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item Thomashow, Linda

Submitted to: Proceedings of Workshop on Global Int Org Biocontrol (IOBC) Working Group
Publication Type: Proceedings
Publication Acceptance Date: 9/1/2005
Publication Date: 2/20/2006
Citation: Mavrodi, D., Khan, S., Farrand, S., Thomashow, L.S. 2006. Regulation of beneficial traits in antagonistic bacteria. Proceedings of Workshop on Global Int Org Biocontrol (IOBC) Working Group. IOBC/WPRS Bulletin Vol. 29(2) The Netherlands. 2006.

Interpretive Summary: Phenazines are a class of bioactive compounds with antifungal and antibacterial activity. The are responsible for the biocontrol activity of many biocontrol agents being tested in agriculture. This review focuses on the synthesis, regulation and activity of phenazines in a broad spectrum of important strains.

Technical Abstract: The phz operon of Pseudomonas fluorescens 2-79, which produces the antibiotic phenazine-1-carboxylic acid (PCA), is preceded by phzR and phzI, a pair of quorum-sensing members of the luxR-luxI gene family. Quantitative analyses showed that strain 2-79 produces six acyl-homoserine lactone (HSL) signaling components, of which N-(3-hydroxyhexanoyl)-L-HSL is the most abundant. Deletion of phzR and phzI led to the loss of production of PCA as well as all acyl-HSLs. Expression of phzI in Escherichia coli or the PCA nonproducer P. fluorescens 1855 enabled the synthesis of all six acyl-HSLs normally produced by strain 2-79. A reporter strain of 2-79 bearing phzA and phzR fused respectively to lacZ and uidA required PhzR and the addition of acyl-HSLs for maximum expression of both gene fusions. Analyses with synthetic acyl-HSLs revealed that the phzA::lacZ fusion responded with highest sensitivity and greatest magnitude to N-(3-hydroxy hexanoyl)-L-HSL. When exposed to extracts of culture supernatants from strain 2-79 containing normal ratios of all six acyl-HSLs, the reporter responded to N-(3-hydroxy-hexanoyl)-L-HSL but not to the other five acyl-HSLs. Mapping of the transcriptional start sites for the divergently-oriented phzA and phzR genes showed that the putative –35 element of the phzA promoter is situated downstream from and adjacent to an 18-bp almost-perfect inverted repeat, the phz-box, which resembles the binding sites of other members of the LuxR family. Disrupting the phz-box abolished PhzR-dependent activation of phzA and phzR. We conclude that PhzI of strain 2-79 synthesizes 3-OH acyl-HSLs and, unlike the N-hexanoyl-L-HSL-based phz systems of P. aureofaciens 30-84 and P. chlororaphis PCL1391, strain 2-79 recognizes N-(3-hydroxy-hexanoyl)-L-HSL as its quorum-sensing signal. We conclude further that PhzR, together with its quormone, activates expression of phzA and phzR and that this activation requires the phz-box present in the divergent promoter region. Considering the differences in spacing of the phz-box relative to the –35 elements of the phzA and phzR promoters, we also suggest that PhzR activates transcription of these genes by different mechanisms.