1887

Abstract

Summary: The locus of encodes functions which catalyse the synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide, from molybdopterin and GTP. Reporter translational fusion mutations in the gene have been constructed using λMu9 mutagenesis. The locus is expressed at very low levels under both aerobic and anaerobic growth conditions. Neither additions to the growth media (nitrate, tungstate or molybdate) nor secondary mutations at the or loci affected the level of expression. Two transcription initiation sites and their associated promoter regions have been identified upstream of . Both of the promoter regions show a poor match to the −35 and −10 consensus sequences for −70 promoters. A 2-2 kb chromosomal DNA fragment which complemented all available mutants has been sequenced. Two ORFs were identified, arranged as a single transcription unit. The encoded polypeptides have predicted molecular masses of 21642 Da and 19362 Da, respectively. The DNA has been subcloned into a T7 overexpression system and the predicted products identified. The gene encodes protein FA, which has been purified to homogeneity and brings about the activation of inactive molybdoenzymes in cell extracts of mutants. The gene encodes a polypeptide with a putative nucleotide binding site. All available mutations which have been selected for by their ability to grow anaerobically in the presence of chlorate are located in the gene.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-141-7-1663
1995-07-01
2021-08-01
Loading full text...

Full text loading...

/deliver/fulltext/micro/141/7/mic-141-7-1663.html?itemId=/content/journal/micro/10.1099/13500872-141-7-1663&mimeType=html&fmt=ahah

