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1 Department of Chemistry and Biochemistry, University of Regina, Regina, Saskatchewan S4S 0A2, Canada
2 Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, DK1307, Denmark
Correspondence
Rod A. Kelln
rod.kelln{at}uregina.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Response Biomedical Corp., 100-8900 Glenlyon Parkway, Burnaby, BC V5J 5J8, Canada.
Present address: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada.
Present address: Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106, USA.
||Present address: Department of Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, CA 94143-2542, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Uridine 5'-monophosphate (UMP) is the parent compound for the biosynthesis of all other pyrimidine (deoxy)ribonucleotides in the cell. UMP is synthesized de novo from carbamoylphosphate and aspartate through a multi-step process involving five enzymes and four intermediate compounds. The order of synthesis and the corresponding genes are: carbamoylaspartate (CAA, or ureidosuccinate; pyrBI), dihydroorotate (DHO; pyrC), orotate (OA; pyrD), orotidine 5'-monophosphate (OMP; pyrE), with the final step being the decarboxylation of OMP (pyrF) to yield UMP (Fig. 1a
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; Hantke, 1976; van Alphen et al., 1978). Inner membrane transport systems for pyrimidine compounds include a uracil permease, UraA (Andersen et al., 1995), a cytosine permease, CodB (Danielsen et al., 1992), and two high-affinity nucleoside transport systems, NupC and NupG (Munch-Petersen & Jensen, 1990). Although wild-type S. enterica serovar Typhimurium is permeable to pyrimidine nucleosides and nucleobases, it is permeable to only a single pyrimidine intermediate, namely OA. In an earlier study (Baker et al., 1996), we showed that transport of OA was dependent on DctA, an inner-membrane protein with a predicted 12-transmembrane-domain structure that is typical of bacterial transport systems, whose primary function is the transport of the C4-dicarboxylates succinate, fumarate and malate. Consistent with this role, mutants defective in dctA are unable to use these compounds as sole carbon source, and are also incapable of utilizing OA as an exogenous pyrimidine source (Baker et al., 1996).
In this study, we have extended our analyses of the uptake and utilization of pyrimidine intermediates in S. enterica serovar Typhimurium. Mutants capable of utilizing exogenous CAA as sole pyrimidine source have been isolated previously (Kelln & Zak, 1980; Legrain et al., 1976; Syvanen & Roth, 1973). These isolates, however, were neither extensively characterized, nor were the genes responsible for the phenotype identified. Here we describe the characterization of the previously isolated S. enterica serovar Typhimurium mutants as well as a newly isolated novel mutant capable of using DHO as sole exogenous pyrimidine source. In this report, we show that the utilization of both CAA and DHO is the result of distinct mutations in the previously uncharacterized yhiT locus, the third gene in the yhiVUTS operon.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Other strains and their construction are described at various points in the text. Plasmid vectors used were: pBR322 (Bolivar et al., 1977), pJQ200 (Quandt & Hynes, 1993), pUC4K (Kokotek & Lotz, 1989), pUC18 (Yanisch-Perron et al., 1985) and pWSK29 (Wang & Kushner, 1991). Plasmids constructed during this study are described in the text in the relevant section.
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) and usp-3 (Kelln & Zak, 1980) were used previously to indicate mutations mediating the use of exogenous ureidosuccinate [preferred name carbamoylaspartate (CAA)] as a pyrimidine source. Accordingly, the phenotype associated with the mutant allele is Caa+; for the wild-type it is Caa–. Correspondingly, the phenotype associated with the utilization of exogenous dihydroorotate (DHO) is indicated as Dho+. The mutation in the parental Dho+ isolate described herein, KR1667, was designated yhi-23, based on the determined map location. For the corresponding chromosomal DNA region, the currently accepted nomenclature for the S. enterica serovar Typhimurium genome was applied (McClelland et al., 2001); therefore, the genes for the four contiguous open reading frames were designated yhiVUTS.
Media and growth conditions.
