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Applied and Environmental Microbiology, February 2006, p. 1288-1294, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1288-1294.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Construction and Complementation of In-Frame Deletions of the Essential Escherichia coli Thymidylate Kinase Gene

David-Nicolas Chaperon^*

Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, CH-1211 Geneva, Switzerland

Received 13 October 2005/ Accepted 13 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
This work reports the construction of Escherichia coli in-frame deletion strains of tmk, which encodes thymidylate kinase, Tmk. The tmk gene is located at the third position of a putative five-gene operon at 24.9 min on the E. coli chromosome, which comprises the genes pabC, yceG, tmk, holB, and ycfH. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded tmk, the tmk gene was replaced by the kanamycin resistance gene kka1, encoding amino glycoside 3'-phosphotransferase kanamycin kinase. The kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The E. coli tmk gene is essential under any conditions tested. To show functional complementation in bacteria, the E. coli tmk gene was replaced by thymidylate kinases of bacteriophage T4 gp1, E. coli tmk, Saccharomyces cerevisiae cdc8, or the Homo sapiens homologue, dTYMK. Growth of these transgenic E. coli strains is completely dependent on thymidylate kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes holB and ycfH with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Metabolic pathways for the synthesis of the deoxynucleotide precursors required for DNA replication in Escherichia coli are well characterized (26, 40). The deoxynucleoside monophosphate kinases are universally conserved key catalysts involved in the turnover of natural nucleotides and are important in the phosphorylation of nucleoside analogues used in therapies against cancer and infectious diseases (35). Thymidylate kinases (EC 2.7.4.9) catalyze the phosphorylation of deoxythymidylate (dTMP) to the corresponding deoxythymidine diphosphate. This kinase is required after the merging of the de novo and salvage pathways of deoxythymidine triphosphate biosynthesis and is the last essential enzyme in the deoxythymidine triphosphate biosynthetic pathway of most organisms.

The E. coli Tmk (Swiss-Prot no. P37345 is expressed at low levels during cell growth; it acts as a dimer (25) and is known to represent ca. 0.01% of the soluble protein in E. coli. This is 10 to 20 times less than the most abundant nucleoside monophosphate kinase, adenylate kinase, Amk (34). The eukaryotic Saccharomyces cerevisiae thymidylate kinase TmpK (Swiss-Prot no. P00572 and H. sapiens thymidylate kinase dTYMK (Swiss-Prot no. P23919 are predominantly cytoplasmic or cell membrane bound, and their activity is cell cycle controlled (1, 16, 38). In view of the importance of dTMP kinase in the phosphorylation of nucleoside analogues (2, 15, 35), the crystal structures of E. coli, yeast, human, and other related monophosphate kinases were solved.

The E. coli tmk gene is located at 24.9 min in a putative five-gene operon on the chromosome (3, 28). The existence of a conditional-lethal, temperature-sensitive (Ts) mutant strain of tmk (9, 34) underlines the importance of Tmk in cellular metabolism and control of DNA replication and is estimated to be essential (9, 13, 34). It has been shown that a Ts strain of S. cerevisiae carrying a mutation in the thymidylate kinase gene cdc8 can be complemented by the E. coli tmk gene (28). However, due to the lack of a suitable genetic system, the reverse situation could not be tested. Interestingly, the human dTYMK is able to complement the loss of dTMP kinase activity of a S. cerevisiae cdc8 Ts strain, but the activity of the human kinase could not be purified from yeast (16). The complementation was explained by assuming a very low, yet sufficient level of human dTMP kinase activity (33). Several attempts to produce wild-type human dTMP kinase activity in E. coli failed, and dTYMK activity could only be efficiently detected by using a baculovirus system (16) or be purified as a fusion protein overproduced in E. coli (5).

