Transcriptional Response of Escherichia coli to Temperature Shift Mugdha Gadgil,† Vivek Kapur,‡ and Wei-Shou Hu*,† Department of Chemical Engineering and Materials Science, and Department of Microbiology, Biomedical Genomics Center, University of Minnesota, 421 Washington Avenue SE, Minneapolis, Minnesota 55455-0132
Temperature shift is often practiced in the cultivation of Escherichia coli to reduce undesired metabolite formation and to maximize synthesis of correctly folded heterologous protein. As the culture temperature is decreased below the optimal 37 °C, growth rate decreases and many physiological changes occur. In this study, we investigated the gene expression dynamics of E. coli on switching its cultivation temperature from 37 to 33 and 28 °C using whole genome DNA microarrays. Approximately 9% of the genome altered expression level on temperature shift. Overall, the alteration of transcription upon the downshift of temperature is rapid and globally distributed over a wide range of gene classes. The general trends of transcriptional changes at 28 and 33 °C were similar. The largest functional class among the differentially expressed genes was energy metabolism. About 12% of genes in energy metabolism show a decrease in their level of expression, and ∼6% show an increase. Consistent with the decrease in the glucose uptake rate, many genes involved in glycolysis and the PTS sugar transport systems show decreased expression. Genes encoding enzymes related to amino acid biosynthesis and transport also have reduced expression levels. Such decrease in expression probably reflects the reduced growth rate and the accompanying reduction in energy and amino acid demand at lower temperatures. However, nearly all genes encoding enzymes in the TCA cycle have increased expression levels, which may well be compensating the reduction of the activity of TCA cycle enzymes at lower temperatures. Temperature shift also results in shift of the cytochromes from the high affinity cytochrome o system to the low affinity cytochrome d system. There is no evidence that protein processing genes are selectively altered to create favorable conditions for heterologous protein synthesis. Our results indicate that the beneficial effect of temperature shift in many biotechnological processes is likely to be attributed to the general effect of reduced growth and metabolism.
Introduction Most bacteria, including Escherichia coli, can grow over a range of temperature of approximately 40 °C (1). The growth rate of E. coli increases with temperature over the normal range from 21 to 37 °C. This relationship breaks down both above and below this range (1). E. coli is sometimes cultured below the optimal 37 °C temperature in recombinant protein production. Heterologous proteins expressed at high levels in E. coli often fail to reach their native conformation and have a tendency to form inclusion bodies. This can be minimized by culturing cells at a reduced growth temperature (2, 3). This approach has been effective in improving the solubility of a number of recombinant proteins including human interferon R-2 (4), subtilisin (5), bacterial luciferase (6), Fab fragments (7), β lactamase (2), soybean lypooxygenase L-1 (8), kanamycin nucleotidyltransferase (9), and rabbit muscle glycogen phosphorylase (10). Plasmid stability has also been reported to be improved when induction of protein expression was carried out at low temperatures (11), as is the degradation of proteolytically sensitive products (12, 13). Synthesis of recombinant * To whom correspondence should be addressed. Phone: (612) 626-7630. Fax: (612) 626-7246. E-mail:
[email protected]. † Department of Chemical Engineering and Materials Science. ‡ Department of Microbiology, Biomedical Genomics Center. 10.1021/bp049630l CCC: $30.25
protein at low temperature also abolished read-through of the UGA stop codon during expression of the Bacillus subtilis gene flgM in E. coli (14). Growth over a large temperature range is accompanied by many physiological changes. The changes occurring at extreme low temperatures (at or below 15 °C), the cold shock response, have been studied extensively in the past two decades (15-19). However, very few studies have analyzed the differences in protein or mRNA expression at different temperatures within the normal range. Herendeen et al. (20) carried out a systematic study of the expression level of 133 proteins in cultures cultivated between 13.5 and 46 °C and found that only 11 proteins changed their expression level more than 2-fold in the normal range (21-37 °C). This is in contrast to 83 proteins whose expression levels differed by more than 2-fold between 13.5 and 46 °C. The results indicate cellular response to temperature change in the normal range is very different from the cold shock or heat shock response. However, the gel system used in the study might not be sensitive enough to reveal many changes. With the frequent use of reduced temperature in the normal range for cell cultivation in the production of heterologous protein, a better understanding of the physiological changes underlining temperature shift may provide insight into the process. Using whole genome
© xxxx American Chemical Society and American Institute of Chemical Engineers Published on Web 00/00/0000 PAGE EST: 10.7
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DNA microarrays, it is now possible to analyze the expression of all genes in the genome with high accuracy. In this paper, we report the transcriptional changes occurring on reducing the growth temperature of E. coli from 37 to 33 and 28 °C. Even a seemingly small change of 4 °C in the growth temperature results in changes in expression levels of ∼9% of the genome, indicating that a large number of changes occur, at least transiently, in response to a change in temperature. Our results show that transcriptional response to the downshift of temperature is rapid and globally distributed over a wide range of functional groups.
