Nucleic Acids Research

Enzymes, reagents and general methods

Calf intestinal alkaline phosphatase and the restriction enzyme HindIII were purchased from Promega Corporation (Madison, WI). Nuclease P1 was purchased from Boehringer-Mannheim (Indianapolis, IN). All other restriction enzymes, T4 DNA ligase, polynucleotide kinase, Vent DNA polymerase and their supplied buffers were purchased from New England Biolabs Inc. (Beverly, MA). All enzyme units in the text are those specified by the manufacturer.

Pyridoxal phosphate and imidazole were purchased from Acros Organics (Pittsburgh, PA). Isopropyl [beta]-d-thiogalactopyranoside (IPTG), dithiothreitol and Tris were purchased from Boehringer-Mannheim. Low molecular weight protein standards were purchased from BioRad Laboratories (Hercules, CA). Ethanol was purchased from McCormick Distilling Co. Inc. (Pekin, IL). All other materials were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise noted.

DNA sequencing was performed using the SilverSequence protocol (Promega). Occasionally, 5[prime]-³³P-labeled primers were used with the same protocol except that autoradiography was employed to visualize the bands. Primers were purchased from the Great American Gene Company (Ramona, CA). [[gamma]-³³P]ATP was purchased from Andotek Life Sciences Co. (Irvine, CA).

Restriction digests, purification of restriction fragments by agarose gel electrophoresis, ligations, transformations and SDS-PAGE analysis were performed according to standard methods. Ni-NTA resin (Qiagen Inc., Valencia, CA) was prepared according to the manufacturer's instructions. Centriplus ultrafiltration devices were purchased from Amicon, Inc. (Cherry Hill, NJ). Sephadex G-10 and Sephadex G-25 (DNA grade) were purchased from Pharmacia (now part of Amersham Pharmacia Biotech, Piscataway, NJ). HPLC was performed with a Beckman System Gold HPLC instrument equipped with a System 125 solvent module, a Rheodyne 7725i injector and a Beckman 168 diode array detector. A Higgins Analytical HAIsil 120 C18 5 µm column (250 × 4.6 mm) was purchased from Bodman Industries (Aston, PA) and used for all HPLC analysis.

Strains and plasmids

Near-UV resistant Escherichia coli VJS2890 is a derivative of E.coli RK4353, which has a kanamycin resistance cassette inserted into the thiJ gene (12). The plasmid pVJS728 is part of an E.coli genetic library constructed by digestion of genomic DNA with HindIII and ligation into pHG165 (13) opened with the same restriction enzyme. The size of the genomic insert in pVJS728 is ~8.7 kb (12). Both VJS2890 and pVJS728 were kindly provided by T.Begley. A [lambda]DE3 lysogen of VJS2890, VJS2890(DE3), was prepared using the [lambda]DE3 lysogenization kit of Novagen according to the manufacturer's instructions. Competent BLR(DE3) pLysS, a strain of E.coli BLR that produces both T7 RNA polymerase and T7 lysozyme, was purchased from Novagen Inc. (Madison, WI).

The plasmids pBH100, pBH101 and pBH104 were constructed by excising fragments of the genomic DNA insert in pVJS728 (KpnI-PstI for pBH100 and PstI-PstI for both pBH101 and pBH104) and ligation into pGEM3Zf(+) (Promega). The composition of each plasmid was confirmed by appropriate restriction analysis (data not shown). The sequences of all plasmids described in this paper can be found at http://www.udel.edu/chem/mueller

To create pBH113, an open reading frame (ORF) in the insert of pVJS728 was amplified by PCR using the following two primers:
   ACTCGACGGCTCATatgaagtttatcattaaattgttcccg
   GCATGGATCCACAAAttacgggcg
where the lower case letters denote the bases that hybridize to the target ORF and the underlined bases denote the restriction sites. The template was pVJS728 opened with ScaI and the PCR amplification was performed using HotStart tubes (Molecular Bio-Products Inc., San Diego, CA) according to the manufacturer's protocol. A Robocycler Gradient 96 thermal cycler (Stratagene, La Jolla, CA) was used with the following program: 3.0 min, 95°C; 30 × (1.5 min, 95°C; 2.5 min, 40°C; 3.0 min, 72°C); 2.0 min, 72°C. The PCR product containing the target ORF was purified using a QIAquick PCR Purification kit (Qiagen), digested with NdeI and BamHI and ligated into pET15b (Novagen) that had been opened with the same restriction enzymes. The structure of pBH113 was confirmed by restriction analysis and sequencing of both ends of the inserted PCR product (data not shown).

