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Gram-positive commensal bacteria, such as Streptococcus gordonii, are currently being developed as live vaccine vectors able to colonize mucosal surfaces and stimulate a secretory immunoglobulin A, as well as a systemic, immune response to a recombinant antigen displayed on the surface of these organisms (4, 7). In order to test these organisms as live vaccine vectors in humans, nonantibiotic selectable markers will be needed for the manipulation of these recombinant organisms in research and clinical laboratories. Previous studies of gram-negative bacteria have suggested it is possible to select for tdk mutations on the basis of resistance to the pyrimidine analog fluorodeoxyuridine (FUDR) (1, 3). All FUdR^r Escherichia coli possess mutations that map to the tdk locus and are deficient in thymidine kinase (TK) (9). Here, we demonstrate that FUdR^r strains of S. gordonii are readily selected, identify the nature of the mutations in the tdk locus, assay for the loss of TK enzyme activity, and test the in vivo consequences of the mutation.

S. gordonii strain GP204, a spontaneous streptomycin-resistant mutant of S. gordonii strain V288 (ATCC 35105) (8), is the parental strain of the FUdR^r S. gordonii strains. Strain GP204 was plated on brain heart infusion agar base containing between 1 and 10 µg of FUDR per ml in the presence of uridine (12.5 µg/ml) and thymidine (2 µg/ml). Two FUdR^r colonies were obtained on the plates containing 1 µg of FUDR and were designated SP204(1-1) and SP204(1-2). One FUdR^r colony grew on the plates containing 10 µg of FUDR per ml and was designated SP204(10-1). Quantitative analyses revealed that the spontaneous mutation rate of S. gordonii GP204 FUdR^r mutants was 10⁶ (data not shown).

PCR was employed to amplify the TK open reading frame (ORF) from chromosomal DNA preparations of S. gordonii strains SP204(1-1), SP204(1-2), SP204(10-1), and GP204. PCR primers were based on the previously published sequence of the S. gordonii tdk gene from strain DL-1 (Challis) (6) and were designed to amplify a 579-bp DNA product encompassing the entire tdk locus. The PCR products were cloned into the plasmid vector pCR2.1 (Invitrogen) and transformed into INVF' cells. The DNA from representative clones was sequenced with the M13 reverse and T7 sequencing primers.

Analysis of the DNA sequence from the FUdR^r strains revealed that the tdk ORFs each contained a single base pair substitution which resulted in the introduction of a translational termination codon in the tdk ORF at a unique position in each strain, as follows: codon 86 of SP204(1-1), codon 155 of SP204(1-2), and codon 88 of SP204(10-1). The derived nucleotide sequence of S. gordonii GP204 differed from that published for DL-1 (Challis) (6) at a single residue, containing a silent A to G mutation at nucleotide 54 of the tdk ORF, as did SP204(1-1), SP204(1-2), SP204(10-1), verifying that S. gordonii GP204 was the parent of these strains.

The predicted molecular mass of the full-length S. gordonii TK polypeptide is 21,843 Da. The predicted molecular mass of the prematurely terminated TK polypeptides encoded by the SP204(1-1), SP204(1-2), and SP204(10-1) FUdR^r mutants are 9,800, 17,800, and 10,100 Da, respectively. To verify these phenotypes, the FUdR^r tdk loci cloned in the pCR2.1 plasmid vector were transcribed using T7 RNA polymerase, the derived transcripts were translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine, and the radiolabeled translation products were analyzed by gel electrophoresis. The data in Fig. 1A indicate that the apparent molecular masses of the labeled in vitro translation products observed agree with the predicted sizes of the TK truncation products of the tdk loci of SP204(1-1), SP204(1-2), and SP204(10-1).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Structural and functional analysis of FUdRr tdk genes. Transcripts of the tdk genes of wild-type S. gordonii and the FUdRr mutants were prepared and then translated in rabbit reticulocyte lysates in the presence or absence of [35S]methionine. (A) The radiolabeled translation products were resolved by electrophoresis on 12% polyacrylamide gels containing sodium dodecyl sulfate and visualized by autoradiography. Lane M, molecular weight markers (46, 30, 21.5, 14.3, 6.5, and 3.4 kDa, respectively); lane 1, no RNA added; lane 2, GP204; lane 3, SP204(1-1); lane 4, SP204(1-2); lane 5, SP204(10-1). (B) The unlabeled translation products were tested for TK enzyme activity using a previously described protocol (5) which measures the conversion of [3H]thymidine to [3H]thymidine monophosphate. The incorporation of the radiolabel is shown for each of the tdk gene products. The bar labeled "H20" represents the incorporation directed by a reticulocyte lysate to which no exogenous RNA had been added. Results are means ± standard deviations (error bars).

