1 Division of Integrated Biosciences, The University of Tokyo, 202 Bioscience Bldg, 5-1-5 Kashiwanoha, Chiba 277-8562, Japan
2 Division of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
3 Department of Agricultural Sciences, School of Agriculture, Ibaraki University, Chuo 3-21-1, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan
Correspondence
Shigetou Namba
snamba{at}ims.u-tokyo.ac.jp
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ABSTRACT |
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The GenBank accession numbers for the tmk-a and tmk-b genes are AB010446 and AB094668, respectively. The accession numbers of tmk-a homologues T01, T03 and T08 are AB100419, AB100420 and AB100421, respectively.
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INTRODUCTION |
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Thymidylate kinase (TMK, EC 2.7.4.9), which catalyses the transfer of a terminal phosphoryl group from ATP to dTMP, is crucial to both the de novo synthetic and the salvage pathways for pyrimidine deoxyribonucleotides. However, mollicutes have only the salvage pathway (Pollack et al., 1997). The cascade of enzymes that convert nucleosides to nucleoside triphosphates and deoxynucleoside triphosphates is of interest in drug discovery, as these molecules are vital precursors in the synthesis of DNA, RNA, and other cellular macromolecules. TMK has been extensively studied as an antiviral target. The variability in the active-site residues and catalytic properties of TMKs in different organisms (viral, eukaryotic and bacterial) has opened the possibility of designing specific and selective inhibitors (Jong & Campbell, 1984
; Darby, 1995
; Griffiths, 1995
; Lavie et al., 1998
). The necessity of TMK for bacterial growth (Li et al., 2000
), and the presence of the enzyme in both Gram-negative and Gram-positive bacterial pathogens, make it an attractive target for the development of novel, broad-spectrum antibacterial agents.
TMK is largely encoded as a single-copy gene in bacteria for which the genomes have been completely sequenced [refer to the Clusters of Orthologous Groups of proteins (COGs) database: http://www.ncbi.nlm.nih.gov/COG/index.html], and tmk genes have been annotated in the genomes of Mycoplasma genitalium (Fraser et al., 1995), Mycoplasma pneumoniae (Himmelreich et al., 1996
), Mycoplasma pulmonis (Chambaud et al., 2001
) and Ureaplasma urealyticum (Glass et al., 2000
).
TMK enzymes are globular dimeric proteins with a folding pattern similar to that of nucleoside monophosphate kinases (Ostermann et al., 2000), which possess three loops crucial for enzyme activity: the phosphate-binding motif at the N-terminus (P-loop), the nucleoside-monophosphate-binding domain, and the region that covers part of the P-loop upon substrate binding (the LID domain).
With the aim of developing an effective therapy for phytoplasmosis, we focused on the catalytic activity of TMK, which may be one of the important and active cellular metabolic enzymes in phytoplasmas. Thus we tried to isolate the tmk gene from the onion yellows (OY) phytoplasma genome, and examined the genomic organization of tmk genes and the catalytic activity of phytoplasma TMK.
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METHODS |
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Extraction of phytoplasma genomic DNA, construction of a genomic library, DNA sequencing, and homology analysis.
Phytoplasma-enriched DNA from infected plants was extracted using a previously reported procedure (Kuboyama et al., 1998; Miyata et al., 2002a
). Purified OY genomic DNA was completely digested with the restriction endonuclease HindIII and ligated into pUC18. The ligated genomic DNA was used to transform E. coli JM109, and several thousand plasmid clones were obtained. The cloned DNA fragments were then excised from pUC18 and prepared as probes for dot-blot hybridization to DNA samples extracted from OY-infected and healthy plants (Kuboyama et al., 1998
). DNA probes were labelled with glutaraldehyde and the hybridization products were detected using the ECL direct nucleic acid labelling and detection system (Amersham Biosciences), following the manufacturer's protocols. Approximately 200 positive clones were obtained.
