Article Figures & Data
Figures
- 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 transinto 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 ofprrB. 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 F113prrBmutant 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.
Tables
- Table 1.
Bacterial strains and plasmids used in this study
Bacterial strain or plasmid Relevant characteristicsa Source or reference Bacterial strains Pseudomonas fluorescens F113 Wild type, Phl+ HCN+ Prt+lac 35 FG9 F113gacAΩ::mini-Tn5-lac Phl−HCN− Prt−lacKmr 8 FL33 F113gacSΩ::mini-Tn5-lac Phl−HCN− Prt−lacKmr 8 FRB1 F113prrB::Ω-Tc, Phl+ HCN+Prt+lac Tcr This study Escherichia coli DH5-α φ80lacZΔM15 Δ(lacZYA-argF)U169 hsdR17 recA1 endA1 thi-1 33 Plasmids pBBR1MCS Cloning vector, BHR, Cmr 16 pSUP106 Cloning vector, BHR, Tcr 36 pK18 Cloning vector, NHR, ColE1, Kmr 28 pMP220 PromoterlesslacZ vector, IncP, Tcr 37 pRK2013 Helper plasmid, Tra+ Mob+ColE1 Kmr 12 pHP45-Tc ColE1 replicon carrying an Ω-Tc; AprTcr 27 pCU300 pSUP106 carrying a 5.4-kb BamHI fragment from P. fluorescens F113; Cmr This study pCU301 pBBR1MCS carrying a 2.8-kbBamHI-HindIII fragment from pCU300; Cmr This study pCU302 pBBR1MCS carrying a 3.3-kb BamHI-SalI fragment from pCU300; Cmr This study pCU303 pBBR1MCS carrying a 2.1-kb BamHI-SalI fragment from pCU300; Cmr This study pCU304 pBBR1MCS carrying a 0.85-kb HindIII-EcoRV fragment amplified from pCU301; Cmr This study pCU305 pBBR1MCS carrying a 228-bp KpnI-BamHI fragment amplified from pCU301; Cmr This study pCU306 pK18 carrying a 5-kb BamHI-XhoI fragment from pCU300 with Ω-Tc cassette inserted into prrB; KmrTcr This study ↵a Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Kmr, kanamycin resistant, Tcr, tetracycline resistant.
