Complementation with the hfq+ plasmid, p415-hfq, into strain PM107 restored the β-galactosidase activity to the wild-type level (Fig. 2a). Quantitative real-time reverse transcriptase-PCR (qRT-PCR) assay also revealed that the levels of the phlA gene transcription were significantly reduced (P<0.01) in the hfq mutant compared with the wild-type strain 2P24 (Fig. S1). The effect of hfq on the production of 2,4-DAPG in P. fluorescens 2P24 and its derivatives ABT-199 mw was evaluated by HPLC. The result (Fig. 2b) was consistent

with the phlA promoter assay and the qRT-PCR assay described above and confirmed the involvement of the hfq gene in the regulation of phlA gene expression in strain 2P24. The effect of the hfq gene on the PcoI–PcoR QS system, another important characteristic contributing to the biocontrol activity of P. fluorescens 2P24 (Wei & Zhang, 2006), was also evaluated. In the hfq-defective mutant PM107, β-galactosidase activity from the plasmid carrying the lacZ gene fused to the pcoI promoter (Yan et al., 2009) was about 30-fold decreased compared with strain 2P24 (Fig. 3a). The qRT-PCR analysis also revealed that the levels Selleckchem Selumetinib of pcoI transcription

were significantly reduced (P<0.01) in the hfq mutant compared with strain 2P24 (Fig. S1). A similar tendency was observed in the experiments quantifying AHL production using the biosensor strain A. tumefaciens NTL4 (pZLR4) (Fig. 3b). The introduction of the complementation plasmid p415-hfq into PM107 restored both pcoI-lacZ transcriptional activity and AHL production, suggesting that Hfq functions as a positive regulator of pcoI gene expression. Biofilm formations by strain 2P24 and its variants Liothyronine Sodium were measured in PVC Eppendorf tubes at 12, 24 and 36 h after inoculation (Fig. 4). Biofilms formed by the hfq mutant PM107 were significantly reduced (P<0.05) compared with those formed by the wild type and the complemented strain

PM107/p415-hfq, indicating that Hfq has a positive effect on the biofilm formation in P. fluorescens 2P24 (Fig. 4). Because the expression of the pcoI gene is under the regulation of the hfq gene (Fig. 3), and the PcoI–PcoR QS system has been known to positively control biofilm formation in strain 2P24 (Wei & Zhang, 2006), we hypothesized that this regulation could be mediated through the QS system. To verify this, the effect of synthetic AHL on biofilm formation by the PM107 mutant was measured. Synthetic 3-oxo-C8-HSL (Sigma) was used because it is the major QS signal produced by strain 2P24 (Wei & Zhang, 2006). Although exogenous 3-oxo-C8-HSL improved pcoI expression in the hfq mutant (data not shown), no significant difference in biofilm formation by PM107 was detected with or without 3-oxo-C8-HSL (Fig. 4). These observations suggested that Hfq may regulate biofilm formation independent of QS in strain 2P24.