Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000

Elsevier Editorial System(tm) for Protein Expression and Purification Manuscript Draft Manuscript Number: Title: Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde- 3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000 Article Type: Regular Article Keywords: Pseudomonas syringae pv tomato DC3000, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Paralogous gap genes, Heterologous protein, Optimized overexpression. Order of Authors: Bouchra Elkhalfi, PhD; José Miguel Araya, PhD; Jorge Rodríguez Castro, PhD; Manuel Rey Méndez, Dr; Abdelaziz Soukri, Dr; Aurelio Serrano, Dr Abstract: The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in E. coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles. -Whole-genome sequence analyses revealed the occurrence in the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC300, of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D- erythrose-4-phosphate dehydrogenase/GAPDH enzyme. -By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy. -We optimized their expression in E. coli BL21 to achieve high yield of the heterologous proteins. -In order to verify a possible toxic character of P. syringae GAPDH2 on the E. coli BL21 host cells, a comparative study was performed concerning the effect of different alternatives carbon sources (glucose, glycerol, succinate, succinate plus glycerol) on the overproduction of the heterologous GAPDHs. -The 36-kDa GAPDH1 band follows the expected pattern for T7-lac promoter-dependent production, namely, heterologous protein decrease by glucose (actually an inhibitor of T7-lac-promoter transcription). -The 52-kDa GAPDH2 band shows a clear increase in the presence of glucose and reduction with succinate and/or glycerol. This finding suggests stabilization of pET21a-gap2 construct by glucose, which is a characteristic feature of the heterologous expression of toxic proteins in E. coli. -In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles. *Highlights (for review) Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000 Bouchra Elkhalfi1, José Miguel Araya-Garay2, Jorge Rodríguez Castro2, Manuel Rey Méndez2, Abdelaziz Soukri1, Aurelio Serrano3 1 Laboratory of Physiology & Genetics Molecular. Faculty of Sciences Ain Chock, Casablanca Morocco, 2 Laboratory of Molecular Systematic. CIBUS University of Santiago de Compostela, Santiago de Compostela, Spain, 3 Institute for Plant Biochemistry and Photosynthesis (CSIC-University. Of Seville), Seville, Spain, Corresponding Authors: Abdelaziz SOUKRI: [email protected] 00212661095495 Faculty of Sciences Ain Chock,Km8 Route Eljadida B;P 5366 Maarif, Casablanca 20100 Morocco *Manuscript Click here to view linked References Protein Expr Purif. 2013 Jun;89(2):146-55. doi: 10.1016/j.pep.2013.02.005 Abstract: The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in E. coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles. Key words: Pseudomonas syringae pv tomato DC3000, glyceraldehyde-3-phosphate dehydrogenase, GAPDH, paralogous gap genes, heterologous protein; optimized overexpression Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IPTG, isopropyl-β-D- thiogalactopyranoside; LB, Luria-Bertani; PCR, Polymerase Chain Reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. Introduction Tomato is an outstanding crop grown in many countries around the world and under diverse climates, including relatively cold regions, due to the development of vegetable crops under cover. By the volume of its production, tomato remains the first vegetable in the world [1,2]. However, tomato fields are potential targets of pathogenic bacteria that cause diseases ranging from small spots on the foliage to the almost total defoliation of the plant, with severe consequences on photosynthesis and yield potential [3,4] One of these diseases is bacterial speck caused by the gram-negative bacteria strain Pseudomonas syringae