Selection

Selection for near-UV sensitivity of VJS2890 and VJS2890(DE3) harboring different plasmids was based on the method described by Backstrom (12). The near-UV resistant strains were first made competent by the method of Hanahan (14) and then transformed with the plasmid to be tested. A single colony of the transformed mutant was suspended in 5 ml LB broth; 50 µl serial dilutions of this cell suspension were then plated on LB agar. These plates were then positioned over semicircular openings in a cardboard mask such that only half of the plate was exposed to the near-UV light emitted by the lamp on which the mask rested. The lamp used was a Spectroline XX-15A (Spectronics Corp, Westbury, NY) with a long wavelength UV bulb ([lambda] = 365 nm, 1100 µW/cm²); it was cycled on (1.5 h) and off (2.5 h) overnight at room temperature. The distance between the plate and the bulb was ~9 cm and the cardboard mask rested on a glass plate (UV cut-off ~315 nm, compared with a UV cut-off of ~295 nm for the Petri dishes). Similar numbers of colonies on both the `dark' and `UV' sides of the plate indicated a continued mutant phenotype (no complementation, near-UV resistance) and a very small number of colonies on the UV side relative to the dark side indicated pseudoreversion to the wild-type phenotype (complementation, near-UV sensitivity).

Isolation of tRNA

The isolation of tRNA was based on the method of Hou and Schimmel (15). A single colony of either VJS2890(DE3) or VJS2890(DE3)/pBH113 was used to inoculate LB broth (1 l) and this culture was incubated overnight at 37°C with moderate shaking. The cells were harvested by centrifugation for 30 min at 6000 g. The supernatant was decanted and the pellet was resuspended in 10 ml 10 mM Tris-HCl buffer, pH 7.4, containing 1 mM MgCl₂.

Using a vortex mixer, the cells were vigorously agitated for 1 min with water-saturated phenol (10 ml) and the mixture was subjected to centrifugation for 30 min at 8000 g. The aqueous phase was decanted and the cells were resuspended, extracted and subjected to centrifugation as just described. The two aqueous phases were combined (13 ml final volume). To precipitate the tRNA, 830 µl 5 M sodium acetate buffer, pH 5.2, (0.3 M final concentration) was added, followed by addition of 40 ml absolute ethanol and incubation for a minimum of 1 h at -20°C.

The tRNA was pelleted by centrifugation for 30 min at 8000 g and this pellet (2 g) was redissolved in 8 ml 50 mM Tris-HCl buffer, pH 7.6. The tRNA was then passed over a Sephadex G-25 column equilibrated in the same buffer. The final tRNA solution was ~0.2 mM (A_{260 nm} [ap] 100, [epsis]_{260 nm} = 5 × 10⁵/M/cm).

tRNA digestion and HPLC analysis

The tRNA digestion was accomplished by the method of Gehrke et al. (16) with slight modification. A solution (20 µl) containing tRNA (4 nmol) was incubated for 2 min at 100°C then cooled to 37°C. To this solution were added 85 µl 30 mM sodium acetate buffer, pH 5.3, 10 µl 20 mM ZnSO₄ and 0.4 U nuclease P1. The solution (196 µl) was incubated for 1 h at 37°C and then for 2 min at 100°C. After cooling to 37°C, more nuclease P1 (0.4 U) was added and the solution was incubated for 1 h at 37°C. To adjust the pH of the solution, 300 µl 500 mM Tris-HCl buffer, pH 7.6, were added, followed by 2 U calf intestine alkaline phosphatase. The solution was incubated for 1 h at 37°C. The resultant nucleosides were analyzed by chromatography over an HPLC C18 column; elution was accomplished using a gradient of acetonitrile in ammonium acetate buffer, pH 6.0, according to the method of Buck et al. (17).

Over-expression and purification of ThiI

A 5 ml saturated overnight culture of BLR(DE3) pLysS/pBH113 was used to inoculate 1 l LB broth containing 50 µg ampicillin, 34 µg chloramphenicol and 1 mM pyridoxal phosphate, which was shaken at 37°C. When A_{600 nm} = 0.5, IPTG was added (to 0.4 mM). Induction of ThiI was monitored by SDS-PAGE analysis of total cellular protein.

