- Email: [email protected]
Heterologous expression of maize chloroplast protein synthesis elongation factor (EF-Tu) enhances Escherichia coli viability under heat stress
Plant Science 163 (2002) 1075 /1082 www.elsevier.com/locate/plantsci
Heterologous expression of maize chloroplast protein synthesis elongation factor (EF-Tu) enhances Escherichia coli viability under heat stress T. Moriarty a, R. West a, G. Small b, D. Rao a, Z. Ristic a,* a
b
Department of Biology, University of South Dakota, Vermillion, SD 57069, USA Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, SD 57069, USA Received 29 March 2002; received in revised form 27 July 2002; accepted 31 July 2002
Abstract A heat-tolerant maize line, ZPBL 1304, synthesizes increased amounts of chloroplast protein synthesis elongation factor (EF-Tu) under heat stress conditions. Previous studies have suggested that maize EF-Tu may be involved in the development of heat tolerance [Planta 212 (2001) 359; J. Plant Physiol. 153 (1998) 497]. In this study, we tested the hypothesis that overexpression of maize EF-Tu enhances the viability of Escherichia coli under heat stress. The approach was to expose E. coli transformed with a maize EF-Tu expression vector (pTrcHis2A-Zmeftu1) to 55 8C and assess viability at 37 8C. Western blots showed E. coli overproduced recombinant EF-Tu protein, and the protein seemed to be in a highly soluble form. E. coli overexpressing maize EFTu showed increased viability after exposure to heat stress, demonstrating that the maize EF-Tu is involved in the development of heat tolerance. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Escherichia coli ; Chloroplast protein synthesis elongation factor (EF-Tu); Heat stress; Heat tolerance; Zea mays
1. Introduction Protein synthesis elongation factors (elongation factors or EFs) are proteins that are involved in the elongation of polypeptides during the translational process [1,2]. The basic process of translation involves three separate factors (the prokaryotic EFs are indicated in parentheses): (1) EF-1a (EF-Tu), (2) EF-1b (EF-Ts), and (3) EF-2 (EF-G). Polypeptide elongation proceeds in three steps: (1) EF-1a (EF-Tu) binds GTP and aminoacyl-tRNA, and leads to the codon-dependent placement of this aminoacyl-tRNA at the A site of the ribosome; (2) following release of EF-1a-GDP (EF-TuGDP) from the ribosome, EF-1b (EF-Ts) facilitates the exchange of bound GDP for GTP; and (3) after peptide bond formation, EF-2 (EF-G) translocates the mRNA one codon to allow for the arrival of the new aminoacyl-
* Corresponding author. Tel.: /1-605-677-6170; fax: /1-605-6776557 E-mail address: [email protected] (Z. Ristic).
tRNA in the A site [2]. Elongation factors have been extensively studied in prokaryotes, and the functional homology between the prokaryotic and eukaryotic factors is quite striking [2]. Prokaryotic protein elongation factor EF-Tu (molecular mass: 43 kDa) may have other functions in addition to the conventional role that it plays in polypeptide elongation [3 /7]. One of these functions is in protein refolding and protection of polypeptides from heat denaturation [7]. Elongation factor EF-Tu, in a manner similar to that of molecular chaperones, increases the refolding of unfolded proteins, protects proteins against thermal denaturation, and forms complexes with unfolded proteins [7]. Our laboratory has recently found that a heat-tolerant maize line, ZPBL 1304, synthesizes increased amounts of chloroplast protein synthesis elongation factor, EFTu, under heat stress conditions [8]; chloroplast protein synthesis elongation factor is encoded by a gene that is of prokaryotic origin and, therefore, this plant protein is commonly referred to with the prokaryotic designation,
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 2 7 3 - X
1076
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
EF-Tu [9]. Three heat stress-induced EF-Tu polypeptides (pI, 5.2 /5.7) of molecular mass of 45 /46 kDa have been identified in this line of maize [8]. N-terminal amino acid sequencing has revealed a high similarity (80 /90%) of these polypeptides with the EF-Tu from other higher plant chloroplasts and from other organisms [8]. In addition, Northern blots have shown an increase (1.85-fold) in the steady-state levels of EF-Tu mRNA during heat stress [8], and the increase in EF-Tu transcript levels during heat stress was accompanied by increased levels of the EF-Tu protein [8]. Isolated chloroplasts from heat-stressed ZPBL 1304 plants have also been shown to have higher levels of EF-Tu as compared with non-stressed chloroplasts [8]. These findings have led to the hypothesis that maize EF-Tu may be involved in the development of heat tolerance [8]. In this study, we tested the hypothesis that overexpression of maize EF-Tu enhances the viability of Escherichia coli under heat stress. The approach was to transform E. coli with maize EF-Tu cDNA, expose the E. coli transformants for maize EF-Tu to 55 8C and assess viability after incubation at 37 8C. Here, we report that E. coli overexpressing maize EF-Tu shows increased viability after exposure to heat stress.
2. Materials and methods 2.1. cDNA subcloning and bacterial transformation A cDNA for maize (Zea mays L.) EF-Tu, designated Zmeftu1, was already on hand in our laboratory. It was previously isolated from a cDNA library, constructed using RNA from aerial tissue of B73 line maize seedlings that were exposed to 10 days of drought stress followed by 24 h heat stress at 45 8C [8]. Zmeftu1 was subcloned into the expression vector pTrcHis2A, which adds Cterminal c-myc and polyhistidine tags to the protein (Invitrogen, Carlsbad, CA) (Fig. 1). Subsequently, the pTrcHis2A vector carrying Zmeftu1 was used to transform competent E. coli cells of the strain DH5a (pTricHis2A-Zmeftu1). As controls, E. coli DH5a cells were transformed with either the pTrcHis2A vector alone (pTricHis2A), or vector carrying an insert for lac Z */the bacterial gene encoding b-galactosidase (pTricHis2A-lac Z). 2.2. Heterologous expression in E . coli cells pTrcHis2A-Zmeftu1, pTrcHis2A, and pTrcHis2Alac Z transformants were grown at 37 8C, with vigorous shaking, to an A600 /1.0 in Luria /Bertani (LB) medium supplemented with ampicillin at 75 mg ml 1. Culture aliquots (100 ml) were then diluted with 10 ml of fresh ampicillin supplemented medium and subcultured to an
A600 between 0.4 and 0.5. These subcultures were split into two groups: (1) a group induced to overexpress heterologous protein (induced group) and (2) a group not induced to overexpress heterologous protein (noninduced group). Induction was achieved by adding isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM. For all induced cultures, induction time was 3 h after induction, both the induced and non-induced cultures were measured again at 600 nm to estimate cell concentration. Culture samples with similar amount of cells were used for subsequent 1D SDS-PAGE and Western blot analysis, and for heat stress viability experiments. 2.3. 1D SDS-PAGE and Western blot analysis To assess maize EF-Tu overexpression in E. coli, equivalent cell culture samples were centrifuged briefly in a microcentrifuge, resuspended in SDS-PAGE sample buffer, and boiled for 3 min [10]. For 1D SDS-PAGE, extracted proteins were separated on 10% polyacrylamide gels with SDS [11], and then stained with Coomassie blue. For Western blot analysis, extracted proteins were separated on 10% polyacrylamide gels with SDS [11], and then transferred to nitrocellulose membrane (Bio-Rad, CA). Blots were probed for recombinant protein using two Western blot methods: (1) a colorimetric development method with primary antibody raised against maize EF-Tu and (2) a chemiluminescent development method with primary antibody raised against the myc epitope, which is included near the C-terminus of recombinant proteins obtained via the pTrcHis2A expression vector (Fig. 1). Western blot analysis with anti-EF-Tu antibody was performed as described by Bhadula et al. [8] with the exception that primary antibody was diluted 1:3000. Anti-myc Western blotting was performed using a 1:2500 dilution of primary antibody and a 1:2000 dilution of secondary antibody, conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Inc., CA). Antimyc blots were developed using a chemiluminescent HRP reaction development kit (Amersham Pharmacia Biotech, UK). Blots were developed for 5 min and then exposed to high performance autoradiography film for approximately 1 s. For samples transfected with pTrcHis2A-Zmeftu1, and pTrcHis2A, both immunoblotting techniques were utilized. pTrcHis2A-lac Z samples were subjected to anti-myc Western blot analysis only. 2.4. Testing for the solubility of recombinant EF-Tu We tested the solubility of overexpressed EF-Tu. Total proteins were extracted from both IPTG-induced and non-induced cultures of E. coli transformants for maize EF-Tu (pTrcHis2A-Zmeftu1) using the extraction
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
1077
Fig. 1. Zmeftu1 expression construct, as inserted into the plasmid expression vector, pTrcHis2A. Vector sequences are indicated in red, boldface indicates ribosomal binding sites. Zmeftu1 cDNA sequence is indicated in black (for complete Zmeftu1 sequence, see [8]). A: pTrcHis forward priming site; B: mini-cistron; C: restriction sites for Nco I endonuclease; D: restriction sites for Kpn I endonuclease; E: myc -epitope tag; F: polyhistidine tag.
