Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Dec;170(12):5409–5415. doi: 10.1128/jb.170.12.5409-5415.1988

Nucleotide sequence of plasmid NAH7 gene nahR and DNA binding of the nahR product.

I S You 1, D Ghosal 1, I C Gunsalus 1
PMCID: PMC211631  PMID: 2848005

Abstract

The nah and sal operons of the 80-kilobase-pair (kb) NAH7 plasmid specify catabolism of naphthalene and salicylate under positive regulation by gene nahR. A 1.75-kb fragment (PstI-HindIII) cloned into the pCP13 derivative of vector RK2 complemented in trans five nahR mutations. The fragment sequence contained a 1,122-base-pair open reading frame with a predicted sequence of 374 residues that was rich in basic amino acids with regions similar to known DNA-binding proteins. Clones from the nahR gene region were expressed in mexicells. Plasmid pY1923, carrying the 1.75-kb PstI-HindIII fragment, expressed a protein of Mr ca. 35,000 which bound to the upstream region of gene nahR in a gel electrophoresis DNA-binding assay. Other clones expressed proteins of currently unknown function; pY1311, with the 1.1-kb HindIII fragment, produced a polypeptide with an Mr of 23,000, and pY1812, with the 1.2-kb PstI-SphI fragment, produced a polypeptide (Mr 41,000) which appeared to be a fused nahR-lacZ product.

Full text

PDF
5409

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arcangioli B., Lescure B. Identification of proteins involved in the regulation of yeast iso- 1-cytochrome C expression by oxygen. EMBO J. 1985 Oct;4(10):2627–2633. doi: 10.1002/j.1460-2075.1985.tb03980.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnsley E. A. The induction of the enzymes of naphthalene metabolism in pseudomonads by salicylate and 2-aminobenzoate. J Gen Microbiol. 1975 May;88(1):193–196. doi: 10.1099/00221287-88-1-193. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Franklin F. C., Bagdasarian M., Bagdasarian M. M., Timmis K. N. Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7458–7462. doi: 10.1073/pnas.78.12.7458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Franklin F. C., Lehrbach P. R., Lurz R., Rueckert B., Bagdasarian M., Timmis K. N. Localization and functional analysis of transposon mutations in regulatory genes of the TOL catabolic pathway. J Bacteriol. 1983 May;154(2):676–685. doi: 10.1128/jb.154.2.676-685.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ghosal D., You I. S., Gunsalus I. C. Nucleotide sequence and expression of gene nahH of plasmid NAH7 and homology with gene xylE of TOL pWWO. Gene. 1987;55(1):19–28. doi: 10.1016/0378-1119(87)90244-7. [DOI] [PubMed] [Google Scholar]
  7. Grund A. D., Gunsalus I. C. Cloning of genes for naphthalene metabolism in Pseudomonas putida. J Bacteriol. 1983 Oct;156(1):89–94. doi: 10.1128/jb.156.1.89-94.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harayama S., Rekik M., Wasserfallen A., Bairoch A. Evolutionary relationships between catabolic pathways for aromatics: conservation of gene order and nucleotide sequences of catechol oxidation genes of pWW0 and NAH7 plasmids. Mol Gen Genet. 1987 Dec;210(2):241–247. doi: 10.1007/BF00325689. [DOI] [PubMed] [Google Scholar]
  9. Inouye S., Nakazawa A., Nakazawa T. Expression of the regulatory gene xylS on the TOL plasmid is positively controlled by the xylR gene product. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5182–5186. doi: 10.1073/pnas.84.15.5182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Inouye S., Nakazawa A., Nakazawa T. Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS. J Bacteriol. 1981 Nov;148(2):413–418. doi: 10.1128/jb.148.2.413-418.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Inouye S., Nakazawa A., Nakazawa T. Molecular cloning of regulatory gene xylR and operator-promoter regions of the xylABC and xylDEGF operons of the TOL plasmid. J Bacteriol. 1983 Sep;155(3):1192–1199. doi: 10.1128/jb.155.3.1192-1199.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Inouye S., Nakazawa A., Nakazawa T. Nucleotide sequence of the regulatory gene xylS on the Pseudomonas putida TOL plasmid and identification of the protein product. Gene. 1986;44(2-3):235–242. doi: 10.1016/0378-1119(86)90187-3. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Martin K., Huo L., Schleif R. F. The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3654–3658. doi: 10.1073/pnas.83.11.3654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  16. Mermod N., Ramos J. L., Bairoch A., Timmis K. N. The xylS gene positive regulator of TOL plasmid pWWO: identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon. Mol Gen Genet. 1987 May;207(2-3):349–354. doi: 10.1007/BF00331600. [DOI] [PubMed] [Google Scholar]
  17. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  18. Pfeifer K., Arcangioli B., Guarente L. Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene. Cell. 1987 Apr 10;49(1):9–18. doi: 10.1016/0092-8674(87)90750-1. [DOI] [PubMed] [Google Scholar]
  19. Ramos J. L., Mermod N., Timmis K. N. Regulatory circuits controlling transcription of TOL plasmid operon encoding meta-cleavage pathway for degradation of alkylbenzoates by Pseudomonas. Mol Microbiol. 1987 Nov;1(3):293–300. doi: 10.1111/j.1365-2958.1987.tb01935.x. [DOI] [PubMed] [Google Scholar]
  20. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schell M. A. Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida. Proc Natl Acad Sci U S A. 1986 Jan;83(2):369–373. doi: 10.1073/pnas.83.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schell M. A., Wender P. E. Identification of the nahR gene product and nucleotide sequences required for its activation of the sal operon. J Bacteriol. 1986 Apr;166(1):9–14. doi: 10.1128/jb.166.1.9-14.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  24. Spooner R. A., Bagdasarian M., Franklin F. C. Activation of the xylDLEGF promoter of the TOL toluene-xylene degradation pathway by overproduction of the xylS regulatory gene product. J Bacteriol. 1987 Aug;169(8):3581–3586. doi: 10.1128/jb.169.8.3581-3586.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Struhl K. The DNA-binding domains of the jun oncoprotein and the yeast GCN4 transcriptional activator protein are functionally homologous. Cell. 1987 Sep 11;50(6):841–846. doi: 10.1016/0092-8674(87)90511-3. [DOI] [PubMed] [Google Scholar]
  26. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  27. Vogt P. K., Bos T. J., Doolittle R. F. Homology between the DNA-binding domain of the GCN4 regulatory protein of yeast and the carboxyl-terminal region of a protein coded for by the oncogene jun. Proc Natl Acad Sci U S A. 1987 May;84(10):3316–3319. doi: 10.1073/pnas.84.10.3316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  29. Yen K. M., Gunsalus I. C. Plasmid gene organization: naphthalene/salicylate oxidation. Proc Natl Acad Sci U S A. 1982 Feb;79(3):874–878. doi: 10.1073/pnas.79.3.874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yen K. M., Gunsalus I. C. Regulation of naphthalene catabolic genes of plasmid NAH7. J Bacteriol. 1985 Jun;162(3):1008–1013. doi: 10.1128/jb.162.3.1008-1013.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. You I. S., Gunsalus I. C. Regulation of the nah and sal operons of plasmid NAH7: evidence for a new function in nahR. Biochem Biophys Res Commun. 1986 Dec 30;141(3):986–992. doi: 10.1016/s0006-291x(86)80141-3. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES