Thermus thermophilus Proteins That Are Differentially Expressed in

Dec 17, 2009 - ... Hangzhou 310058, The People's Republic of China. Phone: Tel.: 86-571-88981129. Fax: 86-571-88981129. E-mail: [email protected]., â...
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Thermus thermophilus Proteins That Are Differentially Expressed in Response to Growth Temperature and Their Implication in Thermoadaptation Hebin Li,‡,# Xinglai Ji,§,# Zhidong Zhou,‡ Yiqian Wang,‡ and Xiaobo Zhang*,† The Key Laboratory of Conservation Genetics and Reproductive Biology for Wild Animals of the Ministry of Education and College of Life Sciences, Zhejiang University, Hangzhou 310058, The People’s Republic of China, Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, The People’s Republic of China, and Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, 650091, The People’s Republic of China Received August 26, 2009

As a kind of important extremophiles to realize the adaptation of life at high temperatures, thermophiles have attracted extensive studies. However, the pathways of thermophile proteins related to thermoadaptation remain to be addressed. Our study showed that there existed two types of protein profiles for the thermophile Thermus thermophilus wl in response to temperature change. One of them came from cultures growing below 65 °C, which was close to the optimal growth temperature, and another from cultures at or above 65 °C. These protein profiles were confirmed by Northern blots. On the basis of the proteomic and computational analyses, it was found that the thermophile proteins related to thermoadaptation might be involved in metabolic pathways as well as the stabilities and modifications of DNA and proteins. Interestingly, for the basic metabolism glycolysis, the phosphoglucomutase was up-regulated at below-optimum temperature, while the glyceraldehyde-3-phophate dehydrogenase was up-regulated at above-optimum temperature, suggesting that different regulations might be used for basic metabolism at different temperatures. To characterize the proteins in response to high temperatures, superoxide dismutase (SOD), an important enzyme in organism to remove free radical produced in stress environment such as high temperature, was selected as a target protein for this investigation. SOD was inactivated to construct a SOD mutant. The results showed that the SOD protein was essential in thermoadaptation of T. thermophilus. Our study, therefore, presented the thermophile proteins required for thermoadaptation and their possible pathways in thermoadaptation. Keywords: Thermoadaptation • Thermus thermophilus • Protein • Pathway

Introduction Extremophilic microorganisms, such as thermophiles, psychrophiles, acidophiles, alcalophiles, halophiles and barophiles, could be found thriving within extreme environments hostile to human beings. Among extremophiles, thermophilic microorganisms have attracted intensive investigations, resulting in some exciting research progresses in recent years. Thermophile is a kind of very important microbial resource with scientific value and applied foreground.1 In these years, thermostable enzymes from extreme thermophiles have led to a special focus due to their intrinsic thermostability and resistance to denaturing physical and chemical factors. Up to date, more than 70 * To whom correspondence should be addressed: College of Life Sciences, Zhejiang University, Hangzhou 310058, The People’s Republic of China. Phone: Tel.: 86-571-88981129. Fax: 86-571-88981129. E-mail: zxb0812@ zju.edu.cn. ‡ Third Institute of Oceanography. § Yunnan University. # These authors had equal contributions. † Zhejiang University. 10.1021/pr900754y

 2010 American Chemical Society

genera and 140 species of thermophiles have been isolated from a variety of thermal environments.2 In 2003, the discovery of archaeon strain 121, which can grow at 121 °C, renews the upper temperature limit for life.3 In contrast to usual mesophiles, thermophiles are a group of microorganisms which can grow and proliferate normally at the temperature over 60 °C. Thermophiles adapt to hot environments by their physiological properties. As a consequence, cell components such as nucleic acids, proteins and membranes have to be stable and even function best at temperatures even around 100 °C.4,5 Evolutionary biology has long attempted to find the adaptive mechanism of nucleic acids molecules under high growth temperatures. Considerable variation in the guanine (G) and cytosine (C) content exists among the microorganisms, suggesting a link between GC content and thermoadaptation.6 As reported by Basak et al., however, it is indicated that any attempt to obtain a generalized relation between genomic GC composition and optimal growth temperature would hardly evolve any satisfactory result.7 To better understand the microbial response and adaptation to Journal of Proteome Research 2010, 9, 855–864 855 Published on Web 12/17/2009