Lennox L broth (LB) was the complex medium (Enquist & Sternberg, 1979). The minimal medium, medium A, has been described previously (Kelln et al., 1975). Carbon sources were added at a final concentration of 0.2 %, except for glycerol (0.3 %). Unless stated otherwise, the final concentrations of supplements (µg ml–1) were: uracil, 25; thiamine, 2; individual amino acids, 50; carbamoylaspartate (CAA), 100; dihydroorotate (DHO), 100; orotate (OA), 100; 5-bromo-4-chloro-3-indoyl β-D-galactopyranoside (XGal), 50. As required, antibiotics were added at the following final concentrations (µg ml–1): ampicillin, 100; chloramphenicol, 15; gentamicin, 20; kanamycin, 120 (minimal medium) or 60 (LB); tetracycline, 20. Solid media were prepared by the addition of 15 g agar l–1. Cultures were grown at 37 °C and liquid cultures were incubated on an air shaker operating at 250 r.p.m. Growth of liquid cultures was monitored by measuring cell turbidity with a Klett–Summerson colorimeter (filter no. 54).
Genetic techniques.
Bacteriophage P22HT105/1int-201 (Hughes & Roth, 1984) was used for all transductions with S. enterica serovar Typhimurium. EGTA (10 mM) was added to the plating medium to limit lysogeny of transductants. Methods for the creation of MudJ transcriptional fusions and Tn10dTc transposon technology were as reported previously (Gillen & Hughes, 1993; Kleckner et al., 1977). For the isolation of loss-of-function mutants following transposition mutagenesis (e.g. MudJ insertion to create a Dho– derivative from a Dho+ strain), the transductants were pooled and grown to stationary phase in a medium containing the required supplement. A sample of washed cells was inoculated into medium without the supplement and used to enrich for the desired isolate by penicillin counterselection (Miller, 1992). Isolates were screened for the corresponding phenotype and appropriate candidates were then used for genetic analyses. Rapid transductional mapping (Benson & Goldman, 1992) was carried out using Kit-22 from the Salmonella Genetic Stock Centre. Genetic recombination of gene fusions and insertional mutations into the S. enterica serovar Typhimurium chromosome using suicide vector pJQ200 were carried out as described previously (Quandt & Hynes, 1993).
Mutagenesis.
Chemical mutagenesis involved treating the culture at a density of 1–2x108 cells ml–1 with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 0.1 mg ml–1 (Miller, 1992). Following mutagenesis, cells were cultured for 24 h in appropriately supplemented minimal medium prior to plating on a selective medium.
Disk diffusion assays.
KR1667 (pyrB137 usp-2 yhi-23 dctA1 : : MudJ) was grown with either CAA or DHO in liquid minimal medium to mid-exponential phase and plated onto the same solid medium. A sterile filter paper disk soaked with a test compound was placed on the plate, which was then incubated for 24 h. The presence of a zone of growth inhibition indicated competition for uptake of either CAA or DHO by YhiT.
Uptake and incorporation assays.
[5-3H]Orotic acid, 250 µCi (9.25 MBq; specific activity 370–740 GBq mol–1), was purchased from Moravek Biochemicals. Radioactive OA-uptake and incorporation assays were carried out essentially as described previously (Yurgel et al., 2000). KR1667 and KRM106 were grown in medium A with glycerol and OA (25 µg ml–1) to 60 Klett units (OD660 0.24); radioactive OA (3.7x104 Bq ml–1) was then added. Samples were taken immediately, then at 15 and 30 min, and subsequently at 30 min intervals for a total of 180 min. The cells were collected by vacuum filtration (0.45 µm pore size; mixed cellulose esters), washed twice with 1 ml medium A salts containing non-radioactive OA (100 µg ml–1), and dried, and the radioactivity was determined by liquid scintillation counting. The effect of CAA on the uptake of radiolabelled OA by KRM106 (and on the corresponding cell growth culture) was examined by adding CAA (200 µg ml–1) after 45 min, followed by sampling of the cultures for 120 min.
DNA techniques.