pv. tomato DC3000, a member of the gamma subgroup of the Proteobacteria[5]. This bacterium has emerged as an important model organism in molecular plant pathology because of its genetic tractability, and its genome has been fully sequenced [6,7]. P syringae pv. Tomato DC3000 causes leaf lesions characterized by small brown-black specks, 1-3 mm in diameter, with a yellow chlorotic halo [8], and it can survive up to 20 years in the crevices and cavities of the tomato seed coat. These lesions constitute a severe blemish on fruit for fresh market and greatly reduce yield potential [9]. Up to now, the virulence molecular mechanisms of P. syringae are not well understood. Previous research reported that this strain encodes a wide range of proteins that are involved in virulence, and 298 genes (ca. 5 % of the total) have been identified in the virulence category [7]. One of these proteins is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a highly conserved enzyme during evolution with a key role in the glycolytic and gluconeogenic pathways. GAPDH catalyzes the redoxlinked reversible phosphorylation of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate, in the presence of NAD(P)+ and inorganic phosphate [10]. GAPDH family members are classified into the ubiquitous class I enzymes that utilize NAD+ (EC 1.2.1.12), NADP+ (EC 1.2.1.13) or either (EC 1.2.1.59), class II of archaeal NAD(P)+-dependent GAPDHs, and class III bifunctional enzymes (erythrose-4-phosphate dehydrogenase/GAPDH) that are prevalent among gamma-proteobacteria [11]. Most glycolytic class I GAPDHs so far studied are homotetrameric proteins with 34–38 kDa subunits, and the native oligomer has a molecular weight of 140-150 kDa [12–14]. In addition to its classic metabolic roles, GAPDH has been claimed to be involved in a number of diverse cellular processes unrelated to glycolysis (“moonlighting protein”). Many of these alternative roles are dependent on the ability of GAPDH to modify its subcellular localization. In particular, it has been reported to bind DNA and RNA [15,16] and regulate transcription in cell nuclei [17,18], to exhibit kinase/phosphotransferase activity [19], to catalyze microtubule formation thus facilitating vesicular transport, and to bind to membrane-associated ion pumps involved in Ca2+ release. Besides, it interacts with a number of small molecules, including tumor necrosis α-factor, ribozymes, glutathione and nitric oxide [20]. Since GAPDH interacts with disease-associated proteins, like huntingtin [21] and the amyloid β-protein precursor [22], it also operates as a fusogenic protein in rabbit brain cytosol [23] and in nuclear membrane assembly in Xenopus eggs [24]. On the other hand, a functional GAPDH has been reported as a cell wall component in yeasts, like Saccharomyces cerevisiae[25]. Usually, GAPDH was used as a model protein, or control, in gene regulation and catalytic mechanism related studies, as well as a standard in Northern and Western blots, because of its high degree of structural conservation across species [18]. Furthermore, despite its general consideration as a housekeeping enzyme, GAPDH recently emerged with the advent of the molecular biotechnology as a useful model for innovative methods to advance our understanding of the mechanisms through which cells organize and express their genetic information [26]. Regarding its role in some pathogenic organisms, the presence of a surface-associated GAPDH has been reported on all streptococcal strains tested so far (21), as well as in microorganisms as diverse as Candida albicans, Schistosoma bovis, Mycoplasma genitalium, and Staphylococcus spp. [27]. GAPDH was shown to be secreted by several pathogenic bacteria, like Streptococcus pyogenes and a number of enteropathogenic Escherichia coli strains [28– 30]. It was claimed that secreted GAPDH has a role in signal transduction to their host cells, rendering them probably more susceptible to