1887

Abstract

Acyl carrier protein (ACP) plays a crucial role in bacterial fatty acid synthesis. Cloning genes encoding ACPs from Gram-negative bacteria in is difficult due to adverse effects of the cloned gene on host cell viability, and we were unsuccessful in cloning the full length ACP gene () from using conventional methods. Therefore, ACP from was purified to homogeneity and a part of the gene was cloned using the polymerase chain reaction (PCR) technique with two primers, one designed from the N-terminal amino acid sequence of the purified ACP and the other from the highly conserved amino acid sequence of bacterial ACPs. The nucleotide sequence of the gene was obtained by cloning and sequencing inverse PCR products containing the region generated by two oppositely oriented internal primers designed from the partial gene sequence using restriction-enzyme-digested, self-circularized chromosomal DNA fragments as templates. Characterization of the purified ACP and analysis of the derived amino acid sequence of the gene of revealed that: (a) the mature ACP, composed of 78 amino acids, is a highly expressed protein (about 2·0–3·0 × 10 molecules per cell), (b) compared to ACP, it has a more compact structure and contains significantly more hydrophobic amino acid residues and (c) the potential mRNA sequence of the ACP gene has some structural features typical of a stable mRNA.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-142-8-2097
1996-08-01
2021-05-07
Loading full text...

Full text loading...

/deliver/fulltext/micro/142/8/mic-142-8-2097.html?itemId=/content/journal/micro/10.1099/13500872-142-8-2097&mimeType=html&fmt=ahah