research articles high temperatures, some researches on the structural basis of enzyme thermostability and the adaptation strategies for survival at high temperatures have been documented.8,9 It is found that the catalytic capacity and thermal stability of Thermus thermophilus glycoside-hydrolyzing enzyme can be improved by an in vivo evolution selection within a thermophilic host cell.9 As reported by Forterre et al.,8 the reverse gyrase from hyperthermophiles may be a probable transfer of a thermoadaptation trait from archaea to bacteria. Except for enzymes, heat-shock proteins are suggested to be strongly associated with heat tolerance and heat adaptation.10 However, it is hard to outline the adaptation strategies of thermophiles in response to high temperatures in studies on individual proteins. One of the emerging principles in biology is that it is generally not individual genes but biological pathways and networks that drive an organism’s response to a wide range of stimuli.11 In this context, to realize the thermoadaptation of thermophiles, it is essential to identify proteins involved in thermophilic metabolism. To this end, a proteomic approach using mass spectrometry (MS) has been proven to be the most effective technology for the identification of potential proteins. Proteomic techniques are now obviously the overwhelming methods in studies of proteins differentially expressed in altered conditions.12,13 To date, the genome sequences of different thermophiles T. thermophilus HB27 (GenBank accession number AE017221) and HB8 (GenBank accession number AP008226) have been sequenced, which will facilitate the proteomic analysis. The genus Thermus belongs to one of the oldest phylogenetic branches of bacterial evolution,14 being among the thermophilic bacteria yet known, which can grow at temperatures over 75 °C. They are usually aerobic, rod-shaped and nonsporulating Gram-negative bacteria. Because of their aerobic characters and rapid growth rates and the biotechnological applications of their thermophilic enzymes, different isolates from this genus are currently being studied as putative hosts for the expression of multimeric thermophilic enzyme complexes and genetic studies. In an attempt to reveal the proteins and their pathways involved in thermoadaptation of T. thermophilus, a universal hot spring bacterium, the proteomic approach and the computational analysis were applied in the present study. The results of SDS-PAGE, Tricine-SDS-PAGE and two-dimensional gel electrophoresis (2-DE) showed that 21 proteins were differentially expressed at different temperatures, producing 20 unique protein identifications as revealed by MS. The proteins related to thermoadaptation might be involved in metabolic pathways as well as the stabilities and modifications of DNA and proteins.

Experimental Procedures Thermophilic Bacterial Strain and Thermophile Culture. T. thermophilus strain wl was collected and identified as described previously.15 Briefly, the strain wl was assigned to T. thermophilus wl on the basis of 16S rRNA sequence analysis (GenBank accession number AY497773). The routine culture of T. thermophilus wl was performed with TM broth medium (0.4% tryptone, 0.2% yeast extract, 0.1% NaCl and basal salt, pH 7.5) at different temperatures (55, 60, 65, 70, 75, and 80 °C).16 Sample Preparations for Protein Electrophoresis. The overnight culture of T. thermophilus wl was diluted at 1:100 with TM medium and inoculated to fresh TM medium for 856

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Li et al. continuous growth to late log phase. Then, cells were harvested by centrifugation at 5000g for 8 min. After three washes with sterile phosphate buffered saline (PBS, pH 7.4), the pellets were resolved in 1× lysis buffer (50 mM Tris-HCl, 10% glycerol, 2% SDS, 1% β-ME, pH 6.8) as samples for SDS-PAGE. The pellets as prepared above were resolved in 1× Tricine-lysis buffer (50 mM Tris-HCl, 12% glycerol, 4% SDS, 2% β-ME, 0.01% Coomassie blue G250, pH 6.8) and used for Tricine-SDS-PAGE. For 2-DE sample preparations, it was prepared as described by Eschenbrenner et al. with modifications.17 The pellets were resuspended in 7 mL of sterile TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.4), followed by the addition of nucleases (2000 U/mL DNAase, 1750 U/mL RNAase A, 50 mM MgCl2) and 35 µL of protease inhibitor Cocktail I (Serological Corporation). Subsequently, cells were disrupted by intermittent sonication with Sonics Vibra Cell (Sonics & Materials, Inc.) for 90 s on ice. After centrifugation at 5000g for 15 min, unbroken cells and cellular debris were removed. The supernatants were further centrifuged at 100 000g for 30 min at 4 °C. The protein samples were precipitated with 3-4 vol of ice-cold acetone (0.05% β-ME) at 4 °C for at least 2 h and subsequently centrifuged for 10 min (15 000g) at 4 °C. The pellets were lyophilized and resuspended in 2-DE sample buffer (8 M urea, 4% CHAPS, 100 mM DTT, 2% 3/10 carrier ampholine (GE Healthcare). The concentrations of the protein samples were determined using Bradford Protein Assay Kit (Bio-Rad). SDS-PAGE. Samples were heated for 5 min in boiling water, followed by protein separation with 12% SDS-PAGE. The protein bands were visualized by staining with Coomassie Blue R-250. Sample preparation and SDS-PAGE separation of proteins from T. thermophilus wl were repeated six times. Tricine-SDS-PAGE. Samples were heated for 10 min in boiling water. Then, Tricine-SDS-PAGE was conducted at 30 V for 1 h, followed by electrophoresis at 30 mA for 16 h.18 After electrophoresis, the gel was fixed in a solution containing 50% ethanol and 10% acetic acid for 1 h and stained with 0.5% brilliant blue R-250 in 10% acetic acid for at least 2 h, followed by shaking in 10% acetic acid for 2 h to destain the gel. The Tricine-SDS- PAGE assay was performed with six biological replicates. 2-DE. In the first dimension, isoelectric focusing (IEF) of the protein samples (about 300 µg each) was performed on immobilized pH gradient gel (IPG) strip (pH 3-10, 13 cm) (GE Healthcare) with IPGphore (GE Healthcare) by soaking the IPG strip in protein samples. The IPG strip, rehydrated at 50 V for 12 h at 20 °C, was focused using a 3-step program (500 V for 1 h with rapid ramping, 1000 V for 1 h with rapid ramping, and 8000 V for 3 h with rapid ramping until 16 000-20 000 Vh was reached). Upon completion of electrophoresis in the first dimension, the IPG gel strip was incubated in equilibration buffer (6 M urea, 50 mM Tris-HCl, 2% SDS, 30% glycerol, 0.002% bromophenol blue, pH 8.8) containing 1% (w/v) DTT for 15 min before being washed for a further 15 min in equilibration buffer containing 2.5% (w/v) iodoacetamide (Sigma). The second-dimensional separation was performed on 12% SDS-polyacrylamide gel by placing the IPG strip at the top of SDS-polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Blue R-250 and digitized using GS710 Calibrated Imaging Densitometer (Bio-Rad). Spot detection and quantification were performed using the PDQUEST 6.2 software package (Bio-Rad). All the above assays including the culture of T. thermophilus wl, the sample preparation and

