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Journal of Bacteriology, May 2005, p. 3238-3248, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3238-3248.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Molecular Determinants for PspA-Mediated Repression of the AAA Transcriptional Activator PspF

Sarah Elderkin,{dagger} Patricia Bordes, Susan Jones,{ddagger} Mathieu Rappas, and Martin Buck*

Imperial College London, Department of Biological Sciences, Sir Alexander Fleming Building, South Kensington Campus, London SW7 2AZ, United Kingdom

Received 20 October 2004/ Accepted 29 January 2005

The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the {sigma}54 subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of {sigma}54 AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal {alpha}-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.

* Corresponding author. Mailing address: Imperial College London, Department of Biological Sciences, Sir Alexander Fleming Building, South Kensington Campus, London SW7 2AZ, United Kingdom. Phone: 44 207 594 5442. Fax: 44 207 594 5419. E-mail: m.buck{at}imperial.ac.uk.

{dagger} Present address: MRC Clinical Sciences Centre, Hammersmith Hospital Campus, London W12 0NN, United Kingdom.

{ddagger} Present address: Nature Reviews Journals, Porters South, London N1 9XW, United Kingdom.

Journal of Bacteriology, May 2005, p. 3238-3248, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3238-3248.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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