The DNA inserts were sequenced using the dideoxynucleotide chain termination method with an automated DNA sequencer (model 377, Applied Biosystems). The similarity of each ORF to known genes was analysed using sequence interpretation tools (Institute for Chemical Research, University of Kyoto, Japan) and the BLAST algorithm (Altschul et al., 1990) via the GenomeNet server (http://www.genome.jp/). The amino acid sequences of proteins that were similar to each ORF were aligned using CLUSTAL W version 1.7 (Thompson et al., 1994
). The sequences used for comparison were TMK of M. genitalium, AAC71222, M. pneumoniae, AE000016, U. urealyticum, AE002101, Bacillus subtilis, D26185, Bacillus halodurans, AP001507, Escherichia coli, AB001340 and Buchnera sp. APS, AP001119.
Southern blotting.
The tmk-a probe DNA was amplified by PCR using primers tmka-N (5'-TTG AAT TCC ATA TGA AAT TAA TCG TTT TTG AAG GAC T-3') and tmka-C (5'-TGA GCT CGA GTT AGT TAT GAT CGC CAT TTG ATA GTA CT-3'). The tmk-b probe DNA was amplified by PCR using primers tmkb-N (5'-TTG AAT TCC ATA TGT TTA TTT CTT TTG AAG GTT GTG A-3') and tmkb-C (5'-TGA GCT CGA GCT ATT TGA AAG ACT TCT TTG AGT TTT GT-3'). PCR amplification was performed in a thermal cycler (Perkin Elmer model 9700) using 30 cycles of denaturation for 15 s at 94 °C, annealing for 30 s at 55 °C, and extension for 1 min at 60 °C. DNA probes were labelled with glutaraldehyde, and hybridization and detection were carried out using the ECL Direct nucleic acid labelling and detection system (Amersham Biosciences). Purified OY phytoplasma genomic DNA and control DNA preparations from healthy plants were completely digested with HindIII, EcoRV or XbaI. Fragments were separated by agarose gel electrophoresis, and transferred to a positively charged nylon membrane (Boehringer Mannheim) (Sambrook et al., 1989). The membrane was prehybridized in hybridization buffer [5x SSC, 2 % (w/v) blocking reagent, 0·1 % (w/v) N-lauroylsarcosine, 7 % (w/v) SDS, 50 mM sodium phosphate buffer (pH 7·0), 50 % (v/v) formamide] at 42 °C for 1 h. Hybridization was carried out at 42 °C overnight in hybridization buffer containing the denatured probe. After hybridization, the membrane was washed twice in primary wash buffer (0·5x SSC, 6 M urea and 0·4 % SDS) at 42 °C for 20 min each, and then washed twice in secondary wash buffer (2x SSC) at room temperature for 5 min each. Hybridized probes were detected following the manufacturer's guidelines.
PCR amplification and cloning of tmk-a homologues.
In order to amplify the tmk-a homologues, we used primers tmk-aF (5'-ATG AAA TTA ATC GTT TTT GA-3') and tmk-aR (5'-TGA GCT CGA GGT TAT GAT CG-3') and performed PCR using OY-W total DNA as a template. PCR amplification was performed under the same conditions as described above. The amplified DNA fragment was cloned in the pGEM-T plasmid vector (Promega) and several cloned DNA fragments were sequenced.
Expression of the phytoplasma tmk genes.
The pET system (Novagen) was used to generate a polyhistidine (polyHis)-tagged TMK fusion protein. We amplified tmk-a and tmk-b gene fragments by PCR using primers tmka-N and tmka-C, and tmkb-N and tmkb-C, respectively. The amplicon was then digested with NdeI and XhoI and inserted into pET30a. The polyHis-tagged TMKs were expressed in Epicurian Coli BL21-CodonPlus (DE3)-RIL (Stratagene). Cell extracts were applied to a nickel NTA-column (Novagen), washed with TBS buffer (20 mM Tris/HCl, pH 7·9 and 500 mM NaCl), and the fusion protein was eluted with TBS buffer containing 1 M imidazole. The purity of the protein was checked by SDS-PAGE and staining by Coomassie brilliant blue.
Measuring TMK activity.