Thermophile Proteins Involved in Thermoadaptation protein separation by 2-D were repeated with six biological replicates to obtain the comparable protein patterns. MALDI-TOF MS. MALDI-TOF of samples was performed as described by Zhang et al. with a small modification.19 Protein bands or spots were excised and dehydrated several times with acetonitrile (ACN). After vacuum drying, the gel bands or spots were incubated with 10 mM DTT in 100 mM ammonium bicarbonate (ABC) buffer at 57 °C for 60 min and subsequently with 55 mM iodoacetamide (Sigma) in 100 mM ABC buffer at room temperature for 60 min. Then the gels were washed with 100 mM ABC buffer and dried. All in-gel protein digestions were performed using sequencing grade modified porcine trypsin (Promega) in 50 mM ABC buffer at 37 °C for 16 h. Digests were centrifuged at 6000g. The supernatants were separated, and the gel pieces were extracted further first with 50% ACN, 5% formic acid and then with ACN. The extracts were combined with the original digesting supernatants, vacuum-dried, and redissolved in 0.5% trifluoroacetic acid (TFA) and 50% ACN. A 1.5 µL aliquot was spotted onto a MALDI-TOF sample plate with equal volume of matrix. The matrix used was a saturated solution of R-cyano-4-hydroxycinnamic acid (CHCA) in 1% TFA and 50% ACN. MALDI-TOF spectra of the peptides were obtained with a time-of-flight delayed extraction MALDI MS (Bruker Autoflex, Germany). A nitrogen laser (337 nm) was used to irradiate the sample. Spectra were acquired in reflectron mode in the mass range of 600-3500 Da. A near point calibration was applied and a mass tolerance of 100 ppm was used. Data mining was performed using Mascot search engine against the ORF database of T. thermophilus HB27. A mass deviation of 100 ppm and modifications such as carbamidomethyl, oxidation and Pyro-glu were usually allowed in the database searches. Northern Blot. The total RNAs were isolated from T. thermophilus wl cultured at different temperatures using Trizol reagent (Promega) according to the manufacturer’s instructions. After treatment with RNase-free DNase I (TakaRa, Japan) for 30 min at 37 °C, RNAs were separated by electrophoresis on a 1.2% agrose gel (containing 3.7% formaldehyde) in 1× TBE buffer and transferred to a nitrocellulose membrane (GE Healthcare). The blots were, respectively, probed with the DIGlabeled T. thermophilus wl genes upregulated at higher temperature, as well as the 16s rRNA gene. The T. thermophilus wl genes and the 16s rRNA gene were amplified by PCR using the T. thermophilus wl genomic DNA and labeled with DIG. In vitro RNA labeling, hybridization, and signal detection were carried out according to the manufacturer’s instructions of DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany). The blots were scanned and the density of blot at 55 °C was designated as 100%. Computational Analysis. BLAST search was performed on the proteins against NCBI NR (Non-Redundant) and Swiss-Prot databases. The BLAST results were then used by Blast2GO20-22 for functional annotation, including GO (Gene Ontology) term annotation and KEGG pathway mapping. GO enrichment analysis was performed based on the GO term annotations. KEGG pathways were also mapped with KAAS server.23 The KEGG pathways were sorted based on the KEGG orthology. The proteins were connected if they were involved in a same pathway or the associated pathways were linked. Construction of T. thermophilus wl SOD Mutant (∆SOD). To characterize the proteins in thermoadaptation, the SOD gene, selected as a target gene, was inactivated by inserting a kanamycin nucleotidyltransferase gene (kat) into its coding

