1887

Abstract

The formate hydrogenlyase complex of catalyses the cleavage of formate to CO and H and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7–37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the operon , although the FhlA9-2 did bind to promoter DNA . The ATPase activity of the FhlA9-2–DNA–formate complex was at least three times higher than that of the native protein–DNA–formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of expression. The FhlA165 protein also had a twofold higher affinity to promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for promoter DNA . Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

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2001-11-01
2019-12-06
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References

  1. Adams, M. D., Wagner, L. M., Graddis, T. J., Landick, R., Antonucci, T. K., Gibson, A. L. & Oxender, D. L. ( 1990; ). Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli. J Biol Chem 265, 11436-11443.
    [Google Scholar]
  2. Altuvia, S., Zhang, A., Argaman, L., Tiwari, A. & Storz, G. ( 1998; ). The Escherichia coli OxyS regulatory RNA represses fhlA translation by blocking ribosome binding. EMBO J 17, 6069-6075.[CrossRef]
    [Google Scholar]
  3. Ames, G. F. & Lever, J. ( 1970; ). Components of histidine transport: histidine-binding proteins and HisP protein. Proc Natl Acad Sci USA 66, 1096-1103.[CrossRef]
    [Google Scholar]
  4. Böck, A. & Sawers, G. (1996). Fermentation. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 262–282. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
  5. Boyington, J. C., Gladyshev, V. N., Khangulov, S. V., Stadtman, T. C. & Sun, P. D. ( 1997; ). Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster. Science 275, 1305-1308.[CrossRef]
    [Google Scholar]
  6. Drummond, M., Whitty, P. & Wootton, J. ( 1986; ). Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. EMBO J 5, 441-447.
    [Google Scholar]
  7. Grunden, A. M. & Shanmugam, K. T. ( 1997; ). Molybdate transport and regulation in bacteria. Arch Microbiol 168, 345-354.[CrossRef]
    [Google Scholar]
  8. Grunden, A. M., Ray, R. M., Rosentel, J. K., Healy, F. G. & Shanmugam, K. T. ( 1996; ). Repression of the Escherichia coli modABCD (molybdate transport) operon by ModE. J Bacteriol 178, 735-744.
    [Google Scholar]
  9. Hasona, A., Ray, R. M. & Shanmugam, K. T. ( 1998a; ). Physiological and genetic analyses leading to identification of a biochemical role for the moeA (molybdate metabolism) gene product in Escherichia coli. J Bacteriol 180, 1466-1472.
    [Google Scholar]
  10. Hasona, A., Self, W. T., Ray, R. M. & Shanmugam, K. T. ( 1998b; ). Molybdate-dependent transcription of hyc and nar operons of Escherichia coli requires MoeA protein and ModE-molybdate. FEMS Microbiol Lett 169, 111-116.[CrossRef]
    [Google Scholar]
  11. Hopper, S. & Böck, A. ( 1995; ). Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli. J Bacteriol 177, 2798-2803.
    [Google Scholar]
  12. Hunke, S., Mourez, M., Jehanno, M., Dassa, E. & Schneider, E. ( 2000; ). ATP modulates subunit–subunit interactions in an ATP-binding cassette transporter (MalFGK2) determined by site-directed chemical cross-linking. J Biol Chem 275, 15526-15534.[CrossRef]
    [Google Scholar]
  13. Kashiwagi, K., Miyamoto, S., Nukui, E., Kobayashi, H. & Igarashi, K. ( 1993; ). Functions of potA and potD proteins in spermidine-preferential uptake system in Escherichia coli. J Biol Chem 268, 19358-19363.
    [Google Scholar]
  14. Keener, J. & Kustu, S. ( 1988; ). Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC. Proc Natl Acad Sci USA 85, 4976-4980.[CrossRef]
    [Google Scholar]
  15. Kim, R., Sandler, S. J., Goldman, S., Yokota, H., Clark, A. J. & Kim, S. H. ( 1998; ). Overexpression of archael proteins in E. coli. Biotechnol Lett 20, 207-210.[CrossRef]
    [Google Scholar]
  16. Korsa, I. & Böck, A. ( 1997; ). Characterization of fhlA mutations resulting in ligand-independent transcriptional activation and ATP hydrolysis. J Bacteriol 179, 41-45.
    [Google Scholar]
  17. Lee, J. H., Patel, P., Sankar, P. & Shanmugam, K. T. ( 1985; ). Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism. J Bacteriol 162, 344-352.
    [Google Scholar]
  18. Leonhartsberger, S., Ehrenreich, A. & Böck, A. ( 2000; ). Analysis of the domain structure and the DNA binding site of the transcriptional activator FhlA. Eur J Biochem 267, 3672-3684.[CrossRef]
    [Google Scholar]