The kinase activities of the purified polyHis-tagged TMKs were measured by the method of Berghauser (1975), using an enzyme-coupled assay involving the following reactions:
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RESULTS |
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In vitro catalytic analysis of phytoplasma TMK proteins expressed in E. coli
In order to determine whether the tmk-a and tmk-b genes were functional, we expressed products of both genes using an E. coli expression system. Each tmk gene and a polyHis sequence were inserted 3' to the E. coli pET30 promoter. Each construct was used to transform E. coli cells, and the fusion proteins were overexpressed at 18 and 37 °C. The soluble fractions of the extracts of the E. coli cells were partially purified on Ni affinity columns. SDS-PAGE detected 25 kDa TMK-apolyHis and TMK-bpolyHis fusion proteins (Fig. 4), which was consistent with their predicted molecular mass (TMK-apolyHis, 25·0 kDa; TMK-bpolyHis, 24·9 kDa). To measure the TMK activity of these fractions, we used the methods described by Berghauser (1975)
. TMK activity was detected in the TMK-bpolyHis fraction (68·7±5·7 U mg-1) and was destroyed (0·6±0·8 U mg-1) by incubation of the fraction at 98 °C. No activity was detected in the TMK-apolyHis fraction (0·2±0·6 U mg-1). The negative control reactions without TMP (data not shown), or with elution buffer only (0·1±0·7 U mg-1), had no activity. These results indicated that TMK-b has catalytic activity. We also tested a mixture of the TMK-apolyHis and TMK-bpolyHis fractions and the catalytic activity of TMK-b was not affected (data not shown). This suggested that there was no inhibitory factor in the TMK-apolyHis fraction.
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DISCUSSION |
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The deduced amino acid sequences of OY phytoplasma TMK-a and TMK-b resembled those of Bacillus spp., Mycoplasma spp. and U. urealyticum more than those of Gram-negative bacteria (E. coli or Buchnera APS sp.) (Table 1, Fig. 2
), which is consistent with the classification of the class Mollicutes in the Bacillus/Clostridium group. In addition, the holB gene, downstream of the tmk gene, which is conserved in several bacterial genomes including M. genitalium, U. urealyticum, B. subtilis, Staphylococcus aureus and E. coli, was also conserved in the tmk-b locus. Further investigation of the regions flanking tmk-a and its homologues provide further information about the origin of tmk-a.
The TMK-bpolyHis fusion protein had kinase activity, indicating that tmk-b encodes a functional TMK protein. However, the TMK-apolyHis fusion protein had no catalytic activity. As all the functional motifs were conserved in TMK-a, it seems likely that TMK-a is functional, and experiments to detect its catalytic activity are being conducted. There may be several reasons why we could not detect catalytic activity for TMK-a. The optimal reaction conditions for TMK-a may differ from those of TMK-b. As phytoplasmas inhabit both plant phloem sieve cells and insect cells, they may switch their metabolic pathways in response to change in habitat and TMK-a and TMK-b may function in independent pathways. As the same conserved motifs are also found in other NTP/NMP kinases, TMK-a might function as another type of kinase in phytoplasmas. For example, in Mycoplasma spp., deoxyadenosine kinase can phosphorylate not only deoxyadenosine but also deoxyguanosine and deoxycytidine (Wang et al., 2001). Contaminating proteins that were not removed from the TMK-apolyHis fraction may have inhibited the catalytic reactions in our experiments, or the fused polyHis-tag protein may have inhibited the reaction. Finally, the two highly conserved regions TKEPGG, downstream of the P-loop motif, and PAL, upstream of the TMP binding motif are absent in TMK-a (Fig. 2
). There are no reports of the function of these motifs, but it is possible that the loss of the TKEPGG and PAL sequences impaired the TMK activity of TMK-a.
Southern blot hybridization analyses suggested that several homologous tmk-a sequences exist in both the OY-W and OY-M genomes, in addition to the one tmk-b gene. The tmk gene is largely encoded as a single copy gene in those organisms for which the genome has been completely determined (http://www.ncbi.nlm.nih.gov/COG/index.html). Two tmk gene homologues have been reported in the B. subtilis (BS-yorR), Methanothermobacter thermoautotrophicus (MTH1100) and Archaeoglobus fulgidus (AF1308) genomes. However, none of the additional homologues possess the TMP-binding motif, which is one of the three functional domains conserved in all known TMKs. This suggests that the OY phytoplasma is unique in encoding more than two copies of tmk genes containing catalytic domains.