buffer [20 mM Tris /HCl (pH 8.0), 0.2 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 7 mM MgCl2, 60 mM NH4Cl, 10% glycerol] of Boon et al. [12]. Protein extracts were centrifuged at 12 000 /g for 25 min, and the resulting pellet and supernatant were separated. The pellet was then resuspended in the volume of the extraction buffer [12] that was equal to the volume of the supernatant. Proteins from the supernatant and resuspended pellet were then analyzed using 1D SDSPAGE and Western blotting [8]. Gel was loaded with equal volumes of protein extracts. Blot was probed for recombinant EF-Tu using anti-myc antibody [8].
2.5. Heat stress viability assays For heat stress experiments, samples of induced and non-induced cultures for each of the three E. coli transformants (pTrcHis2A-Zmeftu1, pTrcHis2A, and pTrcHis2A-lac Z) were diluted to 6 /106 cells ml 1 in fresh LB medium. Subsequently, 1 ml aliquots were transferred to 55 8C for 0, 1 or 2 h. Temperature of 55 8C was chosen because the preliminary experiments indicated that this was the temperature that causes substantial death of bacterial culture. Aliquots of heated samples were taken at each time interval, serially diluted, and plated in duplicate onto LB agar supplemented with ampicillin (75 mg ml 1). Plates were incubated overnight at 37 8C. Cell viability was estimated after incubation by counting colony-forming
units (CFUs). Counting was performed using a manual colony-counter. 2.6. Statistical methods For the cell viability experiments, means of each individual trial were obtained from the appropriate dilution, plates yielding 30/300 CFUs [13], and then expressed as CFUs ml1 of culture exposed to 55 8C. Analyses for E. coli cells containing pTrcHis2AZmeftu1 and pTrcHis2A were derived from the means of four independent heat stress viability experiments. Analysis of E. coli cells transfected with pTrcHis2Alac Z was based on means of five independent experiments. Means of each independent experiment were then calculated as a percentage of the corresponding 0 h heat treatment. Data from the heat stress trials were analyzed by twofactor ANOVA tests using the general linear models procedure [14]. Separate two-factor ANOVAs were conducted for each sampled time interval (1 or 2 h) under 55 8C. Factors used in the ANOVAs were transformant identity (pTrcHis2A-Zmeftu1, pTrcHis2A, or pTrcHis2A-lac Z) and induction status (IPTG-induced or non-induced). Means and standard errors of all individual experiments for each treatment group at each time interval were also determined [14]. When ANOVAs indicated significant differences among treatments, Bonferonni t-tests were used to separate means [14].
1078
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
3. Results 3.1. Heterologous expression in E . coli cells Western blot analysis of induced E. coli samples transformed with pTrcHis2A-Zmeftu1 showed overexpression of the recombinant maize EF-Tu (Fig. 2A). The recombinant protein had molecular mass of approximately 54 kDa. Overproduction of maize EF-Tu was detected in protein extracts from pTrcHis2A-Zmeftu1 cells that were induced for 3 h, using the anti-myc immunoblotting method (Fig. 2A, lane 2). Some baseline expression of maize EF-Tu was evident in noninduced cells (Fig. 2A, lane 1). However, substantial increase in expression of this protein was apparent in IPTG-induced cells (Fig. 2A, lane 2). No recombinant protein production was detected in protein extracts from pTrcHis2A transformed cells, regardless of induction status (Fig. 2A, lanes 3 and 4). Similar results for pTrcHis2A-Zmeftu1 and pTrcHis2A extracts were obtained using the anti-EF-Tu Western blot method (not shown). The results of Western blot analysis were corroborated by 1D SDS-PAGE. Coomassie blue stained gels showed similar protein profiles in induced and non-induced cells of both pTrcHis2A-Zmeftu1 and pTrcHis2A with the exception that the induced cells of pTrcHis2A-Zmeftu1 appeared to have greater amount of protein of approximately 54 kDa, a protein that likely represents recombinant maize EF-Tu (not shown).
Fig. 2. Western blots showing recombinant polypeptide expression by the E. coli transformants pTrcHis2A-Zmeftu1, pTrcHis2A, and pTrcHis2A-lac Z. Blots were probed with anti-myc primary antibody. (A) Lane 1 */extract from non-induced pTrcHis2A-Zmeftu1 cells; lane 2 */extract from IPTG-induced pTrcHis2A-Zmeftu1 cells; lane 3 */ extract from non-induced pTrcHis2A cells; lane 4 */extract from IPTG-induced pTrcHis2A cells. Black arrow indicates recombinant maize EF-Tu polypeptide (54 kDa). (B) Lane 1 */extract from noninduced pTrcHis2A-lac Z cells; lane 2 */extract from IPTG-induced pTrcHis2A-lac Z cells. Black arrow indicates recombinant b-galactosidase polypeptide (molecular mass approximately 116 kDa). In both A and B, equal amount of protein was loaded in each lane.