The methods used were primarily adapted from the manual of Sambrook et al. (1989). Transfer of plasmids between E. coli and S. enterica serovar Typhimurium using strain KR1562 as an intermediate in transformations was performed as described previously (Baker et al., 1996). Sequencing was carried out as a service by either the University Core DNA services (University of Calgary, Calgary, AB, Canada) or the Plant Biotechnology Institute, National Research Council, (Saskatoon, SK, Canada). DNA sequence data were compiled and analysed using the Chromas (Version 1.45), DNA Strider (Version 1.3) and Align software packages, the GenBank network BLAST server, and a modified version of the original Targsearch promoter analysis software (Mulligan et al., 1984). Further analysis was carried out using the GenBank 
BLAST protein server and TopPred II (Version 1.3) (Claros & von Heijne, 1994) protein prediction programs.
RNA isolation and RT-PCR reactions.
RNA was isolated as previously described (Mackie, 1989) from exponential-phase cells (KR1312) grown in LB. Samples were treated with DNase I (Invitrogen) at 37 °C for 45 min to remove contaminating DNA prior to reverse transcription reactions. The cDNA encompassing yhiVUTS was generated using an oligonucleotide, RAKter1, (5'-GGGTGGGCGCACAAGCCTGC-3') complementary to nucleotides just downstream of the yhiS termination codon. Specifically, RNA (3 µg) was incubated with 2 pmol RAKter1 and 1 mM dNTPs at 65 °C for 5 min. Reverse transcription reactions were assembled according to the manufacturer's instructions and cDNA synthesis was carried out using Superscript II reverse transcriptase (Invitrogen) at 42 °C for 60 min, followed by incubation at 70 °C for 15 min. A 300 ng aliquot of the RT reaction was used in the amplification of various DNA fragments using an Expand High Fidelity PCR System (Roche). Reaction mixtures contained 300 µM dNTPs, 15 pmol of each gene-specific DNA oligonucleotide (see below), 1xPCR buffer, and 2.6 units of DNA polymerase in a 50 µl reaction volume. Reactions were incubated at 94 °C for 2 min, followed by 30 cycles at 94 °C for 15 s, 56 °C for 30 s, and 68 °C for 5 min. Following cycling, reactions were incubated for 7 min at 72 °C and then stored at 4 °C. Reaction aliquots were analysed by agarose gel electrophoresis. Gene-specific oligonucleotide primers for PCR were the common 5' primer, RAKmocD4 (5'-CCTCGCCTGTATCATTGATGGAACC-3'), coupled with one of the 3' primers, RAKmocD2 (5'-GGCGTATTACCTTTGTGGGAGGCG-3'), RAKmocD3 (5'-GCAGTTCGACCGCACTCAGCAGGCC-3'), RAKdhp (5'-CGCCTAACCCGCCGACCATCCC-3') or RAKter1 (see above).
5' Random amplification of cDNA ends (RACE).
The 5'-RACE kit (Invitrogen, Version 2.0) was used to define the transcriptional start site of yhiVUTS. For cDNA synthesis, an oligonucleotide complementary to the 5' end of yhiV, RAKmocD1 (5'-CAGACCGACAGTCAACGCTTTCGC-3'), was used. The resulting amplified DNA product of 265 bp was cloned in pPCR-Script AMP SK(+) (Stratagene) then sequenced.
In vitro synthesis and cloning of yhiT wild-type and mutant (yhiT23) alleles.