Materials and Methods Culture Conditions. All cultures were grown in minimal media containing 0.04 M MOPS, 4 mM tricine, 9.5 mM ammonium chloride, 0.28 mM potassium sulfate, 0.5 µM calcium chloride, 0.53 mM magnesium chloride, 1.32 mM K2HPO4, 0.05 M sodium chloride, 10 µM ferrous sulfate, 0.003 µM ammonium molybdate, 0.4 µM boric acid, 0.03 µM cobalt chloride, 0.01 µM cupric sulfate, 0.08 µM manganese chloride, 0.01 µM zinc sulfate, and 0.01 M glucose. 8 mL culture of E. coli K12 was grown overnight in the defined media described above in a 50 mL tube at 37 °C and 250 rpm. 1% (v/v) of this overnight culture was inoculated into 400 mL media in a 2 L Erlenmeyer flask, and the culture was allowed to grow at 37 °C with shaking at 250 rpm until a cell density corresponding to an OD600 of around 0.6 was reached. At this point, a 100 mL sample was taken for RNA extraction (time 0), and the remaining culture was split into two 150 mL cultures in 1 L Erlenmeyer flasks each and transferred to 28 and 33 °C with shaking at 250 rpm. Samples were taken at 5, 10, 20, 30, 60, 90, and 120 min after changing the growth temperature. Immediately after sampling, the cell suspension was centrifuged at 5500 rpm for 3 min at 4 °C. The cell pellet was stored at -80 °C. Glucose Concentration Measurement. Glucose concentration in the culture supernatant was measured using the Infinity Glucose Reagent (Thermo Electron Corporation, Waltham, MA). Oxygen Consumption Rates. Culture sample was quickly transferred to fill an airtight 15 mL tube fitted with a DO probe, and the change of dissolved oxygen concentration over time was recorded. The entire free volume of the tube was occupied by the sample. The tube was maintained at the culture temperature. The rate of decrease in oxygen concentration is the rate of oxygen consumption by the cells. The measured decrease in DO with time was plotted, and the oxygen uptake rate was calculated from the slope of this line. The specific oxygen uptake rate was obtained by dividing the uptake rate calculated above by the cell density of the culture at the time of sampling. RNA Extraction. RNA extraction was carried out using RNeasy Mini Kits (Qiagen, Valencia, CA) according to the manufacturer’s protocol. DNase digestion for removal of genomic DNA contamination was performed on the RNeasy Mini columns according to the manufacturer’s protocol. cDNA Microarray Preparation. 90% of the E. coli genome (3866 successful PCRs) was printed onto a polylysine coated slides using the MicroGridPro Spotting Robot (BioRobotics, Cambridge, UK). Full length PCR products were obtained for a majority of these genes with the following conditions: 0.75 units Taq polymerase, 1X Buffer, 0.2 µM dNTPs, 2 mM MgCl2, 200 ng of genomic
DNA, primers and water to 25 µL. Thermocycling conditions were (a) 94 °C for 5 min; (b) cycle conditions: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; (c) 72 °C for 10 min. 0.5 µL of the PCR product was used as template for a second 100 µL PCR reaction (same conditions) to minimize genomic DNA contamination. The PCR reactions that failed under these conditions (either gave no product or multiple bands were observed on agarose gels) were reamplified at different annealing temperatures. After each round of amplification, the PCR product was run on agarose gels, and the gels were analyzed using BioRad Q1 software. 90% of the genome was successfully amplified this way. Amplified PCR products were cleaned with MultiScreen PCR plates (Millipore, Billerica, MA) as per the manufacturer’s instructions and resuspended in 50 µL of 3X SSC, 0.01% SDS. After printing, slides were postprocessed. Postprocessing involved rehydration, blocking, and denaturation. Blocking was carried out by acylation with succinic anhydride. Details of all protocols related to microarray preparation and hybridization are available at http:// www.agac.umn.edu/microarray/microarrays.htm. Sample Labeling. It has been previously shown that the total RNA content of E. coli is invariant in the temperature range used in this study (21, 22). Hence, using equal amounts of total RNA from samples at different temperatures is equivalent to using RNA from equal number of cells. 10 µg of total RNA was primed with 20 µg of random hexamers (Amersham Biosciences, Piscataway, NJ), incubated at 70 °C for 10 min, and then kept on ice for 10 min. The RNA mixture was then added to the reverse transcription reaction, which included the reverse transcriptase Superscript II (Invitrogen, Carlsbad, CA), DTT (Invitrogen, Carlsbad, CA), and 2 mM dNTPs (4:1 ratio of amino-allyl dUTP:dTTP) (Amersham Biosciences, Piscataway, NJ). The mixture was incubated for 2 h at 42 °C. After incubation, the reaction was stopped by addition of 20 µL of NaOH, 20 µL of 0.5 M EDTA, and incubating at 65 °C for 15 min. The samples were neutralized by addition of 50 µL of 1 M Tris-HCl (pH 7.4). The amino labeled cDNA was then stripped of all extra amine groups before the fluorescent dye was coupled to the amino group using Microcon YM-30 filters as per the manufacturer’s instructions (Millipore, Billerica, MA). The sample was dried down to concentrate cDNA. The cDNA was resuspended in 9 µL of sodium bicarbonate buffer (pH 9.0) and kept at room temperature for 10 min to ensure resuspension. The entire volume was transferred into a tube containing the appropriate dried Cy-dye aliquot (Cy3 or Cy5) and incubated in the dark for an hour. To prevent cross coupling between the probes, 4.5 µL of 4 M hydroxylamine was added and the solution was incubated for 15 min at room temperature in the dark. The Qia-Quick PCR purification kit was used to clean up the hybridization extract as per the manufacturer’s instructions (Qiagen, Valencia, CA) with the addition of one Buffer PE wash. To have the hybridization extract ready for hybridization, the samples were dried down and then eluted in 33.5 µL of dH2O, 6.7 µL of 20x SSC, 1 µL of 10% SDS, and 3.4 µL of 10 mg/mL salmon sperm DNA (Invitrogen, Carlsbad, CA) as a blocker. Hybridization. 10 µL of 3x SSC was added to the ends of each slide to prevent dehydration of the hybridization solution. The hybridization solution was denatured for 2 min and then allowed to cool for 10 min at room temperature. It was then transferred to the postprocessed printed slide, covered with a cover slip, and incubated