Recombinant lac Z polypeptide was also overproduced in E. coli (pTrcHis2A-lac Z cells). Western blots probed with anti-myc antibody showed baseline production of this polypeptide in non-induced samples (Fig. 2B, lane 1) and substantial overproduction (increase in lac Z level) in IPTG-induced cultures (Fig. 2B, lane 2). The 1D SDS-PAGE corroborated the results of Western blot analysis (not shown). 3.2. Solubility of recombinant maize EF-Tu Western blot analysis showed that the recombinant EF-Tu was in a highly soluble form (Fig. 3). The protein band corresponding to EF-Tu was observed only in a soluble fraction of protein extracts (supernatant) (Fig. 3, lanes 1 and 2), and this was apparent in both IPTGinduced and non-induced cultures of E. coli transformants for maize EF-Tu (pTrcHis2A-Zmeftu1). Consistent with the Western blot shown in Fig. 2, the EF-Tu band was more prominent in IPTG-induced (Fig. 3, lane 1) than in non-induced (Fig. 3, lane 2) E. coli cells. 3.3. Heat stress viability assays To evaluate the effect of recombinant maize EF-Tu on E. coli survival, E. coli transformed with pTrcHis2AZmeftu1, pTrcHis2A, or pTrcHis2A-lac Z were subjected to 55 8C, a temperature sufficient to cause cell autolysis (as determined in our preliminary experiments). Although cell viability decreased for all cultures upon heat stress, survival rates were significantly greater for cells overexpressing the recombinant EF-Tu polypeptide (Figs. 4 and 5). Measured as a percentage of their respective 0 h heat treatments, cultures exposed to 1 h heat stress at 55 8C showed significant differences in survival tied to transformant identity (Fig. 5). IPTGinduced cultures for pTrcHis2A-Zmeftu1 were seen to survive at percentages significantly higher than any other culture exposed to 1 h at 55 8C. Percent survival for induced pTrcHis2A-Zmeftu1 cultures was 2.67-fold higher than for non-induced pTrcHis2A-Zmeftu1, over eightfold higher than either pTrcHis2A culture, and
Fig. 3. Western blot showing recombinant maize EF-Tu in soluble (supernatant, lanes 1 and 2) and non-soluble (pellet, lanes 3 and 4) fractions of protein extracts from E. coli transformants pTrcHis2AZmeftu1. Lanes 1 and 3: IPTG-induced pTrcHis2A-Zmeftu1 cells; lanes 2 and 4: non-induced pTrcHis2A-Zmeftu1 cells. Equal volume of protein extracts was loaded in each lane. Blot was probed with antimyc primary antibody. Note that the recombinant EF-Tu is apparent only in the soluble (supernatant) fraction.
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
1079
Fig. 4. LB agar plates inoculated with recombinant E. coli cultures. Cultures were heated at 55 8C for 1 h and then diluted 1:10 (v/v) with PBS. 100 ml of diluted cultures were spread on each plate. Plates were then incubated overnight at 37 8C. A: IPTG-induced pTrcHis2A-Zmeftu1 culture; B: non-induced pTrcHis2A-Zmeftu1 culture; C: IPTG-induced pTrcHis2A culture; D: non-induced pTrcHis2A culture; E: IPTG-induced pTrcHis2Alac Z culture; F: non-induced pTrcHis2A-lac Z culture.
more than twofold higher than either pTrcHis2A-lac Z culture. Cultures exposed to 2 h heat stress at 55 8C also showed significant differences in survival tied to the recombinant insert identity (Fig. 5). Again, IPTGinduced cultures for pTrcHis2A-Zmeftu1 were seen to survive at a significantly higher percentage than any other culture exposed to 2 h at 55 8C. Percent survival for induced pTrcHis2A-Zmeftu1 cultures was 3.7-fold higher than for non-induced pTrcHis2A-Zmeftu1, over fivefold higher than either pTrcHis2A culture, and more than 23-fold higher than either pTrcHis2Alac Z culture.
4. Discussion In this study, we overexpressed the maize chloroplast protein synthesis elongation factor, EF-Tu in E. coli and assessed the viability of EF-Tu expressing E. coli after exposure to 55 8C. The overexpressed EF-Tu protein had molecular mass of approximately 54 kDa (Fig. 2A), which was higher than the molecular mass of the native EF-Tu (45 /46 kDa). This difference is likely due to the presence of chloroplast targeting sequence at the Nterminus end of the recombinant protein; the chloroplast targeting sequence is normally cleaved from the native protein upon import into the chloroplast but it is
1080
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
Fig. 5. Viability of E. coli transformants pTrcHis2A-Zmeftu1, pTrcHis2A, and pTrcHis2A-lac Z, subjected to 1 and 2 h heat shock at 55 8C. Survival rates were estimated by counting CFUs (colonyforming units) present on LB agar culture plates inoculated with aliquots of heated culture, then allowed to incubate overnight at 37 8C. Survival rates for each transformant are expressed as a percentage of their respective 0 h treatments. Bars indicate standard errors (for 1 h: transformant identity F2, 20 /6.02, P B/0.0090; for induction F1, 20 /4.56, P B/0.0453; for interaction F /1.65, N.S. For 2 h: transformant identity F2, 20 /4.96, P B/0.0178; for induction F / 2.71, N.S.; for interaction F/1.92, N.S.). Analyses for E. coli cells containing pTrcHis2A-Zmeftu1 and pTrcHis2A were derived from the means of four independent heat stress viability experiments (n/4). Analysis of E. coli cells transfected with pTrcHis2A-lac Z was based on means of five independent experiments (n/5).
not cleaved from the recombinant protein in E. coli . The molecular mass of the recombinant EF-Tu was also increased by the addition of c-myc and polyhistidine tags at the C-terminus end of the polypeptide (Fig. 1). It is a common occurrence for bacterially expressed proteins to appear in an insoluble form as an inclusion body. Our expressed protein, however, seemed to appear in a highly soluble form, as indicated by Western blot analysis of soluble and insoluble fractions of protein
extracts from E. coli transformants for maize EF-Tu (Fig. 3). The exposure of E. coli (strain DH5a) to 55 8C resulted in a significant reduction of cell viability. This was expected since the temperature of 55 8C was sufficiently high to cause cell death. Other investigators, Yeh et al. [15], Muchowski and Clark [16], Soto et al. [17], have reported that 50 8C causes cell autolysis in E. coli . These investigators have, however, used different strains of E. coli . For our E. coli strain, DH5a, temperature of 55 8C rather than 50 8C was needed to cause substantial reduction in cell viability. Although exposure to 55 8C substantially reduced cell survival, the overexpression of maize EF-Tu enhanced the viability of E. coli under heat stress. E. coli cells induced to overexpress the polypeptide coded for by the Zmeftu1 cDNA survived 55 8C heat stress at a significantly higher rate than their non-induced counterparts, or any of the control transformants (Fig. 5). No protective effect due to transformation itself seemed to be associated with vector-only transformants, pTrcHis2A, regardless of induction status. Also, no significant protective effect due to recombinant polypeptide expression could be seen in the control-protein transformants, pTrcHis2A-lac Z. These results confirm that the protective effect against heat stress in IPTGinduced pTrcHis2A-Zmeftu1 cultures was a consequence of the recombinant EF-Tu polypeptide. The results of this study are similar to previous studies with E. coli transformed to express other recombinant polypeptides implicated in heat tolerance or as having molecular chaperone activity. Muchowski and Clark [16] demonstrated increased viability under heat stress for E. coli expressing recombinant human aB-crystalline. Also, enhanced viability has been shown for heat stressed E. coli expressing heterologous chestnut small (17.5 kDa) heat shock protein (HSP) [17], and for E. coli expressing rice low molecular mass (16.9 kDa) HSP [15]. Previous studies have shown an association between the synthesis of maize EF-Tu and the ability to tolerate heat stress [18 /20] (it should be noted that in these previous studies maize EF-Tu was referred to as a 45/46 kDa HSP since the identity of this protein was not known until the report of Bhadula et al. [8]). Ristic et al. [18] have observed heat-induced synthesis of 45 /46 kDa HSP (EF-Tu) in the leaves of a heat-tolerant maize line, ZPBL 1304, a line that can survive 24 h at 45 8C [21]. Heat-induced synthesis of proteins of molecular mass of 45 /46 kDa, however, was not seen in a heat sensitive maize line, ZPL 389 [18], a line that cannot withstand severe heat stress (24 h at 45 8C) [21]. Furthermore, elevated levels of 45 /46 kDa HSP (EF-Tu) were also seen under heat stress conditions in several maize hybrids bred for tolerance to drought and heat, and hybrids that better withstand heat stress also show greater synthesis of the 45/46 kDa HSP [19]. In