After 3 h, the cells were pelleted by centrifugation for 30 min at 10 000 g then resuspended in 30 ml 20 mM Tris-HCl buffer, pH 7.9, containing 5 mM imidazole and 500 mM NaCl. The cells (3.7 g) were again pelleted by centrifugation, resuspended in 25 ml of the same buffer, quick frozen in liquid nitrogen and stored overnight at -80°C. The cells were thawed in a 37°C water bath with frequent swirling; all subsequent steps were performed at 4°C. Cellular disruption was achieved by double passage through a French pressure cell (5000 p.s.i.) and cell debris was removed by centrifugation for 20 min at 40 000 g.

Figure 1. Localization of the near-UV resistance gene. (Top) Partial restriction map of the parent plasmid pVJS728. (Middle) Portions of pVJS728 excised and ligated into pGEM3Zf(+), with the orientation of the insert in pGEM3Zf(+) indicated by the direction of the arrow. (Bottom) Location of thiI, the s⁴U biosynthetic gene identified in this study, with the direction of transcription indicated. (Right) Reversion of VJS2890 to wild-type phenotype when harboring the particular plasmid.

The supernatant was stirred in a beaker for 1.5 h with 4 ml settled Ni-NTA resin that had been equilibrated in 20 mM Tris-HCl buffer, pH 7.9, containing 5 mM imidazole and 500 mM NaCl. The resin was then allowed to settle and the supernatant was decanted. The resin was then resuspended in the same buffer and loaded into a 0.7 cm column (final bed height ~9.5 cm); the beaker was rinsed with a small amount of buffer and this rinse was applied to the settled column of resin. The column was then washed with 3 × 10 ml 20 mM Tris-HCl buffer, pH 7.9, containing 23 mM imidazole and 500 mM NaCl. The ThiI was eluted by application of a linear gradient (50 + 50 ml) of 23-500 mM imidazole in 20 mM Tris-HCl buffer, pH 7.9, containing 500 mM NaCl; 5 ml fractions were collected. Protein was localized in the fractions by measuring A_{280 nm} and the purity of these fractions was assessed by SDS-PAGE. Purified ThiI was precipitated by addition of ammonium sulfate to 70% saturation for storage at 4°C.

UV-visible spectroscopy of purified ThiI

Dissolution of ThiI in triethanolamine or Tris buffer followed by gel filtration over Sephadex G-10 resulted in precipitation of ThiI. Portions of the ammonium sulfate pellet were therefore dissolved in 50 mM potassium phosphate buffer, pH 7.5, containing 5 mM MgCl₂, 100 mM KCl, 0.1 mM ethylenediamine tetraacetic acid and 1 mM dithiothreitol. Chromatography over Sephadex G-10 pre-equilibrated with the same buffer removed ammonium sulfate. Spectra of desalted ThiI were recorded on a Beckman DU-640 spectrophotometer.

RESULTS

The near-UV resistant mutant VJS2890 had been developed by Begley and co-workers to identify a thiamin biosynthetic gene (see Discussion). Using this mutant, the same investigators screened an E.coli genetic library and discovered that VJS2890 is complemented by pVJS728, a plasmid which contains an 8.7 kb insert of E.coli genomic DNA (12). We began our search for the gene involved in s⁴U biosynthesis with VJS2890 and pVJS728.

Fragments of the genomic insert of pVJS728 were subcloned into pGEM3Zf(+) (Fig. 1). VJS2890 was separately transformed with each of these plasmids and the transformants were analyzed for complementation of the near-UV resistance by growth under cycling near-UV light. The gene of interest was localized to the PstI-PstI fragment and complementation by the same fragment ligated into pGEM3Zf(+) in either orientation (compare pBH101 and pBH104, Fig. 1) suggested that the gene of interest (later named thiI, see Discussion) retained its regulatory cis elements in the fragment.

Figure 2. The near-UV selection. (A) VJS2890(DE3), the near-UV resistant mutant (growth on both the `UV' and the `dark' sides). (B) VJS2890(DE3)/pBH113, the mutant harboring a plasmid containing the ORF that complements the mutation (growth only on the `dark' side).

