Cloning and Transcriptional Analysis of groES and groEL in Ethanol-producing Bacterium Zymomonas mobilis TISTR 548

INTRODUCTION

The inhibitory effect of ethanol during fermentation is complex and is the main reason for slow and incomplete enological fermentation in many organisms. In Saccharomyces cerevisiae and Escherichia coli, for example, ethanol retards the specific growth rate, their viability and their fermentative rate (Jones and Greenfield, 1985; Ansanay-Galeote et al., 2001). Ethanol also modifies plasma membrane fluidity, stimulates the activity of plasma membrane H⁺-ATPase and inhibits glucose transport (Salmon et al., 1993; Alexandre et al., 1993, 1994; Piper et al., 1994). Furthermore, ethanol also induces an increase in the proportion of acidic phospholipids and 18:1 fatty acid at the expense of 16:0 and a decrease in the lipid-to-protein ratio in E. coli (Ingram, 1977; Carey and Ingram, 1983). Recently, ethanol has been shown to have an effect identical to that of heat in stimulating the synthesis of specific proteins known as heat shock proteins (HSPs) or stress proteins (Michel and Starka, 1986; An et al., 1991; Michel, 1993). These evolutionarily proteins were originally characterized on the basic of their strong induction by heat shock, however they are also induced by a variety of stresses other than heat including ethanol, nalidixic acid, UV irradiation and viral infection (Craig, 1986).

Zymomonas mobilis is an obligatorily fermentative gram-negative bacterium that converts glucose, fructose and sucrose to ethanol and CO₂ via the Entner-Doudoroff (ED) pathway (Rogers et al., 1982; Sprenger, 1996). Unlike S. cerevisiae and E. coli, Z. mobilis is capable of growth in the presence of up to 10% ethanol and of fermentation in media with up to 25% glucose. Thus, Z. mobilis may have evolved specialized features to allow survival under these environmental stresses. Previous studies have reported that heat and ethanol induce the synthesis of stress proteins in Z. mobilis. This biological change has been proposed as being adaptive for growth and survival of this organism under environmental stresses. The first stress gene cloned and sequenced from Z. mobilis encoded alcohol dehydrogenase II (adhB), the most abundant alcohol dehydrogenase in this organism, which serves as a stress protein and a primary function in central metabolism (An et al., 1991). The promoter region for this gene shared homology with other stress promoters of E. coli. Recently, several stress-responsive proteins provisionally identified as dnaJ and dnaK in two-dimensional PAGE of cytoplasmic extracts from Z. mobilis have been cloned and sequenced. Furthermore, the cloning and expression in E. coli of the Z. mobilis dnaK gene have been performed (Arfman et al., 1992; Michel, 1993). In this study we present some evidences for the existence of a typical stress response induced by heat and ethanol in ethanol-producing bacterium Z. mobilis TISTR 548. We report here that both heat and ethanol stresses have affected not only on bacterial growth and cell viability but also on gene expression. The synthesis of several stress-responsive genes was remarkably increased, of which at least two identified as groES and groEL were cloned and sequenced. Expression analysis of these genes under different stresses including ethanol, heat and salt stresses by RT-PCR was also demonstrated.

MATERIALS AND METHODS

Strains, plasmids and culture conditions: Z. mobilis TISTR 548 (The culture collection of the Thailand Institute of Scientific and Technological Research, Bangkok) was used in this study. The culture was grown at 30°C in the YPG medium containing (per liter): 3 g of malt extract, 3 g of yeast extract, 5 g of peptone and 20 g of glucose (Michel et al., 1985). Solid medium was prepared for culture maintenance by adding 2% agar. E. coli strain DH5α and pGEM T-easy vector were used for all cloning procedures.

Stress induction and cell viability: An overnight culture was grown at 30°C in the YPG medium described previously. Ethanol or heat stress was provoked according to the following procedures. Ethanol stress; cells were cultivated to an optical density (OD 550 nm) of 0.8, ethanol was added to a final concentration of 3.5, 7, 10.5 and 14% (v/v): heat stress; at an optical density of 0.8 cultures were shifted from 30°C to 35, 40, 45 and 50°C. The time of the shift was set as zero and samples were taken at the time indicated in the relevant figure legends. Viability of cell was measured by plating the cell on solid YPG medium after stress treatments. Cell viability was estimated by counting colonies after 72 h incubation at 30°C.