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
addition, Ristic et al. [20] have provided genetic evidence linking the ability to synthesize 45/46 kDa HSP (EFTu) and heat tolerance. Although evidence supports the hypothesis that maize EF-Tu plays a role in the development of heat tolerance, the mechanism by which this protein could confer heat tolerance is not clear. We hypothesize, however, that maize EF-Tu may act as a molecular chaperone. Two lines of evidence support this hypothesis. First, in prokaryotes, EF-Tu has been shown to exhibit chaperone properties [7,22]. Kudlicki et al. [22] have demonstrated chaperone-like activity for bacterial EF-Tu in the renaturation of rhodanese. Also, Caldas et al. [7] have reported chaperone properties for prokaryotic EFTu; E. coli EF-Tu has been found to form stable complexes with denatured proteins, protecting them against thermal aggregation. Second, prokaryotic and eukaryotic EF-Tu are strikingly similar [2,8]. Maize EFTu, for example, exhibits /80% amino acid identity with bacterial EF-Tu [8], and this is not surprising since, as stated earlier, the gene coding for chloroplast EF-Tu is of prokaryotic origin [9]. Thus, it seems reasonable to postulate the molecular chaperone function as a possible mechanism for the role of maize EF-Tu in heat tolerance. Indeed, our preliminary in vitro experiments showed that the purified recombinant maize EF-Tu displays chaperone properties as it protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation (Rao et al., unpublished). Furthermore, maize EF-Tu may possibly be involved in heat tolerance through its well-characterized function in polypeptide chain elongation [1,2,23,24]. Increase in the level of EF-Tu under heat stress may enhance the overall efficiency of protein synthesis and this, in turn, may have an impact on heat tolerance. Further studies are needed to investigate the mechanism of action of maize EF-Tu in possible relation to heat tolerance. In summary, the results of this study showed that E. coli overexpressing maize chloroplast protein synthesis elongation factor, EF-Tu, shows increased tolerance to heat stress. This finding complements previous studies on prokaryotic [7,22] and eukaryotic [8,18/20] EF-Tu and supports the hypothesis that this protein may play a role in the development of heat tolerance in maize. We hypothesize that maize EF-Tu may act as a molecular chaperone and/or may improve protein synthesis under heat stress conditions.
Acknowledgements We acknowledge financial support for this research from the United States Department of Agriculture grant (Agreement no. 99-35100-8550) to Z. Ristic. The authors are thankful to Dr. Karen Olmstead, Department of Biology, University of South Dakota, for her
1081
help with statistical analysis; and to Dr. Keith Weaver, Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, for the use of his colony-counting equipment.
References [1] D.L. Miller, H. Weissbach, Molecular Mechanisms of Protein Biosynthesis, Academic Press, New York, 1977, pp. 323 /373. [2] B. Riis, S.I.S. Rattan, B.F.C. Clark, W.C. Merrick, Eukaryotic protein elongation factors, TIBS 15 (1990) 420 /424. [3] G.R. Jacobson, J.P. Rosenbusch, Abundance and membrane association of elongation factor Tu in E . coli , Nature 261 (1976) 23 /26. [4] C.C. Young, R.W. Bernlohr, Elongation factor Tu is methylated in response to nutrient depravation in E . coli , J. Bacteriol. 173 (1991) 3096 /3100. [5] H. Gonen, C.E. Smith, N.R. Siegel, C. Kahana, W. Merrick, K. Chakraburtty, A.L. Schwartz, A. Ciechanover, Protein synthesis elongation factor EF-1a is essential for the ubiquitin-dependent degradation of certain Na-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu, PNAS 91 (1994) 7648 /7652. [6] M. Kjeldgaard, J. Nyborg, Refined structure of elongation factor EF-Tu from Escherichia coli , J. Mol. Biol. 223 (1992) 721 /742. [7] T.D. Caldas, A.E. Yaagoubi, G. Richarme, Chaperone properties of bacterial elongation factor, J. Biol. Chem. 273 (1998) 11478 / 11482. [8] S.K. Bhadula, T.E. Elthon, J.E. Habben, T.G. Helentjaris, S. Jiao, Z. Ristic, Heat-stress-induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line, Planta 212 (2001) 359 /366. [9] S.L. Baldauf, J.D. Palmer, Evolutionary transfer of the chloroplast tuf A gene to the nucleus, Nature 344 (1990) 262 /265. [10] G.J. Lee, E. Vierling, Molecular Chaperones, Academic Press, San Diego, CA, 1998, pp. 350 /365. [11] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680 /685. [12] K. Boon, E. Vijgenboom, L.V. Madsen, A. Talens, B. Kraal, L. Bosch, Isolation and functional analysis of histidine-tagged elongation factor Tu, Eur. J. Biochem. 210 (1992) 177 /183. [13] T.D. Brock, K.M. Brock, D.M. Ward, Basic Microbiology with Applications, 3rd ed., Prentice-Hall, Englewood Cliffs, NJ, 1986. [14] SAS Institute, Inc, SAS/Stat User’s Guide, Version 6, 4th ed., SAS Institute, Inc, Cary, NC, 1989. [15] C.H. Yeh, P.L. Chang, K.W. Yeh, W.C. Lin, Y.M. Chen, C.Y. Lin, Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshp 16.9, in Escherichia coli enhances thermotolerance, PNAS 94 (1997) 10967 /10972. [16] P.J. Muchowski, J.I. Clark, ATP-enhanced molecular chaperone functions of the small heat shock protein human aB crystalline, PNAS 95 (1998) 1004 /1009. [17] A. Soto, I. Allona, C. Collada, M.A. Guevara, R. Casado, E. Roderiguez-Cerezo, A. Cipriano, L. Gomez, Heterologous expression of a plant small heat-shock protein enhances Escherichia coli viability under heat and cold stress, Plant Physiol. 120 (1999) 521 /528. [18] Z. Ristic, D.J. Gifford, D.D. Cass, Heat shock proteins in two lines of Zea mays L. that differ in drought and heat resistance, Plant Physiol. 97 (1991) 1430 /1434. [19] Z. Ristic, G. Williams, G. Yang, B. Martin, S. Fullerton, Dehydration, damage to cellular membranes and heat-shock
1082
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
proteins in maize hybrids from different climates, J. Plant Physiol. 149 (1996) 424 /432. [20] Z. Ristic, G. Yang, B. Martin, S. Fullerton, Evidence of association between specific heat-shock protein(s) and the drought and heat tolerance phenotype in maize, J. Plant Physiol. 153 (1998) 497 /505. [21] Z. Ristic, D.D. Cass, Dehydration avoidance and damage to the plasma and thylakoid membranes in lines of maize differing in endogenous levels of abscisic acid, J. Plant Physiol. 142 (1993) 759 /764.
[22] W. Kudlicki, A. Coffman, G. Kramer, B. Hardesty, Renaturation of rhodanese by translational elongation factor (EF) Tu: protein refolding by EF-Tu flexing, J. Biol. Chem. 272 (1997) 32206 / 32210. [23] P. Nissen, M. Kjeldgaard, S. Thirup, G. Polekhina, L. Reshetnikova, B.F.C. Clark, J. Nyborg, Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog, Science 270 (1995) 1464 /1472. [24] K.S. Willson, H.F. Noller, Molecular movement inside the translational engine, Cell 92 (1998) 337 /349.