Sequencing of the PstI-PstI fragment was undertaken. Shortly thereafter, this portion of the E.coli genome became available (GenBank accession nos U82664 and AE000148) and the sequence was scrutinized for ORFs of unknown function, all of which were considered as candidates for the s⁴U biosynthetic gene. Of the five identified candidate ORFs, one is severely truncated even in pVJS728 (the codons for only 204 of 620 amino acids are present). The next three candidate ORFs are sequential, as expected for an operon; however, no control sequences for transcription or translation are obvious. Furthermore, the PstI-PstI fragment is truncated at the 5[prime]-end of the first ORF of the putative operon and this truncation would likely make expression of these candidate ORFs dependent upon the orientation of the fragment in pGEM3Zf(+), which is not observed. The last candidate ORF possesses discernible Shine-Dalgarno and -10 sequences and retains them in the PstI-PstI fragment, making it the prime candidate even though it is slightly truncated at the C-terminus in pBH101 and pBH104 (missing 18 of 482 amino acids). This candidate ORF (GenBank accession no. U82664, bases 20999-22447) was amplified by PCR and positioned behind the T7 promoter and ribosomal binding site of pET15b to afford pBH113. This plasmid was used to transform VJS2890(DE3), a [lambda]DE3 lysogen of VJS2890 suitable for expression of genes behind a T7 promoter.

As shown in Figure 2, pBH113 complemented the near-UV resistance of VJS2890(DE3), which demonstrates the involvement of the subcloned ORF in the generation of s⁴U at position 8 of certain tRNAs. Two of the other candidate ORFs exist on the strand complementary to the target ORF and one (or both) of these could potentially encode the gene product necessary for complementation of the mutation in VJS2890. VJS2890 itself, however, was not complemented by pBH113 (data not shown), which indicates a requirement for the T7 RNA polymerase encoded by the [lambda]DE3 lysogen of VJS2890(DE3).

Figure 3. The UV-visible spectra of tRNA isolated from the near-UV resistant mutant and the near-UV resistant mutant complemented with thiI. a, Spectrum of tRNA from the mutant, which lacks s⁴U in tRNA; b, spectrum of tRNA from the mutant harboring pBH113, which expresses ThiI. The peak at 334 nm arises from s⁴U in the tRNA.

We sought independent biochemical confirmation of the proposed role of the cloned ORF. Total tRNA was isolated from both VJS2890(DE3) and VJS2890(DE3)/pBH113 and the tRNA from the latter displayed an absorbance peak at 334 nm (the [lambda]_max of s⁴U), while tRNA from VJS2890(DE3) displayed no such peak (Fig. 3). Both samples of tRNA were then subjected to total digestion and the resultant nucleosides were analyzed chromatographically (17). As shown in Figure 4, the tRNA from VJS2890(DE3) contains no s⁴U, as expected for a UV-resistant strain, while the tRNA from VJS2890(DE3) harboring pBH113 clearly contains s⁴U.

Figure 4. Analysis of the nucleosides resulting from total digestion of tRNA from the UV-resistant mutant and the mutant harboring pBH113, which expresses ThiI. (A) Chromatogram, detection at 260 nm, of the tRNA from the complemented mutant. (B) Chromatogram, detection at 330 nm, of the tRNA from the mutant (lower trace) and the complemented mutant (upper trace). In (A) and (B), the star denotes the position of s⁴U, which is the only nucleoside that absorbs significantly at 330 nm. (C) Spectrum of the peak denoted with the star, which matches the spectrum of s⁴U ([lambda]_max = 334 nm).

Over-production of ThiI was readily achieved in BLR(DE3) pLysS/pBH113. Because the subcloning placed a His₆ tag at the N-terminus of ThiI, chromatography over a column of agarose impregnated with nickel(II) allowed purification to homogeneity in one step (Fig. 5). Approximately 15 mg purified ThiI were obtained from 1 l bacterial culture. The UV/visible spectrum of purified ThiI showed only the expected peak due to protein at 280 nm; the spectrum revealed no evidence of bound pyridoxal phosphate (data not shown).

