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Journal of Bacteriology, October 2009, p. 6123-6135, Vol. 191, No. 19
0021-9193/09/$08.00+0     doi:10.1128/JB.00744-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

NtrC-Dependent Regulatory Network for Nitrogen Assimilation in Pseudomonas putida{triangledown}

Ana B. Hervás,1 Inés Canosa,1* Richard Little,2 Ray Dixon,2 and Eduardo Santero1

Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/CSIC, Carretera de Utrera, Km. 1, 41013 Seville, Spain,1 Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom2

Received 9 June 2009/ Accepted 24 July 2009

Pseudomonas putida KT2440 is a model strain for studying bacterial biodegradation processes. However, very little is known about nitrogen regulation in this strain. Here, we show that the nitrogen regulatory NtrC proteins from P. putida and Escherichia coli are functionally equivalent and that substitutions leading to partially active forms of enterobacterial NtrC provoke the same phenotypes in P. putida NtrC. P. putida has only a single PII-like protein, encoded by glnK, whose expression is nitrogen regulated. Two contiguous NtrC binding sites located upstream of the {sigma}N-dependent glnK promoter have been identified by footprinting analysis. In vitro experiments with purified proteins demonstrated that glnK transcription was directly activated by NtrC and that open complex formation at this promoter required integration host factor. Transcription of genes orthologous to enterobacterial codB, dppA, and ureD genes, whose transcription is dependent on {sigma}70 and which are activated by Nac in E. coli, has also been analyzed for P. putida. Whereas dppA does not appear to be regulated by nitrogen via NtrC, the codB and ureD genes have {sigma}N-dependent promoters and their nitrogen regulation was exerted directly by NtrC, thus avoiding the need for Nac, which is missing in this bacterial species. Based upon these results, we propose a simplified nitrogen regulatory network in P. putida (compared to that in enterobacteria), which involves an indirect-feedback autoregulation of glnK using NtrC as an intermediary.

* Corresponding author. Mailing address: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/CSIC, Carretera de Utrera, Km. 1, 41013 Seville, Spain. Phone: 34-954349052. Fax: 34-954349376. E-mail: icanper{at}upo.es

{triangledown} Published ahead of print on 31 July 2009.

Journal of Bacteriology, October 2009, p. 6123-6135, Vol. 191, No. 19
0021-9193/09/$08.00+0     doi:10.1128/JB.00744-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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