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at 63 °C for 16 h. Images of the fluorescent activity were measured with the ScanArray 5000 microarray scanner (PerkinElmer, Boston, MA). Image Quantification. The fluorescent intensities were quantified using the GenePix Software (Axon Instruments, Union City, CA). Six replicate measurements were obtained for each gene from two replicate hybridizations. The local background was subtracted from the spot intensity to obtain the final signal intensity. Signal intensities that were greater than at least 1.5 standard deviation of the local background were used for further analysis. The spot intensities were normalized using Lowess normalization in the GeneSpring software (Silicon Genetics, Redwood City, CA). After normalization, the average and standard deviation (SD) of the log2 transformed ratio for all of the replicate spots for each gene was calculated. Spots outside the range [mean 1.5 SD, mean + 1.5 SD] were considered outliers and therefore discarded. The average of the log2 ratios for the remaining spots was calculated. Any gene that had only one valid data point was discarded. To ensure statistical significance of the data, a p-value was calculated to test the null hypothesis that the expression level is unchanged using the Student t-test. The Ecocyc database was used to obtain preliminary annotation for genes (23). Real-Time PCR. 10 µg of total RNA was treated for DNA removal using the DNA-free kit from Ambion (Austin, TX) according to the manufacturer’s protocol. 1 µg of the “cleaned” RNA was reverse transcribed using random hexamers as primers similar to the reverse transcription reaction for the generation of the probe for the microarray hybridization, as described above. Amplification, detection, and real-time analysis were performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). SYBR Green I (Applied Biosystems, Foster City, CA) was used for detection of the amplified product. Primers were designed to produce amplicons of approximately 100 bp using the Primer3 software. The cleaned RNA was also used as a template in the amplification reaction to check for any amplification from genomic DNA contamination. The real-time PCR assay was performed for the 0 and 20 min time-points from 28 and 33 °C. The n-fold difference in expression of each gene between time 0 and 20 min was calculated from the threshold cycle (CT) as 2-x, where x ) (CT for 20 min sample - CT for 0 min sample). Because comparisons were made between products of PCRs using identical primers, and not between PCR products derived from different genes, the amplification efficiencies of different primer pairs were eliminated as a variable in the interpretation of the data.
Results and Discussion E. coli K12 was cultured at 37 °C in minimal media until the cell density reached an OD600 of 0.6. The culture was then divided into two and cultivated at 33 and 28 °C, respectively. Shifting the temperature from 37 to 33 or 28 °C led to a 40% and 70% decrease in the growth rate, respectively. Figure 1 depicts the growth curves of the cultures. The pH remains unchanged during the experiment. The specific glucose consumption rate and oxygen uptake rates also decreased at both lower temperatures. At 37, 33, and 28 °C, the glucose consumption rates were 8.7, 5.1, and 4.3 µg/(109 cells min), while the oxygen uptake rates were 1.2, 1.1, and 0.9 µg/(109 cells min), respectively. Samples for transcriptional analysis were taken at 5, 10, 20, 30, 60, 90, and 120 min after each temperature shift, and each sample was compared
Figure 1. Growth curve after temperature shift. E. coli is cultured in MOPS-based minimal media at 37 °C (b) until an OD600 of approximately 0.6. The culture is then split into two parts, which are cultured at 33 °C (2) and 28 °C (9), respectively.
to the sample taken before the temperature shift using DNA microarrays. Overall Transcriptional Changes on Temperature Shift. Genes whose expression levels changed more than 2-fold at a minimum of three out of the seven timepoints were classified as differentially expressed. To ensure that the observed expression changes were statistically relevant, only data points with a p-value less than 0.1 for the null hypothesis that the expression level is unchanged were considered. A p-value less than 0.05 is usually used for single data-point analysis. Because we are analyzing a time series and require three timepoints to be differentially expressed, we have chosen the less stringent p-value of 0.1. We have previously carried out experiments in which three control RNA species were spiked into both the total RNA samples used in a hybridization experiment. These three species were spiked at different concentrations to represent mRNA species expressed at low, intermediate, and high levels in a cell. Based on these results (unpublished), we found that the expression ratio, which should ideally be 1, was less than 1.5-fold for 90% of the data points. Some genes with a 2-fold change in expression level have also been verified to be differentially expressed using real-time PCR. Hence, we believe that a 2-fold cutoff results in reducing false positives in the data and have used this value in the present work. Using these criteria, 401 (∼9% of the genome) genes were found to be differentially expressed at 28 °C and 389 (∼9% of the genome) genes at 33 °C. In our microarray assay, the amount of total RNA was kept constant in all hybridizations and the transcript levels of individual genes were compared on that basis. Ribosomal RNA is the major constituent of total RNA. It has been shown that the levels of ribosomal RNA are constant in the temperature range used in this study (22). If a decrease in growth rate with temperature only causes all mRNA levels to be proportionately decreased, the expression of all genes would proportionately decrease. The differentially expressed genes, which are ∼9% of the genome at both of the temperatures, are regulated in both directions, with 180 and 172 having increased expression at 28 and 33 °C, and 221 and 217 with decreased expression levels at 28 and 33 °C, respectively. This distribution suggests that the observed differential ex-
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Figure 2. Distribution of differentially expressed genes among the various functional classes. The numbers indicate the percentage of genes in that functional class that are differentially expressed. % 28 °C Increased expression. * 28 °C Decreased expression. Gray % 33 °C Increased expression. Gray * 33 °C Decreased expression.
