Abstract
We determined, under several growth conditions, the kinetics of mRNA synthesis from the four Pseudomonas putida pWW0 plasmid promoters involved in degradation of xylenes and methylbenzyl alcohols via toluates. Transcription by XylS of the meta-cleavage pathway operon promoter (Pm) for the metabolism of alkylbenzoates was stimulated immediately after the addition of an effector, both in Luria-Bertani (LB) medium and in minimal medium. Activation of the sigma 54-dependent upper-pathway operon promoter (Pu) and the xylS gene promoter (Ps) by effector-activated XylR was dependent on the growth medium used: on minimal medium, activation of transcription from Pu and Ps occurred immediately after the addition of a XylR effector; in contrast, activation appeared only after several hours when cells were growing on LB medium. When Pm was induced through the physiological overexpression of XylS, mediated by XylR when this regulator was activated by upper-pathway effectors, the kinetics of transcription from Pm was similar to that of Pu and Ps: maximum values were reached after delays of several hours in rich medium and after several minutes in minimal medium. The delay in the induction of transcription of sigma 54-dependent promoters reflects catabolite inhibition exerted by LB components, since the addition of yeast extracts, Casamino Acids, or several combinations of amino acids dramatically inhibited the synthesis of XylR-controlled sigma 54-dependent promoters. Expression from xylR gene tandem promoters occurred independently of the growth medium used.
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- Abril M. A., Buck M., Ramos J. L. Activation of the Pseudomonas TOL plasmid upper pathway operon. Identification of binding sites for the positive regulator XylR and for integration host factor protein. J Biol Chem. 1991 Aug 25;266(24):15832–15838. [PubMed] [Google Scholar]
- Abril M. A., Michan C., Timmis K. N., Ramos J. L. Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J Bacteriol. 1989 Dec;171(12):6782–6790. doi: 10.1128/jb.171.12.6782-6790.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- BAUCHOP T., ELSDEN S. R. The growth of micro-organisms in relation to their energy supply. J Gen Microbiol. 1960 Dec;23:457–469. doi: 10.1099/00221287-23-3-457. [DOI] [PubMed] [Google Scholar]
- Dixon R. The xylABC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli. Mol Gen Genet. 1986 Apr;203(1):129–136. doi: 10.1007/BF00330393. [DOI] [PubMed] [Google Scholar]
- Duetz W. A., Marqués S., de Jong C., Ramos J. L., van Andel J. G. Inducibility of the TOL catabolic pathway in Pseudomonas putida (pWW0) growing on succinate in continuous culture: evidence of carbon catabolite repression control. J Bacteriol. 1994 Apr;176(8):2354–2361. doi: 10.1128/jb.176.8.2354-2361.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Harayama S., Rekik M. The meta cleavage operon of TOL degradative plasmid pWW0 comprises 13 genes. Mol Gen Genet. 1990 Mar;221(1):113–120. doi: 10.1007/BF00280375. [DOI] [PubMed] [Google Scholar]
- Harayama S., Rekik M., Wubbolts M., Rose K., Leppik R. A., Timmis K. N. Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products. J Bacteriol. 1989 Sep;171(9):5048–5055. doi: 10.1128/jb.171.9.5048-5055.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holtel A., Timmis K. N., Ramos J. L. Upstream binding sequences of the XylR activator protein and integration host factor in the xylS gene promoter region of the Pseudomonas TOL plasmid. Nucleic Acids Res. 1992 Apr 11;20(7):1755–1762. doi: 10.1093/nar/20.7.1755. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hugouvieux-Cotte-Pattat N., Köhler T., Rekik M., Harayama S. Growth-phase-dependent expression of the Pseudomonas putida TOL plasmid pWW0 catabolic genes. J Bacteriol. 1990 Dec;172(12):6651–6660. doi: 10.1128/jb.172.12.6651-6660.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inouye S., Ebina Y., Nakazawa A., Nakazawa T. Nucleotide sequence surrounding transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1688–1691. doi: 10.1073/pnas.81.6.1688. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inouye S., Gomada M., Sangodkar U. M., Nakazawa A., Nakazawa T. Upstream regulatory sequence for transcriptional activator XylR in the first operon of xylene metabolism on the TOL plasmid. J Mol Biol. 1990 Nov 20;216(2):251–260. doi: 10.1016/S0022-2836(05)80317-1. [DOI] [PubMed] [Google Scholar]
- Inouye S., Nakazawa A., Nakazawa T. Determination of the transcription initiation site and identification of the protein product of the regulatory gene xylR for xyl operons on the TOL plasmid. J Bacteriol. 1985 Sep;163(3):863–869. doi: 10.1128/jb.163.3.863-869.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Inouye S., Nakazawa A., Nakazawa T. Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida. Gene. 1984 Sep;29(3):323–330. doi: 10.1016/0378-1119(84)90061-1. [DOI] [PubMed] [Google Scholar]
- Inouye S., Nakazawa A., Nakazawa T. Overproduction of the xylS gene product and activation of the xylDLEGF operon on the TOL plasmid. J Bacteriol. 1987 Aug;169(8):3587–3592. doi: 10.1128/jb.169.8.3587-3592.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kessler B., de Lorenzo V., Timmis K. N. A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet. 1992 May;233(1-2):293–301. doi: 10.1007/BF00587591. [DOI] [PubMed] [Google Scholar]
- Köhler T., Harayama S., Ramos J. L., Timmis K. N. Involvement of Pseudomonas putida RpoN sigma factor in regulation of various metabolic functions. J Bacteriol. 1989 Aug;171(8):4326–4333. doi: 10.1128/jb.171.8.4326-4333.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marqués S., Ramos J. L., Timmis K. N. Analysis of the mRNA structure of the Pseudomonas putida TOL meta fission pathway operon around the transcription initiation point, the xylTE and the xylFJ regions. Biochim Biophys Acta. 1993 Nov 16;1216(2):227–236. doi: 10.1016/0167-4781(93)90149-8. [DOI] [PubMed] [Google Scholar]
- Marqués S., Ramos J. L. Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways. Mol Microbiol. 1993 Sep;9(5):923–929. doi: 10.1111/j.1365-2958.1993.tb01222.x. [DOI] [PubMed] [Google Scholar]
- Mermod N., Lehrbach P. R., Reineke W., Timmis K. N. Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters. EMBO J. 1984 Nov;3(11):2461–2466. doi: 10.1002/j.1460-2075.1984.tb02156.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakazawa T., Inouye S., Nakazawa A. Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants. J Bacteriol. 1980 Oct;144(1):222–231. doi: 10.1128/jb.144.1.222-231.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Worsey M. J., Williams P. A. Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol. 1975 Oct;124(1):7–13. doi: 10.1128/jb.124.1.7-13.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Lorenzo V., Cases I., Herrero M., Timmis K. N. Early and late responses of TOL promoters to pathway inducers: identification of postexponential promoters in Pseudomonas putida with lacZ-tet bicistronic reporters. J Bacteriol. 1993 Nov;175(21):6902–6907. doi: 10.1128/jb.175.21.6902-6907.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Lorenzo V., Herrero M., Metzke M., Timmis K. N. An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid. EMBO J. 1991 May;10(5):1159–1167. doi: 10.1002/j.1460-2075.1991.tb08056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]