In the present study, a replacement of the E. coli tmk gene by the kanamycin resistance (Km^r) gene kka1, which encodes the amino glycoside 3'-phosphotransferase kanamycin kinase (Swiss-Prot no. P00551 (EC 2.7.1.95), is presented. The Km^r gene kka1 is expressed under the natural promoter(s) of the putative operon. The in-frame substitution construct of tmk shows no polar effects on the downstream genes holB and ycfH with respect to cell viability. The essential E. coli tmk gene was complemented by plasmids expressing either the bacteriophage T4 gene 1, encoding a less-specific monophosphate kinase (Swiss-Prot no. P04531 (4, 6), the S. cerevisiae dTMP kinase gene cdc8 (17, 30), or the human dTYMK homologue (20, 33). The last enzyme is of special interest, since this is the first time that the human thymidylate kinase protein dTYMK has been functionally produced as wild-type protein in bacteria in the absence of its bacterial counterpart. The presented transgenic bacteria, expressing various thymidylate kinases in a simplified cellular system, could be of interest for testing the phosphorylation of thymidine-like nucleoside analogues that are used in therapies against cancer and infectious diseases.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and bacteriophages.
The various bacterial strains, plasmids, and phages used and constructed in the present study are listed in Table 1.

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TABLE 1. Strains, plasmids, and bacteriophages used or constructed in this study

Bacterial media and culture conditions.
Luria-Bertani (LB) medium (10 g of tryptone [Difco], 5 g of yeast extract [Difco], and 5 g of NaCl per liter; pH 7.0) was used for bacterial growth. For LB agar plates, 10 g of agar (Difco) were added per liter. For selection of antibiotic resistance, 100 µg of ampicillin, 50 µg of kanamycin, 20 µg of chloramphenicol, and 10 µg of tetracycline ml^–1 were used as required. For the tmk replacement strains and plasmid constructs, 25 µg of kanamycin ml^–1 was added. Growth was always performed at 30°C unless otherwise indicated.

Cultivation of human embryonic kidney 293T cells and mRNA preparation.
Human embryonic kidney 293T cells (gift of A. Melotti) were cultivated by standard laboratory techniques using Dulbecco modified Eagle medium, fetal calf medium, and antibiotics. About 10⁸ cells were centrifuged for 3 min at room temperature at 900 rpm and then kept frozen at –20°C. Using QIAGEN RNeasy mRNA mini-prep columns, mRNA of 5 x 10⁷ 293T cells was purified and diluted in 200 µl of distilled H₂O.

PCR, RT-PCR, and oligonucleotides.
Standard PCR was performed with Pfu DNA polymerase according to the manufacturer's instructions (Promega) using a MiniCycler (MJ Research). PCR amplifications were cycled 30 times for 1 min at 94°C, 1 min at 53°C, and between 1 and 4 min at 72°C, depending on the template and the primer pair used. The human dTMP kinase gene dTYMK (dTYMK-2) was amplified and cloned from mRNA of human embryonic kidney 293T cells by using reverse transcription-PCR (RT-PCR) DNA amplification techniques (SuperScript one-step RT-PCR, Platinum Taq; Gibco-BRL/Life Technologies). The reaction mixture was incubated for 30 min at 50°C to allow the RT to produce cDNA from the human mRNA, followed by a 40-cycle standard PCR amplification at an annealing temperature of 53°C and an elongation time of 1 min at 72°C using primers 9 and 10.