Wild-type and mutant yhiT alleles were amplified from plasmids pRSB4 and pRSB3 respectively (see Results) using oligonucleotides 5'-CGGAATTCCACTCACTCCAGATAACCCAGG-3' and 5'-CCGGGATCCTAAGTGGCGTGTGCTATAAAAAC-3', with Phusion High-Fidelity DNA Polymerase from Finnzymes (New England Biolabs) in accordance with the manufacturer's instructions. The 1.3 kb DNA fragments were cloned into the NruI site of plasmid pBR322 to generate pCLS41 (harbouring the yhiT23 mutant allele) and pCLS42 (harbouring the wild-type yhiT allele). Both plasmids contained the insert in the same direction with the distal end of the gene proximal to the tet promoter of the vector.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Syvanen & Roth, 1973), but characterization of these mutants was not undertaken. In this study, the original usp-2 and usp-3 mutations were transduced into a pyrimidine auxotroph, KR1312 (Table 1
), by selecting for growth on CAA as sole pyrimidine source. Mutants capable of using dihydroorotate (DHO) as sole pyrimidine source were isolated by treatment of KR1650 (usp-2 pyrB137 dctA1 : : MudJ) with MNNG, followed by selection on minimal medium supplemented with DHO. One Dho+ isolate, KR1667, was chosen for further analysis. Based on the results of gene mapping studies (see below), the mutation was designated yhi-23.
DHO can spontaneously hydrolyse to produce CAA (R. A. Kelln & J. Neuhard, unpublished results), and therefore the utilization of DHO by KR1667, which harbours the usp-2 allele, may have been a consequence of CAA utilization arising from the hydrolysis of DHO. To test this possibility, pyrC691 : : Tn10 was transduced into KR1667, yielding KRM106, a strain incapable of metabolizing CAA as a pyrimidine source. KRM106, KR1667 and several other strains were re-evaluated for growth on pyrimidine intermediates. KRM106 grew with exogenous DHO (Table 2), confirming that the growth on DHO pertained to the novel gain-of-function mutation, yhi-23, and was not simply a consequence of uptake and utilization of CAA arising from hydrolysis of DHO. Notably, both KRM106 and KR1667 grew with exogenous OA (Oa+) despite harbouring a disruption in dctA. Considering that a functional dctA is normally required for utilization of OA (Baker et al., 1996), the growth of these strains on OA indicated an additional capability imparted by the yhi-23 mutation. The individual usp-2 (Table 2) and usp-3 (data not shown) mutations, in contrast, did not afford the additional capabilities to use exogenous DHO or OA. Collectively, the results confirmed that the Dho+ (and Oa+) phenotype was a consequence of the yhi-23 mutation.
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), whereby the location of the insertion was determined to be near 78 centisomes on the S. enterica serovar Typhimurium chromosome. To confirm that the Tc-resistance marker of KRM115 was integral to yhi-23, the parental Dho+ strain, KR1667, was used as the recipient for transductional analysis. Importantly, the resulting transductants were not only Dho–, but also Caa–. Moreover, transducing the Tc marker into the usp-2 (KR1647) or usp-3 (KRM123) strain also resulted in a Caa– phenotype. A number of explanations were consistent with the results, with the simplest interpretations being that the yhi-23 and usp mutations were very closely linked, were in themselves allelic, or that the Tc-resistance element imparted a polarity effect on the expression of both the usp-2 and usp-3 alleles.
Cloning and sequencing of DNA harbouring the yhi-23 mutation
To facilitate the cloning of the DNA region corresponding to yhi-23 (as well as providing a facile system for gene expression studies), the MudJ-insertion mutant KRM104 was constructed (see Methods). As observed for the mini-tet insertion, introduction of the MudJ element in yhi-23 not only resulted in a Dho– phenotype, but simultaneously led to a Caa– phenotype as well. DNA both 5' and 3' to the insertion site was cloned, based on knowledge of restriction sites within the MudJ element (Castilho et al., 1984). More than 4400 bp of genomic DNA sequence encompassing a region corresponding to yhiVUTS and flanking DNA was determined, and the analysis established that MudJ was inserted into yhiT, the third gene of a putative four-gene operon. (Fig. 1b). Sequence differences between the yhi-23 mutant DNA and the published wild-type sequence (McClelland et al., 2001) pertained to only two residues, and both were located within yhiT (see below). Additionally, these studies confirmed that the cloned DNA was from the 78.5 centisomes region of the S. enterica serovar Typhimurium chromosome, as had been approximated by transductional mapping.