pression was not merely due to possible changes of mRNA content in total RNA, but an adjustment of the gene expression levels to the particular culture condition of lower growth temperatures. Figure 2 shows the distribution of differentially expressed genes among the various functional classes. The number of genes in each functional class with decreased expression is similar to the number of genes with increased expression. Exceptions to this symmetric change are amino acid biosynthesis genes and transport genes, which have a higher number of genes with reduced expression levels. The functional class with the highest number of differentially expressed genes is energy metabolism (71 and 64 for 33 and 28 °C, respectively), followed by transport (40 and 43 for 33 and 28 °C, respectively) and amino acid biosynthesis (26 for both 33 and 28 °C). These are also the functional classes (other than hypotheticals and unclassified proteins) with the highest percentage of genes within that class changing their expression level. For the majority of genes whose expression levels changed, the change occurs rapidly in the first 5-10 min, and the expression levels remain at a steady level thereafter. To quantify this behavior, the differential expression ratio of later time-points (t )10, 20, 30, 60, 90, and 120 min) were compared to that at 5 min. Note that in the microarray hybridization, samples from all time-points were hybridized to the time 0 sample taken just before temperature shift. The differential expression ratios are thus all relative to time 0. Genes whose differential expression ratios had a p-value less than 0.01 at 5 min (898 at 28 °C and 666 at 33 °C) were included in the analysis. The ratios, Rt, of the differential expression ratio at different time-points to that at 5 min were calculated. For those genes whose expression levels are relatively invariant after the initial change, the values of Rt for subsequent time-points will remain close to 1. The standard deviation of the six Rt values was calculated for each gene at both of the temperatures. 798 out of 898 for 28 °C and 608 out of 666 at 33 °C had a standard deviation less than 1, indicating that the expression levels remain at a steady level after the initial change. In general, we find that the transcriptional changes due to temperature shift to 28 and 33 °C were very similar. To quantify the differences in the time profile of
transcript expression at the two temperatures, we calculated the Euclidean distance between the time profiles of transcript of every gene at 28 and 33 °C. The Euclidean distance Ej for gene j is calculated as Ej )∑7i)1 (xji - yji)2, where i represents one of the seven samples over the seven time-points, and x and y are the log ratios of the relative expression level of samples for the 28 and 33 °C cultures, respectively. The average Euclidean distance for all genes is 1.5 with a standard deviation of 1. This indicates that the gene expression profiles at 28 and 33 °C are similar for a vast majority of genes. The three functional classes with the highest fraction of genes changing their expression level are discussed in detail in the following section. Because of the relevance of temperature shift in recombinant protein production, changes in expression level of genes related to protein synthesis are discussed. Furthermore, change in growth temperature inevitably causes changes in the fatty acid composition of the bacterial cell membrane; hence the changes in transcripts related to fatty acid biosynthesis are also discussed. A notable change among the other classes not discussed is the statistically significant upregulation of the majority of the genes involved in purine and pyrimidine ribonucleotide biosynthesis. Energy Metabolism. Glycolysis. Temperature shift to 33 and 28 °C does not cause major changes in the expression profile of genes involved in the glycolysis and pentose phosphate pathway. The expression level of cra, regulating many genes involved in the central pathways of carbon metabolism, is unchanged. The effect of temperature shift on the expression profiles of the glycolysis genes is shown in Figure 3. Many of these reactions are catalyzed by isozymes, which are preferentially used under different conditions. The analysis of gene expression profile will thus need to consider all related isozymes together. Four genes (glyceraldehydes-3-phosphate dehydrogenase (gapA), phosphoglycerate kinase (pgk), phosphoglycerate mutase (pgmI), and pyruvate kinase II (pykA)) among the 14 genes involved in the nine reaction steps in the conversion of glucose-6-phosphate to pyruvate (glycolysis) show a rapid decrease in their expression levels. There are three phosphoglycerate mutase isozymes in E. coli. Two, encoded by the gpmA and gpmB genes, are expressed at moderate and low levels with intensity of 5000 and 1000, respectively. The third one, encoded by
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Figure 3. Expression profiles of genes in glycolysis. Data shown are log2 ratio of transcript level at time t to that at time 0. The dotted lines indicate a 2-fold change in expression level. - 33 °C, - - - 28 °C.
the pgmI gene, is expressed at the highest level among the three (intensity 20 000) and shows a 4-fold decrease in expression level at 5 min after the temperature is shifted to 28 and 33 °C. This indicates that pgmI is likely to be the enzyme that plays the most significant catalytic role among the three isozymes. The enzyme catalyzes the reversible conversion of glycerol-3-phosphate to glycerol2-phosphate. Glycerol-3-phosphate is the downstream product of another reversible reaction from glyceraldehyde-3-phophate. Glyceraldehyde-3-phosphate in turn is closely linked to the precursor pool for lipid metabolism through phosphoglycerol and to pentose phosphate pathway. It is possible that this perturbation plays a role in modulating the intracellular pool of metabolic intermediates. For pyruvate kinase, the two isozymes, encoded by pykA and pykF, are expressed at rather different levels. pykA (pyruvate kinase II), the one expressed at the higher intensity (∼15 000), shows a reduction in its expression. The other, pykF (intensity ∼2000), did not have significant change in its expression level upon temperature shift. Pyruvate kinase II (pykA) is regulated by AMP levels in the cell, while pyruvate kinase I (pykF) is activated by fructose-1,6-bisphosphate. At the lower temperatures, the glucose consumption rate and glycolysis flux are reduced. It is thus not surprising that genes in the glycolysis pathway show a
reduced level of expression. None of the genes in the pentose phosphate pathway changed more than 2-fold at more than one time-point after temperature shift. TCA Cycle. In contrast to glycolysis, temperature shift has a markedly different effect on genes of TCA cycle enzymes. With a decrease in glucose consumption rates at lower temperatures, a reduced flux through the TCA cycle is anticipated. Hence, should the gene expression level change, one would predict a decrease in their expression level and the corresponding activity (or abundance). Unexpectedly, the majority of the TCA cycle genes show an increase in expression level after temperature shift (Figure 4). A similar increase in the protein level of enzymes in TCA at reduced temperatures has also been reported previously (20, 24). Glyoxylate bypass related genes aceA and aceB also increase ∼2-fold in expression level at both temperatures. The maximum change is seen in the genes coding for the 4 subunits of succinate dehydrogenase (sdhABCD) and malate dehydrogenase gene (mqo). After a rapid increase, the expression level of the succinate dehydrogenase genes at 33 °C decreases with time, although it never reaches the same level as that before the temperature shift. mqo encodes a membrane-associated malate:quinone-oxidoreductase and does not seem to play a significant role in malate oxidation (25). Among all of the TCA cycle-related enzymes, aconitase and fumarase have multiple isozymes, and we
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Figure 4. Expression profiles of genes in TCA cycle. Data shown are log2 ratio of transcript level at time t to that at time 0. - 33 °C, - - - 28 °C.