Heterologous expression of maize chloroplast protein synthesis elongation factor (EF-Tu) enhances Escherichia coli viability under heat stress T. Moriarty a, R. West a, G. Small b, D. Rao a, Z. Ristic a,* a
b
Department of Biology, University of South Dakota, Vermillion, SD 57069, USA Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, SD 57069, USA Received 29 March 2002; received in revised form 27 July 2002; accepted 31 July 2002
Abstract A heat-tolerant maize line, ZPBL 1304, synthesizes increased amounts of chloroplast protein synthesis elongation factor (EF-Tu) under heat stress conditions. Previous studies have suggested that maize EF-Tu may be involved in the development of heat tolerance [Planta 212 (2001) 359; J. Plant Physiol. 153 (1998) 497]. In this study, we tested the hypothesis that overexpression of maize EF-Tu enhances the viability of Escherichia coli under heat stress. The approach was to expose E. coli transformed with a maize EF-Tu expression vector (pTrcHis2A-Zmeftu1) to 55 8C and assess viability at 37 8C. Western blots showed E. coli overproduced recombinant EF-Tu protein, and the protein seemed to be in a highly soluble form. E. coli overexpressing maize EFTu showed increased viability after exposure to heat stress, demonstrating that the maize EF-Tu is involved in the development of heat tolerance. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Escherichia coli ; Chloroplast protein synthesis elongation factor (EF-Tu); Heat stress; Heat tolerance; Zea mays
1. Introduction Protein synthesis elongation factors (elongation factors or EFs) are proteins that are involved in the elongation of polypeptides during the translational process [1,2]. The basic process of translation involves three separate factors (the prokaryotic EFs are indicated in parentheses): (1) EF-1a (EF-Tu), (2) EF-1b (EF-Ts), and (3) EF-2 (EF-G). Polypeptide elongation proceeds in three steps: (1) EF-1a (EF-Tu) binds GTP and aminoacyl-tRNA, and leads to the codon-dependent placement of this aminoacyl-tRNA at the A site of the ribosome; (2) following release of EF-1a-GDP (EF-TuGDP) from the ribosome, EF-1b (EF-Ts) facilitates the exchange of bound GDP for GTP; and (3) after peptide bond formation, EF-2 (EF-G) translocates the mRNA one codon to allow for the arrival of the new aminoacyl-
* Corresponding author. Tel.: /1-605-677-6170; fax: /1-605-6776557 E-mail address: [email protected] (Z. Ristic).
tRNA in the A site [2]. Elongation factors have been extensively studied in prokaryotes, and the functional homology between the prokaryotic and eukaryotic factors is quite striking [2]. Prokaryotic protein elongation factor EF-Tu (molecular mass: 43 kDa) may have other functions in addition to the conventional role that it plays in polypeptide elongation [3 /7]. One of these functions is in protein refolding and protection of polypeptides from heat denaturation [7]. Elongation factor EF-Tu, in a manner similar to that of molecular chaperones, increases the refolding of unfolded proteins, protects proteins against thermal denaturation, and forms complexes with unfolded proteins [7]. Our laboratory has recently found that a heat-tolerant maize line, ZPBL 1304, synthesizes increased amounts of chloroplast protein synthesis elongation factor, EFTu, under heat stress conditions [8]; chloroplast protein synthesis elongation factor is encoded by a gene that is of prokaryotic origin and, therefore, this plant protein is commonly referred to with the prokaryotic designation,
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 2 7 3 - X
1076
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
EF-Tu [9]. Three heat stress-induced EF-Tu polypeptides (pI, 5.2 /5.7) of molecular mass of 45 /46 kDa have been identified in this line of maize [8]. N-terminal amino acid sequencing has revealed a high similarity (80 /90%) of these polypeptides with the EF-Tu from other higher plant chloroplasts and from other organisms [8]. In addition, Northern blots have shown an increase (1.85-fold) in the steady-state levels of EF-Tu mRNA during heat stress [8], and the increase in EF-Tu transcript levels during heat stress was accompanied by increased levels of the EF-Tu protein [8]. Isolated chloroplasts from heat-stressed ZPBL 1304 plants have also been shown to have higher levels of EF-Tu as compared with non-stressed chloroplasts [8]. These findings have led to the hypothesis that maize EF-Tu may be involved in the development of heat tolerance [8]. In this study, we tested the hypothesis that overexpression of maize EF-Tu enhances the viability of Escherichia coli under heat stress. The approach was to transform E. coli with maize EF-Tu cDNA, expose the E. coli transformants for maize EF-Tu to 55 8C and assess viability after incubation at 37 8C. Here, we report that E. coli overexpressing maize EF-Tu shows increased viability after exposure to heat stress.
2. Materials and methods 2.1. cDNA subcloning and bacterial transformation A cDNA for maize (Zea mays L.) EF-Tu, designated Zmeftu1, was already on hand in our laboratory. It was previously isolated from a cDNA library, constructed using RNA from aerial tissue of B73 line maize seedlings that were exposed to 10 days of drought stress followed by 24 h heat stress at 45 8C [8]. Zmeftu1 was subcloned into the expression vector pTrcHis2A, which adds Cterminal c-myc and polyhistidine tags to the protein (Invitrogen, Carlsbad, CA) (Fig. 1). Subsequently, the pTrcHis2A vector carrying Zmeftu1 was used to transform competent E. coli cells of the strain DH5a (pTricHis2A-Zmeftu1). As controls, E. coli DH5a cells were transformed with either the pTrcHis2A vector alone (pTricHis2A), or vector carrying an insert for lac Z */the bacterial gene encoding b-galactosidase (pTricHis2A-lac Z). 2.2. Heterologous expression in E . coli cells pTrcHis2A-Zmeftu1, pTrcHis2A, and pTrcHis2Alac Z transformants were grown at 37 8C, with vigorous shaking, to an A600 /1.0 in Luria /Bertani (LB) medium supplemented with ampicillin at 75 mg ml 1. Culture aliquots (100 ml) were then diluted with 10 ml of fresh ampicillin supplemented medium and subcultured to an
A600 between 0.4 and 0.5. These subcultures were split into two groups: (1) a group induced to overexpress heterologous protein (induced group) and (2) a group not induced to overexpress heterologous protein (noninduced group). Induction was achieved by adding isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM. For all induced cultures, induction time was 3 h after induction, both the induced and non-induced cultures were measured again at 600 nm to estimate cell concentration. Culture samples with similar amount of cells were used for subsequent 1D SDS-PAGE and Western blot analysis, and for heat stress viability experiments. 2.3. 1D SDS-PAGE and Western blot analysis To assess maize EF-Tu overexpression in E. coli, equivalent cell culture samples were centrifuged briefly in a microcentrifuge, resuspended in SDS-PAGE sample buffer, and boiled for 3 min [10]. For 1D SDS-PAGE, extracted proteins were separated on 10% polyacrylamide gels with SDS [11], and then stained with Coomassie blue. For Western blot analysis, extracted proteins were separated on 10% polyacrylamide gels with SDS [11], and then transferred to nitrocellulose membrane (Bio-Rad, CA). Blots were probed for recombinant protein using two Western blot methods: (1) a colorimetric development method with primary antibody raised against maize EF-Tu and (2) a chemiluminescent development method with primary antibody raised against the myc epitope, which is included near the C-terminus of recombinant proteins obtained via the pTrcHis2A expression vector (Fig. 1). Western blot analysis with anti-EF-Tu antibody was performed as described by Bhadula et al. [8] with the exception that primary antibody was diluted 1:3000. Anti-myc Western blotting was performed using a 1:2500 dilution of primary antibody and a 1:2000 dilution of secondary antibody, conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Inc., CA). Antimyc blots were developed using a chemiluminescent HRP reaction development kit (Amersham Pharmacia Biotech, UK). Blots were developed for 5 min and then exposed to high performance autoradiography film for approximately 1 s. For samples transfected with pTrcHis2A-Zmeftu1, and pTrcHis2A, both immunoblotting techniques were utilized. pTrcHis2A-lac Z samples were subjected to anti-myc Western blot analysis only. 2.4. Testing for the solubility of recombinant EF-Tu We tested the solubility of overexpressed EF-Tu. Total proteins were extracted from both IPTG-induced and non-induced cultures of E. coli transformants for maize EF-Tu (pTrcHis2A-Zmeftu1) using the extraction
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
1077
Fig. 1. Zmeftu1 expression construct, as inserted into the plasmid expression vector, pTrcHis2A. Vector sequences are indicated in red, boldface indicates ribosomal binding sites. Zmeftu1 cDNA sequence is indicated in black (for complete Zmeftu1 sequence, see [8]). A: pTrcHis forward priming site; B: mini-cistron; C: restriction sites for Nco I endonuclease; D: restriction sites for Kpn I endonuclease; E: myc -epitope tag; F: polyhistidine tag.