Protein extraction and SDS-polyacrylamide gel electrophoresis: Ethanol- and heat-shocked cultures were harvested, washed and suspended in 10 mM Tris-HCl (pH 7.0) buffer. Proteins were extracted by sonicating cell suspension in Bioruptor (Cosmo Bio, USA) for 10 min at 50% pulser duty cycle and output power 5. The extracts were centrifuged at 12,000 g for 2 min. The supernatant fractions were removed and lyophilized. Protein concentration of the cell free extracts was measured using Lowry reagent. For electrophoresis, approximately 20 μg of protein sample was heated at 100°C for 5 min and separated by SDS electrophoresis on 12% acrylamide gel with constant voltage of 50 V. After electrophoresis, proteins separated on the gel were visualized using Coomassie Brilliant Blue R250 and fixed in 10% ethanol. For sequencing of the N-terminal amino acids, proteins separated in the gel were electroblotted onto an Immobilon-P transfer membrane (Millipore, USA) and stained with ponceau S. The stained protein of interest was excised and the N-terminal amino acid sequences were determined by using a model PPSQ-21A protein sequencer (Shimadzu, Japan).

DNA manipulations, cloning and sequencing: Genomic DNA of Z. mobilis was extracted using CTAB-DNA precipitation method (Del Sal et al., 1989). The genomic DNA was quantified spectrophotometrically. All restriction enzymes, calf intestine phosphatase, T4 DNA ligase and Taq DNA polymerase were used as recommended by the manufacturer (Takara, Japan). Agarose gel electrophoresis and colony hybridization were performed following standard protocols (Sambrook and Russell, 2001).

The Z. mobilis groES and groEL genes were generated by PCR using primers designed based on the groESL sequence of Z. mobilis (GenBank Accession number: L11654). The forward ZM1 (5’-TTCGTCCGCTACATGAT-3’) and reverse ZM2 (5’-CTTCATGATGAGGAGGT-3’) were used to amplify the groES gene and the forward ZM3 (5’-GATATTCTTGCCGATGC-3’) and reverse ZM4 (5’- CGACAGATAGCCGCGAT-3’) were used to amplify the groEL gene. For 100 μL of PCR reaction, 50 ng of Z. mobilis genomic DNA was used. Thermal cycling conditions were initial denaturation at 94°C for 3 min followed by 30 cycles at 94°C for 1 min, 58°C for 2 min and 72°C for 5 min and final extension at 72°C for 10 min using Ex. Taq DNA polymerase (Takara, Japan). The PCR products of approximately 250 and 540 bp for groES and groEL, respectively, were purified using QIAquick PCR purification kit (QIAGEN, Germany). The purified PCR product was cloned into pGEM T-easy vector and was transformed into E. coli DH5α using the Rapid DNA ligation and transformation kit (Fermentas). Sequencing of the groES and groEL region of Z. mobilis was performed by the dideoxynucleotide chain termination method with a SQ3000 nucleotide sequencer (Hitachi Electronics Engineering, Tokyo, Japan). The complete groES and groEL of Z. mobilis was PCR amplify by forward ZM5 (5’-AGGTGAAGATATGAAT-3’) and reverse ZM6 (5’-GCGTAATTCAACCCATC-3’) and cloned into pGEM T-easy vector. The sequence of the groES and groEL and deduced amino acid sequence were analyzed using GENETYX (Software Development, Tokyo, Japan). Homology searching was performed using FASTA and BLAST program in the GenBank and DDBJ databases.

RT-PCR analysis: Reverse transcription-PCR (RT-PCR) was carried out with 0.1 μg of total RNA, prepared as described previously (Aiba et al., 1981) and the mRNA Selective PCR kit (Takara Shuzo, Japan). The primers for groES region were the forward ZM1 and reverse ZM2 and groEL region were the forward ZM3 and reverse ZM4. The RT-PCR products were then electrophoresed on a 0.9% agarose gel and after staining with ethidium bromide, the relative amounts of the products were densitometrically estimated by using a Bio-Rad molecular imager. As a control, 10 μg samples of total RNA were subjected to agarose gel electrophoresis (1.2% agarose) and stained with ethidium bromide. Linearity of the amplification was observed at least up to the 23nd cycle. The experiment was repeated at least twice. Under our conditions, the RNA-selective RT-PCR was able to specifically detect mRNA because no band was observed when reverse transcriptase was omitted.