examined the effect on the different isozymes on the basis on their function and abundance level under these conditions. There are two aconitases in E. coli; both catalyze the reversible isomerization of citrate and iso-citrate via cisaconitate. The main function of the acnA enzyme is of maintenance during nutritional or oxidative stress. The acnB enzyme functions as the main catabolic enzyme. Consistent with this, acnB transcript’s intensity is higher (∼20 000) than acnA (∼2000), and it has a higher increase in expression level (∼8-fold) than acnA transcript (∼3-fold). Fumarase has three isozymes, fumarase A, B, and C encoded by fumA, fumB, and fumC, respectively. fumA and fumB are used under microaerobic and anaerobic conditions, respectively, while fumC is highly active under aerobic conditions. fumC shows an increase in expression after temperature shift to both 33 and 28 °C, while the expression levels of fumA and fumB remain unchanged. fumC has been previously shown to have increased expression as the growth rate was decreased under aerobic conditions (26). The general trend of regulation among those differentially expressed genes for enzymes with multiple isozyme thus emerges; the likely metabolically most active one has reduced expression in glycolysis, but increased expression in TCA cycle. The increased level of the transcripts of TCA cycle genes does not necessarily reflect an increase in the flux through TCA cycle. As noted at the beginning of this section, the oxygen consumption rates were lower at the lower temperatures. This points toward a reduced carbon flux through TCA cycle and a reduced electron flux through the electron transport pathway. This seemingly inconsistent behavior can be explained if the activity of these enzymes decreases at lower temperatures. With the activity of the TCA cycle enzymes decreasing at lower temperatures, it is plausible that their expression level has to increase to maintain the required flux through TCA cycle. Such a decrease in activity has been reported earlier for succinate dehydrogenase (27) and malate dehydrogenase (28).
The expression levels of TCA cycle enzymes isocitrate dehydrogenase, citrate synthase, succinate dehydrogenase, and malate dehydrogenase have also been previously reported to vary inversely with growth rate (29-32). With a decrease in temperature, the growth rate of E. coli decreases, and the increase in expression level of the genes encoding for TCA cycle enzymes could be caused by the change in growth rate. Respiratory Chain. The respiratory chain of E. coli has a modular structure and consists of many dehydrogenases serving as electron donors to the quinones, and reductases that transfer the electrons from the quinones to the final electron acceptors. Different groups of dehydrogenases and reductases are expressed depending on the available electron donors and final electron acceptors. Out of the 69 genes involved in the respiratory chain, 28 and 24 °C at 33 and 28 °C, respectively, exhibit significant change in their expression level. However, the expression levels of arcA and arcB, components of the ArcAB two-component signal transduction system involved in sensing aerobic and anaerobic growth conditions, are unchanged. The majority of the dehydrogenases and reductases that are differentially expressed have similar fluorescence intensities on the microarray. Among the differentially expressed respiratory genes, nine are dehydrogenases. Two dehydrogenases with increased expression are subunits of formate dehydrogenase (fdoG ∼4-fold) and succinate dehydrogenase (sdhABCD), discussed above in energy metabolism. Those with decreased expression are hybB, required for the formation of all hydrogenases (∼4-fold), subunits of glycerol-3-phosphate dehydrogenase (glpA ∼16-fold, glpB ∼4-fold), and pyruvate oxidase (poxB ∼4-fold). The rest of the differentially expressed genes are reductases. Many reductases except cytochrome o ubiquinol oxidase (whose final electron acceptor is oxygen, and hence is used under aerobic conditions) have decreased expression. These include components of fumarate reductase (frdA ∼4-fold, frdB ∼8-fold), dimethyl sulfoxide reductase (dmsAC ∼4-fold), nitrite reductase complex (nrfA ∼4-fold), nitrate reductase A (narH ∼4-fold), nitrate
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Figure 5. Expression profiles of cyo and cyd genes. Data shown are log2 ratio of transcript level at time t to that at time 0. - 33 °C, - - - 28 °C.