The following PCR primers were used: primer 1 (5'-kka1 5'3'; 5'-ATCTGCATGCTAAGTTATGAGCCATATCAAC-3'), primer 2 (3'-kka1 3'5'; 5'-ATCTGCATGCCATTTAGAAAAACTCATCGAGCA-3'), primer 3 (5'-kka1 RBS 5'3'; 5'-ATCTGCATGCTAAGGAGAATTCATATGAGCCATATTCAAC-3'), primer 4 (3'-kka1 RBS 3'5'; 5'-ATCTGCATGCCATATGAATTCTCCTTAGAAAAACTCATCGAGCA-3'), primer 5 (5'-T4 gene 1 5'3'; 5'-GGAGGAATTCATATGAAACTAATCTTTTTAAGCG-3'), primer 6 (3'-T4 gene 1 3'5'; 5'-ACTATCTAGATTATAGTACCTTTAGTGTATTTT-3'), primer 7 (5' of the operon; 5'-GTAGTGGCGGGCGAGG-3'), primer 8 (SalI ycfH 3'5'; 5'-GCCAGAACGTCATCCACGTC-3'), primer 9 (5'-dTYMK 5'3'; 5'-GGAGGCATGCTAAGGAGAATTCATATGGCGGCCCGGCGC-3'), primer 10 (3'-dTYMK 3'5'; 5'-ACTATCTAGAGGCCGGCCTCACTTCCATAGCTCCCC-3'), primer 11 (5'-tmk 5'3'; 5'-GGAGGAATTCACCATGCGCAGTAAGTATATCGT-3'), primer 12 (3'-tmk 3'5'; 5'-ACGCGCATGCTCATGCGTCCAACTCCTTC-3'), primer 13 (3'-myc dTYMK 3'5'; 5'-ACTATCTAGATCACAGATCCTCTTCTGAGATGAGTTTTTGTTCCTTCCATAGCTCCCCA-3'), and primer 14 (3'-myc tmk 3'5'; 5'-ACTATCTAGATCACAGATCCTCTTCTGAGATGAGTTTTTGTTCTGCGTCCAACTCCTTCAC-3'). Lyophilized primers were dissolved in H₂O and stored at –20°C.

Plasmid construction.
General techniques for plasmid DNA preparation, restriction enzyme manipulation, molecular cloning, and agarose gel electrophoresis were carried out as described in Sambrook and Russell (29).

The plasmids pDC11 and pDC12, used for the genomic replacement constructs to generate the tmk::kka1 null strains, were prepared by inserting the SphI-digested kka1 PCR products of pACYC177 (7) (primer pair 1 and 2 for pDC11 and 3 and 4 for pDC12, respectively) into the unique SphI site of plasmids pDC7 and pDC6, respectively. Plasmid pSR1613 (a gift from S. Raina) was digested with BglII and HindIII, and a fragment of approximately 4.8-kb harboring the five-gene operon was cloned into the BamHI- and HindIII-digested vector pFYZ1 (18), resulting in pDC13.

Plasmid pDC14 was constructed by inserting the EcoRI/XbaI-digested PCR amplification product (primer pair 5 and 6) of genomic bacteriophage T4 Do DNA (gift of D. Ang) into the EcoRI/XbaI-digested vector pMPM-A6 (23). Plasmid pDC15 was constructed by ligating a fragment containing the S. cerevisiae cdc8 gene from the plasmid pScTmpK (19) into pMPM-A6. The above-mentioned two vectors were linearized with NdeI and EcoRI, respectively, followed by filling the ends by using T4 DNA polymerase. Plasmid pScTmpK was digested with BamHI, and pMPM-A6 with BglII, the appropriate fragments were gel purified and ligated together. The PCR product, using the template pHsTmpK (5) and the primers 9 and 10, was digested with EcoRI/XbaI and inserted into the EcoRI/XbaI-digested vectors pMPM-A6, pMPM-A4 (23), or pGP189 (11), resulting in plasmids pDC16, pDC17, and pDC18, respectively. Plasmid pDC19 was constructed by ligating the EcoRI/XbaI-digested PCR product (primer pair 11 and 12, template pDC3) into the EcoRI/XbaI-digested vector pGP189. Plasmid pDC20 (primers 9 and 10) was constructed by inserting the EcoRI/XbaI-digested PCR fragments into the EcoRI/XbaI-digested vector pGP189. The cDNA product of the RT-PCR amplification of human embryonic kidney 293T mRNA served as a template for the PCRs (primer pair 9 and 10), resulting in pDC20. The plasmids overproducing C-terminal myc tag proteins were constructed in a similar way and are all derivatives of pGP189. Vector and PCR products were digested with EcoRI/XbaI prior to ligation. The tmk insertion in plasmid pDC21 was amplified from the template pDC19 using primers 11 and 14. The DNA insertion in plasmid pDC22 was amplified from pDC20 by using the primers 9 and 13. The PCR amplified thymidylate kinase genes were sequenced and proved to be identical to database sequences of E. coli (gb U00096.2), bacteriophage T4 (gb AF158101.6), and H. sapiens (dTYMK-2) (gb BT020055.1), respectively.