Figure 5. SDS-PAGE analysis of the induction and purification of ThiI. (A) S, molecular weight standards; 0-3, total cellular protein analysis before (0) and 1, 2 and 3 h (1-3 respectively) after addition of IPTG to induce production of ThiI. (B) a, supernatant of cell extract treated with Ni-NTA resin; b, column flow-through of the treated Ni-NTA resin; c, wash of Ni-NTA column; S, molecular weight standards; d-i, fractions 3, 5, 7, 9, 11 and 13 respectively after application of the imidazole gradient (denoted by the triangle). The arrow denotes the ThiI band. The molecular weights of the standards are denoted in the column marked MW; the predicted size of expressed ThiI is 57.1 kDa. The bottom of the gels fanned out during staining and destaining.

DISCUSSION

Previous workers had selected for mutants in s⁴U biosynthesis by growing the bacteria with cyclic exposure to near-UV light: only bacteria lacking s⁴U grow under these conditions (10,11,18). These mutants were divided into classes and the genes nuvA and nuvC were mapped (18,19). The gene names derive from the near-UV resistance of the mutants and from correspondence to factor A and factor C, heterogeneous protein isolates reported by Lipsett and co-workers in seminal investigations of the enzymology of s⁴U generation (20). Begley's group utilized the reported thiamin auxotrophy of nuvC^- mutants to screen for thiamin biosynthetic genes. These efforts produced both the nuv^- mutant VJS2890 and pVJS728, a plasmid from an E.coli genetic library that complemented VJS2890. Begley and co-workers identified thiJ (GenBank accession no. U34923), an ORF that was reported to complement the thiamin auxotrophy but not the near-UV resistance of VJS2890 (12). A recent re-examination, however, has revealed that the identification of thiJ as a thiamin biosynthetic gene resulted from an artifactual recombination event involving a portion of thiI that remained on the plasmid bearing thiJ. No function is currently assigned to thiJ (T.Begley, personal communication).

Scant days before our demonstration of a role for the subcloned ORF in s⁴U generation, Downs' group (21) reported that the Salmonella typhimurium homolog of this gene plays an essential role in thiamin biosynthesis and christened it thiI. Therefore, it seems likely that thiI is identical to the gene named nuvC by Lipsett, for Lipsett had reported that nuvC^- E.coli were also thiamin auxotrophs (18). However, nuvC was mapped to 42-46 min on the E.coli chromosome (18), while thiI is found at ~9 min. Strikingly, nuvA was mapped to 9.3 min (19), generating uncertainty regarding the identity of thiI as nuvA or nuvC. Furthermore, isolated ThiI protein displays no spectroscopic sign of pyridoxal phosphate, which is a cofactor reported by Lipsett to be essential for NuvC activity (22). Experiments are underway to determine whether or not pyridoxal phosphate can be loaded onto ThiI using the conditions of Lipsett (22) and the results of these experiments may clarify the identity of thiI as nuvA or nuvC.

Lipsett and co-workers reported that NuvC preparations were unstable to more than rudimentary purification (20) and preliminary results from our continuing efforts to purify NuvA and NuvC from E.coli also indicate instability of the enzymes necessary to effect the overall transfer of sulfur from cysteine to s⁴U in tRNA. Because of the greater difficulty inherent in isolating multiple enzymes that must act together in order to achieve an observed overall activity, it seems likely that more than two gene products may be involved in the generation of s⁴U at position 8 of tRNA. To resolve these issues, we have begun the isolation of more nuv mutants and complementation experiments with genuine nuvA mutants (11). Experiments are also currently underway to determine the discrete chemical transformation catalyzed by ThiI. The discovery of the substrate(s) and product(s) of this one step in the s⁴U generation pathway should greatly aid in the elucidation of this pathway as well as the pathway of sulfur incorporation in thiamin biosynthesis.

ACKNOWLEDGEMENTS

We are grateful to T.Begley for providing pVJS728 and the mutant strain VJS2890. This work was supported by grants from the University of Delaware Research Foundation (LTR 19960415 and LTR 19970410), the National Science Foundation (CHE-9633445) and the Exxon Education Foundation. P.M.P. was supported by USPHS grant T32 GM08550. Support for this research was provided by a grant from the Howard Hughes Medical Institute through the Undergraduate Biological Sciences Education Program.

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*To whom correspondence should be addressed. Tel: +1 302 831 2739; Fax: +1 302 831 6335; Email: emueller@udel.edu

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