reductase Z (narZY ∼4-fold), nitrate reductase, periplasmic (napA ∼8-fold napBC ∼4-fold), cytochrome d ubiquinol oxidase (cydA ∼8-fold, cydB ∼4-fold), and cytochrome bd-II terminal oxidase (appB ∼2-fold). Cytochrome d ubiquinol oxidase, with a higher affinity for oxygen, is the predominant form expressed under low oxygen conditions, while cytochrome o ubiquinol oxidase, having a low affinity for oxygen, is the predominant form under high oxygen conditions. Notably, the cytochrome d ubiquinol oxidase (cydA, cydB) genes show a decrease in expression, and the expression of genes encoding for cytochrome o ubiquinol oxidase (cyoA ∼16-fold, cyoB ∼8-fold, cyoC ∼4-fold, cyoD ∼2-fold) is increased (Figure 5). Consistent with the observed changes in cyo and cyd genes, Wall et al. (33) also describe a transient increase in expression level of cydB on changing growth temperature from 30 to 42 °C. A reduced level of cytochrome o was reported at elevated temperatures. They also showed that a cydB- mutant has a growth defect at 37 °C as compared to 30 °C under aerobic conditions due to energy limitation, indicating cydB is required at 37 °C, but not as much at 30 °C. Our results are consistent with this interpretation. This implies that the low affininty cytochrome o is required to a greater extent at lower temperatures and the large affinity cytochrome d at higher temperatures, although the exact reason for this is currently unknown. Other Energy Metabolism Genes. Another set of energy metabolism genes also affected by temperature shift are pta (phosphotransacetylase) and poxB (pyruvate oxidase) involved in acetate formation. Cultivating E. coli at low temperatures has been reported to reduce acetate production (34). Phosphotransacetylase (pta) catalyses the conversion of acetyl coA to acetyl phosphate and coenzymeA, and in conjunction with acetate kinase (ackA, ackB) forms the branch diverting acetyl coA to acetate. Pyruvate can also be converted to acetate directly by pyruvate oxidase (poxB). PoxB has been shown to contribute significantly to the aerobic growth efficiency of E. coli in glucose minimal media (35). Both pta and poxB have reduced expression upon downshift of temperature. poxB shows a 32-fold and 8-fold decrease in expression at 28 and 33 °C, respectively, at the 5 min time-point, and the transcript levels steadily increase thereafter, but do not reach the same level of expression as that before temperature shift. pta shows an ∼3-fold decrease in expression at both temperatures. Also, acetyl coA syn-
thetase (acs), converting acetate to acetyl coA, has an ∼4-fold increase in expression at both temperatures. E. coli grown in high glucose medium diverts some of its carbon to acetate. Our results suggest a reduced production of acetate and greater conversion of acetate to acetyl coA at lower temperatures. Production of acetate is undesirable in E. coli cultivation, especially for recombinant protein production. Our results indicate that the reduced acetate production at lower temperatures reported earlier (34) is a concerted effect of changes in the level of pta, poxB, and acs transcripts. Amino Acid Biosynthesis. In this study, cells were grown in minimal media with NH4Cl as the sole nitrogen source. They thus acquire all amino acids through biosynthesis. The demand of amino acids for protein synthesis decreases with the decreasing growth rate at low temperature. Consistent with the decreasing demand, many genes involved in amino acid biosynthesis show reduced expression (Figure 2). Many of the enzymes with a decrease in transcript levels are the “ratecontrolling” enzymes in different pathways as will be described below. A notable exception to this overall decrease in expression level is the expression of genes related to glutamate synthesis, which did not show the same trend of downregulation. Glutamate is the major node for NH4+ assimilation into the cells. The amino group of glutamate is transferred to various amino acids through transamination reactions. Ammonium assimilation via glutamate is accomplished by two routes: (1) glutamate dehydrogenase at high NH4+ concentrations, and (2) glutamine synthetase and glutamate synthase, at low NH4+ concentrations. These enzymes are among the ones with the highest intensities (and hence abundance) among the amino acid biosynthesis genes. Considering that these assimilation reactions supply all of the nitrogen required by the cell, their high level of expression is not unexpected. Our results indicate that the reduced amino acids synthesis and NH4+ assimilation does not result from reduced transcription of genes involved in those two pathways. Histidine and Arginine Pathway. Nearly all of the genes coding for enzymes in the histidine and arginine pathways show reduced expression. The histidine biosynthesis genes had a 2-4-fold decrease in expression level within the first 20 min, and stayed at that level thereafter. The first gene in the pathway coding for ATP phosphoribosyltransferase (hisG), which is the regulated enzyme in the histidine synthesis pathway, shows a 4-fold decrease in expression at the first time-point sampled (5 min) after temperature shift, indicating that it is among the first gene to exhibit detectable regulation. Genes encoding for other enzymes in the histidine biosynthesis pathway show smaller changes in their expression levels as compared to hisG. This suggests that the activity of enzymes throughout the histidine synthesis pathway decreases with temperature. Similar to the histidine pathway, the transcript for the first enzyme N-acetylglutamate synthase (argA) in the arginine biosynthesis pathway, which is inhibited by arginine, has reduced expression. However, some other genes downstream of argA in the pathway change more than argA. These include argC (N-acetylglutamyl phosphate reductase), argI, argF (ornithine carbomyltransferase), and argG (arginosuccinate synthase), which have an ∼8-fold decrease in expression at both temperatures. The changes for these genes also occur rapidly in the first 5 min. The only genes in the arginine pathway to increase their expression level after temperature shift are carA and carB, involved in the synthesis of carbamoyl phos-
H