research articles region to construct a SOD mutant of T. thermophilus wl. The insertion of kat, which encoded a thermostable protein resistant to kanamycin, would facilitate the screening for ∆SOD. A SOD-gene- containing DNA fragment was amplified from T. thermophilus wl genomic DNA using primers 5′-AAGGCCCTCAAGGAGAAG-3′ and 5′-TCGCCCGGGCCTGCCTGG-3′. This fragment consisted of the entire ORF encoding SOD, a 500-bp DNA upstream of the start codon and a 500-bp DNA downstream of the stop codon. After PCR amplification from the Escherichia coli/T. thermophilus wl shuttle vector pMK18 with primers 5′-GGATCCCCG GGAGTATAAC-3′ and 5-GGATCCTCTAGAGTCGAC-3′,24 the kat gene was inserted into the 303-304 site of SOD ORF by PCR. Subsequently, the recombinant DNA fragment, containing the disrupted SOD ORF and its upstream and downstream 500-bp DNAs and the kat gene, was obtained in quantity by PCR. After purification and addition in the TM medium, the recombinant DNA fragment was transformed into T. thermophilus wl by natural transformation at 65 °C. The transformants were screened on the TM medium containing 40 µg/mL kanamycin at 65 °C to obtain ∆SOD. The ∆SOD was further confirmed by DNA sequencing, Southern blot, Northern blot and Western blot. Southern Blot. The genomic DNAs of T. thermophilus wl and ∆SOD strains were extracted with SQ Tissue DNA Kit (OMEGA). Subsequently, they were incompletely digested by EcoRI and transferred onto Hybond-N+ membrane (GE Healthcare), respectively. The membrane was probed with DIGlabeled kat fragment. The DIG labeling and detection were conducted according to the recommended protocol of DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany). Protein Recombinant Expression in E. coli and Antibody Preparation. The SOD-encoding gene of T. thermophilus wl was cloned into pET-28a vector (Novagen, Germany) and expressed in E. coli BL21 (DE3) as a 6× His-tagged fusion protein with primers 5′-CGCGGATCCCCGTACCCGTTCAAGCT3′ (BamHI site, italic) and 5′-CGG CGGCCGCGGCCTTCTTGAAGAACT-3′ (NotI, italic). The recombinant bacteria, confirmed by DNA sequencing, were induced by isopropyl-β-D-thiogalactoside (IPTG) when the OD600 reached 0.5. After further incubation for 6 h at 37 °C, the induced cells were harvested by centrifugation at 4000g for 5 min. The recombinant protein was purified by affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) resins under native conditions according to the recommended protocol (Qiagen). The purified SOD fusion protein was used as antigen to immunize mice according to standard procedure.25 The immunoglobulin (IgG) fraction of antiserum was purified by protein A-Sepharose (Bio-Rad) and stored at -80 °C. As determined by enzyme-linked immunosorbent assay (ELISA), the titers of antisera were 1:10 000. The specificity of antibody was confirmed by Western blot with the recombinant SOD protein. Western Blot. Proteins separated by SDS-PAGE were transferred to nitrocellulose membrane (Bio-Rad) in electroblotting buffer (25 mM Tris, 190 mM glycine, 20% methanol) for 70 min. The membrane was immersed in blocking buffer (0.1% skimmed milk, 20 mM Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.2) at 4 °C overnight, followed by incubation with a polyclonal mouse antiHis-SOD for 3 h. Subsequently, the membrane was incubated in alkaline phosphate-conjugated goat anti-mouse IgG (Pierce Biotechnology, Inc.) for 1 h and detected with NBT and BCIP solutions (Amresco, Inc.). Journal of Proteome Research • Vol. 9, No. 2, 2010 857

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Figure 1. Separation of T. thermophilus wl proteins differentially expressed at different temperatures by SDS-PAGE (A), Tricine-SDSPAGE (B) and 2-DE (C and D). The gels were stained with Coomassie blue. For 2-DE of T. thermophilus wl cultured at 55 °C (C) or 75 °C (D), the protein samples were separated in the first dimension by IEF, followed by separation in the second dimension by 12% SDS-PAGE. The numbered bands and spots were excised and digested with trypsin. Peptides from the unfractionated tryptic digests were analyzed by MS. All the above assays including the culture of T. thermophilus wl, the sample preparation and protein separation by electrophoresis were repeated six times. The major differential bands/spots with consistent expression patterns in all of the six independent assays or with more than 50% intensity difference at 55 and 75 °C were excised and subjected to mass spectral analysis. M: protein marker.

Statistical Analysis. The numerical data from independent experiments were analyzed by one-way ANOVA to calculate the mean and standard deviation of repeated assays.

Results Separation and Identification of T. thermophilus wl Proteins Involved in Thermoadaptation. The growth assays indicated that T. thermophilus wl grew within a temperature 858

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range of 45-80 °C with an optimum at 70-75 °C. To identify the bacterial proteins involved in thermoadaptation, T. thermophilus wl cultured at different temperatures was subjected to SDS-PAGE analysis. The results showed that there appeared two protein profiles (Figure 1A), one of which came from cultures growing below 65 °C, which was close to the optimal growth temperature, and another from cultures at or above 65 °C. As revealed in Figure 1A and Table 1, many differentially

pI

5.7 8.3 8.2 9 6.2 4.2 6.8 7.2 6.3 5.4

metabolism

methylation stability, sulfur metabolism, methylation metabolism stability, SOD stability ferritin metabolism stability

metabolism

metabolism metabolism, sulfur metabolism

metabolism stability stability n/a ferritin

TA2

TA3 TA4 TA5

TA6 TA7 TA8 TA9 TA10 TA12

TA13

TA14 TA15 TA16

TA17 TA18 TA19 TA20 TA21

524 466 496 285 296 284 204 97 330 317 97 658 395 361 404 331 378 228 186 181 134

aa

56.3 50.8 52.6 32.9 33 30.7 23.2 10.4 36.1 35.6 10.4 72.5 43.4 39.6 45.3 35.9 41.4 25.7 21.6 20.5 14.1

mass

sizea

8 7.2 6.5 9 7.1 6.6 5.9 6.4 5.3 5.2

pI

enzyme codes

18 37 13 20 44 15 41 40 17 31 26 37 36 22 51 28 19 67 58 13 25

sequence coverage (%)

0 0 276.7 ( 11.1 149 ( 14 287.7 ( 8.5 0 286.3 ( 8.14 157 ( 7.21 131 ( 3.61 120.6 ( 6.01 131.6 ( 13.01 123 ( 8.2 54.6 ( 4.0 91.3 ( 3.1 99.3 ( 2.51 119.7 ( 3.8 130 ( 4.4 144.7 ( 7.6 103.7 ( 4.5 49.7 ( 1.5 309.3 ( 11.2