bacterial infection [28,31]. Three GAPDH encoding genes are present in the genome of P. syringae pv. tomato DC3000 [5,7]. It remains to be established whether any of these bacterial GAPDHs are extracellular proteins involved in the plant infection process. Their possible secretion and association to the cell membrane might have biotechnological implications that are worth to be further investigated. In the present work, we successfully cloned and optimized overexpression in E. coli of three paralogous gap genes encoding different GAPDHs identified in the genome of Pseudomonas syringae pv. tomato DC300. Some molecular and catalytic parameters of the recombinant GAPDHs of this phytopathogenic bacterium have been determined revealing the diverse structural and functional features of these enzymes. Materials and Methods Bioinformatics research: search, alignment and sequences analysis Searches for sequences of gap genes in EMBL/DDBJ/GeneBank databases, and multiple alignments of GAPDH sequences, were performed using the bioinformatics programs Blast and Clustal X v.2.0, respectively. Domain structures of the predicted proteins were analyzed with the ProDom (http://prodom.prabi.fr/prodom/current/html/home.php), SUPERFAMILY (http://supfam.cs.bris.ac.uk/ SUPERFAMILY/) and Pfam (http://pfam.sanger.ac.uk/) bioinformatics tools Organisms and growth conditions P. syringae was cultured in LB medium with glucose (3 g/l) at 28 °C in the presence of rifampicin (50 µg/ml) with agitation (200 rpm) in the dark. Unless otherwise indicated, E. coli Top10 and BL21 strains were grown in LB medium at 37 °C with agitation (200 rpm), and ampicillin was employed for selection of transformants. Where indicated, an additional carbon source -glycerol, succinate or glucosewas added at a final concentration of 2 % (w/v) to the LB medium. When necessary, ampicillin and isopropyl-β-D-thiogalactopyranoside (IPTG) were added at concentrations of 50-100 µg/ml and 1 mg/ml, respectively. DNA manipulation and cloning strategy -DNA isolation and purification. A modified phenol-chloroform method was used for the extraction of chromosomal DNA from Pseudomonas syringae pv. tomato DC3000 cells. Purified DNA was eventually washed with 70% (v/v) ethanol, air dried and resuspended in 100-200 µl of TE buffer. DNA concentration was estimated after 100-fold dilution in 1 ml of TE and measuring ultraviolet absorbance at 260 nm. -Primers Design. Specific primers for each putative gap gene of P. syringae were designed using Primer Premier 5.0 (Biosoft International). At each primer end, a new restriction site was added as shown in Table 1 to facilitate directional cloning. -Cloning strategy Step 1. Amplification by PCR. To check primers, a PCR amplification of the complete open reading frames (ORF) of the three gap genes was carried out with their specific complementary oligonucleotides, using the genomic DNA from P. syringae pv tomato as a template. The reaction mixture contained 100 mM Tris-HCl buffer (pH 8.3), 1.5 mM MgCl2, 0.8 mM of each deoxynucleoside triphosphate, 0.08 µM of each primer, 50 ng of DNA, and 1 U of GoTaq DNA Polymerase (Promega). Amplification was performed in a DNA thermal cycler (Perkin–Elmer Cetus, Norwalk, CT) programmed for 2 min at 95 °C and 35 cycles of 40 s at 94 °C, 40 s at 60 °C, and 1 min at 72 °C. In the end, a final cycle of 7 min at 72 °C was added. In this way, new restriction sites were created to facilitate directional one-step cloning of the amplified DNA fragments. Amplification products were resolved by electrophoresis in a 0.8% agarose gel according to Sambrook [32] and detected by staining with ethidium bromide. After agarose gel electrophoresis the PCR-amplified DNA fragments were purified with Wizard SV Gel and PCR Clean-Up System kits (Promega). Step 2. Digestion of inserts and plasmid, and ligation. The plasmid pET-21a (Novagen, Cambridge, UK) and the three PCR-amplified DNA fragments (1-1.5 kb) were