Production of Acyl-Homoserine Lactone Quorum-Sensing Signals by Gram-Negative Plant-Associated Bacteria

Abstract

Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL). Signal specificity is conferred 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 tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. 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 bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.

Binding Events of (S)-N-(3-Oxo-octanoyl)-homoserine Lactone with Agrobacterium tumefaciens Mutant Cells Studied by Saturation Transfer Difference NMR

“…Thus, b-galactosidase enzyme cleaves the X-gal reagent added in colorimetric bioassays, producing a blue-green indigo derivative. 6 This interesting biological test reveals that there is a cascade of biochemical reactions induced by the interaction of 1 with the mutated A. tumefaciens cells, as also observed for Ti oncogenic plasmid transfer in the crown gall disease in wild type strains. 5,6 The interaction of 1 with TraR receptor protein was solved based on X-ray diffraction of a crystallized complex dimer.…”

“…6 This interesting biological test reveals that there is a cascade of biochemical reactions induced by the interaction of 1 with the mutated A. tumefaciens cells, as also observed for Ti oncogenic plasmid transfer in the crown gall disease in wild type strains. 5,6 The interaction of 1 with TraR receptor protein was solved based on X-ray diffraction of a crystallized complex dimer. The study revealed the main anchoring sites of 1 with different aminoacid residues and the protein structural changes induced by complexation that increases the complex affinity to specific DNA-binding sites.…”

“…This interaction causes TraR monomers to dimerise thereby masking the hydrophobic region of these proteins and allowing them to enter the cytoplasm and interact with specific DNA promoters and then coordinate phenotype expression, including the transfer of oncogenic Ti plasmid. 5 The mutant A. tumefaciens NTL4(pZLR4) was built by Cha and co-workers 6 and is extensively employed as reporter for acyl-HSLs detection in colorimetric bioassays. Architecture of pZLR4 plasmid includes a traG:lacZ fusion and traR.…”

“…This mutant was provided by Dr. Welington L. Araújo (originally from Dr. Stephen K. Farrand, University of Illinois). 6 …”

Interação entre Methylobacterium extorquens e cana-de-açúcar (Saccharum sp.)

“…Estas moléculas apresentam baixo peso molecular, são produzidas por diversas bactérias e apresentam a capacidade de atuar na comunicação celular bactéria-bactéria e bactéria-planta (CHA et al, 1998;GRAM et al, 1999) A formação de biofilme é determinada por uma seqüência de genes que podem induzir as células bacterianas a se aderirem a uma superfície e posteriormente estruturar um biofilme. Para X. fastidiosa, tem sido sugerido que o biofilme seja importante para obstrução dos vasos, fato este que poderia ser um importante fator de patogênese (MARQUES et al, 2002).…”