309.3 ( 11.2 363.7 ( 11.1 0 0 0 375.3 ( 20.7 166 ( 7.6 0 60.7 ( 2.5 55.7 ( 4.0 0 80.7 ( 2.1 0 0 0 72.3 ( 4.5 0 72 ( 3 42.7 ( 2.5 0 0

2.1.2.11:3-methyl-2-oxobutanoate hydroxymethyltransferase 1.15.1.1:Superoxide dismutase n/a n/a 4.99.1.1:Ferrochelatase 3.6.5.1:Heterotrimeric G-protein GTPase 3.6.5.3:Protein-synthesizing GTPase 3.6.5.2:Small monomeric GTPase 3.6.5.4:Signal-recognition-particle GTPase 2.3.1.29:Glycine C-acetyltransferase 2.6.1.-:Transaminases (aminotransferases) 2.3.1.47:8-amino-7-oxononanoate synthase 3.5.1.-:In linear amides 1.8.3.1:Sulfite oxidase 1.2.1.12:Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) 1.3.99.2:Butyryl-CoA dehydrogenase n/a n/a n/a n/a

n/a 2.8.1.1:Thiosulfate sulfurtransferase 1.5.1.20:Methylenetetrahydrofolate reductase (NAD(P)H)

e

40 n/a 9 n/a 13

20 22 20

31

10 13 6 8 11 12

20 19 14

11

32

number of GO terms

75 °C

intensityb 55 °C

5.4.2.2:Phosphoglucomutase 5.4.2.-:Phosphotransferases (phosphomutases) 4.2.1.2:Fumarate hydratase

TA1 TA2 TA3 TA4 TA5 TA6 TA7 TA8 TA9 TA10 TA8 TA12 TA13 TA14 TA15 TA16 TA17 TA18 TA19 TA20 TA21

gene

6 n/a n/a n/a n/a

2 1 14

4

4 1 n/a 1 4 n/a

n/a n/a 6

13

14

characteristics of deduced proteins

Below optimum Below optimum Above optimum Above optimum Above optimum Below optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum Above optimum

upregulation at temperature

Metabolism f Amino Acid Metabolism MetabolismfEnergy Metabolism Human Diseases f Neurodegenerative Diseases Metabolism f Carbohydrate Metabolism Metabolism f Xenobiotics Biodegradation and Metabolism n/a n/a n/a n/a

Metabolism f Amino Acid Metabolism

Metabolism f Biosynthesis of Secondary Metabolites Metabolism f Carbohydrate Metabolism Human Diseases f Cancers Metabolism f Energy Metabolism Metabolism f Carbohydrate Metabolism n/a n/a Metabolism f Energy Metabolism Metabolism f Metabolism of Cofactors and Vitamins Metabolism f Metabolism of Cofactors and Vitamins Human Diseases f Neurodegenerative Diseases n/a Environmental Information Processing f Membrane Transport Metabolism f Metabolism of Cofactors and Vitamins n/a

pathway orthologyd

phosphoglucomutase fumarate hydratase methyltransferase thiosulfate sulfurtransferase methylenetetrahydrofolate reductase 3-methyl-2-oxobutanoate hydroxymethyltransferase superoxide dismutase putative DNA binding protein iron(III)-binding protein ferrochelatase putative DNA binding protein translation elongation and release factors probable glycine C-acetyltransferase putative N-acetyllysine deacetylase probable sulfite reductase glyceraldehyde 3-phosphate dehydrogenase acyl-CoA dehydrogenase, short-chain specific heat-stable protein putative ribosomal associated protein hypothetical conserved protein bacterioferritin

number of KEGG pathways

AAS81972 AAS80538 AAS80421 AAS81014 AAS81998 AAS80387 AAS80537 AAS81326 AAS81606 AAS80579 AAS81326 AAS81476 AAS81561 AAS81738 AAS81303 AAS80897 AAS80586 AAS81461 AAS81271 AAS81085 AAS80966

GenBank accession no.

a The predicted molecular masses (in kilodaltons) and pIs from the sequence. aa, amino acids. b Intensity was represented as mean ( standard deviation within ( 1% standard deviation. c For space limit, the detailed annotations were available at http://rich.yunda.org/test/ttwl02/. d The classification of the KEGG pathways. e n/a: not available.

metabolism

TA1

categoriesc

59 55 53 36 35 28 26 13 32 30 12 70 46 42 42 40 39 27 20 18 15

Band 1 Band 2 Band 3 Band 4 Band 5 Band 6 Band 7 Band 8 Band 9 Band 10 Band 11 Spot 1 Spot 2 Spot 3 Spot 4 Spot 5 Spot 6 Spot 7 Spot 8 Spot 9 Spot 10

gene

mass

bands/ spot nos.

gel data

Table 1. T. thermophillus wl Proteins Involved in Thermoadaptation

Thermophile Proteins Involved in Thermoadaptation

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Thermophile Proteins Involved in Thermoadaptation