sequencially cleaved, first with NdeI endonuclease overnight and purified; then, the second cleavage was carried out also overnight by BamH1 for plasmid pET-21a and the gap1 and gap3 inserts, or by HindIII for plasmid pET-21a and the gap2 insert. The reaction mixtures were carried out according to the manufacturer’s recommendations of each restriction enzyme. All DNA ligations were carried out with T4 DNA ligase (New England BioLabs, MA), as recommended by the manufacturer. Ligation reactions between the vector and the inserts were analyzed by PCR using T7 vector-specific primers and a combination of vector-specific and insert-specific primers. Step 3. Transformation into non-expressing host E. coli Top10. After DNA ligation, recombinant plasmids were transformed into chemically-competent E. coli Top 10 “One shot” (Invitrogen), which was grown on low salt LB agar plates containing 50 μg/ml ampicillin. Plates were then incubated overnight at 37 °C and recombinant colonies were selected. Step 4. Colony PCR and sequencing. Before growing colonies for plasmid isolation, subcloning was verified by analyzing the presence of the appropriate insert and its orientation by single-colony PCR using T7 vector-specific primers and a combination of vector-specific and insert-specific primers. Then, plasmids were isolated by E.Z.N.A R Plasmid Mini Kit I, and reading frame verified by sequencing. Step 5. Heterologous expression by transformation into host E. coli BL21 and induction. Recombinant pET-21a (Novagen, Cambridge, UK) expression vectors containing the complete ORF of each P. syringae gap gene (pET21a-gap1, pET21a-gap2 and pET21a-gap3) was transformed into E. coli BL21 (DE3) (Invitrogen) and grown on low-salt LB media containing 50 μg/ml ampicillin, until cultures reached an OD of 0.5–1. The cultures were then inducted by addition of IPTG. After four hours and overnight incubation at 37 or 22 °C, cells were harvested by centrifugation at 4 °C (8,000 g, 10 min). Cell pellets were washed twice in 25 mM Tris–HCl buffer, pH 7.5, and resuspended in the same buffer supplemented with 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10% (v/v) glycerol. The cells were then disrupted by sonication in a chilling water bath, using a Branson model B12 Sonifier at medium strength. The resulting broken-cell suspension was centrifuged at 20,000 g for 30 min at 4 °C to obtain the soluble protein fraction, or crude extract. Protein techniques Protein concentration was estimated by the Bradford technique [33], using ovalbumin as a standard. -Protein electrophoresis (SDS-PAGE). Prior to polyacrylamide gel electrophoresis, samples were incubated at 100 °C for 3min in solubilization buffer containing 2.5 % (w/v) SDS, 125 mM dithiothreitol, 25 % (v/v) glycerol and 112.5 mM Tris–HCl, pH 6.8. The protein samples were then loaded onto a 12 % polyacrylamide gel and separated on a Mini Protean II cell (Bio-Rad, Hercules, CA), according to the method of Laemmli [34]. The gel was run at 120 V for 1 h, using Tris–Glycine buffer (25 mM Tris; 192 mM Glycine; 0.1 % SDS; pH: 8.3). The molecular mass of the proteins was estimated using Precision Plus Protein Standard (Bio-Rad) and the analysis software Quantity One (Bio-Rad). Western blots after SDSPAGE and transfer to nitrocellulose membranes were carried out as previously described[35]. -Enzyme assays. The phosphorylating GAPDH activity was measured spectrophotometrically, using a Jenway 6405 UV/Visible spectrophotometer (Bibby Scientific Ltd, Stone, UK). The reaction was started by adding the enzyme preparation to the assay mixture containing 50 mM Tricine buffer (pH 8.5), 10 mM sodium arsenate (AsO4 3–), 1mM NAD+ or NADP+, and 2 mM D-glyceraldehyde-3- phosphate (D-G3P) at 25 °C, The total volume of the reaction mixture was 1 ml. Absorbance changes at 340 nm were monitored. One unit of enzyme (U) was defined as the amount that catalyzes the reduction of 1 µmol of NAD+ or