Transcriptional and post-transcriptional regulation of pst2 operon expression in Vibrio cholerae O1

• Published in: Infection, genetics and evolution Vol. 51; pp. 10 - 16
• Main Authors: da C. Leite, Daniel M., Barbosa, Livia C., Mantuano, Nathalia, Goulart, Carolina L., Veríssimo da Costa, Giovani C., Bisch, Paulo M., von Krüger, Wanda M.A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-07-2017
• Subjects: Bacterial gene regulation; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Binding Sites; Biological Transport; Codon - chemistry; Codon - metabolism; Computational Biology; Endoribonucleases - genetics; Endoribonucleases - metabolism; Gene Expression Regulation, Bacterial; Inverted Repeat Sequences; Operon; Periplasmic Binding Proteins - genetics; Periplasmic Binding Proteins - metabolism; Pho regulon; Phosphate limitation response; Phosphate-Binding Proteins - genetics; Phosphate-Binding Proteins - metabolism; Phosphates - metabolism; Promoter Regions, Genetic; Protein Binding; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Alignment; Transcription, Genetic; Vibrio cholerae O1 - genetics; Vibrio cholerae O1 - metabolism; Vibrio cholerae O1 - pathogenicity; Virulence; Pho regulon; Phosphate limitation response; Bacterial gene regulation
• ISSN: 1567-1348; 1567-7257
• DOI: 10.1016/j.meegid.2017.02.017

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.
•V. cholerae pst2 operon transcription in response to Pi limitation depends on PhoBR.•pst2 polycistronic mRNA is processed into minor transcripts of distinct stabilities.•Bioinformatics and experimental data were combined to refine genomic annotation.