Cloning and characterization of wild-type and mutant yhiT alleles
A plasmid, pNJK203, containing the Km-resistance cassette from pUC4K inserted into yhiV was constructed. The yhiV1 : : KmR insertion was subcloned into the suicide vector pJQ200 and this construct was used to recombine the insertion in wild-type S. enterica serovar Typhimurium, or strains harbouring the various yhi mutant alleles. The Km-resistant strains KRNJK14 (yhiV1 : : KmR), KRNJK11 (usp-2 yhiV1 : : KmR), KRNJK12 (usp-3 yhiV1 : : KmR) and KRNJK10 (yhi-23 yhiV1 : : KmR) were obtained by this approach (Table 1). A restriction digestion/cloning strategy based on known sequence data enabled the joint cloning of the KmR marker and DNA encompassing yhiU and yhiT into pWSK29, yielding plasmids pRSB1 through pRSB4 (Fig. 2a), corresponding to cloned DNA from usp-2, usp-3, yhi-23 and wild-type cells, respectively. Transformation of the plasmids containing the individual mutant alleles into KR1312 (pyrB137) conferred the ability to utilize CAA (pRSB1, 2 and 3) or CAA and DHO (pRSB3) as sole pyrimidine source, whereas the wild-type yhiT+ plasmid (pRSB4) did not. It was noteworthy that the Km insertion strains (i.e. KNR strains) lost the ability to utilize exogenous pyrimidine intermediates, consistent with a polarity effect due to the insertion of the Km-resistance cassette. Since the plasmids were capable of mediating utilization, their multicopy nature apparently sufficed to overcome the polarity effect of the insertion element in single copy.
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To determine whether the T89P and Y383 alterations encoded by the yhiT23 allele exhibited a cooperative effect on DHO utilization, the mutations were separated by subcloning either the proximal or distal DNA region of the yhi-23 allele with the counterpart region of wild-type DNA (Fig. 2b). As anticipated, the T89P amino acid substitution (pSSL47) rendered the cell Caa+, but not Dho+. Notably, the Y383C alteration (pSSL63) alone conferred the Dho+ phenotype as well as imparting a Caa+ phenotype. When the two mutations within the usp-3 allele were separately analysed, either point mutation, G124D or A397T, conferred a Caa+ phenotype (pSSL49 and 50).
Mutant YhiT mediates utilization of pyrimidine intermediates
Inspection of the E. coli genome failed to identify any homologous elements to yhiVUT, and thus E. coli was an ideal test system to determine if the phenotype could be mediated by only the introduction of the cloned DNA. Accordingly, pRSB3 containing the cloned mutant yhiVUT region was transformed to KUR1182 (a pyrB deletion strain; Table 1) and found to impart a Caa+ phenotype. This result was extended by examining the growth of KUR1182 with CAA after introducing a plasmid containing only yhiT, either the mutant yhiT23 allele (pCLS41) or wild-type (pCLS42). The pCLS41 construct imparted a Caa+ and Dho+ phenotype, whereas the pCLS42 transformant failed to grow with CAA or DHO as a pyrimidine source, demonstrating that the mutant allele alone confers the ability for the cells to grow with exogenous pyrimidine intermediates as a pyrimidine source.
The operon nature of yhiVUTS
It was noted that following insertion of the yhiV1 : : KmR marker to create strains KRNJK10–14, the strains were no longer able to utilize the respective pyrimidine intermediate(s). The foregoing was consistent with a polarity effect on gene expression imposed by the insertion element and where the promoter expressing yhiT would be located upstream of yhiV. Moreover, while a plasmid harbouring the yhi-24 : : MudJ fusion from KRM104 plus a region of 130 nucleotides directly upstream of yhiV maintained expression of the lacZ gene within MudJ, deletion of the region upstream of yhiV led to the loss of lacZ expression, indicative of a functional promoter element having been removed. Further support for the existence of a promoter upstream of yhiV was gained by inspection of the sequence, which led to the identification of a putative promoter having a –35 sequence (ATGTCT) and a –10 sequence (AATAAT) separated by 19 nucleotides (Fig. 3a). In contrast, inspection and analysis of the intergenic regions of yhiVUTS failed to reveal sequence elements characteristic of a promoter element. The presence of a promoter element was confirmed using 5'-RACE, which identified the transcriptional start site to be an adenosine residue, 6 nucleotides downstream of the deduced –10 region and 55 residues upstream of the AUG initiation codon of yhiV (Fig. 3a). Together, these findings supported the notion that yhiVUTS is an operon, expressed as a single mRNA from a promoter located upstream of yhiV. Consistent with this interpretation was the observation that deletion of DNA upstream of the promoter-proximal BamHI site resulted in a twofold reduction in expression from a plasmid-borne yhiT–lacZ transcriptional fusion (data not shown).