NADP+ per minute. All experiments and assays were carried out in triplicate. Results and discussions Occurrence of three distinct paralogous gap genes of diverse molecular phylogeny in P. syringae pv. tomato DC3000 Bioinformatic searches in databases identified three putative gap genes that encode distinct GAPDH proteins and are located in different regions of the P. syringae pv. tomato DC3000 genome: PSPTO_1287 (gi|28868497), PSPTO_2102 (gi|28869306), and PSPTO_0386 (gi|28867616), which were named as gap1, gap2 and gap3, respectively. Gap1 gene encodes a class I glyceraldehyde-3-phosphate dehydrogenase (GAPDH1) of 333 aminoacids, with a predicted molecular mass and a pI value of 36,260 Da and 6.22, respectively. These are the expected values for archetypical glycolytic-gluconeogenic GAPDHs. The two structural domains proper of GAPDHs, namely NAD(P)-binding Rossmann-fold and C-terminal GAPDH domain were clearly identified by using several structural bioinformatics tools (Figure 1). This gene takes place in the genome of P. syringae near to genes encoding a 6-phosphogluconate dehydratase (edd), a RNA polymerase sigma70 factor (PSPTO_1286), a glucokinase (glk), a glucose transporter, permease proteins and several hypothetical proteins (Figure 2). It should be noted that adjacent divergent genes gap1 and edd (involved in the same KEGG metabolic pathways) arrange in a head-to- head manner in opposite orientations suggesting regulation by a single bidirectional promoter. This genomic organization is conserved in most Pseudomonas strains (both pathogenic and nonpathogenic), and other gammaproeobacteria (e.g. Marinobacter sp. Chromohalobacter salexigens, and Marinomonas spp.). However, gap1 appears in an operon with edd and a pyruvate kinase (pyk) encoding gene in other gammaproteobacteria (e.g. Nitrosococcus watsoni, Marinobacter spp.). It is interesting to note that two protein spots have been identified as GAPDH1 (PSPTO_1287) in 2D- PAGE gels of the extracellular proteins of P. syringae pv. tomato DC3000, showing a virtually identical pI but different molecular masses (http://www.leelab.org/wildtype_extracellular.html, Dr. Kelvin Lee's Research Group, Delaware Biotechnology Institute, University of Delaware, USA;). This result, obtained in a massive proteomic study, strongly suggests that two GAPDH isoforms generated by post-translational modification are secreted by this bacterium, although no detailed studies on this subject have been performed so far. A similar scenario has been reported for pathogenic E. coli strains, namely GAPDHA is secreted to the periplasmic compartment and the extracellular medium [29,30]. Moreover, post-translational modification of the secreted GAPDH has been described in the parasitic protist Entamoeba histolytica [36]. 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Perham, Identification, molecular cloning and sequence analysis of a gene cluster encoding the Class II fructose 1, 6-bisphosphate aldolase, 3-phosphoglycerate kinase and a putative second glyceraldehyde 3-phosphate dehydrogenase of Escherichia coli, Molecular Microbiology, 3 (2006) 723–732. Figures Legends: Figure 1. Domain architecture of the class I glyceraldehyde 3-phosphate dehydrogenase protein encoded by the gap1 gene of Pseudomonas syringae pv. tomato str. DC3000 (gi|28868497|ref|NP_791116.1|), as predicted by ProDom (top), SUPERFAMILY (middle) and Pfam (bottom) bioinformatics tools. Figure 2. Structure of the genomic region (ca. 20 kb) around the gap1 gene of P. syringae pv. tomato DC3000. Sequence data were from the Pseudomonas Genome Database (top) and JGI microbial genomes (bottom) websites. Gene designation of indicated ORFs is based on the closest BLAST hit. Note that adjacent divergent genes gap1 and edd arrange in a head-to-head manner in opposite orientations. Figure 3. Domain architecture of the class I glyceraldehyde 3-phosphate dehydrogenase protein (GAPDH2, 482 aa) encoded by the