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Figure 2. Transcriptions of T. thermophilus wl genes involved in thermoadaptation by Northern blot. The total RNAs extracted from T. thermophilus wl cultured at different temperatures were probed by 17 DIG-labeled genes up-regulated at higher temperatures, respectively. The 16s rRNA gene was used as control. Each column represented the mean of triplicate assays within (1% standard deviation. The headings indicated the growth temperatures of T. thermophilus wl. The density at 55 °C was designated as 100%.

expressed proteins (more than 5-fold change) could be found at temperatures below and above 65 °C. In an attempt to obtain the differentially expressed proteins with lower molecular masses, Tricine-SDS-PAGE of T. thermophilus wl was conducted and yielded essentially the same results as those of SDSPAGE (Figure 1B, Table 1). The protein bands with consistent expression patterns in all of the six independently repeated experiments were picked up for MS identification (Table 1). Among the differentially expressed proteins, eight protein bands from SDS-PAGE and three bands from Tricine-SDSPAGE were excised for MS identification. The protein electrophoresis data of SDS-PAGE and Tricine-SDS-PAGE suggested that the optimal growth temperature was a threshold point for proteins of T. thermophilus wl in response to temperature change. At or above the optimal growth temperature, the thermophile would initiate the expressions of proteins involved in thermoadaptation. On the basis of SDS-PAGE and Tricine-SDS-PAGE analyses, the T. thermophilus wl cultured at 55 or 75 °C was analyzed by 2-DE to reveal more differentially expressed proteins. After protein separation, up to 400 polypeptide spots could be detected by Coomassie staining. By comparison of the protein profiles at 55 and 75 °C, many differentially expressed protein spots were observed (Figure 1C and 1D, Table 1). The major differential spots with consistent expression patterns in all of the six independent assays at 55 and 75 °C were excised and subjected to mass spectral analysis (Table 1). Peptide mass fingerprinting (PMF) was obtained by tryptic in-gel digestion and MALDI-TOF MS analysis. After searches in the T. thermophillus HB27 ORF database, a total of 11 differentially expressed protein bands from SDS-PAGE or Tricine-SDS-PAGE and 10 differentially expressed protein spots from 2-DE were identified by their peptide fingerprints, covering 13-67% of amino acid sequences (Table 1). The results indicated that band 8 from SDS-PAGE and band 11 from Tricine-SDS-PAGE matched the same protein encoded by TA8 gene, therefore, yielding 20 unique protein identifications as revealed by MS. Among the 20 differentially expressed proteins, 17 proteins were significantly up-regulated at temperatures above the

optimal growth temperature of T. thermophilus wl, while 3 proteins were up-regulated at temperatures below the optimal temperature. Transcription Analyses of Genes Involved in Thermoadaptation. In an attempt to confirm the mass spectral data, the transcription analyses of T. thermophilus wl genes encoding the 17 up-regulated proteins at higher temperatures were conducted by Northern blot. After labeling with DIG, these genes were hybridized with total RNAs extracted from T. thermophilus wl cultured at different temperatures, respectively. To facilitate quantitative analysis, the 16S rRNA gene was included in the hybridizations as a reference gene.26-29 The results indicated that the 17 genes were significantly upregulated (more than 50%) when T. thermophilus wl grew above the optimal growth temperature, while the transcription level of the 16S rRNA gene remained constant (Figure 2, Supporting Information Figure S1). The transcription analyses yielded the similar results as those of protein electrophoresis. On the basis of the statistic analyses, the upregulations for the 17 genes were significant. The gene transcription analysis, therefore, confirmed the data as revealed by mass spectrometry analyses. Pathways of Thermophile Proteins Involved in Thermoadaptation. On the basis of homology searches using BLAST and PROSITE analyses in GenBank, 19 of the 20 differentially expressed proteins shared homologies to known proteins, except for a hypothetical conserved protein (TA20, AAS81085) revealed in spot 9 (Table 1). The computational analyses showed that 18 proteins were assigned a total of 192 GO terms (Table 1). Two proteins, annotated as heat stable protein (TA18, AAS81461) and hypothetical conserved protein (TA20, AAS81085), were not assigned any GO terms. GO enrichment analysis revealed that the majority of the proteins had catalytic activity or binding function. Thirteen proteins were annotated with enzyme codes. In addition, most of the proteins were involved in primary or cellular metabolic process or biosynthetic process. The results of KEGG pathway mapping were consistent with GO term annotations (Table 1). Twelve of the 20 proteins were mapped in 50 KEGG reference pathways, including both general and KO reference pathways. The remaining 8 proteins were not mapped in any KEGG pathways, including the 2 Journal of Proteome Research • Vol. 9, No. 2, 2010 861

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Figure 3. Construction and characterization of SOD mutant of T. thermophilus wl (∆SOD). (A) Southern blot of ∆SOD and wild-type strains with DIG-labeled kat gene. (B) Northern blot of ∆SOD and wild-type strains with DIG-labeled SOD gene. (C) Western blot of ∆SOD and wild-type strains with SOD-specific antibody. (D) Growth curves of ∆SOD and wild-type strains. Each point represented the mean of triplicate assays (n ) 3) within (1% standard deviation. M, protein marker.