Replacement of tmk on the E. coli chromosome.
Plasmid pDC11 was used for the replacement of the tmk gene by the kka1 gene using the Ts-plasmid integration method as described by Link et al. (21). Strain B178 (12) was transformed with the plasmid pDC11 and selected at 43°C in the presence of chloramphenicol and kanamycin on LB agar plates. Colonies isolated at 43°C were then grown at 37°C overnight. The next day, they were diluted and grown at 37°C for the preparation of competent cells. Cultures were transformed with pFYZ1 as a control, or with the plasmid pDC13 carrying the tmk operon, and selected on plates containing ampicillin, chloramphenicol, and kanamycin at 37°C overnight. From overnight cultures grown at 37°C, dilutions of equal numbers of cells were spread on LB agar plates at 30°C containing 5% sucrose, kanamycin, and ampicillin. DC11 shows Km^r, ampicillin resistance (Ap^r), and sucrose resistance (Suc^r) but chloramphenicol sensitivity (Cm^s) (Table 1).

Alternatively, using the plasmid pDC12 (tmk::kka1 [RBS]), a second tmk::kka1 deletion strain containing additional RBS before and after kka1 was constructed by linear transformation as described by Yu et al. (39) (Fig. 2b and data not shown). DC3 transformed with pDC3 in the presence of 0.05% L-arabinose was transformed with a PCR amplification product of plasmid pDC12 using the primer pair 7 and 8. P1 lysates were grown on candidates with the tmk::kka1 (RBS) deletion at 30°C. Cotransduction experiments using B178 previously transformed with pDC3 as recipient strain resulted in strain DC12.

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FIG. 2. DNA sequences present on plasmid pDC11 (a) and plasmid pDC12 (b), as well as their translational products: sequence in italics, SphI site; (RBS) or RBS, potential RBS or RBS based on sequence analysis (31). Lines and arrows indicate translation products. Capital triplets represent start and stop codons. Dashed lines represent potential translational products.

Bacteriophage P1 lysates, transduction, and cotransduction.
Lysate preparations and transduction were performed as described by Miller (24). A lysate grown on strain DC1 was used for the transduction of DC11 to yield strain DC13 (Table 1). The frequency of cotransduction between tmk::kka1 and the linked tetracycline resistance (Tc^r) marker miniTn10 (Tc^r) was determined with lysates grown on the strains DC13 or DC14. These P1 lysates were used to infect B178 carrying various plasmids. Transductants were first selected on LB agar plates containing sodium citrate, tetracycline, ampicillin, and various concentrations of L-arabinose or IPTG (isopropyl-ß-D-thiogalactopyranoside). To determine the linkage frequency, candidates were then screened on the same plates additionally containing kanamycin. A transductant of B178 previously transformed with pDC15 in the presence of 0.05% L-arabinose indicating Tc^r, Km^r, and Ap^r resulted in DC14.

Protein expression.
For protein expression, overnight cultures were diluted to an optical density of 0.1 at 600 nm and cultivated at 37°C. When the cultures reached an optical density of 0.5, they were induced with 1 mM IPTG and grown for an additional 1.5 h at 37°C. Matching uninduced cultures were also grown for 1.5 h at 37°C. The cultures were centrifuged, and the pellets boiled for 10 min in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Standard SDS-PAGE was performed and gels were stained with Coomassie blue (29).

Western blot analysis.
Western blots were prepared as described by Harlow and Lane (14). Blots were first treated with a mouse anti-myc tag antibody, followed by treatment with a goat anti-mouse antibody linked to horseradish peroxidase. For imaging, the SuperSignal West Pico Trail-Kit (Pierce) was used with X-ray films (Kodak).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of tmk::kka1 E. coli strains by the plasmid-based gene replacement method.
The tmk gene was replaced in strain B178 by the Km^r gene kka1, whose expression is under the control of the promoter(s) of the putative five-gene operon (Fig. 1a). Since the structure in this operon is relatively complex (Fig. 1b and c), two different replacement strains were constructed by using plasmids pDC11 and pDC12, respectively (Fig. 2). The basic strategy for the replacement of tmk was to construct deletion strains in which the genomic organization of the operon stays intact. In plasmid pDC11 (Fig. 2a), the Km^r gene kka1 was flanked by short 5' and 3' sequences of tmk, which overlap with the yceG and holB genes, but has no clear RBS for the translation initiation of the Km^r gene kka1. Plasmid pDC12 (Fig. 2b) was identical to pDC11 except for the addition of RBS 5' upstream and 3' downstream of kka1. However, no difference in results with either of the two tmk replacement constructs was observed.