gap2 gene of Pseudomonas syringae pv. tomato str. DC3000 (gi|28869306|ref|NP_791925.1|), as predicted by ProDom (top), SUPERFAMILY (middle) and Pfam (bottom) bioinformatics tools. Note the N-terminal extra region of ca. 130 aminoacids upstream the GAPDH-specific domains. Figure 4. Structure of the genomic region (ca. 20 kb) around the gap2 gene of P. syringae pv. tomato DC3000. Sequence data were from the Pseudomonas Genome Database (top) and JGI microbial genomes (bottom) websites. Gene designation of indicated ORFs is based on the closest BLAST hit. Note that adjacent divergent genes gap2 and mfd arrange in a head-to-head manner in opposite orientations. Figure 5. Domain architecture of the putative class III glyceraldehyde 3-phosphate dehydrogenase protein (GAPDH3, 347 aa) encoded by the gap3 gene of Pseudomonas syringae pv. tomato str. DC3000 (gi|28867616|ref|NP_790235.1), as predicted by ProDom (top), SUPERFAMILY (middle) and Pfam (bottom) bioinformatics tools. This protein belongs to a group of bifunctional D-erythrose 4- phosphate dehydrogenase/GAPDH enzymes prevailing among gammaproteobacteria. Figure 6. Structure of the genomic region (ca. 20 kb) around the gap3/epd gene of P. syringae pv. tomato DC3000. Sequence data were from the Pseudomonas Genome Database (top) and JGI microbial genomes (bottom) websites. Gene designation of indicated ORFs is based on the closest BLAST hit. Note the occurrence of gap3 gene in an operon with other genes encoding glycolysis/pentoses phosphate pathway enzymes (tkt, pgk and fba). Figure 7. Multiple sequence alignment of the three GAPDHs of P. syringae pv. tomato DC3000 and selected prototypical orthologs of class I (E. coli GapA and Corynebacterium glutamicum GapB) and class III (E. coli GapB-Epd) GAPDHs. Alignment of aminoacid sequences was performed with ClustalX software. Shadowing displaying fully conserved positions in black and conservative changes in gray was made with Boxshade v 3.3 (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::boxshade). The essential Cys residue that directly participates in the catalytic mechanism (thiohemiacetal/thioester intermediate with substrate) is marked with an asterisk. Figure 8. Molecular phylogenetic analysis of the three GAPDHs of P. syringae pv. tomato DC3000. Full amino acid sequences derived from the paralogous gap genes studied in this work were aligned with selected sequences of class I and class III GAPDHs of other bacteria and eukaryotes using CLUSTAL X program, and then subjected to phylogenetic analysis by distance (Neighborg-Joining) method with the MEGA4 program. Possible clusters of functional equivalent orthologs of class I and class III GAPDHs are presented. Biochemical characterized enzymes are boxed and the three bacterial paralogs studied in this work are marked with arrows. Numbers in nodes indicate the statistical support (bootstrap values for 1000 replicates) of selected clusters of homologs. The bar represents amino acid substitutions per site. Figure 9. Electrophoretic analysis of PCR-amplified DNA fragments corresponding to the three gap genes of P. syringae pv. tomato DC3000. Amplifications were performed with specific primers pairs and bacterial genomic DNA as a template, as described in Material and Methods, and subsequently loaded onto 1% agarose-TBE gels using EcoRI/HindIII-cleaved lambda phage DNA as a marker (M). Single DNA bands of ca. 1.5, 1.1 and 1.0 kb obtained in four independent PCR experiments for gap3 (1,446 bp), gap2 (1,041 bp) and gap1 (999 bp), respectively, are shown. Figure 10. Coomassie blue stained SDS–PAGE electrophoretogram showing the protein patterns of cell-free extracts (ca. 50 µg protein per lane) from E. coli BL21 transformed clones. Overproduction of the three GAPDHs from P. syringae was achieved under different conditions of induction. The molecular masses of protein markers (M) are shown on the left. Arrows with the expected molecular masses of