proteins without GO term annotations. It was found that 10 proteins were involved in metabolic pathways. Interestingly, protein linkage analysis showed that these 10 proteins were involved in a same pathway or linked pathways. Among the other proteins, the bacterioferritin (TA21, AAS80966) and iron(III)-binding protein (TA9, AAS81606) might be involved in energy metabolism. The putative DNA binding protein (TA8, AAS81326) and putative ribosomal associated protein (TA19, AAS81271) were supposed to be related to chromosome stability. These proteins might form a more compact structure on the DNA strands at high temperature to protect thermophiles from insult by high temperature. The heat-stable protein (TA18, AAS81461) might be involved in promoting and maintaining the stability of macromolecules at high temperature. The SOD (superoxide dismutase, TA7, AAS80537) was important in antioxidant defense, which might play a very important role in the response to environmental stress such as high temperature. Interestingly, for the basic metablism glycolysis, the phosphoglucomutase was up-regulated at below-optimum temperature while the glyceraldehyde-3-phophate dehydrogenase was up-regulated at above-optimum temperature, suggesting that different regulations might be used for basic metabolism at different temperatures. Function of SOD in Thermoadaptation. As indicated in the above studies, 20 proteins of T. thermophilus wl were potentially related to thermoadaptation. To further characterize these proteins in response to higher temperatures, SOD, an important enzyme in organism to remove free radical produced in stress environment such as high temperature, was selected as a target protein for this investigation. The SOD gene in T. thermophilus wl was inactivated by inserting a kanamycin resistance gene (kat) in its coding region to construct a SOD mutant of T. thermophilus wl (∆SOD). After screening on TM broth medium containing kanamycin for three times at 65 °C, many SOD mutants were obtained. The ∆SOD genomic DNA was probed with DIG-labeled kat gene. 862

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As revealed by Southern blot analysis, only one positive band was observed (Figure 3A). The ∆SOD strain was further confirmed by DNA sequencing, showing the correct insertion of kat gene into SOD gene of ∆SOD strain. To detect the transcription and expression of SOD gene in the ∆SOD strain, Northern and Western blots were conducted. Northern blots indicated that the mRNA of SOD gene was detected in wildtype strain, but not in the ∆SOD strain (Figure 3B), indicating that the SOD gene was inactivated in ∆SOD. As demonstrated by Western blots, the protein encoded by SOD gene was only detectable in wild-type strain (Figure 3C), showing that there was no expression of SOD protein in ∆SOD. On the basis of the above results, the growth curve of the ∆SOD strain was examined at different temperatures. It was presented that the maximal growth temperature of ∆SOD decreased from 80 °C, the maximal growth temperature of wild-type strain, to 75 °C (Figure 3D). At the same time, the optimal growth temperature of ∆SOD also had a decrease of 5-10 °C. However, no decrease of the minimal growth temperature was observed. The results suggested that the SOD protein was essential in thermoadaptation of T. thermophilus wl. At least three replicas were executed for each growth curve analysis. To evaluate the effect of SOD protein on ∆SOD growth in response to temperature change, the SOD gene from T. thermophilus wl was expressed in E. coli as a fusion protein with only 6× His tag. After induction with IPTG, the recombinant SOD protein was purified (Figure 4A). The temperaturedependent growth curve revealed that the ∆SOD strain slowly retrieved its growth at 80 °C (Figure 4B), when the recombinant SOD protein was supplemented into the medium at a final concentration of 0.25 µg/mL. However, the addition of the recombinant SOD protein took no effect on the wild-type strain in response to temperature change. As control, the purified recombinant glutathione S-transferase (GST) (0.25 µg/mL) was added to the medium of ∆SOD strain. But the mutant strain could not retrieve its growth at 80 °C. Therefore, it could be

Thermophile Proteins Involved in Thermoadaptation

Figure 4. Effect of recombinant SOD on the growth curve of ∆SOD strain. (A) Recombinant expression of SOD gene in E. coli. Lanes: M, protein marker; 1, uninduced control bacteria (vector only); 2, induced control bacteria; 3, uninduced recombinant bacteria (containing the SOD gene); 4, induced recombinant bacteria; 5, purified SOD-His fusion protein. (B) The growth curve of ∆SOD strain. The SOD was expressed as a fusion protein with 6× His tag and purified. After supplement of the purified SOD in the medium, the growth rate of ∆SOD strain was determined at different temperatures to obtain the growth curve. Each point represented the mean of triplicate assays (n ) 3) within (1% standard deviation.

inferred that the SOD protein played an important role in the thermoadaptation of thermophile.

Discussion To adapt to hot environments, thermophiles have developed specific strategies for thermoadaptation by their physiological, nutritional and metabolic requirements including the cell components such as lipids, nucleic acids and proteins, thermal resistance of the DNA helixes and the structure of ribonucleic acids.30,31 Some asymmetric amino acid substitutions between the thermophiles and the mesophiles may be associated with the thermoadaptation.31 By comparing the genomic DNA sequences of two T. thermophilus strains HB27 and HB8, it is found that 32 genes encoded by megaplasmids of the two strains exhibit sequence and domain composition similarities to the predicted DNA repair systems, suggesting the importance of DNA repair systems in thermoadaptation.32 The comparative genomics analysis of T. thermophilus HB27 and Deinococcus radiodurans indicates divergent routes of adaptation to thermophily.33 The thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity

