ORIGINAL PAPER Alternative promoters in the pst operon of Escherichia coli Beny Spira • Meire Aguena • Juliana Velasco de Castro Oliveira • Ezra Yagil Received: 26 August 2010 / Accepted: 4 October 2010 / Published online: 21 October 2010 Ó Springer-Verlag 2010 Abstract The pst operon of Escherichia coli is com- posed of five genes pstS, pstC, pstA, pstB and phoU, that encode a high-affinity phosphate transport system and a negative regulator of the PHO regulon. Transcription of pst is induced under phosphate shortage and is initiated at the promoter located upstream of the first gene of the operon, pstS. Here, we show by four different technical approaches the existence of additional internal promoters upstream of pstC, pstB and phoU. These promoters are not induced by Pi-limitation and do not possess PHO-box sequences. Plasmids carrying the pst internal genes par- tially complement chromosomal mutations in their cor- responding genes, indicating that they are translated into functional proteins. Keywords Pst operon Á PHO regulon Á Transcription start point Á Internal promoters Introduction Operons are chromosomal genetic units of two or more open reading frames in tandem transcribed as a single RNA molecule. Transcription of the operon genes usually initiates at a promoter upstream of the proximal gene of the operon. The pst operon of Escherichia coli, com- prised of five reading frames (pstS, pstC, pstA, pstB and phoU; Fig. 1), encodes a high-affinity transport system for orthophosphate (Pi). Transcription of the pst operon is initiated at the pstS promoter, which consists of a -10 region and two PHO-boxes (Fig. 1). Though the full- length pstSCAB–phoU transcript is formed, it is imme- diately processed by RNase E resulting in smaller, but well-defined mRNA species (Aguena et al. 2002; Aguena and Spira 2008). The presence of a repetitive extragenic palindrome sequence (REP, Fig. 1) located in the inter- genic region between pstS and pstC stabilises the pstS transcript. Hence, the levels of pstS mRNA and of its protein are in excess compared with the other cognate genes of the pst operon (Aguena et al. 2009). Other putative regulatory sequences, such as a second REP element and a conventional r 70 -dependent promoter in the intergenic region between pstA and pstB have been postulated, but have hitherto not been confirmed (Amemura et al. 1985). The pst operon belongs to the PHO regulon, a group of more than 40 genes associated with the uptake and metabolism of Pi (Baek and Lee 2006). Pst is a typical ABC transport system composed of four characterized proteins: PstS is a periplasmic Pi-binding protein; PstC and PstA are integral membrane proteins that mediate the translocation of Pi through the inner membrane (Webb et al. 1992) and PstB is a dimer that binds ATP and energizes the transport (Chan and Torriani 1996). The Communicated by D. Andersson. Electronic supplementary material The online version of this article (doi:10.1007/s00438-010-0584-x) contains supplementary material, which is available to authorized users. B. Spira (&) Á M. Aguena Á J. V. de Castro Oliveira Departamento de Microbiologia, Instituto de Cie ˆncias Biome ´dicas, Universidade de Sa ˜o Paulo, Av. Prof. Lineu Prestes, 1374, Sa ˜o Paulo, SP 05508-900, Brazil e-mail: benys@usp.br E. Yagil Department of Biochemistry, Tel-Aviv University, 69978 Tel-Aviv, Israel 123 Mol Genet Genomics (2010) 284:489–498 DOI 10.1007/s00438-010-0584-x