Regulation of polyphosphate kinase production by antisense RNA in Pseudomonas fluorescens Pf0-1

Abstract

Pseudomonas spp. adapt rapidly to environmental fluctuations. Loss or overproduction of polyphosphate reduces the fitness of Pseudomonas fluorescens Pf0-1, indicating the importance of the fine-tuning of polyphosphate production. An antisense RNA was investigated and shown to regulate the polyphosphate kinase gene (ppk) by a posttranscriptional mechanism reducing ppk transcript abundance.

Fig 1

Accumulation of PolyP in P. fluorescens Pf0-1ΔrecA carrying pHERDiiv8 or the vector (pHERD). Induction of iiv8 transcription from the P_BAD promoter in pHERDiiv8 resulted in PolyP accumulation that was approximately 25% of that obtained with the uninduced vector control (P < 0.01). The y axis shows PolyP accumulation as a percentage of that in uninduced Pf0-1 carrying the pHERD26 vector after normalization for cell number. Data are the average of at least three independent experiments. Error bars show the standard deviations.

Fig 2

(A) Diagrammatic representation of the transcriptional fusion construct used to assess ppk transcription in the presence or absence of induced iiv8 expression. Numbers indicate the coordinates in the P. fluorescens Pf0-1 genome sequence (GenBank accession number CP000094). This construct, carried by a mini-Tn7 element, was used to create a single-copy transcriptional fusion in Pf0-1. (B) Expression of iiv8 does not reduce hemB/ppk promoter activity. The activity of the hemB/ppk promoter fused to lacZ was measured using the method of Miller (17). The promoter activity in Pf0-1 carrying pHERDiiv8 did not differ significantly between arabinose-induced and noninduced cultures (P = 0.537). The data shown are averages from three independent experiments. Error bars show the standard deviations.

Fig 3

Absolute numbers of transcripts per nanogram of total RNA from rplU (control) and ppk in Pf0-1 carrying either the vector pHERD26 or iiv8 asRNA-expressing pHERDiiv8. Induction of iiv8 significantly reduced transcript ppk abundance (t test) relative to that in the control sample. These data are the average and standard error of the mean from three biological replicate experiments, each of which consisted of three technical replicates.