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). The cDNA was used as the template for the amplification of various DNA fragments by PCR. Although a PCR product of 4.4 kbp encompassing the entire region defined by yhiVUTS was not observed, amplification of 290 bp, 470 bp and 2 kbp DNA fragments was achieved using oligonucleotides complementary to a region directly 5' proximal of the initiation codon of yhiV and various regions within the coding region of yhiV or yhiT (Fig. 3b, lanes 1–3). Importantly, amplification products were not observed when reverse transcriptase was omitted from the cDNA synthesis reaction, or when RNA samples were treated with RNase A prior to cDNA synthesis (data not shown). Notably, the 290 bp, 470 bp and 2 kbp products were generated only in the presence of cDNA that was primed downstream of yhiS. Collectively, the data strongly support the interpretation that the yhiVUTS genes are organized as an operon and expression occurs from a promoter region located immediately upstream of yhiV.
Probing the regulation of expression of yhiVUTS
The regulation of yhiVUTS expression was analysed by monitoring the expression of lacZ from the yhiT : : MudJ transcriptional fusion in KRM104 following the introduction of mutations in a series of common regulatory loci. The global regulatory genes cya, cra, crp, oxrA and arcA (Iuchi & Lin, 1988; Kolb et al., 1993; Spiro & Guest, 1990), each with an inactivating Tn10dTc insertion, were introduced into KRM104 by transduction. Expression of lacZ in the parental strain, KRM104, was consistently twofold lower when cells were grown in glucose than for glycerol-grown cells (as shown in Table 3), indicating that yhiVUTS is a target of catabolite repression. Individual inactivation of the three carbon source regulatory loci cya, crp and cra resulted in a modest lowering of the basal level of lacZ expression under either growth condition (data not shown). No significant change in LacZ activity was observed with the introduction of mutations in oxrA (Fnr) or arcA (ArcA), which are involved in the adaptation to anaerobic cell growth and metabolism, but it must be noted that the analysis was limited to aerobically grown cells.
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). A more dramatic threefold or fivefold increase in LacZ activity was observed for cells grown on fumarate or citrate, respectively (Table 3).
Uptake and incorporation of pyrimidine intermediates
Growth studies of mutants harbouring the yhiT23 mutation indicated that, in addition to conferring a Dho+ phenotype, the cells were also able to transport exogenous OA (Table 2). The ability to transport OA was further examined through uptake and incorporation studies of radiolabelled OA by KR1667 (yhi-23 dctA1 : : MudJ pyrB137) and KR1654 (dctA1 : : MudJ pyrB137), with the latter serving as the control. As shown in Fig. 4(a), incorporation of labelled OA by KR1667 increased with the length of exposure to the compound. In contrast, uptake and incorporation of the radiolabelled substrate did not occur with KR1654. Since OA uptake by the DctA transport system is precluded in both strains, the growth and incorporation of radiolabelled OA in KR1667 resulted from the capability imparted by the yhiT23 mutation.
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). The inhibition of OA uptake and incorporation by CAA demonstrated that transport of these two pyrimidine intermediates was occurring through a shared mechanism in which the mutant YhiT is an integral component.