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FIG. 1. (a) Construction of tmk::kka1 replacement strains: pabC, 4-amino-4-deoxy-chorismate lyase; yceG, gene of unknown function; tmk, dTMP kinase; holB, DNA polymerase III subunit; ycfH, conserved gene of uncertain function; kka1, Kmr gene. (b and c) Overlapping genes at 24.9 min on the E. coli genome and translation products of the mRNA(s) at the 5' (b) and 3' (c) termini of the tmk gene; the RBS location is based on sequence analysis (28, 31). Lines and arrows indicate translation products; capital triplets indicate start and stop codons.

Plasmid pDC11 was used to replace tmk on the genome following the procedure of Link et al. (21). First, pDC11 was introduced into the chromosome of B178 by selecting for both Cm^r and Km^r at 43°C. Next, the vector pFYZ1 (control) or the plasmid pDC13 (five-gene operon of tmk) were transformed into candidates that already had integrated the plasmid pDC11 into the genome due to its Ts pSC101 origin of replication. Excision and loss of pDC11 was selected in the presence of sucrose, since sucrose was toxic to the cell due to the pDC11-encoded levansucrase sacB gene product (10). Dilutions of an equal number of transformants were plated at 30°C on LB agar plates containing 5% sucrose, ampicillin, and kanamycin. The efficiency of plating was approximately 1,000-fold lower for cells carrying the pFYZ1 control plasmid than for transformants harboring a second copy of tmk (pDC13). This was the first indication that the Tmk protein was essential for E. coli. Colonies showing Suc^r, Ap^r, and Km^r phenotypes, harboring either the control plasmid pFYZ1 or the plasmid pDC13, were tested for Cm^s. Of the pFYZ1-transformant cells, all colonies (15 of 15) indicated Cm^r and Km^r, whereas all of the pDC13-transformants (104 of 104) had efficiently lost the pDC11 plasmid and therefore showed Cm^s and Km^r. This was a further indication for tmk being an essential gene. Replacement of tmk by the kka1 gene (tmk::kka1) using the above method and the plasmid pDC11 resulted in strain DC11.

Alternatively, plasmid pDC12 was used to construct a tmk::kka1 strain with 5'- and 3'-RBS flanking kka1 (Fig. 2b). Using the method of linear DNA fragment transformation as described by Yu et al. (39), the construction of the tmk::kka1 (RBS) was only possible in the presence of a second, plasmid-encoded tmk (pDC3). The second procedure resulted in strain DC12.

Cotransduction experiments with the tmk::kka1 construct.
To independently confirm the essential role of tmk, the miniTn10 (Tc^r) marker in DC1 (8) was transduced into DC11 by bacteriophage P1 transduction, selecting for Km^r, Ap^r, and Tc^r. This resulted in strain DC13. Phage P1 lysates grown on DC13 or DC14 were used to infect E. coli B178 strains, previously transformed with various L-arabinose-inducible plasmids harboring parts of the genomic region of tmk. This was done in the presence of 0.05% L-arabinose and at 30°C (Table 2). For the tmk::kka1 replacement construct (Fig. 2a), cotransductants were isolated only in the presence of a second copy of tmk at an average frequency of 25% (96 of 390 candidates). In the absence of a second tmk gene, no cotransduction was observed (0 of 322 candidates). The cotransduction experiments confirmed that the Tmk protein was absolutely essential and indicated no severe polarity effects of the replacement construct on viability due to altered expression of the up- or downstream genes yceG, holB, and ycfH. The variable level of gene expression of the complementing plasmid constructs may explain the difference in the frequency of cotransduction (22 to 43%) compared to the expected 40% cotransduction between the linked miniTn10 (Tc^r) and a yceG::miniTn10 (Km^r) marker insertion in the gene yceG upstream of tmk in the operon (Fig. 1) (8, 27). Using two independent tmk::kka1 replacement constructs (Fig. 2a and b), no difference in the cotransduction frequency was observed (data not shown). The deletions of tmk and its replacement by kka1 were confirmed by PCR amplification, as well as by Southern blot analysis (data not shown).