phate from HCO3-. Their expression level increases 2-4-fold. Carbamoyl phosphate is however also used in other pathways such as pyrimidine synthesis, and this might be the reason it does not show a reduction in expression like the other genes in the pathway. Serine Family. The serine and glycine biosynthesis pathways are distinctive in having none of the genes change their expression level greater than 2-fold at more than one time-point. The gene coding for serine acetyl transferase (cysE), the enzyme catalyzing the first step in the conversion of serine to cysteine, has reduced expression at both temperatures (∼2-fold at 33 °C and ∼4-fold at 28 °C), while o-acetylserine (thiol)-lyase A (cysK), one of the two enzymes catalyzing the second step, shows an ∼3-fold increase in expression at 33 °C and is unchanged at 28 °C. Aromatic Amino Acid Family. The aromatic amino acid pathway genes also remained largely unchanged. However, the expression level of anthranilate synthetase component 1 (trpE) decreases 4-8-fold. Anthranilate synthetase, which catalyzes the first step in the pathway of tryptophan synthesis from chorismate, is known to be the primary target of regulation in tryptophan synthesis. Component 1 catalyzes formation of anthranilate using ammonia (rather than glutamine), which is the nitrogen source in the media, and hence is probably the functional component of anthranilate synthetase in these culture conditions. Pyruvate Family. The expression level of many of the genes responsible for the synthesis of amino acids in the pyruvate family (alanine, leucine, and valine) is reduced after temperature shift. Among the genes involved in the first step in the synthesis of alanine, valine, and leucine, involving the conversion of pyruvate to 2-aceto-lactate, only one (ilvM) is differentially expressed. ilvM, a component of acetolactate synthase II, has a 2-fold decrease in expression at both temperatures, indicating that the flux through the entire pathway is probably reduced. The exceptions to this reduction in expression level are the 3-isopropylmalate dehydrogenase gene (leuB) and valinepyruvate aminotransferase (avtA). The expression level of leuB decreased by ∼2-fold after shifting to 33 and 28 °C but subsequently increases expression at 120 min to 4-fold higher and similar levels as compared to the initial level in the 33 °C sample and 28 °C sample, respectively. The valine-pyruvate aminotransferase (avtA) transcript has an ∼3-fold increase in expression at both temperatures. Aspartate Family. A similar effect is seen in the pathway for amino acids in the aspartate family (lysine, threonine, methionine, and isoleucine). The initial step in this pathway, conversion of aspartate to L-aspartyl4-P, is catalyzed by three isozymes, aspartokinase I (thrA), aspartate kinase II (metL), and aspartokinase (lysC). The intensities for all three isozymes are similar. Out of these, lysC, known to be inhibited by lysine, has a 2-4-fold decrease in expression, while thrA (inhibited by threonine) and metL are unchanged. This regulation of the first biosynthetic step again signifies a decreased flux through the entire pathway. Also, as before, many other genes in the pathway have reduced expression. Aspartate-ammonia ligase (asnA) and asparagine synthase B (asnB) related to interconversion between aspartate and asparagine have an ∼3-fold decrease in expression at both temperatures. Aspartate ammonialyase (aspA), which catalyzes the reversible conversion of aspartate into fumarate with the release of NH3, has an ∼16-fold decrease in expression at both 33 and 28 °C.
Figure 6. Expression profiles of pssA and psd involved in phospholipid metabolism. Data shown are log2 ratio of transcript level at time t to that at time 0. 9 pssA, b psd. - 33 °C, - - - 28 °C.
Transport. Forty-five transport related genes are differentially expressed at 28 °C and 42 at 33 °C. Out of these, the number of transporters with increased expression is relatively small: 12 and 16, respectively, at 28 and 33 °C, while those with decreased expression are 33 and 26, respectively. The expression profiles of the differentially expressed transport related genes are shown in Figure 7. Among the genes with increased expression are those related to choline, proline, and putrescine transport (betT, putP, potF, ∼2-fold); sulfate and thiosulfate transport (cysA, cysW, cysU, cysP, sbp, ∼3-4-fold); leucine transport (livK, livJ, ∼2-fold); uracil transport (uraA, ∼6-fold); ferric citrate transport (fecA); and R-ketoglutarate permease (kgtP, ∼16-fold) and a putative lipid A/glycerophospholipid transporter across the inner membrane (msbA). The R-ketoglutarate permease transporter has the highest increase in expression. In fact, it is the only carbohydrate transporter to have an increase in expression. Transport genes with decreased expression at 28 and 33 °C are mostly related to amino acid transport or sugar transport. In general, the intensities (and hence by inference, the abundance) of the genes for carbohydrate transport are much higher than those for amino acid transport. The amino acid transporters with reduced expression include the transporters of arginine (artJ, ∼6-fold, artP, ∼2-fold), glutamine (glnH, ∼2-fold), and serine (sdaC, ∼2-fold). The majority of the transporters with reduced expression at both temperatures belong to carbohydrate transport. A large fraction among the carbohydrate transporters is part of the PTS sugar transport system for glucose, fructose, and mannose. The PTS system components specific for glucose transport (ptsG, crr) have an ∼2-fold decrease in expression. Glucokinase, glk, which under normal conditions plays a minor role in glucose metabolism, also has reduced expression (∼4-fold). The extent of decrease in expression of fructose and mannose specific components (4 and 4-8-fold, respectively) of the PTS system (fruA, fruB, manX, manY, manZ) is more than that for the glucose specific components at both temperatures. At the reduced temperature, glucose consumption rate decreased by 41% and 51% at 33 and 28 °C, respectively. The downregulation is effected through the reduced expression of transporters. If protein expression is also altered at a similar ratio of ∼2-fold as the transcripts for the glucose transport component of PTS, the maximum transport rate will be reduced by one-half. This modulation is certainly sufficient to accommodate the reduced flux
I
Figure 7. Expression profiles of transport genes that are differentially expressed. Data shown are log2 ratio of transcript level at time t to that at time 0. - 28 °C, - - - 33 °C.