References

  1. Arai K., Clark B.F.C., Duffy L., Jones M.D., Kaziro Y., Laursen R.A., L’ltalien J., Miller D.L., Nagarkatti S., Nakamura S., Nielsen K.M., Petersen T.E., Takahashi K., Wade M. 1980; Primary structure of elongation factor Tu from Escherichia coli. . Proc Natl Acad Sci USA 771326–1330
    [Google Scholar]
  2. Baker K.P., Boxer D.H. 1991; Regulation of the chlA locus of Escherichia coli K12: involvement of molybdenum cofactor.. Mol Microbiol 5:901–907
    [Google Scholar]
  3. Blasco F., lobbi C., Ratouchniak J., Bonnefoy V., Chippaux M. 1990; Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJ1 operon.. Mol & Gen Genet 222:104–111
    [Google Scholar]
  4. Dever T.E., Glynias M.J., Merrick W.C. 1987; GTP-binding proteins: three consensus sequence elements with distinct spacing.. Proc Natl Acad Sci USA 841814–1818
    [Google Scholar]
  5. Figueroa N., Wills N., Bossi L. 1991; Common sequence determinants of the response of a prokaryotic promoter to DNA bending and supercoiling.. EMBO J 10:941–949
    [Google Scholar]
  6. Glaser J.H., DeMoss J.A. 1972; Comparison of nitrate reductase mutants of Escherichia coli selected by alternative procedures.. J Bacteriol 108:854–860
    [Google Scholar]
  7. Inoue T., Cech T.R. 1985; Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.. Proc Natl Acad Sci USA 82648–652
    [Google Scholar]
  8. Irby R.B., Adair W.L. Jr 1994; Intermediates in the folic acid biosynthetic pathway are incorporated into molybdopterin in the yeast, Pichia canadensis. . J Biol Chem 269:23981–23987
    [Google Scholar]
  9. James R., Dean D., Debbage J. 1993; Five open reading frames upstream of the dnaK gene of E. coli. . J DNA Sequencing & Mapping 3:327–332
    [Google Scholar]
  10. Johann S., Hinton S.M. 1987; Cloning and nucleotide sequence of the chlD locus.. J Bacteriol 169:1911–1916
    [Google Scholar]
  11. Johnson J.L., Hainline B.E., Rajagopalan K.V., Arison B.H. 1984; The pterin component of the molybdenum cofactor. Structural characterization of two fluorescent derivatives.. J Biol Chem 259:5414–5422
    [Google Scholar]
  12. Johnson J.L., Indermaur L.W., Rajagopalan K.V. 1991; Molybdenum cofactor biosynthesis in Escherichia coli. Requirement of the chlB gene product for the formation of molybdopterin guanine dinucleotide.. J Biol Chem 266:12140–12145
    [Google Scholar]
  13. Lee J.H., Wendt J.C., Shanmugam K.T. 1990; Identification of a new gene, molR, essential for utilization of molybdate by Escherichia coli. . J Bacteriol 172:2079–2087
    [Google Scholar]
  14. Mandrand-Berthelot M.-A., Wee M.Y.K., Haddock B.A. 1978; An improved method for the identification and characterization of mutants of Escherichia coli deficient in formate dehydrogenase activity.. FEMS Microbiol Eett 4:37–40
    [Google Scholar]
  15. Maniatis T., Fritsch E.F., Sambrook J. 1982 Molecular Cloning: a Eaboratory Manual Cold Spring Harbor, New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  16. Messing J., Crea R., Seeburg P.H. 1981; A system for shotgun DNA sequencing.. Nucleic Acids Res 9:309–321
    [Google Scholar]
  17. Miller J.B., Amy N.K. 1983; Molybdenum cofactor in chlorate- resistant and nitrate reductase-deficient insertion mutants of Escherichia coli. . J Bacteriol 155:793–801
    [Google Scholar]
  18. Miller J.B., Scott D.J., Amy N.K. 1987; Molybdenum- sensitive transcriptional regulation of the chlD locus of Escherichia coli. . J Bacteriol 169:1853–1860
    [Google Scholar]
  19. Miller J.H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  20. Nohno T., Kasai Y., Saito T. 1988; Cloning and complete nucleotide sequence of the Escherichia coli chlEN operon involved in molybdopterin biosynthesis.. J Bacteriol 170:4097–4102
    [Google Scholar]
  21. Palmer T., Vasishta A., Whitty P.W., Boxer D.H. 1994; Isolation of protein FA: a product of the mob locus required for molybdenum cofactor biosynthesis in Escherichia coli. . Eur J Biochem 222:687–692
    [Google Scholar]
  22. Pitterle D.M., Rajagopalan K.V. 1993; Biosynthesis of molybdopterin in Escherichia coli. Purification and characterisation of the converting factor.. J Biol Chem 268:13499–13505
    [Google Scholar]
  23. Pitterle D.M., Johnson J.L., Rajagopalan K.V. 1993; In vitro synthesis of molybdopterin from precursor Z using purified converting factor.. J Biol Chem 268:13506–13509
    [Google Scholar]
  24. Plunkett G. III Burland V., Daniels D.L., Blattner F.R. 1993; Analysis of the Escherichia coli genome. III. DNA sequence of the region from 87-2 to 89-2 minutes.. Nucleic Acids Res 21:3391–3398
    [Google Scholar]
  25. Rajagopalan K.V., Johnson J.L. 1992; The pterin molybdenum cofactors.. J Biol Chem 267:10199–10202
    [Google Scholar]
  26. Reiss J., Klingmuller W. 1987; Direct selection of recombinant plasmids with chlorate.. FEMS Microbiol Eett 43:201–205
    [Google Scholar]
  27. Reiss J., Kleinhofs A., Klingmuller W. 1987; Cloning of seven differently complementing DNA fragments with chi functions from Escherichia coli K-12.. Mol & Gen Genet 206:352–355
    [Google Scholar]
  28. Rivers S.L. 1991; A physical and genetic analysis of the chi A locus of Escherichia coli.. PhD thesis University of Dundee;
    [Google Scholar]
  29. Rivers S.L., McNairn E., Blasco F., Giordano G., Boxer D.H. 1993; Molecular genetic analysis of the moa operon of Escherichia coli K-12 required for molybdenum cofactor biosynthesis.. Mol Microbiol 8:1071–1081
    [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A.R. 1977; DNA sequencing with chain-terminating inhibitors.. Proc Natl Acad Sci USA 745463–5467
    [Google Scholar]
  31. Santini C.-L., lobbi-Nivol C., Romane C., Boxer D.H., Giordano G. 1992; Molybdoenzyme biosynthesis in Escherichia coli: in vitro activation of purified nitrate reductase from a chlB mutant.. J Bacteriol 174:7934–7940
    [Google Scholar]
  32. Schagger H., von Jagow G. 1987; Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.. Anal Biochem 166:368–379
    [Google Scholar]
  33. Silhavy T.J., Berman M.L., Enquist L.E. 1984 Experiments with Gene Fusions Cold Spring Harbor, New York: Cold Spring Harbor Laboratory;
  34. Stewart V., MacGregor C.H. 1982; Nitrate reductase in Escherichia coli K-12: involvement of the chlC, chlE and chlG loci.. J Bacteriol 151:788–799
    [Google Scholar]
  35. Studier F.W., Moffatt B.A. 1986; Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.. J Mol Biol 189:113–130
    [Google Scholar]
  36. Tabor S., Richardson C.C. 1985; A bacteriophage T7 poly- merase/promoter system for controlled exclusive expression of specific genes.. Proc Natl Acad Sci USA 821074–1078
    [Google Scholar]
  37. Wootton J.C., Nicolson R.E., Cock J.M., Walters D.E., Burke J.F., Doyle W.A., Bray R.C. 1991; Enzymes depending on the pterin molybdenum cofactor: sequence families, spectroscopic properties of molybdenum and possible cofactor binding domains.. Biochim Biophys Acta 1057:157–185
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-141-7-1663
Loading
/content/journal/micro/10.1099/13500872-141-7-1663
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error