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TABLE 2. Frequencies of cotransduction between the miniTn10 (Tcr) marker and the tmk::kka1 (Kmr) markera

Complementation of the genomic E. coli tmk gene by plasmid-encoded thymidylate kinases of bacteriophage T4, E. coli, and S. cerevisiae.
Several expression systems and vectors of various copy numbers were tested for the expression of genes presumed to complement the E. coli tmk gene. Since tmk is expressed at low levels (34), these genes were generally first cloned into the low-copy-number plasmid pMPM-A6 (p15A origin of replication) and tightly regulated by the L-arabinose-inducible promoter (23). If the complementation of genomic E. coli tmk was not successful, they were cloned into the L-arabinose-inducible high-copy-number vector pMPM-A4 (pUC-like origin of replication) (23). Later, the genes of highest interest were cloned into a derivative of the IPTG-inducible vector pSE380 (Invitrogen) using the restriction enzymes EcoRI and XbaI. The derivative of pSE380, called pGP189 (11), has a 39-bp deletion near the lac promoter site of pSE380. This deletion replaces the NcoI site of pSE380 by an EcoRI site, so that genes originally cloned into pMPM-A6 have an optimal translation initiation in pGP189.

Plasmids pMPM-A6 (control), pDC3 (tmk), pDC14 (T4 gene 1) and pDC15 (cdc8) were transformed into B178, and the resulting strains were used as recipients in cotransduction experiments in the presence of 0.05% L-arabinose. Table 3 indicates that thymidylate kinase activity of the bacteriophage T4 gene 1, E. coli tmk, and S. cerevisiae cdc8 can complement the absence of the genomic E. coli tmk gene. The control plasmid pMPM-A6 gave no cotransduction. The absence of genomic E. coli tmk was confirmed by PCR analysis. A B178 tmk::kka1 candidate complemented with the S. cerevisiae cdc8 gene (pDC15) resulted in DC14 and was further used for the preparation of lysates for cotransduction experiments. In contrast to DC11, which harbors the tmk operon of plasmid pDC13, a recombination of the genomic tmk::kka1 construct with the plasmid-encoded yeast cdc8 of pDC15, can be excluded in DC14. All following work was therefore done with the tmk::kka1 construct present in DC14.

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TABLE 3. Complementation of the E. coli tmk gene by homologues of various originsa

Complementation by the human dTMP kinase gene dTYMK.
The human dTYMK gene was subcloned from pHsTmpK (dTYMK-1) (5) to create plasmids pDC16, pDC17, and pDC18, existing at various copy numbers per cell. To prove that the complementation activity of the human dTMP kinase is not allele specific, a second allele of the human dTYMK gene was amplified from mRNA of human embryonic kidney 293T cells and cloned into pGP189 resulting in pDC20 (dTYMK-2). Indeed, the two dTYMK proteins proved to be identical in their amino acid sequence.