buffer [20 mM Tris /HCl (pH 8.0), 0.2 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 7 mM MgCl2, 60 mM NH4Cl, 10% glycerol] of Boon et al. [12]. Protein extracts were centrifuged at 12 000 /g for 25 min, and the resulting pellet and supernatant were separated. The pellet was then resuspended in the volume of the extraction buffer [12] that was equal to the volume of the supernatant. Proteins from the supernatant and resuspended pellet were then analyzed using 1D SDSPAGE and Western blotting [8]. Gel was loaded with equal volumes of protein extracts. Blot was probed for recombinant EF-Tu using anti-myc antibody [8].
2.5. Heat stress viability assays For heat stress experiments, samples of induced and non-induced cultures for each of the three E. coli transformants (pTrcHis2A-Zmeftu1, pTrcHis2A, and pTrcHis2A-lac Z) were diluted to 6 /106 cells ml 1 in fresh LB medium. Subsequently, 1 ml aliquots were transferred to 55 8C for 0, 1 or 2 h. Temperature of 55 8C was chosen because the preliminary experiments indicated that this was the temperature that causes substantial death of bacterial culture. Aliquots of heated samples were taken at each time interval, serially diluted, and plated in duplicate onto LB agar supplemented with ampicillin (75 mg ml 1). Plates were incubated overnight at 37 8C. Cell viability was estimated after incubation by counting colony-forming
units (CFUs). Counting was performed using a manual colony-counter. 2.6. Statistical methods For the cell viability experiments, means of each individual trial were obtained from the appropriate dilution, plates yielding 30/300 CFUs [13], and then expressed as CFUs ml1 of culture exposed to 55 8C. Analyses for E. coli cells containing pTrcHis2AZmeftu1 and pTrcHis2A were derived from the means of four independent heat stress viability experiments. Analysis of E. coli cells transfected with pTrcHis2Alac Z was based on means of five independent experiments. Means of each independent experiment were then calculated as a percentage of the corresponding 0 h heat treatment. Data from the heat stress trials were analyzed by twofactor ANOVA tests using the general linear models procedure [14]. Separate two-factor ANOVAs were conducted for each sampled time interval (1 or 2 h) under 55 8C. Factors used in the ANOVAs were transformant identity (pTrcHis2A-Zmeftu1, pTrcHis2A, or pTrcHis2A-lac Z) and induction status (IPTG-induced or non-induced). Means and standard errors of all individual experiments for each treatment group at each time interval were also determined [14]. When ANOVAs indicated significant differences among treatments, Bonferonni t-tests were used to separate means [14].
1078
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
3. Results 3.1. Heterologous expression in E . coli cells Western blot analysis of induced E. coli samples transformed with pTrcHis2A-Zmeftu1 showed overexpression of the recombinant maize EF-Tu (Fig. 2A). The recombinant protein had molecular mass of approximately 54 kDa. Overproduction of maize EF-Tu was detected in protein extracts from pTrcHis2A-Zmeftu1 cells that were induced for 3 h, using the anti-myc immunoblotting method (Fig. 2A, lane 2). Some baseline expression of maize EF-Tu was evident in noninduced cells (Fig. 2A, lane 1). However, substantial increase in expression of this protein was apparent in IPTG-induced cells (Fig. 2A, lane 2). No recombinant protein production was detected in protein extracts from pTrcHis2A transformed cells, regardless of induction status (Fig. 2A, lanes 3 and 4). Similar results for pTrcHis2A-Zmeftu1 and pTrcHis2A extracts were obtained using the anti-EF-Tu Western blot method (not shown). The results of Western blot analysis were corroborated by 1D SDS-PAGE. Coomassie blue stained gels showed similar protein profiles in induced and non-induced cells of both pTrcHis2A-Zmeftu1 and pTrcHis2A with the exception that the induced cells of pTrcHis2A-Zmeftu1 appeared to have greater amount of protein of approximately 54 kDa, a protein that likely represents recombinant maize EF-Tu (not shown).
Fig. 2. Western blots showing recombinant polypeptide expression by the E. coli transformants pTrcHis2A-Zmeftu1, pTrcHis2A, and pTrcHis2A-lac Z. Blots were probed with anti-myc primary antibody. (A) Lane 1 */extract from non-induced pTrcHis2A-Zmeftu1 cells; lane 2 */extract from IPTG-induced pTrcHis2A-Zmeftu1 cells; lane 3 */ extract from non-induced pTrcHis2A cells; lane 4 */extract from IPTG-induced pTrcHis2A cells. Black arrow indicates recombinant maize EF-Tu polypeptide (54 kDa). (B) Lane 1 */extract from noninduced pTrcHis2A-lac Z cells; lane 2 */extract from IPTG-induced pTrcHis2A-lac Z cells. Black arrow indicates recombinant b-galactosidase polypeptide (molecular mass approximately 116 kDa). In both A and B, equal amount of protein was loaded in each lane.