RESULTS AND DISCUSSION

Effect of ethanol and heat stress on growth: Growth of Z. mobilis as measured by optical density was largely decreased when initial concentration of ethanol in fermentation medium or incubation temperature was increased (Fig. 1A and B). More than 50% of growth was decreased when concentration of ethanol or incubation temperature higher than 7% and 35°C, respectively. In addition, no growth was observed when ethanol was added to a final concentration of 10.5 and 14% as well as when temperature was shifted from 30 to 45°C. The results suggest that both heat and ethanol had an effect on growth of Z. mobilis, like that observed in other organisms such as S. cerevisiae (Jones and Greenfield, 1985) and Bacillus subtilis (Li and Wong, 1992). The present results also similar to those of Carey and Ingram (1983) who observed decreased in growth rate of Z. mobilis when grown in medium containing ethanol higher than 1.04 M. One possible explanation for the inhibitory effect of heat and ethanol on growth of Z. mobilis is that these stresses decreased the effectiveness of the plasma membrane as a semipermeable barrier, allowing leakage of cofactors and coenzymes necessary for the activity of enzymes involved in central metabolism particularly the glycolysis and alcohol production pathway (Osman and Ingram, 1985).

Fig. 1:

Effect of ethanol or heat stress on growth of Z. mobilis TISTR548. Cells were grown in YPG medium and exposed to ethanol or heat stress. A, each stress of 3.5, 7.0, 10.5 and 14% ethanol at an initial concentration is represented by squares (■), triangles (▲), circles (●) and crosses (

), respectively. Control of 0% ethanol at an initial concentration is shown by diamond (♦). B, each stress at 35, 40 and 45°C is represented by squares (■), triangles (▲) and crosses (

), respectively. Control at 30°C is shown by diamonds (♦)

Effect of heat and ethanol stress on cell viability: Viability of Z. mobilis was slightly decreased when cells were incubated at 40°C for 1 h or addition of 7% ethanol. However, higher temperatures or ethanol concentrations caused dramatically decreased viability, resulting in only 81% at 45°C, 22% at 50°C and 59% at 10.5% and 10% at 14% ethanol, respectively (Table 1).

As expected, the lethality of exposure to stressful heat and ethanol levels was greatly reduced when cells were exposed to preheat treatment or sublethal ethanol. When cells were shifted from 30 to 45°C for 30 min and then transferred to 50°C for 30 min before plating, 67% of cells remained viable as compared to control cells without pretreatment.

Table 1:	Viability of Z. mobilis TISTR548 under heat or ethanol stress

^aTreatment at each condition is for 1 h unless otherwise stated

Fig. 2:

SDS-polyacrylamide gel electrophoresis analysis of protein extracted from Z. mobilis TISTR548 after heat- or ethanol-stressed. A, proteins were extracted from cells incubated at 30°C (lane 1) and after exposed to heat at 45°C for 2 (lane 2), 4 (lane 3), 6 (lane 4), 8 (lane 5) and 10 h (lane 6). B, proteins were extracted from cells grown in YPG without addition of ethanol (lane 1) and in YPG with 10.5% ethanol and incubated for 2 (lane 2), 4 (lane 3), 6 (lane 4) and 8 h (lane 5). Positions of molecular mass markers (lane M) are indicated on the left and arrows indicate proteins of 58 and 10 kDa

Likewise, 60% of cells shifted to 50°C for 30 min retained viability when they were preincubated at 30°C for 30 min in YPG medium containing 7% ethanol. Moreover, 43% of cells subjected to 7% ethanol for 30 min prior to cultivate in YPG medium containing 14% ethanol retained viable. These results clearly indicate that heat and ethanol pretreatment increases tolerance of Z. mobilis to stressful heat and ethanol levels, which may be related to biological functions of some heat shock proteins synthesized during preexposure to a mild heat and ethanol shock.