To test whether the human dTYMK-1 gene can complement the tmk::kka1 allele, E. coli B178 transformed with various plasmids was cotransduced by using a phage P1 lysate grown on DC14 in the presence of either 0.05 or 0.5% L-arabinose. The presence of plasmid pDC3 (containing E. coli tmk) allows deletion of tmk at a concentration of 0.05% L-arabinose, whereas cotransduction was not observed in the presence of pDC16 (human dTYMK-1) (data not shown). Therefore, the human dTMP kinase gene was cloned into the high-copy-number vector pMPM-A4, resulting in plasmid pDC17 (dTYMK-1). This plasmid was transformed into B178, and cotransduction experiments were carried out as before. Plasmid pDC17 (dTYMK-1) allowed deletion of tmk at 0.5% L-arabinose. Since this concentration of L-arabinose is rather high, the two human dTYMK alleles (expressed from pDC18 and pDC20) and the E. coli tmk gene (pDC19) were cloned into the IPTG-inducible high-copy-number vector pGP189. These plasmids were then transformed into B178, which was again cotransduced. The plasmids pDC18 (dTYMK-1) and pDC20 (dTYMK-2) were able to complement the tmk allele in the presence of 0.1 mM IPTG (Table 4). Plasmid pDC19 (tmk) complemented even in the absence of any inducer since its promoter is leaky. The replacement of tmk on the chromosome was again confirmed by PCR analysis (data not shown). The data resulting from these experiments confirm the basic proposition that E. coli may grow with dTMP kinase activity of various origins.

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TABLE 4. Complementation of the E. coli tmk gene by human thymidylate kinase gene dTYMKa

Overexpression of E. coli and human dTMP kinases and Western blot analysis.
Strain B178 was transformed with plasmids pGP189 (control), pDC19 (tmk), pDC18 (dTYMK-1), and pDC20 (dTYMK-2). Cultures were induced with IPTG, and total cellular protein extracts were analyzed by SDS-PAGE. Whereas E. coli Tmk was visible as a band of approximately 28 kDa (28, 34), the human kinases could not be detected by Coomassie blue staining (data not shown).

After induction by IPTG, B178 carrying plasmids pGP189 (control), pDC21 (tmk-C-myc) and pDC22 (dTYMK-2-C-myc) were prepared for Western blot analysis, detecting expressed protein with an anti-myc-tag antibody. The bacterial Tmk could be visualized even without IPTG induction, whereas the human protein appeared only as a weak band upon induction with IPTG (Fig. 3).

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FIG. 3. Western blot analysis of E. coli Tmk and human dTYMK kinases modified with a C-terminal myc tag. B178 transformed with various high-copy-number vectors was grown in the presence of 1 mM IPTG as described in Materials and Methods. The proteins were detected by a primary antibody specific for the C-terminal myc-tag followed by a secondary horseradish peroxidase-linked antibody for imaging. Lanes: 1, pGP189 (control) without IPTG; 2, pGP189 (control) with 1 mM IPTG; 3, pDC21 (tmk-C-myc) without IPTG; 4, pDC21 (tmk-C-myc) with 1 mM IPTG; 5, pDC22 (dTYMK-2-C-myc) without IPTG; 6, pDC22 (dTYMK-2-C-myc) with 1 mM IPTG.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of a tmk deletion strain.
Up to now, no E. coli tmk deletion strain has been constructed. Such a strain is thought to be a useful tool for complementation studies using thymidylate kinases that might be of medical or biotechnological interest. The tmk gene is located in the third position of a putative five-gene operon, and therefore a simple insertion inactivation by a cassette encoding an antibiotic resistance is not possible. The ycfH gene was proposed to be nonessential (37), but holB, the gene next to tmk downstream in the operon, is essential (8, 32). Therefore, the goal of the present study was to replace the tmk gene by a genetic marker without influencing the expression of up- or downstream genes. Although a conditional lethal mutant of tmk already existed, this Ts tmk strain has a point mutation (G146A) in the tmk gene and loses its essential activity only at 42°C (9, 34). The fact that thymidylate kinase is a key enzyme for phosphorylation of thymidine nucleotides underlines the importance of constructing a stable null mutant of tmk, excluding the possibility of true revertants.