The uptake associated with the yhiT23 mutation was further analysed by determining the doubling times for cells growing in the presence of the individual pyrimidine intermediates or combinations thereof. When KRM106 (DctA– and unable to metabolize endogenous CAA) was grown with DHO or OA as the sole pyrimidine source, adding CAA to the growth medium resulted in a dramatic increase in doubling time (Table 4). Similarly, for a strain incapable of metabolizing CAA and DHO (KRM108; Table 1), the addition of either compound to a culture growing with exogenous OA caused a significant increase in the doubling time (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Herein, we have extended the previous work to include an analysis of the utilization of additional pyrimidine nucleotide intermediates, namely CAA and DHO, as exogenous pyrimidine sources. Mutations conferring the Dho+ and Caa+ phenotypes were found to be closely linked, and cloning and sequencing of the chromosomal region conferring these phenotypes revealed the uncharacterized yhiVUTS locus to be the relevant genetic element. Notably, a corresponding region is not found within the genome of the closely related bacterium, E. coli, and thus the results are specific to S. enterica serovar Typhimurium.
Five ORFs encoding high molecular mass polypeptides are predicted to exist within the analysed genetic region, with one of the five (yhiW; STM3602) being well separated from the other four (yhiVUTS; Fig. 1b). The first ORF (yhiV; STM3601) of the putative four-gene operon is deduced to encode a 36 kDa hydrophobic polypeptide with a conserved phosphosugar isomerase domain commonly observed in glucose phosphate isomerases. YhiV shows similarity to gene products involved in the catabolism of sugar–amino acid conjugates, specifically, the conversion of fructosamine 6-phosphates to glucose 6-phosphate and free amino acids in Bacillus subtilis (YurP; 58 %) (Wiame et al., 2004) and the conversion of mannopine to glutamine and mannose in Agrobacterium tumefaciens (MocD; 46 %) (Kim & Farrand, 1996). ORF2 (yhiU, STM3600) is predicted to encode a 31 kDa hydrophobic polypeptide, which displays 48 % similarity to the putative kinase MocE from A. tumefaciens (Kim & Farrand, 1996) and 54 % similarity to the kinase YurL of B. subtilis (Fujita et al., 1986; Wiame et al., 2004).
The DNA coding for ORF3 (yhiT, STM3599) harboured the MudJ insertion and, notably, exhibits no significant nucleotide similarity with any sequence currently in the genome database. However, the deduced 47 kDa polypeptide contains 12 transmembrane domains typical of bacterial transport proteins and has similarity to various anaerobic C4-dicarboxylate transporters (Ullmann et al., 2000), including those from Bacillus spp. (
55 %), Erwinia carotovora (50 %) (Bell et al., 2004), and members of the Campylobacterales (50 %). In particular, YhiT possesses conserved domains characteristic of the transporters DcuA and DcuB and shows similarity to DcuB of Bacteroides spp. (54 %) (Kuwahara et al., 2004; Xu et al., 2003), Mannheimia succiniciproducens (53 %) (Hong et al., 2004) and a variety of Campylobacterales (46 %) (Fouts et al., 2005; Suerbaum et al., 2003), and to DcuA of Photorhabdus luminescens (54 %) (Duchaud et al., 2003) and members of the Campylobacterales (45 %) (Fouts et al., 2005; Suerbaum et al., 2003; Tomb et al., 1997). Significantly, yhiT harboured the only nucleotide changes observed between the cloned DNA and published sequences, providing strong evidence that the mutant YhiT polypeptides manifest the pyrimidine utilization phenotype.
The final ORF (yhiS, STM3598) is predicted to encode a 37 kDa polypeptide with similarity to various prokaryotic asparaginases (Bonthron, 1990). The predicted product exhibits a conserved asparaginase domain with 75 % similarity to the L-asparaginase of Erwinia chrysanthemi (Minton et al., 1986). Significant similarity is found between YhiS and L-asparaginases (I and II) from a variety of Campylobacterales (60 %) and also to the glutaminase-asparaginase enzymes of Pseudomonas and Acinetobacter spp. (55 %). Notably, YhiS displays 58 % similarity to AsnB, an L-asparginase II from M. succiniciproducens (Hong et al., 2004) and 41 % similarity to the L-asparginase of A. tumefaciens (Wood et al., 2001).
