A regulatory RNA (PrrB RNA) modulates expression of secondary metabolite genes in Pseudomonas fluorescens F113

Abstract

The GacS-GacA two-component signal transduction system, which is highly conserved in gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. Screening of a Pseudomonas fluorescens F113 gene bank led to the isolation of a previously undefined locus which could restore secondary metabolite production to both gacS and gacA mutants of F113. Sequence analysis of this locus demonstrated that it did not contain any obvious Pseudomonas protein-coding open reading frames or homologues within available databases. Northern analysis indicated that the locus encodes an RNA (PrrB RNA) which is able to phenotypically complement gacS and gacA mutants and is itself regulated by the GacS-GacA two-component signal transduction system. Primer extension analysis of the 132-base transcript identified the transcription start site located downstream of a sigma(70) promoter sequence from positions -10 to -35. Inactivation of the prrB gene in F113 resulted in a significant reduction of 2, 4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) production, while increased metabolite production was observed when prrB was overexpressed. The prrB gene sequence contains a number of imperfect repeats of the consensus sequence 5'-AGGA-3', and sequence analysis predicted a complex secondary structure featuring multiple putative stem-loops with the consensus sequences predominantly positioned at the single-stranded regions at the ends of the stem-loops. This structure is similar to the CsrB and RsmB regulatory RNAs in Escherichia coli and Erwinia carotovora, respectively. Results suggest that a regulatory RNA molecule is involved in GacA-GacS-mediated regulation of Phl and HCN production in P. fluorescens F113.

FIG. 1

Effect of pCU304 on Phl (A) and HCN (B) production by wild-type F113 and F113gacS (FL33) and F113gacA (FG9) mutants. Introduction of the plasmid pCU304 in trans into both FL33 and FG9 mutants restored Phl and HCN production and increased Phl and HCN production in the wild type.

FIG. 2

(A) Nucleotide sequence of prrB. The first line of the sequence shows the transcription start (str) and promoter organization; −10 and −35 sequences are boxed. Putative KDGR recognition sites are boxed. The potential hairpin loop structures are identified by arrows and the 5′-AGGA-3′ imperfect repeat motifs are in boldface type. The nucleotide sequence of primers P3 and P4 used for the construction of pCU305 are underlined. (B) Secondary structure prediction for PrrB RNA generated using mfold. Note that all the repeated elements are predicted to reside in the single-stranded regions and five of the nine are specifically found in the hairpin stem-loop. (C) Nucleotide sequence alignment of Erwinia KdgR boxes with putative prrB KdgR sequences.

FIG. 3

Northern blot analysis of total RNA isolated from 7 × 10⁹ cells of wild-type P. fluorescens F113 and mutant derivatives grown for 18 h in minimal medium with sucrose as the carbon source. The wild type and mutants used are indicated above the lanes. Lane M contains an RNA marker of 145 bases obtained by in vitro transcription. The exposure time for the RNA marker was reduced in order to reduce the band intensity.

FIG. 4

Mapping of the transcription start site of prrB. A 22-nucleotide primer (prrBSP1) complementary to sequence between positions −126 and −104 from the TAATAT sequence was 5′ end labeled and hybridized to total RNA isolated from the wild-type strain F113. The hybrid was extended using avian myeloblastosis virus reverse transcriptase, and the extension product was resolved by denaturing polyacrylamide gel electrophoresis and autoradiography. The PrrB lane contains the extension product; the T, G, C, and A lanes contain the sequencing product obtained using prrBSP1. The transcription start site is indicated by an asterisk.

FIG. 5

Time course of Phl production by the F113prrB mutant compared with that of the wild type. There was a significant reduction in Phl production during the mid- to late log phase of growth. Phl production of wild-type F113 () and F113prrB (▭) is shown by the bars and the left y axis, and OD₆₀₀ of wild-type F113 (■) and F113prrB (⧫) is shown by the curves and the right y axis.