thiBPQ Encodes an ABC Transporter Required for Transport of Thiamine and Thiamine Pyrophosphate inSalmonella typhimurium

In Salmonella typhimurium, thiamine pyrophosphate (TPP) is a required cofactor for several enzymes in central metabolism. Herein we identify a new thi operon,thiBPQ (designated sfuABC in Escherichia coli), required for the transport of thiamine and TPP into the cell. Insertions in the operon result in strains that are phenotypically and biochemically defective in thiamine and TPP transport. Data presented herein show that this operon is transcriptionally repressed in the presence of exogenous thiamine, with TPP the likely regulatory molecule. This work represents the first identification of thiamine transport genes in bacteria and demonstrates the function of a proposed ABC transporter in E. coli. In Salmonella typhimurium, thiamine pyrophosphate (TPP) is a required cofactor for several enzymes in central metabolism. Herein we identify a new thi operon,thiBPQ (designated sfuABC in Escherichia coli), required for the transport of thiamine and TPP into the cell. Insertions in the operon result in strains that are phenotypically and biochemically defective in thiamine and TPP transport. Data presented herein show that this operon is transcriptionally repressed in the presence of exogenous thiamine, with TPP the likely regulatory molecule. This work represents the first identification of thiamine transport genes in bacteria and demonstrates the function of a proposed ABC transporter in E. coli. Thiamine pyrophosphate (TPP) 1The abbreviations used are: TPP, thiamine pyrophosphate; TMP, thiamine monophosphate; THZ, 4-methyl-5-(β-hydroxyethyl) thiazole; HMP, 4-amino-5-hydroxymethyl-2-methyl pyrimidine; NCE media, no-carbon source E media; NB, nutrient broth; Cs, centisome; PCR, polymerase chain reaction. 1The abbreviations used are: TPP, thiamine pyrophosphate; TMP, thiamine monophosphate; THZ, 4-methyl-5-(β-hydroxyethyl) thiazole; HMP, 4-amino-5-hydroxymethyl-2-methyl pyrimidine; NCE media, no-carbon source E media; NB, nutrient broth; Cs, centisome; PCR, polymerase chain reaction. is a required cofactor synthesized de novo in Salmonella typhimurium.The primary role for TPP is in central metabolism as an electron carrier and nucleophile for such enzymes as pyruvate dehydrogenase (EC1.2.4.1), acetolactate synthase (EC 4.1.3.18), and α-ketoglutarate dehydrogenase (EC 1.2.4.2). Despite its importance in cellular physiology, neither the de novo biosynthetic pathway nor the salvage systems for thiamine are fully understood in any organism. Thiamine monophosphate (TMP) is generated by the condensation of two independently synthesized moieties: 4-amino-5-hydroxy-methyl pyrimidine pyrophosphate (HMP-PP) and 4-methyl-5-(β-hydroxyethyl) thiazole phosphate (THZ-P). TMP is then phosphorylated by the action of thiamine monophosphate kinase, ThiL (1Webb E. Downs D.M. J. Biol. Chem. 1997; 272: 15702-15707Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), to form the physiologically relevant form of the vitamin, TPP. Recently several studies in the enteric bacteria S. typhimurium and Escherichia coli have elucidated many steps in the formation of thiamine (2Begley T.P. Nat. Prod. Rep. 1996; 13: 178-186Crossref Scopus (50) Google Scholar, 3Webb E. Claas K. Downs D. J. Bacteriol. 1997; 179: 4399-4402Crossref PubMed Google Scholar, 4Petersen L.A. Downs D.M. J. Bacteriol. 1997; 179: 4894-4900Crossref PubMed Google Scholar, 5Backstrom A.D. Austin R. McMordie S. Begley T.P. J. Am. Chem. Soc. 1995; 117: 2351-2352Crossref Scopus (42) Google Scholar), but much of the pathway remains unknown. Mutants defective in various steps in de novo synthesis can be supplemented exogenously with THZ, HMP, thiamine, TMP, or TPP. These results suggested that S. typhimurium had the ability to take up and incorporate these compounds into the de novothiamine biosynthetic pathway. It was demonstrated several years ago that thiamine was actively transported in E. coli, and this transport was shown to involve a thiamine-binding protein whose activity was repressed by excess thiamine (6Iwashima A. Matsuura A. Nose Y. J. Bacteriol. 1971; 108: 1419-1421Crossref PubMed Google Scholar, 7Kawasaki T. Esaki K. Arch. Biochem. Biophys. 1971; 142: 163-169Crossref PubMed Scopus (9) Google Scholar, 8Kawasaki T. Miyata I. Esaki K. Nose Y. Arch. Biochem. Biophys. 