Several lines of evidence support the conclusion that the yhiVUTS region of the S. enterica serovar Typhimurium chromosome constitutes an operon. First, inspection of the DNA within this region identified a single promoter element upstream of yhiV. Second, 5'-RACE analysis confirmed a transcriptional start site at the deduced promoter element (Fig. 3a). Third, a polarity effect on yhiT expression as a consequence of insertional inactivation of yhiV was observed. Fourth, and most importantly, RT-PCR demonstrated that the yhiVUTS genes can be transcribed as a single mRNA (Fig. 3b).
We inferred that the point mutations identified within yhiT were solely responsible for the pyrimidine utilization phenotypes since no additional mutations were found in the DNA cloned from the mutant strains. This was confirmed by demonstrating that cloned DNA harbouring the mutant alleles of yhiT (pRSB plasmids) imparts the respective Dho+ or Caa+ phenotype to transformed cells. Furthermore, a plasmid harbouring only the yhiT23 allele rendered an E. coli pyrB mutant capable of growing with CAA or DHO.
The studies on the regulation of yhiVUTS expression (Table 3) indicated that catabolite repression was present. Furthermore, a significant increase in expression (approx. fivefold) was observed when cells were grown with citrate as carbon source. Inspection of the DNA region upstream of yhiV revealed two weakly conserved cAMP-CRP protein-binding sites, one centred at –70 (AAtcGgGATCcAGATCgCggaT) and one centred at –167 (AAAgGaGAaCTctcctgCgTTT). A putative Cra protein-binding site located at –189 (AGTGAAAtGATTgA) was also identified, as was another region exhibiting strong conservation as a Fnr protein-binding site centred at –41 (TTGgTTTCCATCtt). However, introducing null mutations into crp or cra had little or no impact on expression of yhiVUTS. Furthermore, no evidence for the involvement of other known regulatory proteins in the control of the operon was observed.
Using the TopPred program (Claros & von Heijne, 1994), T89P and Y383C correspond to amino acid substitutions in regions of YhiT predicted to be within extracellular loops exposed to the periplasmic space. The effect of the substitutions can be envisioned as altering the substrate-binding affinity of the predicted transport protein for the uptake of additional small molecules found within the periplasm, i.e. pyrimidine intermediates. In contrast, the amino acid substitutions associated with usp-3 are predicted to be within a region exposed to the cytosol (G124D) and within a transmembrane domain proximal to the cytosol (A397T). Accordingly, these amino acid substitutions would not be expected to alter substrate binding, but, alternatively, may influence substrate release on the cytosolic face, or have more profound effects on the folding and/or function of the YhiT protein.
The results presented here demonstrate that utilization of the three pyrimidine intermediates, CAA, DHO and OA, can arise through gain-of-function mutations associated with a single gene, yhiT, resulting in a multifunctional mutant YhiT protein. In addition, the mutant YhiT proteins displayed affinity for succinate, malate and the amino acids aspartate and asparagine. Based on the common structural features of these molecules, we propose that the mutant YhiT protein has affinity for compounds with linear or cyclic four carbon backbones with a carboxyl group present at the C-1 position and a carbonyl group on C-4. One inference from these observations is that wild-type YhiT may function to transport groups of compounds such as C4-dicarboxylates, assuming that the characterized mutations do not impart a dramatic departure from the function of the native protein. Indeed, the structural similarities between YhiT and DcuA/DcuB are consistent with the hypothesis that wild-type YhiT is involved in the utilization of C4-dicarboxylates in S. enterica serovar Typhimurium, perhaps during anaerobic growth. Investigations aimed at characterizing the native function of YhiT and the regulation of the yhiVUTS operon are ongoing.
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ACKNOWLEDGEMENTS |
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Edited by: P. H. Everest
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REFERENCES |
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Received 27 February 2007;
revised 21 April 2007;
accepted 26 April 2007.
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