Recombinant lac Z polypeptide was also overproduced in E. coli (pTrcHis2A-lac Z cells). Western blots probed with anti-myc antibody showed baseline production of this polypeptide in non-induced samples (Fig. 2B, lane 1) and substantial overproduction (increase in lac Z level) in IPTG-induced cultures (Fig. 2B, lane 2). The 1D SDS-PAGE corroborated the results of Western blot analysis (not shown). 3.2. Solubility of recombinant maize EF-Tu Western blot analysis showed that the recombinant EF-Tu was in a highly soluble form (Fig. 3). The protein band corresponding to EF-Tu was observed only in a soluble fraction of protein extracts (supernatant) (Fig. 3, lanes 1 and 2), and this was apparent in both IPTGinduced and non-induced cultures of E. coli transformants for maize EF-Tu (pTrcHis2A-Zmeftu1). Consistent with the Western blot shown in Fig. 2, the EF-Tu band was more prominent in IPTG-induced (Fig. 3, lane 1) than in non-induced (Fig. 3, lane 2) E. coli cells. 3.3. Heat stress viability assays To evaluate the effect of recombinant maize EF-Tu on E. coli survival, E. coli transformed with pTrcHis2AZmeftu1, pTrcHis2A, or pTrcHis2A-lac Z were subjected to 55 8C, a temperature sufficient to cause cell autolysis (as determined in our preliminary experiments). Although cell viability decreased for all cultures upon heat stress, survival rates were significantly greater for cells overexpressing the recombinant EF-Tu polypeptide (Figs. 4 and 5). Measured as a percentage of their respective 0 h heat treatments, cultures exposed to 1 h heat stress at 55 8C showed significant differences in survival tied to transformant identity (Fig. 5). IPTGinduced cultures for pTrcHis2A-Zmeftu1 were seen to survive at percentages significantly higher than any other culture exposed to 1 h at 55 8C. Percent survival for induced pTrcHis2A-Zmeftu1 cultures was 2.67-fold higher than for non-induced pTrcHis2A-Zmeftu1, over eightfold higher than either pTrcHis2A culture, and
Fig. 3. Western blot showing recombinant maize EF-Tu in soluble (supernatant, lanes 1 and 2) and non-soluble (pellet, lanes 3 and 4) fractions of protein extracts from E. coli transformants pTrcHis2AZmeftu1. Lanes 1 and 3: IPTG-induced pTrcHis2A-Zmeftu1 cells; lanes 2 and 4: non-induced pTrcHis2A-Zmeftu1 cells. Equal volume of protein extracts was loaded in each lane. Blot was probed with antimyc primary antibody. Note that the recombinant EF-Tu is apparent only in the soluble (supernatant) fraction.
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
1079
Fig. 4. LB agar plates inoculated with recombinant E. coli cultures. Cultures were heated at 55 8C for 1 h and then diluted 1:10 (v/v) with PBS. 100 ml of diluted cultures were spread on each plate. Plates were then incubated overnight at 37 8C. A: IPTG-induced pTrcHis2A-Zmeftu1 culture; B: non-induced pTrcHis2A-Zmeftu1 culture; C: IPTG-induced pTrcHis2A culture; D: non-induced pTrcHis2A culture; E: IPTG-induced pTrcHis2Alac Z culture; F: non-induced pTrcHis2A-lac Z culture.
more than twofold higher than either pTrcHis2A-lac Z culture. Cultures exposed to 2 h heat stress at 55 8C also showed significant differences in survival tied to the recombinant insert identity (Fig. 5). Again, IPTGinduced cultures for pTrcHis2A-Zmeftu1 were seen to survive at a significantly higher percentage than any other culture exposed to 2 h at 55 8C. Percent survival for induced pTrcHis2A-Zmeftu1 cultures was 3.7-fold higher than for non-induced pTrcHis2A-Zmeftu1, over fivefold higher than either pTrcHis2A culture, and more than 23-fold higher than either pTrcHis2Alac Z culture.
4. Discussion In this study, we overexpressed the maize chloroplast protein synthesis elongation factor, EF-Tu in E. coli and assessed the viability of EF-Tu expressing E. coli after exposure to 55 8C. The overexpressed EF-Tu protein had molecular mass of approximately 54 kDa (Fig. 2A), which was higher than the molecular mass of the native EF-Tu (45 /46 kDa). This difference is likely due to the presence of chloroplast targeting sequence at the Nterminus end of the recombinant protein; the chloroplast targeting sequence is normally cleaved from the native protein upon import into the chloroplast but it is
1080
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
Fig. 5. Viability of E. coli transformants pTrcHis2A-Zmeftu1, pTrcHis2A, and pTrcHis2A-lac Z, subjected to 1 and 2 h heat shock at 55 8C. Survival rates were estimated by counting CFUs (colonyforming units) present on LB agar culture plates inoculated with aliquots of heated culture, then allowed to incubate overnight at 37 8C. Survival rates for each transformant are expressed as a percentage of their respective 0 h treatments. Bars indicate standard errors (for 1 h: transformant identity F2, 20 /6.02, P B/0.0090; for induction F1, 20 /4.56, P B/0.0453; for interaction F /1.65, N.S. For 2 h: transformant identity F2, 20 /4.96, P B/0.0178; for induction F / 2.71, N.S.; for interaction F/1.92, N.S.). Analyses for E. coli cells containing pTrcHis2A-Zmeftu1 and pTrcHis2A were derived from the means of four independent heat stress viability experiments (n/4). Analysis of E. coli cells transfected with pTrcHis2A-lac Z was based on means of five independent experiments (n/5).
not cleaved from the recombinant protein in E. coli . The molecular mass of the recombinant EF-Tu was also increased by the addition of c-myc and polyhistidine tags at the C-terminus end of the polypeptide (Fig. 1). It is a common occurrence for bacterially expressed proteins to appear in an insoluble form as an inclusion body. Our expressed protein, however, seemed to appear in a highly soluble form, as indicated by Western blot analysis of soluble and insoluble fractions of protein
extracts from E. coli transformants for maize EF-Tu (Fig. 3). The exposure of E. coli (strain DH5a) to 55 8C resulted in a significant reduction of cell viability. This was expected since the temperature of 55 8C was sufficiently high to cause cell death. Other investigators, Yeh et al. [15], Muchowski and Clark [16], Soto et al. [17], have reported that 50 8C causes cell autolysis in E. coli . These investigators have, however, used different strains of E. coli . For our E. coli strain, DH5a, temperature of 55 8C rather than 50 8C was needed to cause substantial reduction in cell viability. Although exposure to 55 8C substantially reduced cell survival, the overexpression of maize EF-Tu enhanced the viability of E. coli under heat stress. E. coli cells induced to overexpress the polypeptide coded for by the Zmeftu1 cDNA survived 55 8C heat stress at a significantly higher rate than their non-induced counterparts, or any of the control transformants (Fig. 5). No protective effect due to transformation itself seemed to be associated with vector-only transformants, pTrcHis2A, regardless of induction status. Also, no significant protective effect due to recombinant polypeptide expression could be seen in the control-protein transformants, pTrcHis2A-lac Z. These results confirm that the protective effect against heat stress in IPTGinduced pTrcHis2A-Zmeftu1 cultures was a consequence of the recombinant EF-Tu polypeptide. The results of this study are similar to previous studies with E. coli transformed to express other recombinant polypeptides implicated in heat tolerance or as having molecular chaperone activity. Muchowski and Clark [16] demonstrated increased viability under heat stress for E. coli expressing recombinant human aB-crystalline. Also, enhanced viability has been shown for heat stressed E. coli expressing heterologous chestnut small (17.5 kDa) heat shock protein (HSP) [17], and for E. coli expressing rice low molecular mass (16.9 kDa) HSP [15]. Previous studies have shown an association between the synthesis of maize EF-Tu and the ability to tolerate heat stress [18 /20] (it should be noted that in these previous studies maize EF-Tu was referred to as a 45/46 kDa HSP since the identity of this protein was not known until the report of Bhadula et al. [8]). Ristic et al. [18] have observed heat-induced synthesis of 45 /46 kDa HSP (EF-Tu) in the leaves of a heat-tolerant maize line, ZPBL 1304, a line that can survive 24 h at 45 8C [21]. Heat-induced synthesis of proteins of molecular mass of 45 /46 kDa, however, was not seen in a heat sensitive maize line, ZPL 389 [18], a line that cannot withstand severe heat stress (24 h at 45 8C) [21]. Furthermore, elevated levels of 45 /46 kDa HSP (EF-Tu) were also seen under heat stress conditions in several maize hybrids bred for tolerance to drought and heat, and hybrids that better withstand heat stress also show greater synthesis of the 45/46 kDa HSP [19]. In