1969; 131: 223-230Crossref PubMed Scopus (45) Google Scholar, 9Kawasaki T. Miyata I. Nose Y. Arch. Biochem. Biophys. 1969; 131: 231-237Crossref Scopus (15) Google Scholar). The transport of TPP was not addressed in these previous studies. The presence of the thiamine-binding protein led to the hypothesis that thiamine was transported via a periplasmic binding protein-dependent ABC-type transporter (10Boos W. Lucht J.M. Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 1175-1209Google Scholar). We report here the identification of an operon (thiBPQ) at centisome (Cs) 1.5 on the S. typhimurium and E. coli chromosomes involved in the specific translocation of thiamine and its phosphoesters across the inner membrane. Analysis of the E. coli sequence (designated sfuABC) in addition to phenotypic analysis in S. typhimurium suggested that thiBPQ encoded thiamine binding protein, inner membrane channel, and energy-transducing ATPase, respectively. Transcriptional fusions in this operon were regulated in response to exogenous thiamine. All strains used in this study are derivatives of S. typhimurium LT2 and are listed in Table I. MudJ is used throughout the paper to refer to the MudI 1734 transposon, which has been described (11Castilho B.A. Olfson P. Casadaban M.J. J. Bacteriol. 1984; 158: 488-495Crossref PubMed Google Scholar), and Tn10d(Tc) refers to the transposition defective mini-Tn10(Tn10Δ16Δ17) (12Way J.C. Davis M.A. Morisato D. Roberts D.E. Kleckner N. Gene (Amst .). 1984; 32: 369-379Crossref PubMed Scopus (357) Google Scholar).Table IStrainsStrainGenotypeLT2Wild typeBL21/λDE3hsdS gal (λclts857 ind1 Sam7 nin5 lacUV5-T7 gene 1) (E. coli)DM62thi-924::MudJDM460thiH910::MudJ1-2001MudJ is used throughout the text to refer to the Mud dl1734 transposon (11).DM1683thiL933::Tn10d (Tc)DM1688thi-935::Tn10d(Tc)thiH910::MudJ1-bTn 10d (Tc) refers to the transposition-defective mini-Tn10 (Tn10Δ-16Δ-17) (12).DM2275thiH942::Tn10d (Tc)DM2572BL21/λDE3/pThiL-6DM3340thi-935::Tn10d (Tc)thi-995ΔDM3403zac-8602::MudJthi-995ΔDM3408–3411thi-(1010–1013)::Tn10d(Tc) thi-924::MudJDM3412zac-8603::Tn10d (Tc)thi-1014 thi-924::MudJDM3413zac-8603::Tn10d (Tc)thi-1015 thi-924::MudJDM3616leu-485Δ thiH942::Tn10d (Tc)DM3617–3656thi-(1027–1066)::MudJthiH942::Tn10d (Tc)DM3670zaj-8048::Tn10d (Tc)thiL927thiQ1054::MudJDM3671zaj-8048::Tn10d (Tc)thiL927thiB1062::MudJDM3781zaj-8048::Tn10d (Tc)thiQ1054::MudJDM3782zaj-8048::Tn10d (Tc)thiB1062::MudJDM3922thiB1062::MudJDM3924thiB1062::MudJDM3925thiB1062::MudJthiB1012::Tn 10d (Tc)DM3930thiQ1054::MudJDM3931thiQ1054::MudJthiB1012::Tn10d (Tc)1-2001 MudJ is used throughout the text to refer to the Mud dl1734 transposon (11Castilho B.A. Olfson P. Casadaban M.J. J. Bacteriol. 1984; 158: 488-495Crossref PubMed Google Scholar).1-b Tn 10d (Tc) refers to the transposition-defective mini-Tn10 (Tn10Δ-16Δ-17) (12Way J.C. Davis M.A. Morisato D. Roberts D.E. Kleckner N. Gene (Amst .). 1984; 32: 369-379Crossref PubMed Scopus (357) Google Scholar). Open table in a new tab No-carbon source E media (NCE) supplemented with 1 mm MgSO4 and 11 mm glucose was used as minimal media (13Davis R.W. Botstein D. Roth J.R. Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1980: 207Google Scholar, 14Vogel H.J. Bonner D.M. J. Biol. Chem. 1956; 218: 97-106Abstract Full Text PDF PubMed Google Scholar). Difco nutrient broth (NB, 8 g/liter) with NaCl (5 g/liter) added was used as rich medium. Difco BiTek agar (15 g/liter) was added for solid medium. Antibiotics were added as needed to the following concentrations in rich and minimal media respectively: kanamycin (50, 125 μg/ml), tetracycline (20, 10 μg/ml), and chloramphenicol (40, 4 μg/ml). Radiolabeled thiamine (C2-14C-THZ-thiamine) with a specific activity of 24 mCi/mmol was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). All other chemicals were purchased from Sigma. All transductional crosses were performed by using the high frequency transducing bacteriophage P22 mutant HT 105/1 int-201 (15Schmieger H. Mol. Gen. Genet. 1972; 119: 75-88Crossref PubMed Scopus (460) Google Scholar) as described (16Downs D.M. Petersen L. J. Bacteriol. 1994; 176: 4858-4864Crossref PubMed Google Scholar). Transductants were purified and identified as phage-free by cross-streaking on green plates (17Chan R.K. Botstein T. Watanabe T. Ogata Y. Virology. 