottering and Streips 1995; Roberts and Inniss 1992; Whyte and Inniss 1992a), suggesting that the production of csps may be relatively common in nature and important for bacterial survival at lower temperatures.
Although the function of csps in psychrotrophs is still undeter- mined, research on mesophilic bacteria indicates that they may be involved in one or more functions related to translation, transcription (Jones et al. 1987), or chaperone activity (Lelivelt and Kawula 1995).
As previously found for two psychrotrophic bacteria (Potier et al. 1990; Whyte and Inniss 1992a) and a psychrophilic bacterium (Araki 1991), constant growth of P. putida Q5 and its transconjugant at low temperatures resulted in the produc- tion of caps. The presence of csps and caps was greater in the transconjugant as compared with the parent, and when the transconjugant was grown with toluate, the greatest number of csps and caps occurred. It would appear that the production of these additional proteins in the transconjugant is not essential for growth at low temperatures as suggested by their absence in the parent P putida Q5. As previously indicated, it is consid- ered that these additional csps and caps may result from a stress response due to a metabolic load imposed by the maintenance and expression of the inserted TOL plasmid (Glick 1995). A number of csps found in the transconjugant were also desig- nated as caps, suggesting that these proteins were required during both cold shock and constant low temperature growth.
Growth of the transconjugant with toluate as the sole carbon source induced seven proteins. Since growth of the parent with both salicylate and toluate failed to induce any of these proteins, it is possible that they may be encoded on the TOL pWWO plasmid and are responsible for the toluate utilization. Three of these proteins also showed increased synthesis during constant growth of the transconjugant with toluate at 5°C. This may result from partial inactivation of three toluate-utilizing enzymes at the low temperature and thus is an adaptive response by the bacterium to maintain activity for the degrada- tion of toluate.
In conclusion, the production of csps and caps further sup- ports the growing evidence that these proteins may be involved in the adaptation of bacteria to cold temperatures. The insertion of the TOL plasmid into the psychrotroph P. putida Q5 exerted an effect on protein synthesis. The production of certain addi- tional csps and caps may reflect an increased metabolic load imparted by the inserted plasmid.
Acknowledgment
This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to W.E.I.
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