References

  1. Beyreuther K., Bohmer H., Dimroth P. 1978; Amino-acid sequence of citrate-lyase acyl-carrier protein from Klebsiella aerogenes. . Eur J Biochem 87:101–110
    [Google Scholar]
  2. Bradford M.M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
    [Google Scholar]
  3. Chattopadhyay S., Mukherjee A., Ghosh S. 1994; Molecular cloning and sequencing of an operon, carRS of Azpspirillum brasilense that codes for a novel two-component regulatory system: demonstration of a positive regulatory role of carR for global control of carbohydrate catabolism. J Bacteriol 176:7484–7490
    [Google Scholar]
  4. Cooper C.L, Boyce S.G., Lueking D.R. 1987; Purification and characterization of Rhodobacter sphaeroides acyl carrier protein. Biochemistry 26:2740–2746
    [Google Scholar]
  5. Froehlich J.E., Poorman R., Reardon E., Barnum S.R., Jaworski G.J. 1990; Purification and characterization of acyl carrier protein from two cyanobacteria species. Eur J Biochem 193:817–825
    [Google Scholar]
  6. Geiger O., Spaink H.P., Kennedy E.P. 1991; Isolation of the Rhizobium leguminosarum NodF nodulation protein: NodF carries a phosphopantetheine prosthetic group. J Bacteriol 173:2872–2878
    [Google Scholar]
  7. Higgins C.F. 1991; Stability and degradation of mRNA. Curr Opin Cell Biol 3:1013–1018
    [Google Scholar]
  8. Hirel P.-H., Schmitter J.M., Dissen P., Fayat G., Blanquet S. 1989; Extent of N-terminal methionine excision from E. coli protein is governed by the side chain length of the penultimate amino acid. Proc Natl Acad Set USA 868247–8251
    [Google Scholar]
  9. Innis M.A., Gelfand D.H., Sninsky J.J., White T.J. 1990 PCR Protocols: a Guide to Methods and Applications. New York: Academic Press.;
    [Google Scholar]
  10. Jackowski S., Rock C.O. 1983; Ratio of active to inactive forms of acyl carrier protein in Escherichia coli. . J Biol Chem 258:15186–15191
    [Google Scholar]
  11. Kawakami Y., Ohmori S. 1994; Microidentification of N- terminal blocked amino acid residues of proteins and peptides. Anal Biochem 320:66–72
    [Google Scholar]
  12. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  13. Magnuson K., Jackowski S., Rock C.O., Cronan J.E. JR 1993; Regulation of fatty acid biosynthesis in Escherichia coli. . Microbiol Rev 57:522–542
    [Google Scholar]
  14. Marmur J. 1961; A procedure for the isolation of DNA from microorganisms. J Mol Biol 3:208–218
    [Google Scholar]
  15. Matsudaira P. 1987; Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038
    [Google Scholar]
  16. Mukherjee A., Ghosh S. 1987; Regulation of fructose uptake and catabolism by succinate in Azospirillum brasilense. . J Bacteriol 169:4361–4367
    [Google Scholar]
  17. Nakamura K., Pirtle R.M., Pirtle I.L, Takeishi K., Inouye M. 1980; Messenger ribonucleic acid of the lipoprotein of the Escherichia coli outer membrane. II. The complete nucleotide sequence. J Biol Chem 255:210–216
    [Google Scholar]
  18. Niki H., Ryu I., Yamanaka K., Ogura T., Hiraga S. 1992; E. coli MukB protein involved in chromosome partition forms a homodimer with a rod-and-hinge structure having DNA-binding and ATP/GTP-binding activities. EMBO J 11:5101–5109
    [Google Scholar]
  19. Platt M.W., Miller K.J., Lane W.S., Kennedy E.P. 1990; Isolation and characterization of the constitutive acyl carrier protein from R hizobium meliloti. . J Bacteriol 172:5440–5444
    [Google Scholar]
  20. Rawlings M., Cronan J.E. JR 1992; The gene encoding Escherichia coli acyl carrier protein lies within a cluster of fatty acid biosynthetic genes. J Biol Chem 267:5751–5754
    [Google Scholar]
  21. Revill W.P., Leaday P.F. 1991; Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopoljspora erjthraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis. J Bacteriol 173:4379–4385
    [Google Scholar]
  22. Rock C.O., Cronan J.E. JR 1979; Solubilization, purification and salt activation of acyl-acyl carrier protein synthetase from E. coli. . J Biol Chem 254:7116–7122
    [Google Scholar]
  23. Rock C.O., Cronan J.E. JR 1980; Improved purification of acyl carrier protein. Anal Biochem 102:362–364
    [Google Scholar]
  24. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Eaboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A.R. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 745463–5467
    [Google Scholar]
  26. Shearman C.A., Rossen L., Johnson A.W.B., Downie J.A. 1986; The Rhizobium leguminosarum nodulation gene nodF encodes a polypeptide similar to acyl carrier protein and is regulated by nodD plus a factor in pea root exudate. EMBO J 5:647–652
    [Google Scholar]
  27. Singh L., Jones K.W. 1984; The use of heparin as a simple cost effective means of controlling background in nucleic acid hybridization procedures. Nucleic Acids Res 12:5627–5638
    [Google Scholar]
  28. Therisod H., Kennedy E.P. 1987; The function of the acyl carrier protein in the synthesis of membrane derived oligosaccharides does not require its phosphopentathein prosthetic group. Proc Natl Acad Sci USA 848235–8238
    [Google Scholar]
  29. Triglia T., Peterson M.G., Kemp D.J. 1988; A procedure for in vivo amplification of DNA fragments that lie outside the boundaries of known sequences. Nucleic Acids Res 16:8186
    [Google Scholar]
  30. Vanaman T.C., Wakil S.J., Hill R.L. 1968; The complete amino acid sequence of the acyl carrier protein of Escherichia coli. . J Biol Chem 243:6420–6431
    [Google Scholar]
  31. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103–119
    [Google Scholar]
  32. Zimmer W., Aparica C., Elmerich C. 1991; Relationship between tryptophan biosynthesis and indole-3-acetic acid production in Azospirillum : identification and sequencing of a trpGDC cluster. Mol Gen Genet 229:41–51
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-142-8-2097
Loading
/content/journal/micro/10.1099/13500872-142-8-2097
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