1972; 50: 883-898Crossref PubMed Scopus (188) Google Scholar). Strains defective for TPP transport were isolated by insertional mutagenesis with one of two transposons, Tn10d(Tc) or MudJ. To facilitate mutant isolation, a pool of cells containing >80,000 independent insertions was generated as described elsewhere (18Hughes K.T. Roth J.R. Genetics. 1985; 109: 263-282Crossref PubMed Google Scholar, 19Kleckner J. Roth J.R. Botstein D. J. Mol. Biol. 1977; 116: 125-159Crossref PubMed Scopus (265) Google Scholar). A P22 lysate was grown on these cells to generate either a MudJ or Tn10d(Tc) phage pool. To isolate Tn10d(Tc) insertion mutants, the Tn10d(Tc) phage pool described above was used to transduce a strain defective in de novo thiamine synthesis (either DM62 (thi-924::MudJ) or DM460 (thiH910::MudJ)) to tetracycline resistance (Tcr) on NB-tetracycline plates. The Tcrtransductants were screened for those that were able to grow with 1 μm thiamine but not 1 μm TPP. Putative insertion mutants defective in high affinity TPP transport (defining the thiP locus) were streaked for phage sensitivity and saved for further analysis. Point mutations defective in high affinity TPP transport were isolated as described above with the following exception. The Tn10d(Tc) pool utilized had been mutagenized with hydroxylamine as described (13Davis R.W. Botstein D. Roth J.R. Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1980: 207Google Scholar, 20Hong J.S. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 3158-3162Crossref PubMed Scopus (127) Google Scholar), resulting in the isolation of point mutations linked to a Tn10d(Tc) element. After mapping, MudJ insertions in the thiP locus were identified using co-transduction with the leu locus in strain DM3616 (Δleu-485thiH942::Tn10d(Tc)) and the MudJ pool described above. Isolation of MudJ insertions linked to the thiP locus Tn10d(Tc) in strain DM3340 (thi-935::Tn10(d)thi-995Δ) utilized a positive screen, as described (19Kleckner J. Roth J.R. Botstein D. J. Mol. Biol. 1977; 116: 125-159Crossref PubMed Scopus (265) Google Scholar). One highly linked insertion, DM3403 (zac-8602::MudJ thi-995 Δ) (97%), was saved for further analysis. DNA was sequenced at the University of Wisconsin-Madison Biotechnology Center-Nucleic Acid and Protein Facility. DNA sequence analysis program BLAST (21Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69694) Google Scholar) was used to compare this sequence with known sequences from the data base. The TPP transport-deficient mutations were mapped on the S. typhimurium chromosome via sequencing MudQ phage DNA from strain DM3468 (zac-8602::MudQ thi-995Δ) which had been generated from strain DM3403 (zac-8602::MudJ thi-995 Δ) as described (22Youderian P. Sugiono P. Brewer K.L. Higgins N.P. Elliot T. Genetics. 1988; 118: 581-592Crossref PubMed Google Scholar). The resulting locked-in P22 phage was induced, and DNA was isolated as described (23Benson N.P. Goldman B.S. J. Bacteriol. 1992; 174: 1673-1681Crossref PubMed Google Scholar). The purified DNA was then used as a template for DNA cycle sequencing using a Sequitherm (Epicentre Madison, WI) kit. The primer used was MuR (5′-GAAACGCTTTCGCGTTTTTCGTGC-3′) which hybridizes to the right end of the MudQ insertion. The location of four insertions in the thiP operon were determined via a PCR-based protocol (24Chen P. Ailion M. Weyland N. Roth J. J. Bacteriol. 1995; 177: 1461-1469Crossref PubMed Google Scholar). Amplification between the insertions was done using Vent (exo−) polymerase (New England Biolabs, Inc., Beverly, MA) in a Thermolyne Temp-Tronic Thermocycler (Dubuque, IA). Reaction conditions were as follows: 95 °C denaturation for 1 min, 55 °C annealing for 1 min, and 72 °C extension for 2 min. Primers used were: Tn10-I (5′-GACAAGATGTGTATCCACCTTAAC-3′), which hybridizes to the 66-base pair inverted repeat Tn10 sequence; MuL (5′-ATCCCGAATAATCCAATGTCC-3′), which hybridizes to the left end of the MudJ insertion; and MuR (defined above). Additional MgSO4was added to all reaction mixes to a final concentration of 1 mm. Amplified products were visualized via agarose gel electrophoresis, purified using Qiaquick gel extraction kit (Qiagen, Chatworth, CA), and sequenced at the University of Wisconsin-Madison Biotechnology Center-Nucleic Acid and Protein Facility. Assays were performed using the Miller method (25Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 47-49Google Scholar) as described previously (26Escalante-Semerena J.C. Roth J.R. J. Bacteriol. 