E. coli tmk is an essential gene and can be complemented with thymidylate kinases of various origins.
I obtained evidence for an essential function of the E. coli tmk gene product by various experiments: the first tmk::kka1 replacement strain was constructed according to the method of Link et al. (21). This method tests the frequency of excision and loss of the integrated plasmid harboring the tmk::kka1 construct or the tmk gene. The frequency was approximately 1,000-fold higher for the strains carrying pDC13 (five-gene tmk operon) compared to the one with the control vector pFYZ1. In addition, the colonies thus obtained were tested for loss of chromosome-released plasmid by checking for Cm^s. All of the pFYZ1 (control), but none of the pDC13 (five-gene tmk operon) colonies were Cm^r, Km^r, and Suc^r. This also indicates that tmk is essential, since without a plasmid-encoded copy, tmk cannot be deleted from the chromosome. In a second independent approach, using the linear transformation system of Yu et al. (39), a replacement of tmk could only be achieved in the presence of a second copy of tmk. Finally, cotransduction of a nearby miniTn10 (Tc^r) marker with the tmk::kka1 (Km^r) marker solely occurred in the presence of a second copy of tmk, underlining again the essential role of the Tmk protein in E. coli. Furthermore, cotransduction experiments were repeated in various other genetic backgrounds with similar results (data not shown).

In addition, plasmids overproducing thymidylate kinase activity of bacteriophage T4, S. cerevisiae or human origin could complement the tmk deletion; however, under different conditions. In the case of the human dTMP kinase, various explanations can be given for the low overproduction of human dTYMK protein. Since dTYMK does not have an unusually rare codon usage, this may not be inferred as a reason for poor overproduction. The human protein may not be correctly folded in E. coli and gets degraded or, for proper functioning, the kinase may have to undergo posttranslational modifications. Also, the dTYMK mRNA could be unstable in bacteria, or the human protein contains signals for its degradation or export out of E. coli. Furthermore, the human dTYMK protein may only be stable and active in supra-molecular complexes, such as the proposed "metabolon" multienzyme complex (22).

Advantages of a tmk deletion construct.
The E. coli tmk::kka1 replacement strains presented in this study are clearly advantageous compared to conditional-lethal mutants. For example, the Ts strain of Daws and Fuchs (9) can only be used for complementation studies at high temperature such as 42°C. Although the tmk Ts allele of this strain does not support E. coli growth at high temperature, this does not exclude that there could be residual, yet insufficient Tmk activity to allow E. coli to grow at high temperature (9). Most likely, if this strain was used to test complementation by homologues from other species, it would not be clear whether the residual E. coli tmk does, or does not, play a role in providing the cell with mutant dTMP phosphorylation activity. It is noteworthy that the Tmk(G146A) mutant enzyme present in this E. coli Ts strain (9) is not characterized by lower thermal stability but by significantly lower catalytic efficiency that is thought to be insufficient to support growth at high temperature (34). Furthermore, complementation studies with bacterial Tmks could lead to the formation of heterodimers of the endogenous Ts E. coli kinase and the complementing kinase, restoring the function of the otherwise inactive Tmk(G146A) mutant. Moreover, since the human thymidylate kinase is able to complement the genomic tmk deletion at 37°C, but not at 42°C (data not shown), the E. coli Ts strain is not suitable for complementation studies based on the human enzyme. Therefore, the tmk replacement strain (B178 tmk::kka1) has significant advantages in any in vivo selection strategies, since there is no residual E. coli Tmk activity present and revertants can be excluded. Indeed, this is the first time that the wild-type human thymidylate kinase dTYMK has been functionally expressed in E. coli in the complete absence of the E. coli tmk gene. The described recombinant bacteria, overproducing bacteriophage, bacterial, fungal or mammalian thymidylate kinases from plasmids, will be of interest for testing in a simplified cellular system effects of thymidine-like nucleoside analogues that are used in therapies against cancer and infectious diseases.

 
I thank Debbie Ang, Pierre Genevaux, Costa Georgopoulos, Manfred Konrad, Astrid Melotti, and Satish Raina for the gift of genetic material and Manfred Konrad for discussions and help in editing the manuscript.

This study was supported by grant FN 31-65403 (to the laboratory of C. Georgopoulos) from the Swiss National Science Foundation and the canton of Geneva, Switzerland.

 
* Corresponding author. Mailing address: Max Planck Institute for Biophysical Chemistry, Department of Molecular Genetics, Am Fassberg 11, D-37077 Göttingen, Germany. Phone: 49-551-201-1599. Fax: 49-551-201-1074. E-mail: david.chaperon{at}medecine.unige.ch.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, February 2006, p. 1288-1294, Vol. 72, No. 2
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