  19. Linton, K. J. & Higgins, C. F. ( 1998; ). The Escherichia coli ATP-binding cassette (ABC) proteins. Mol Microbiol 28, 5-13.
    [Google Scholar]
  20. Lopilato, J., Bortner, S. & Beckwith, J. ( 1986; ). Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives. Mol Gen Genet 205, 285-290.[CrossRef]
    [Google Scholar]
  21. Maupin, J. A. & Shanmugam, K. T. ( 1990; ). Genetic regulation of formate hydrogenlyase of Escherichia coli: role of the fhlA gene product as a transcriptional activator for a new regulatory gene, fhlB. J Bacteriol 172, 4798-4806.
    [Google Scholar]
  22. Maupin-Furlow, J. A., Rosentel, J. K., Lee, J. H., Deppenmeier, U., Gunsalus, R. P. & Shanmugam, K. T. ( 1995; ). Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli. J Bacteriol 177, 4851-4856.
    [Google Scholar]
  23. Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  24. Nixon, B. T., Ronson, C. W. & Ausubel, F. M. ( 1986; ). Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc Natl Acad Sci USA 83, 7850-7854.[CrossRef]
    [Google Scholar]
  25. Pistocchi, R., Kashiwagi, K., Miyamoto, S., Nukui, E., Sadakata, Y., Kobayashi, H. & Igarashi, K. ( 1993; ). Characteristics of the operon for a putrescine transport system that maps at 19 minutes on the Escherichia coli chromosome. J Biol Chem 268, 146-152.
    [Google Scholar]
  26. Rosenfeld, S. A., Stevis, P. E. & Ho, N. W. ( 1984; ). Cloning and characterization of the xyl genes from Escherichia coli. Mol Gen Genet 194, 410-415.[CrossRef]
    [Google Scholar]
  27. Rosentel, J. K., Healy, F., Maupin-Furlow, J. A., Lee, J. H. & Shanmugam, K. T. ( 1995; ). Molybdate and regulation of mod (molybdate transport), fdhF, and hyc (formate hydrogenlyase) operons in Escherichia coli. J Bacteriol 177, 4857-4864.
    [Google Scholar]
  28. Rossmann, R., Sawers, G. & Böck, A. ( 1991; ). Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon. Mol Microbiol 5, 2807-2814.[CrossRef]
    [Google Scholar]
  29. Sanger, F., Nicklen, S. & Coulson, A. R. ( 1977; ). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[CrossRef]
    [Google Scholar]
  30. Sankar, P., Lee, J. H. & Shanmugam, K. T. ( 1988; ). Gene-product relationships of fhlA and fdv genes of Escherichia coli. J Bacteriol 170, 5440-5445.
    [Google Scholar]
  31. Sauter, M., Böhm, R. & Böck, A. ( 1992; ). Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli. Mol Microbiol 6, 1523-1532.[CrossRef]
    [Google Scholar]
  32. Schlensog, V. & Böck, A. ( 1990; ). Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli. Mol Microbiol 4, 1319-1327.[CrossRef]
    [Google Scholar]
  33. Schlensog, V., Lutz, S. & Böck, A. ( 1994; ). Purification and DNA-binding properties of FHLA, the transcriptional activator of the formate hydrogenlyase system from Escherichia coli. J Biol Chem 269, 19590-19596.
    [Google Scholar]
  34. Self, W. T. (1998). Genetic and biochemical analysis of the molybdate-dependent expression of hyc operon in Escherichia coli. PhD thesis, University of Florida, USA.
  35. Self, W. T. & Shanmugam, K. T. ( 2000; ). Isolation and characterization of mutant FhlA proteins which activate transcription of hyc operon (formate hydrogenlyase) of Escherichia coli in the absence of molybdate. FEMS Microbiol Lett 184, 47-52.[CrossRef]
    [Google Scholar]
  36. Self, W. T., Grunden, A. M., Hasona, A. & Shanmugam, K. T. ( 1999; ). Transcriptional regulation of molybdoenzyme synthesis in Escherichia coli in response to molybdenum: ModE-molybdate, a repressor of the modABCD (molybdate transport) operon is a secondary transcriptional activator for the hyc and nar operons. Microbiology 145, 41-55.[CrossRef]
    [Google Scholar]
  37. Shuman, H. A. & Silhavy, T. J. ( 1981; ). Identification of the malK gene product. A peripheral membrane component of the Escherichia coli maltose transport system. J Biol Chem 256, 560-562.
    [Google Scholar]
  38. Tabor, S. & Richardson, C. C. ( 1985; ). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82, 1074-1078.[CrossRef]
    [Google Scholar]
  39. Wedel, A. & Kustu, S. ( 1995; ). The bacterial enhancer-binding protein NTRC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation. Genes Dev 9, 2042-2052.[CrossRef]
    [Google Scholar]
  40. Weiss, D. S., Batut, J., Klose, K. E., Keener, J. & Kustu, S. ( 1991; ). The phosphorylated form of the enhancer-binding protein NTRC has an ATPase activity that is essential for activation of transcription. Cell 67, 155-167.[CrossRef]
    [Google Scholar]
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