Our findings suggest that gene duplication events have generated multiple copies of tmk-a in the phytoplasma genome. Ohno (1970) suggested that gene duplication was a major force in genome evolution and that large-scale gene duplication may enable the evolution of novel functions, an important step for the evolution of complex phenotypes. However, it is still unclear how often gene duplications arise and how frequently they evolve new functions. Indeed, there are only a few examples of the development of completely new functions (Cheng & Chen, 1999
; Manzanares et al., 2000
). Thus it is generally thought that the fate of most duplicated genes is degeneration into pseudogenes. Lynch & Conery (2000)
analysed the rate of gene duplication by examining several eukaryotic species and suggested that duplicate genes arise at a very high rate, on average 0·01 per gene per million years, but that most of the duplicates are silenced within a few million years.
Gene duplication has mainly been studied in eukaryotes. However, recently the complete genome sequences of several organisms, including prokaryotes, have been analysed to examine the relationships between gene function and the propensity of a gene to duplicate, and the number of genes in a gene family and the family's rate of sequence evolution (Conant & Wagner, 2002). For example, ribosomal genes and transcriptional factors appear less likely to undergo gene duplication than other genes. In the case of the OY phytoplasma tmk-a homologues, some ORFs were destroyed by mutations. However, the tmk-a gene has a complete ORF and highly conserved functional domains, suggesting that TMK-a might have some other nucleotide kinase activity. Thus it is possible that some tmk genes have degenerated, and some have gained a new function. In mollicutes, there are some homologous genes in the COG database; there are two copies of the 50S ribosomal gene (MPN069 and 471) that encodes ribosomal protein L33 (RPL33) in M pneumoniae. In the same organism MPN140 and 549 code for a 28 kDa protein that is part of the P1 operon. Large families of lipoprotein genes, such as the vsp genes of Mycoplasma bovis (Pfützner & Sachse, 1996
), vsa genes of M. pulmonis (Simecka et al., 1992
) and avg genes of M. agalactiae (Flitman-Tene et al., 2000
), appear to play roles in adaptive systems for survival (Razin et al., 1998
). These surface lipoproteins are recognized by host immune systems, and variation in expression generates antigenic variation on the cell surface. However, TMK, RPL33 and other proteins encoded by duplicated genes may not play a role in host adaptation, and hence the reasons for maintenance of duplications of these genes may be different from those for lipoproteins. Thus the tmk genes of OY phytoplasma might be useful for further investigations of gene duplication in prokaryote genomes.
Southern blot analyses indicated that many tmk-a homologues are located in the OY phytoplasma genome and that the organization of these sequences may have been affected when OY-M was isolated from OY-W (Shiomi et al., 1998). OY-M is a mildly pathogenic variant line in which about 130 kbp of the OY-W genome has been deleted (Oshima et al., 2001
). The regions flanking the tmk-a gene homologues may have been involved in the deletion.
Recently, several phytoplasma proteins were successfully expressed in E. coli, and used as antigens to obtain phytoplasma-specific antibodies. These proteins were unidentified membrane proteins (Yu et al., 1998; Blomquist et al., 2001
), the SecA protein of the secretion system (Kakizawa et al., 2001
), and Rep proteins encoded in extrachromosomal DNA (Nishigawa et al., 2001
). These successes were based on the finding that phytoplasmas use the same universal codon system as E. coli and other bacteria for protein synthesis, unlike most other mycoplasmas (Lee et al., 2000
; Miyata et al., 2002b
). Similarly in this study we could express TMK-apolyHis and TMK-bpolyHis fusion proteins in E. coli, and detected TMK-b catalytic activity. This is believed to be the first demonstration of catalytic activity of a phytoplasma protein using a heterologous expression system. Other phytoplasma proteins, such as many of enzymes involved in the metabolic pathways, may be obtained using the same approach. This information will be valuable for exploring new horizons in the study of the biological characteristics of phytoplasmas. Further analyses of the functions of phytoplasma-encoded proteins will answer many questions about the factors related to phytoplasma infection, plant symptoms, host factors involved in phytoplasma infection, and ways to cure phytoplasma-infected plants by using novel antimicrobial therapeutics.
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ACKNOWLEDGEMENTS |
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Received 13 March 2003;
revised 28 April 2003;
accepted 6 May 2003.
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