Characterization of the putative promoter of the pyocin operon from Pseudomonas fluorescens SF4c

VIRGINIA RIGO; ANALÍA PRÍNCIPE; JOSÉ MIGUEL QUESADA; AGUSTINA GODINO; LLAMAS MA; MANUEL ESPINOSA-URGEL; EDGARDO JOFRÉ; SONIA FISCHER

Workshop; II Workshop Latinoamericano sobre Rizobacterias Promotoras de Crecimiento Vegetal; 2014

Pseudomonas Lamiaceae SF4c, a strain isolated from the wheat rhizosphere, produces a phage-like bacteriocin (R-pyocin) which inhibits the growth of closely related bacteria. In this study, we analyzed the non-coding sequences upstream of the piocin operon with the aim to identify and characterize the putative promoter region.P. Lamiaceae SF4c was grown in LB medium to early stationary phase. The cells were collected and frozen. Total RNA from frozen cells was extracted with TRI Reagent (Ambion). Reverse-transcription PCR (RT-PCR) was performed using 0.5 μg RNA by means Titan OneTube RT-PCR kit (Roche). The primers were designed using sequences located between and within of orf1, orf2 and hol genes. Promoter was predicted with the BPROM software. The putative promoter region was fused to the lacZ gene in the promoterless low-copy-number pMP220 vector. The resulting plasmid (pMP220::Ppyo) and empty vector pMP220 were transformed into P. Lamiaceae SF4C. The cells were grown in LB until DO= 0.6; mitomycin C was then added and the incubation continued until early stationary phase. Promoter activity was analysed by measuring β-galactosidase activity. The ARF-TSS method was used to identify the transcription start sites (TSSs) in pyocin operon. To define the first gene of the pyocin cluster (orf1, orf2 or hol), RT-PCR experiments were performed. Our results indicated that those genes were co-transcribed. Therefore, orf1 is the first gene of cluster. The promoter prediction software BPROM was used to identify a possible promoter upstream to orf1. Two putative promoters were predicted and designated PpyoI and PpyoII. A region of 486 bp containing both putative promoters was cloned into pMP220, and their activity was measured by β-galactosidase in the strain Pseudomonas SF4C grown in LB medium (either untreated or treated with mitomycin C). When cells bearing Ppyo were cultured in LB medium without mitomycin C, a basal level of β-galactosidase (below 25 Miller units) was observed. This result was similar to those obtained with the empty vector pMP220. However, the activity of promoter increased more than 10 times when the cells were induced by mitomycin C. The start point of the transcript (nucleotide +1) was located 45 nucleotides upstream of the start codon of orf1. Therefore, the second predicted promoter appears to be the functional promoter for the pyocin operon. A promoter sequence was located upstream of the pyocin operon. The promoter was active when the cells was exposed to mitomycin C, suggesting that the SOS response is involved in bacteriocin expression.