1987; 169: 2251-2258Crossref PubMed Google Scholar). Curves were done aerobically as described (16Downs D.M. Petersen L. J. Bacteriol. 1994; 176: 4858-4864Crossref PubMed Google Scholar). Final concentrations of THZ, thiamine, TMP, and TPP were as indicated. [32P]TPP was generated using cell-free extracts of a strain overproducing ThiL as described (1Webb E. Downs D.M. J. Biol. Chem. 1997; 272: 15702-15707Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) with the following exceptions. The ThiL reaction was initiated with the addition of 15 μl of ATP (10 μl of 100 mm ATP + MgCl2 and 5 μl of [γ-32P]ATP (specific activity 6000 Ci/mmol)). Radiolabeled [β-32P]TPP was purified from the ThiL reaction mix via column chromatography as described by Matsuda and Cooper (27Matsuda T. Cooper J.R. Anal. Biochem. 1981; 117: 203-207Crossref PubMed Scopus (24) Google Scholar) with the following exceptions. Twenty fractions (3 ml each) from the 60 ml of 0.1 m citrate buffer (pH 3.5) were collected. The TPP elution profile was tested by bioautography with strain DM1683 (thiL933::Tn10d(Tc)), which qualitatively determined the fractions containing significant TPP. To determine the radiochemical purity and concentration of the TPP, high pressure liquid chromatography analysis was performed on an aliquot of this fraction, as described previously (1Webb E. Downs D.M. J. Biol. Chem. 1997; 272: 15702-15707Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 28Kawasaki T. Chytil F. McCormick D.B. Vitamins and Coenzymes, Part G. 122. Academic Press, Inc., New York1986: 15-29Google Scholar). The high pressure liquid chromatography fraction containing the TPP peak was collected and scintillation counted for 1 min in a Packard Instruments Model 4530 Scintillation Counter (Downers Grove, IL), demonstrating that the TPP accounted for ∼80% of the label. The specific activity of the TPP was calculated to be 9400 μCi/mmol. The protocol for the uptake assay used for thiamine and TPP was a combination of previously described methods (7Kawasaki T. Esaki K. Arch. Biochem. Biophys. 1971; 142: 163-169Crossref PubMed Scopus (9) Google Scholar, 29Bellion E. Lash T.D. McKellar R. Biochim. Biophys. Acta. 1983; 735: 331-336Crossref PubMed Scopus (6) Google Scholar) and is summarized below. Overnight cultures grown in NB were pelleted and resuspended in an equal volume of 0.85 m NaCl. 0.5 ml of resuspended cells were inoculated into 10 ml of minimal medium and incubated with shaking at 37 °C until the optical density at 560 nm was ∼0.4. Cultures were then pelleted, resuspended in 2 ml of minimal medium, separated into 1-ml aliquots, and on until The cultures were at 37 °C for 10 min, and were initiated by the addition of concentration of nm for [32P]TPP or to 1 ml of were at and with ml of NCE medium. were a with 5 ml of scintillation and counted min for [32P]TPP and 2 min for in a Packard Instruments Model 4530 Scintillation independently isolated mutations a were 2 point and MudJ P22 co-transduction analysis mapped all of the insertions to the thiP in two significant mutants defective in novo synthesis and the required TPP nm for the mutant and and these strains with μm exogenous thiamine A and thiP mutations in a had in minimal medium. The location of locus on the S. typhimurium chromosome was determined by sequencing the DNA of a MudQ insertion known to be linked to BLAST (21Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69694) Google Scholar) data analysis determined that the insertion was to the end of at 1.5 on sequence to the E. coli analysis with P22 determined that thiP was linked to the biosynthetic at 1.5 in S. typhimurium and that the gene was with the from coli insertions in the thiP locus were mapped with a PCR-based Primers to the of the insertions were used to DNA in strains containing a MudJ and insertion in the Amplified products from strains and were purified and with the E. coli data using determined that the two insertions in strains were in the at and MudJ at the insertions in strain were in at and at The sfuABC genes had been identified by the E. coli sequencing between the biosynthetic genes and This was to the significant sequence to the uptake operon ABC and other ABC we the S. typhimurium genes the of this gene in thiamine transport to known suggested that a thiamine binding protein, an inner membrane channel, and an energy-transducing ATPase, respectively. Data were performed to determine the transport was in and the