title: Transcriptional and post-transcriptional regulation of pst2 operon expression in Vibrio cholerae O1.
abstract: One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions that are known to block 3'-5' exonucleolytic attacks.
authors.affiliation: Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, G1-043, Rio de Janeiro-RJ CEP 21941-902, Brazil.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: da C Leite
authors.affiliation: Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, G1-043, Rio de Janeiro-RJ CEP 21941-902, Brazil. Electronic address: lbarbosa@biof.ufrj.br.
authors.forename: Livia C
authors.initials: LC
authors.lastname: Barbosa
authors.affiliation: Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, G1-043, Rio de Janeiro-RJ CEP 21941-902, Brazil.
authors.forename: Nathalia
authors.initials: N
authors.lastname: Mantuano
authors.affiliation: Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, G1-043, Rio de Janeiro-RJ CEP 21941-902, Brazil. Electronic address: cgoulart@biof.ufrj.br.
authors.forename: Carolina L
authors.initials: CL
authors.lastname: Goulart
authors.affiliation: Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, G1-043, Rio de Janeiro-RJ CEP 21941-902, Brazil.
authors.forename: Giovani C
authors.initials: GC
authors.lastname: Veríssimo da Costa
authors.affiliation: Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, G1-043, Rio de Janeiro-RJ CEP 21941-902, Brazil. Electronic address: pmbisch@biof.ufrj.br.
authors.forename: Paulo M
authors.initials: PM
authors.lastname: Bisch
authors.affiliation: Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, G1-043, Rio de Janeiro-RJ CEP 21941-902, Brazil. Electronic address: wmavk@biof.ufrj.br.
authors.forename: Wanda M A
authors.initials: WMA
authors.lastname: von Krüger
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D003062:Codon / Q000737:chemistry / Q000378:metabolism; D019295:Computational Biology; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D055029:Inverted Repeat Sequences; D009876:Operon; D033902:Periplasmic Binding Proteins / Q000235:genetics* / Q000378:metabolism; D028044:Phosphate-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014158:Transcription, Genetic; D041241:Vibrio cholerae O1 / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D033902:Periplasmic Binding Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D012333:RNA, Messenger; C061278:PhoB protein, Bacteria; D004722:Endoribonucleases; C022537:ribonuclease E
keywords: Bacterial gene regulation; Pho regulon; Phosphate limitation response