through glycolysis even with only moderate changes in glycolysis genes. Protein Synthesis. As the cultivation temperature decreases after temperature shift, the corresponding growth rate decreases, and so should the rate of protein synthesis. As can be seen from Figure 2, a surprisingly small number of genes related to protein synthesis and folding have reduced expression. Two heat shock genes, clpB and groEL, show a decrease in expression on reducing temperature. hscA, a chaperone involved in the maturation of iron-sulfur cluster-containing proteins previously shown to be induced at low temperatures (36), has increased expression. It has a slightly higher increase in expression at 28 °C (∼4-fold) than at 33 °C (∼3-fold). Translation initiation is thought to be the rate-limiting step of translation at lower temperatures (37, 38). Transcripts of translation initiation factor 2 and 3 (infB, infC) remain unchanged with temperature. However, infA, encoding translation initiation factor 1, has an ∼2-fold increase in expression at both temperatures. infC has the lowest fluorescent intensity (∼700) followed by infA (∼3000) and infB (∼12 000) (for reference, the average fluorescent intensity for the ribosomal subunit proteins is 11 000). infA has been shown to be an essential gene (39), but no clear function has been assigned to it. Our results indicate that temperature might be one of the signals regulating expression of infA. Also, its increased expression suggests an important role in translation initiation at lower temperatures. Fatty Acid and Phospholipid Metabolism. Overall, the level of changes in fatty acid and lipid metabolic
pathways is relatively small upon temperature shift. The expression level of the fabA gene product, which catalyzes a key reaction at the point of divergence of unsaturated fatty acid biosynthesis from saturated fatty acid biosynthesis, remains unchanged after temperature shift. fabB (β-ketoacyl-ACP synthase I), which is also required for unsaturated fatty acid biosynthesis, has an ∼1.4-fold decrease in expression initially for 20 min and then increases expression to 1.4-fold upregulation at 2 h. Thermal regulation of fatty acid composition is achieved through β-ketoacyl-ACP synthase II (fabF), which is more active at low temperatures than at high temperatures (40). The fabF gene has increased expression at both temperatures (more at 28 °C then at 33 °C) although not more than 2-fold. Also, fabH (β-ketoacyl-ACP synthase III), involved in the initial steps of fatty acid elongation, shows an ∼1.6-fold increase in expression level at both temperatures. The changes in fabB and fabF are consistent with their role in the synthesis of unsaturated fatty acids, while the changes in fabH observed have not been reported before. Lai et al. (41) have reported that fabH is an essential gene. They have also shown that in Gram positive Lactococcus lactis, a fabH- requires only longchain unsaturated fatty acids for growth; long-chain saturated fatty acids are not required. This suggests that fabH might play some role in unsaturated fatty acids synthesis and is regulated at a transcriptional level by temperature. Three genes involved in phospholipid synthesis are differentially expressed. These include a subunit of ethanolamine ammonia-lyase (eutB) catalyzing the con-
J Table 1. Comparison of Results from Real-Time PCR and Microarray Hybridizationa gene
temp (°C)
real-time PCR
microarray
cydA pykA sdhB sucB cydA cyoA pykA sdhB sucB
28 28 28 28 33 33 33 33 33
-1.8 -2.7 2.3 3.5 -1.4 1.8 -1.8 3.9 2.8
-2.0 -1.5 3.3 1.2 -3.3 3.8 -2.0 3.2 0.9
a
All values are log2 (ratio).
version of ethanolamine to acetaldehyde and phosphatidylserine decarboxylase (psd), involved in the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE). Both of these genes have an ∼3-4-fold decrease in expression at both temperatures, while phosphatidylserine synthase (pssA), involved in the formation of PS, has an ∼3-4-fold increase in expression at both temperatures (Figure 6). This suggests a likely decrease in the synthesis of PE in the cell and an increase in the accumulation of PS. PE plays a key role in modulating membrane proteins. Inverted vesicles from membranes of PE-deficient mutants had a depressed type II NADH dehydrogenase-dependent oxidase activity reducing their ability to generate a proton gradient using NADH (42). These membranes were also shown to have reduced levels of cytochrome d. Thus, the phospholipids content of the membrane appears to have an effect on membrane mediated energy transduction. The gene expression changes described above indicate a likely change in PE and PS composition in the membrane, which might lead to the need to reorganize the cytochrome d and o content in the cell, as described earlier.
Overall Analysis of the Effect of Temperature Downshift on Gene Epxression This study examined the effect of temperature downshift, a practice commonly followed in the production of rDNA proteins, on global gene expression in E. coli. Reducing the temperature to both 28 and 33 °C gave very similar patterns of alteration in gene expression. This consistency gives credence to the results obtained from DNA microarray. We further verified the results by independent measurements of relative gene expression of five selected genes by the real-time PCR assay using samples at the 0 and 20 min time-points for 28 and 33 °C. Comparison of the results from microarray hybridization and real-time PCR is shown in Table 1. Each value presented in Table 1 is an average of two replicate measurements. The results of these analyses are reassuring in that the changes in gene expression obtained from the two methods agree well in both direction and magnitude of the observed changes. The technique of shifting temperature to enhance the proper folding of recombinant proteins is widely used. Overall, the changes in gene expression after temperature shift are rapid and globally distributed over all classes of cellular activities. A reduction in growth temperature causes a decrease in the growth rate and in the OUR and glucose uptake rate. The decrease in glucose uptake rates is consistent with the decreased expression level of transcripts of genes involved in the glucose transport components of the PTS system and glycolysis. Despite the reduced OUR and glucose uptake rates at lower temperatures, TCA cycle-related genes show increased expression at lower temperatures. It is
possible that the increased expression level of these enzymes is to compensate for the decreased activity of the TCA cycle enzymes at the lower temperatures (27, 28) and to maintain a sufficient flux through TCA cycle. We also observed a switch of cytochrome systems from the high affinity cytochrome d to low affinity cytochrome o upon temperature downshift, the reason for which is unknown. The gene expression survey results do not demonstrate increased expression of protein folding machinery. The extent of temperature shift carried out in this study and in rDNA protein production does not appear to greatly affect the expression of stress related genes, such as heat shock proteins. The results may indicate that the main beneficial effect of reduced cultivation temperature for E. coli resides in the overall reduction of metabolic rate without resorting to limiting the growth rate through nutrient limitation.
Acknowledgment The bioinformatic support for this work was provided by the University of Minnesota Supercomputing Institute.
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Accepted for publication February 25, 2005. BP049630L