and ATPase, significant with all ABC was used as a (21Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69694) Google Scholar) analysis with the E. coli determined that all of the transport were in other significant for were in any other listed in were performed on the using the data A. P. K. 1997; PubMed Scopus Google Scholar). These determined that and a binding protein and one binding respectively. To the thiBPQ operon encoded the mutants defective in the operon were tested for ability to transport thiamine and TPP These data that the insertions in thiBPQ a in the transport of thiamine and TPP. LT2 of uptake of of nm and of insertions in or had that were mutants in de novo synthesis and thiBPQ were able to grow in the presence of 1 μm thiamine but not TPP. This result suggested was an for transport of thiamine that was independent of To that thiamine in a these uptake were performed with and μm thiamine not in of thiamine be transport at nm to of nm at an operon was on the E. coli a strain containing a MudJ in used to this Strains and and of grown in minimal medium, with an operon for gene Data presented in Table that thiBPQ to the of TPP transcriptionally regulated genes in S. typhimurium L.A. Downs D.M. J. Bacteriol. 1997; 179: 4894-4900Crossref PubMed Google Scholar, E. F. Downs D.M. J. Bacteriol. 1996; PubMed Google Scholar). This was by the that the fusions in the genes of were defective for the transport of thiamine and TPP. We were able to this we had determined that thiamine in a mutant at concentrations shown in from to were in minimal medium using insertions in the in the presence of exogenous thiamine was and To this response was to thiamine or TPP, the previously point was utilized (1Webb E. Downs D.M. J. Biol. Chem. 1997; 272: 15702-15707Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Strains containing this point and a thi are repressed by TPP, but not thiamine to thiamine monophosphate kinase, ThiL (EC insertions and were into point strains and respectively. shown in Table strains containing point were not repressed in response to thiamine, the strain These data are with of operon in response to TPP, as are other previously described thiamine biosynthetic of 1 mm Open table in a new tab presented here a new ABC transporter (thiBPQ) in S. We show here that the thiBPQ operon at 1.5 is not for the transport of thiamine but TPP in S. This with previous work in E. demonstrated that the thiamine transport was independent of de and repressed by thiamine T. Miyata I. Esaki K. Nose Y. Arch. Biochem. Biophys. 1969; 131: 223-230Crossref PubMed Scopus (45) Google Scholar, H. R. J. Bacteriol. 1972; PubMed Google Scholar). was shown in E. coli that the protein has high affinity for the binding of thiamine, TMP, and TPP, is for TMP transport. This operon is in E. coli at the chromosome we that the gene be from sfuABC to thiBPQ to the role of this operon in thiamine metabolism. A 1997; PubMed Scopus Google Scholar) has identified the thiamine transport gene the transport for thiamine and TPP in E. coli and S. this gene is a of the of 1994; PubMed Google Scholar). The of from ABC-type in two significant the translocation is encoded by a and the for translocation not from ATP but is carrier S. is to transport TPP K. Y. H. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). It is that a for thiamine transport has and that these two systems not in but in The that strains containing mutations in thiBPQ and novo in thiamine were with thiamine TPP suggested that was for thiamine uptake in S. In we show at concentrations thiamine in a We that this was to a affinity thiamine transport or transport by translocation The of this operon was addressed using MudJ insertions in and These determined that this operon was repressed in response to exogenously thiamine Additional that TPP was the likely as has been shown for other transcriptionally regulated thi genes in S. thiBPQ represents the operon shown to be regulated in response to TPP in S. These genes throughout the S. typhimurium and at and and genes involved in and transport. Recently has been shown that many genes involved in thiamine metabolism in bacteria have a highly pair the to the of J. H. A. S. J. M. J. Bacteriol. 1997; 179: PubMed Google Scholar). Analysis of the thiBPQ sequence from E. that this operon this element. The presence of the thi in to in all genes to be regulated by TPP the of a regulatory