Previously, Black and Hruby (2) identified seven functional domains (I to VII) that are highly conserved in both eukaryotic and prokaryotic TK enzymes. The truncated TK enzymes encoded by SP204(1-1), SP204(1-2), and SP204(10-1) would lack the essential domain VII, a four-amino-acid sequence near the carboxyl terminus. To test this prediction, unlabeled translation reactions programmed with FUdR^r tdk-derived in vitro transcription products were tested for TK activity, as measured by the ability of the extracts to convert [³H]thymidine to [³H]-TMP (5). As is evident in Fig. 1B, no [³H]thymidine-phosphorylating activity was present above background level in the SP204(1-1), SP204(1-2), and SP204(10-1) TK translations, whereas a high level of TK activity was evident in the parental GP204 TK translation reaction.

The TK-deficient phenotype of the FUdR^r S. gordonii mutants had no effect on the growth of S. gordonii cells in culture or in recipient animals. Compared to GP204 in brain heart infusion broth, SP204(1-1) grew at a similar rate and to a similar cell density (data not shown). Likewise, there was no significant reduction in the number of mice colonized or the average duration of colonization for inoculated BALB/c mice (data not shown).

The results presented here have confirmed an apparent commonality in nucleotide metabolism between gram-negative bacteria, such as E. coli, and the gram-positive commensal bacterium S. gordonii. When grown in the presence of inhibitory concentrations of FUDR, S. gordonii produces FUDR-resistant mutants with a frequency of about 10⁶. Identification of the genomic locus responsible for the acquisition of FUdR^r revealed that three independent mutations [SP204(1-1), SP204(1-2), and SP204(10-1)] all mapped to the same gene, tdk, which encodes the nucleoside salvage enzyme TK. Each of the three FUdR^r mutants acquired a TK-deficient phenotype by virtue of the introduction of a nonsense mutation within the tdk ORF to produce truncated proteins lacking one or more motifs known to be essential for other TK enzymes. Enzyme assays confirmed that none of the truncated enzymes encoded by the SP204(1-1), SP204(1-2), and SP204(10-1) tdk genes retained any enzymatic activity.

The original impetus for attempting to isolate FUdR^r S. gordonii mutants was to enable this marker to be used for the in vitro selection for recombinant candidate vaccine strains and to facilitate the detection of implanted organisms in recipient animals in vivo. Selective conditions could be established (1 µg of FUDR per ml, 12.5 µg of uridine per ml, and 2 µg of thymidine per ml) which allowed the growth of FUdR^r mutants while inhibiting the growth of wild-type S. gordonii. Furthermore, the growth of FUdR^r S. gordonii was not compromised in rich media, indicating that recombinants derived in this manner can be easily grown to high density for use as vaccine inocula. In vivo implantation experiments suggested that the ability of S. gordonii to establish colonization and persist in the oral cavity of the mouse is not compromised by the presence of the FUdR^r mutation.

Taken together, the results obtained here indicate that FUdR^r should have utility as a selection scheme and phenotypic marker in S. gordonii-based recombinant vaccines. This approach has two advantages. First, since FUDR is not routinely used to treat human disease there should not be a significant reservoir of FUdR^r oral bacteria to complicate detection of implanted vaccines. Second, FUDR is not an antibiotic and FUdR^r is not plasmid-borne. If live bacterial strains are to be used as vaccine vectors, they should not contain any engineered, or selected, markers of resistance to drugs of clinical relevance in a configuration (such as a plasmid) whereby they could be passed from the implanted commensal to an indigenous pathogen. The FUdR^r genomic marker should satisfy both of the above-mentioned criteria.