Effect of heat and ethanol on protein expression pattern: It has been reported that induction of thermotolerance in many organisms such as S. cerevisiae, B. subtilis and Neurospora crassa is directly correlated with induction of specific set of HSPs (Plesofsky-Vig and Brambl, 1985). We therefore examine the effect of induced heat and ethanol tolerance on protein synthesis in Z. mobilis. Bacterial cells growing at 30°C in YPG medium were shifted to different high temperatures or cultivated in medium containing different levels of ethanol. Cells were collected at different times of exposure and proteins in the cellular extracts were separated by SDS-12% polyacrylamide gel. As shown in Fig. 2A and B, proteins, indicated by an arrow, with the apparent molecular weight of approximately 58 and 10 kDa, relative to protein standards, were remarkably induced in heat and ethanol-treated cells. These proteins were also detected as a faint signal particularly the 10 kDa polypeptide in non-treated cells growing at normal growth conditions (panel 1), indicating that the expression of stress proteins occurs not only in the heat or ethanol responses but also in the normal developmental process. The differential expression of the 58 and 10 kDa polypeptides in Z. mobilis suggests that these polypeptides are stress proteins play an important role in the cellular physiology and that they are involved in some essential functions in normal and shocked cells, like that observed in other organisms such as Drosophila melanogaster (Mason et al., 1984; Kurtz et al., 1986), E. coli (Fayet et al., 1989) and S. cerevisiae (Lindquist, 1984). In the figure, the protein patterns of heat and ethanol treated cells at different times of exposure to 45°C and 10.5%, respectively, are shown.

The N-terminal amino acid analysis of the 58 and 10 kDa polypeptides revealed distinct sequence, AAKDVKFSFD and MNFRPLLDDV, respectively. Homology search was performed and it was found that the sequences are identical to a region of polypeptides, the groEL and groES gene products, which are previously reported as the HSPs in Z. mobilis (Barbosa et al., 1994). Therefore, we designated 58 and 10 kDa polypeptides of Z. mobilis as GroEL and GroES, respectively.


Fig. 3:	Nucleotide sequences of the Z. mobilis groES and groEL gene and deduced amino acid sequence. Putative ribosome binding sites for groES and groEL are marked by double underlines. An asterisks mark the stop codon. The GGM motif is indicated by light shade. A palindromic sequence which may serve as a transcriptional terminator is marked with facing arrows


Fig. 4:	Sequence alignments of Z. mobilis groES (Zm) A. aceti groES (Aa) B. subtilis groES (Bs) L. paracasei groES (Lp) and S. cerevisiae hsp10 (Sc). Amino acids residues identical and similar between each other are shown by asterisks and dots, respectively. Gaps introduced for alignment are indicated by a horizontal dash

Cloning and sequence analysis of the Z. mobilis groES and groEL genes: The Z. mobilis groES and groEL genes was cloned and sequenced as described in materials and methods. Sequencing analysis revealed the presence of two contiguous Open Reading Frames (ORFs) encoding the entire amino acid sequence of the groES and groEL gene product. The nucleotide sequence of these ORFs, its flanking region and the deduced amino acid sequence are shown in Fig. 3. The groES and groEL are present as a single copy in the genome of Z. mobilis, as demonstrated by Barbosa et al. (1994). The groES encodes a polypeptide of 95 amino acid residues with calculated relative molecular masses of 10, 290 Da. This polypeptide exhibits significant sequence identity to the GroES of Acetobacter aceti (71%) (Okamoto-Kainuma et al., 2002), B. subtilis (53%) (Schmidt et al., 1992), Lactobacillus paracasei (46%) (Desmond et al., 2004) and hsp10 of S. cerevisiae (34%) (Hohfeld and Hartl, 1994) (Fig. 4). The groEL encodes a polypeptide of 549 amino acid residues with calculated relative molecular masses of 58,428 Da. The start codon of groEL is 106 nucleotides downstream from the stop codons of groES. The groEL contains a canonical ribosomal-binding site with a GGAG Shine-Dalgarno region while that for groES is less conserved (Barbosa et al., 1994). The strongly conserved carboxyl-terminus Gly-Gly-Met (GGM) motif as seen in GroEL proteins of various species was also present in the Z. mobilis GroEL protein. Its biological function remains unclear, however, it is appealing to speculate from the GroEL protein encoded by thermophilic bacterium PS3 that the number of GGM motifs at the C-terminus of GroEL will prove to be correlated with the thermostability of the protein (Schon and Schumann, 1993). The encoded protein sequences for Z. mobilis groEL exhibits significant sequence identity to the GroEL of A. aceti (70%) (Okamoto-Kainuma et al., 2002), B. subtilis (58%) (Schmidt et al., 1992), L. paracasei (56%) (Desmond et al., 2004) and hsp60 of S. cerevisiae (52%) (Johnson et al., 1989) (Fig. 5). Two small segments in the C-terminus in which more conservation of sequence is evident between Z. mobilis and S. cerevisiae were found, suggesting that its may be related to stress responses in high ethanol environment (Barbosa et al., 1994).

