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Journal of Bacteriology, May 2002, p. 2603-2613, Vol. 184, No. 10
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.10.2603-2613.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Functional Characterization and Regulation of gadX, a Gene Encoding an AraC/XylS-Like Transcriptional Activator of the Escherichia coli Glutamic Acid Decarboxylase System{dagger}

Angela Tramonti,1 Paolo Visca,2 Michele De Canio,1 Maurizio Falconi,3 and Daniela De Biase1*

Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università di Roma La Sapienza, 00185 Rome,,1 Dipartimento di Biologia, Università di Roma Tre, 00146 Rome,,2 Dipartimento di Biologia MCA, Università di Camerino, 62032 Camerino, Italy3

Received 14 December 2001/ Accepted 14 February 2002

The Escherichia coli chromosome contains two distantly located genes, gadA and gadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad). The Gad reaction contributes to pH homeostasis by consuming intracellular H+ and producing {gamma}-aminobutyric acid. This compound is exported via the protein product of the gadC gene, which is cotranscribed with gadB. Here we demonstrate that transcription of both gadA and gadBC is positively controlled by gadX, a gene downstream of gadA, encoding a transcriptional regulator belonging to the AraC/XylS family. The gadX promoter encompasses the 67-bp region preceding the gadX transcription start site and contains both RpoD and RpoS putative recognition sites. Transcription of gadX occurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of the gad structural genes. However, PT5lacO-controlled gadX expression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when the gad system is normally repressed. Autoregulation of the whole gad system is inferred by the positive effect of GadX on the gadA promoter and gadAX cotranscription. Transcription of gadX is derepressed in an hns mutant and strongly reduced in both rpoS and hns rpoS mutants, consistent with the expression profile of gad structural genes in these genetic backgrounds. Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX binds gadA and gadBC promoters at different sites and with different binding affinities.

* Corresponding author. Mailing address: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università di Roma ‘La Sapienza', Piazzale Aldo Moro 5, 00185 Rome, Italy. Phone: (06) 4991-7686. Fax (06) 4991-7566. E-mail: debiase{at}caspur.it.

{dagger} This manuscript is dedicated to the memory of Franco Tatò, prematurely deceased 7 July 2001.

Journal of Bacteriology, May 2002, p. 2603-2613, Vol. 184, No. 10
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.10.2603-2613.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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