research articles in order to preserve specific protein structure while allowing the conformational flexibility required for activity.30 Up to date, however, the thermophile proteins and their pathways involved in thermoadaptation remain unclear. In the present study, the proteomic approach and computational analysis revealed that a majority of the proteins involved in thermal adaptation were involved in metabolic pathways or related to heat stability. As indicated by Northern blots, the expression profiles of mRNAs yielded similar tendencies to those of differentially expressed proteins, which confirmed the mass spectrometric data. However, for several differentially expressed proteins (spots 6, 7 and 10), the transcriptional analyses did not absolutely correspond to the protein profiles of 2D gel electrophoresis. The discrepancy might be caused by the sensitivities of various detection methods. In our study, the DNA repair-related genes encoded by the mageplasmids of two T. thermophilus strains HB27 and HB832 were not revealed. This might be caused by the different T. thermophilus strains used. Our study revealed that all the 10 proteins involved in metabolic pathways were assigned enzyme codes. At above-optimum or below-optimum temperature, these proteins were up-regulated to balance the loss of catalytic activity for maintaining normal metabolism. There was another possibility that the metabolism should even be promoted to provide more energy or necessary products for other pathways. Interestingly, all the 3 proteins up-regulated at below-optimum temperature were metabolism associated, including the phosphoglucomutase (TA1) involved in glycolysis, the fumarate hydratase (TA2) involved in citrate cycle (TCA) and the 3-methyl-2-oxobutanoate hydroxymethyltransferase (TA6) involved in CoA biosynthesis. These 3 proteins were involved in closely related basic metabolisms, suggesting that it was very important to maintain or even promote the basic metabolisms at below-optimum temperature. In addition, different proteins were up-regulated in metabolism at different temperatures, even in a same pathway like glycolysis in which the phosphoglucomutase was up-regulated at below-optimum temperature while the glyceraldehyde-3-phophate dehydrogenase (TA16) was up-regulated at above-optimum temperature. All the proteins associated with heat stability were up-regulated at above-optimum temperature, which was reasonable since it was necessary to maintain stability at high temperature. These proteins might not be required at below-optimum temperature. As evidence, the maximal and optimal growth temperatures of ∆SOD had, respectively, been decreased by 5 °C and 5-10 °C; however, there was no significant change for the minimal growth temperature. In addition to the fact that more proteins were found upregulated at above-optimum temperature, it was suggested that different regulations might be used for basic metabolism at different temperatures, and that the regulation at aboveoptimum temperature should be more complicated as additional pathways might be involved for heat stability. In this context, to well understand the adaptive mechanism, further studies merited to be conducted to reveal the regulation of pathways concerning thermoadaptation by molecular approaches, for example, protein interactions strategies which would be very helpful for the construction of the interaction net of proteins involved in thermoadaptation of thermophiles.34 In our study, to understand the roles of thermoadaptationrelated proteins in response to the environmental stress of high temperature, SOD was selected as a target protein for this investigation due to its importance in oxidative/antioxidative Journal of Proteome Research • Vol. 9, No. 2, 2010 863

research articles 35,36

system in bacteria. Mutant construction of a thermophile requires thermostable antibiotic-resistant markers functioning above 55 °C for the screening of mutants at high temperature. However, these markers are very limited. So far only several thermostable markers are available, including kanamycin nucleotidyltransferase gene and bleomycin-binding protein gene.37,38 By inserting the kanamycin nucleotidyltransferase gene into the SOD gene of T. thermophilus wl, the SOD mutant (∆SOD) demonstrated the decrease of thermoadaptation. On the other hand, the extracellular addition of the recombinant SOD in the media of ∆SOD culture significantly increased the growth temperature of ∆SOD. The recombinant SOD might be taken by ∆SOD through endocytosis.39 The results obtained in this study provided a clue to discover the molecular mechanism of SOD on thermoadaptation. In the future work, the proteome of ∆SOD, as well as the protein profile of ∆SOD upon addition of recombinant SOD protein, would be revealed to obtain the proteins which functioned together with SOD.

Conclusions The proteomic and computational analyses presented here indicated that the optimal temperature for thermophile growth was a threshold point which would initiate different pathways in response to the change of environmental temperature. The thermophile proteins responsible for thermoadaptation were involved in metabolic pathways as well as the stabilities and modifications of DNA and proteins. For the basic metabolism such as glycolysis, different regulations might be used for growing at different temperatures. Among the proteins involved in thermoadaptation, SOD was further revealed to be crucial by constructing the ∆SOD mutant and its function identification. The SOD protein might function to eliminate many free radical groups appearing in high-temperature environments. Our study, therefore, provided a clue to understand the thermal adaptive mechanism such as the regulation of thermoadaptation-related pathways and laid a solid foundation for further investigation. Abbreviations: SOD, superoxide dismutase; ABC, ammonium bicarbonate; IgG, immunoglobulin; ACN, acetonitrile; CHCA, R-cyano-4-hydroxycinnamic acid; TFA, trifluoroacetic acid; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; NBT, nitroblue tetrazolium chloride; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes; KAAS, KEGG automatic annotation server.

Acknowledgment. We appreciate Dr. Beate Averhoff for her kindly present of the pMK 18 plasmid and her instructive suggestions. This investigation was financially supported by National Natural Science Foundation of China (40876070), SRF for ROCS, SEM, Science Foundations of Yunnan University (KL070002) and Yunnan Educational Committee (08Y0026). Supporting Information Available: Figure S1 showed the transcription images of T. thermophilus wl genes involved in thermoadaptation by Northern blot. This material is available free of charge via the Internet at http://pubs.acs.org.

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