GAPDH subunits on the right side indicate the band positions of overproduced proteins. Lane 1: BL-21 transformed with pET21a-gap1 construct after 4 h of IPTG induction; lane 2: BL-21 transformed with pET21a-gap1 construct after overnight IPTG induction; lane 3: BL-21 transformed with pET21a-gap2 construct after 4 h of IPTG induction; lane 4: BL-21 transformed with pET21a- gap2 construct after overnight IPTG induction; lane 5: BL-21 transformed with pET21a-gap3 construct after 4 h of IPTG induction at 22 ºC; lane 6: BL-21 transformed with pET21a-gap3 construct after overnight IPTG induction at 22 ºC; lane 7: BL-21 transformed with plasmid pET21a without insert and after overnight IPTG induction; lane 8: not transformed BL-21 cells. Figure 11. Effect of alternative carbon sources on the overproduction levels of P. syringae pv. tomato DC3000 GAPDH1 and GAPDH2 in E. coli BL21 cells. SDS-PAGE analysis of cell-free extracts (ca. 50 µg protein per lane) from E. coli BL21 transformed clones grown in LB medium supplemented with succinate, glycerol, succinate plus glycerol or glucose is shown. Induction was carried out with IPTG, as described in the text. NA: no alternative carbon source added, used as control cultures. Figure 12. Immunoblot analysis of cell-free extracts from E. coli BL21 transformed clones overproducing the three GAPDHs of P. syringae pv. tomato DC3000 strain. Left panel: immunodetection of recombinant GAPDH1 and GAPDH3 using monospecific antibodies against the GAPDH GapC of the protist Tetrahymena pyriformis [41]. Right panel: immunodetection of recombinant GAPDH2 with monospecific polyclonal antibodies against the GAPDH2 of P. syringae pv. tomato DC3000. Lanes 1 and 4: BL-21 transformed with pET21a-gap2 construct after 4 h of IPTG induction; lane 2: BL-21 transformed with pET21a-gap3 construct after 4 h of IPTG induction at 22 ºC; lane 3: BL-21 transformed with pET21a-gap1 construct after overnight IPTG induction; lane 5: BL-21 transformed with pET21a-gap2 construct after 4 h of IPTG induction; lane 6: BL-21 transformed with pET21a-gap1 construct after overnight IPTG induction; lane 7: BL-21 transformed with pET21a-gap3 construct after 4 h of IPTG induction at 22 ºC. About 50 µg of protein were loaded per lane and transferred to nitrocellulose membrane after SDS-PAGE. The molecular masses of prestained protein markers (M) are shown on the left of each panel. The expected molecular masses of GAPDH subunits are shown on the right side of arrows indicating the positions of the immunodetected bands of overproduced proteins. Table 1. Complementary primers used for PCR amplifications of P. syringae gap genes Gene Primers (new restriction site, underlined) gap1 Pseudomonas syringae pv tomatoDC3000 F (NdeI) 5’-GGAATTCCATATGACTCTCCGTATCGCAATC-3’ R (BamH1) 5’-CGGGATCCTTACTCGGCGTTATGCAGCGC-3’ gap2 Pseudomonas syringae pv. tomato DC3000 F (NdeI) 5’-GGAATTCCATATGACTCAGAAGCCCGACCAG-3’ R (HindIII) 5’-CCCAAGCTTTTACTGCGGGAACGCTGGCGG-3’ gap3/epd Pseudomonas syringae pv. tomato DC3000 F (NdeI) 5’-GGAATTCCATATGCCCCAGCCCCGCCCTTAC-3’ R (BamH1) 5’-CGGGATCCTTATTGGTCAGCGACGCGCAA-3’ Table 2. Glyceraldehyde-3-phosphate dehydrogenase enzymatic activity levels with nicotinamide nucleotide coenzymes in cell-free extracts of transformed E. coli BL21 cells E. coli strain P. syringae recombinant gene GAPDH activity (U mg_1)a NAD+ NADP+ BL21 0.60 ± 0.04 ≤ 0.01 BL21 Transformed, not induced 0.66 ± 0.06 ≤ 0.01 BL21 Transformed and induced gap1 74.28 ± 0,02 ≤ 0.01 gap2 0.78 ± 0.07 10.80± 0.10 gap3 7.35 ± 0.14 ≤ 0.01 a Means and standard errors of three independent determinations of activity levels, carried out in cell crude extracts (soluble protein fraction), are shown. Tables Fig. 1 Figure Fig. 2 Fig. 3 Fig. 10 52-kDa GAPDH2 NA 36-kDa GAPDH1 NA pET21a (gap1) pET21a (gap2) Fig. 11 38-kDa GAPDH3 14 1 2 3 4 200 97 66 45 31 21 6.5 116 kDa 36-kDa GAPDH1 M 5 6 7 52-kDa GAPDH2 200 97 66 45 31 21 14 6.5 116 kDa M Fig. 12