PRODUCTION OF ACYL-HOMOSERINE LACTONE QUORUM-SENSING SIGNALS BY GRAM-NEGATIVE PLANT-ASSOCIATED BACTERIA

Many gram-negative bacteria regulate expression of specialized gene se ts in response to population density. This regulatory mechanism, calle d autoinduction or quorum-sensing, is based on the production by the b acteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated deri vatives of L-homoserine lactone (acyl-HSL). Signal specificity is conf erred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tr a of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudom onas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HS Ls, and alkanoyl-HSLs with chain lengths ranging from C-4 to C-12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacter ia that associate with plants. Most of the Agrobacterium, Rhizobium, a nd Pantoea isolates and about half of the Erwinia and Pseudomonas isol ates gave positive reactions. Only a few isolates of Xanthomonas produ ced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudom onads and erwinias, most produced a single dominant activity chromatog raphing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a fe w of the erwinias, and the P. fluorescens and Ralstonia solanacearum i solates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlik e any of our standards. The few positive xanthomonads, and almost all of the agrobacteria, produced small amounts of a compound with the chr omatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the ge nus Rhizobium showed the greatest diversity, with some producing as fe w as one and others producing as many as seven detectable signals. Sev eral isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that qu orum-sensing is common as a gene regulatory mechanism among gram-negat ive plant-associated bacteria.