Analysis of the groES and groEL expression: The expression of Z. mobilis groES and groEL was investigated by RT-PCR using total RNA prepared either from 3 hours after the cells had been transferred to heat shock at 37, 40 and 45°C or to ethanol shock at 3.5, 7.0, 10.5 and 14.0% or to salt stress at 1.5, 3.0 and 6.0% NaCl as a template. The RT-PCR experiments showed that groES and groEL expression was about three to sixfold increased in heat and ethanol treated cells, as compared to non-treated cells (Fig. 6A and B). These results suggest that groES and groEL expression is relatively low in non-stressed cells, but increase rapidly in cell upon heat and ethanol stressed. The present results also revealed that induction of the groES and groEL is regulated at the level of transcription. This can be accomplished by increased synthesis of mRNA as proposed by Schon and Schumann (1993). In some certain organisms, such as Enterococcus faecalis (Laport et al., 2004), the groES and groEL had been shown to highly express under salt stress conditions. This led us to further investigate the expression of groES and groEL under salt stress. As we found in the present study, there was no significant difference in the expression of groES and groEL mRNA between stressed and non-stressed cells under the experimental conditions followed in this assay (Fig. 6C).


Fig. 5:	Sequence alignments of Z. mobilis groEL (Zm) A. aceti groEL (Aa) B. subtilis groEL (Bs) L. paracasei groEL (Lp) and S. cerevisiae hsp60 (Sc). Amino acids residues identical and similar between each other are shown by asterisks and dots, respectively. Gaps introduced for alignment are indicated by a horizontal dash

Fig. 6:

RT-PCR analysis of the groES and groEL expression under heat, ethanol and salt stresses. The logarithmic phase of cells grown in YPG medium was exposed to heat (A), ethanol (B) and salt stress (C) for 3 hours and total RNAs were then prepared and subjected to RT-PCR analysis with primers specific for groES and groEL as described in Materials and Methods. The numbers above the lanes represent cycles of PCR. (D) rRNA extracted from heat-, ethanol- and salt-treated cells was used as a control. Total RNAs (10 μg) in panels A to C were used

These results suggest that the expression of Z. mobilis groES and groEL is not correlated to salt stress response, likes that observed for E. coli (Gross et al., 1990) and B. subtilis (Volker et al., 1992).

Proteins induced by heat as well as ethanol shock have been shown to play an essential role in bacterial physiology under heat or ethanol stresses. In E. coli, for example, the heat shock proteins groES and groEL are well known for their roles in protein folding as molecular chaperones, promoting the assembly, disassembly or translocation of other proteins (Weissman et al., 1995, 1996). Furthermore, the groES and groEL genes products in particular are essential for E. coli growth at all temperature (Fayet et al., 1989). In B. stearothermophilus, the groES and groEL genes had function homology to known GroE proteins, promoting growth of bacteria at high temperature and propagation of phage lambda (Schon and Schumann, 1993). With respect to physiological function of the Z. mobilis groES and groEL, it is not clear at present. The direct involvement of these genes as molecular chaperones could be elucidated by gene disruption experiments.

The present study clearly demonstrated that heat and ethanol stresses had an affect not only on growth and cell viability but also on protein synthesis. The stress of Z. mobilis by heat and ethanol increased the synthesis of several polypeptides, of which at least two, designated as GroES and GroEL, were heat shock proteins. The physiological significance of these proteins in the stress response is still unknown, but there is reason to believe that they are involved in the mechanism of tolerance of Z. mobilis and probably of other cells to heat and ethanol. Further experiments are needed to determine the mechanism of transcription regulation and cellular location of the Z. mobilis groES and groEL.

ACKNOWLEDGMENTS

This study was done by collaboration in a core university program between Yamaguchi University and Kasetsart University, which was supported by the Scientific Cooperation Program agreed to by the Japan Society for the Promotion of Science (JSPS) and the National Research Council of Thailand (NRCT). The authors would like to thank the Energy Conservation Promotion Fund for a grant to K.S.