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
addition, Ristic et al. [20] have provided genetic evidence linking the ability to synthesize 45/46 kDa HSP (EFTu) and heat tolerance. Although evidence supports the hypothesis that maize EF-Tu plays a role in the development of heat tolerance, the mechanism by which this protein could confer heat tolerance is not clear. We hypothesize, however, that maize EF-Tu may act as a molecular chaperone. Two lines of evidence support this hypothesis. First, in prokaryotes, EF-Tu has been shown to exhibit chaperone properties [7,22]. Kudlicki et al. [22] have demonstrated chaperone-like activity for bacterial EF-Tu in the renaturation of rhodanese. Also, Caldas et al. [7] have reported chaperone properties for prokaryotic EFTu; E. coli EF-Tu has been found to form stable complexes with denatured proteins, protecting them against thermal aggregation. Second, prokaryotic and eukaryotic EF-Tu are strikingly similar [2,8]. Maize EFTu, for example, exhibits /80% amino acid identity with bacterial EF-Tu [8], and this is not surprising since, as stated earlier, the gene coding for chloroplast EF-Tu is of prokaryotic origin [9]. Thus, it seems reasonable to postulate the molecular chaperone function as a possible mechanism for the role of maize EF-Tu in heat tolerance. Indeed, our preliminary in vitro experiments showed that the purified recombinant maize EF-Tu displays chaperone properties as it protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation (Rao et al., unpublished). Furthermore, maize EF-Tu may possibly be involved in heat tolerance through its well-characterized function in polypeptide chain elongation [1,2,23,24]. Increase in the level of EF-Tu under heat stress may enhance the overall efficiency of protein synthesis and this, in turn, may have an impact on heat tolerance. Further studies are needed to investigate the mechanism of action of maize EF-Tu in possible relation to heat tolerance. In summary, the results of this study showed that E. coli overexpressing maize chloroplast protein synthesis elongation factor, EF-Tu, shows increased tolerance to heat stress. This finding complements previous studies on prokaryotic [7,22] and eukaryotic [8,18/20] EF-Tu and supports the hypothesis that this protein may play a role in the development of heat tolerance in maize. We hypothesize that maize EF-Tu may act as a molecular chaperone and/or may improve protein synthesis under heat stress conditions.
Acknowledgements We acknowledge financial support for this research from the United States Department of Agriculture grant (Agreement no. 99-35100-8550) to Z. Ristic. The authors are thankful to Dr. Karen Olmstead, Department of Biology, University of South Dakota, for her
1081
help with statistical analysis; and to Dr. Keith Weaver, Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, for the use of his colony-counting equipment.
References [1] D.L. Miller, H. Weissbach, Molecular Mechanisms of Protein Biosynthesis, Academic Press, New York, 1977, pp. 323 /373. [2] B. Riis, S.I.S. Rattan, B.F.C. Clark, W.C. Merrick, Eukaryotic protein elongation factors, TIBS 15 (1990) 420 /424. [3] G.R. Jacobson, J.P. Rosenbusch, Abundance and membrane association of elongation factor Tu in E . coli , Nature 261 (1976) 23 /26. [4] C.C. Young, R.W. Bernlohr, Elongation factor Tu is methylated in response to nutrient depravation in E . coli , J. Bacteriol. 173 (1991) 3096 /3100. [5] H. Gonen, C.E. Smith, N.R. Siegel, C. Kahana, W. Merrick, K. Chakraburtty, A.L. Schwartz, A. Ciechanover, Protein synthesis elongation factor EF-1a is essential for the ubiquitin-dependent degradation of certain Na-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu, PNAS 91 (1994) 7648 /7652. [6] M. Kjeldgaard, J. Nyborg, Refined structure of elongation factor EF-Tu from Escherichia coli , J. Mol. Biol. 223 (1992) 721 /742. [7] T.D. Caldas, A.E. Yaagoubi, G. Richarme, Chaperone properties of bacterial elongation factor, J. Biol. Chem. 273 (1998) 11478 / 11482. [8] S.K. Bhadula, T.E. Elthon, J.E. Habben, T.G. Helentjaris, S. Jiao, Z. Ristic, Heat-stress-induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line, Planta 212 (2001) 359 /366. [9] S.L. Baldauf, J.D. Palmer, Evolutionary transfer of the chloroplast tuf A gene to the nucleus, Nature 344 (1990) 262 /265. [10] G.J. Lee, E. Vierling, Molecular Chaperones, Academic Press, San Diego, CA, 1998, pp. 350 /365. [11] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680 /685. [12] K. Boon, E. Vijgenboom, L.V. Madsen, A. Talens, B. Kraal, L. Bosch, Isolation and functional analysis of histidine-tagged elongation factor Tu, Eur. J. Biochem. 210 (1992) 177 /183. [13] T.D. Brock, K.M. Brock, D.M. Ward, Basic Microbiology with Applications, 3rd ed., Prentice-Hall, Englewood Cliffs, NJ, 1986. [14] SAS Institute, Inc, SAS/Stat User’s Guide, Version 6, 4th ed., SAS Institute, Inc, Cary, NC, 1989. [15] C.H. Yeh, P.L. Chang, K.W. Yeh, W.C. Lin, Y.M. Chen, C.Y. Lin, Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshp 16.9, in Escherichia coli enhances thermotolerance, PNAS 94 (1997) 10967 /10972. [16] P.J. Muchowski, J.I. Clark, ATP-enhanced molecular chaperone functions of the small heat shock protein human aB crystalline, PNAS 95 (1998) 1004 /1009. [17] A. Soto, I. Allona, C. Collada, M.A. Guevara, R. Casado, E. Roderiguez-Cerezo, A. Cipriano, L. Gomez, Heterologous expression of a plant small heat-shock protein enhances Escherichia coli viability under heat and cold stress, Plant Physiol. 120 (1999) 521 /528. [18] Z. Ristic, D.J. Gifford, D.D. Cass, Heat shock proteins in two lines of Zea mays L. that differ in drought and heat resistance, Plant Physiol. 97 (1991) 1430 /1434. [19] Z. Ristic, G. Williams, G. Yang, B. Martin, S. Fullerton, Dehydration, damage to cellular membranes and heat-shock
1082
T. Moriarty et al. / Plant Science 163 (2002) 1075 /1082
proteins in maize hybrids from different climates, J. Plant Physiol. 149 (1996) 424 /432. [20] Z. Ristic, G. Yang, B. Martin, S. Fullerton, Evidence of association between specific heat-shock protein(s) and the drought and heat tolerance phenotype in maize, J. Plant Physiol. 153 (1998) 497 /505. [21] Z. Ristic, D.D. Cass, Dehydration avoidance and damage to the plasma and thylakoid membranes in lines of maize differing in endogenous levels of abscisic acid, J. Plant Physiol. 142 (1993) 759 /764.
[22] W. Kudlicki, A. Coffman, G. Kramer, B. Hardesty, Renaturation of rhodanese by translational elongation factor (EF) Tu: protein refolding by EF-Tu flexing, J. Biol. Chem. 272 (1997) 32206 / 32210. [23] P. Nissen, M. Kjeldgaard, S. Thirup, G. Polekhina, L. Reshetnikova, B.F.C. Clark, J. Nyborg, Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog, Science 270 (1995) 1464 /1472. [24] K.S. Willson, H.F. Noller, Molecular movement inside the translational engine, Cell 92 (1998) 337 /349.