A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra-high affinity ABC system distinct from the molybdate transporter.
The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300-0303 and Cj1538-1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate (K(D) values for both ligands of 4-8 nM), Cj1540 binds tungstate with a K(D) of 1.0 +/- 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 (tupA) mutant compared with a cj0303c (modA) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased approximately 50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium.
Smart
Jonathan P
JP
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Cliff
Matthew J
MJ
Kelly
David J
DJ
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D016123:Campylobacter jejuni / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005051:Eubacterium / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D008982:Molybdenum / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D013379:Substrate Specificity; D014414:Tungsten / Q000378:metabolism* / Q000494:pharmacology*; D017973:Tungsten Compounds / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005561:Formates; D033902:Periplasmic Binding Proteins; D012329:RNA, Bacterial; D017973:Tungsten Compounds; C044659:molybdate; D008982:Molybdenum; D005560:Formate Dehydrogenases; C045951:tungstate; D014414:Tungsten