Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. With the exception of N-butanoyl-L-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed. Each of the 3-oxo-and the 3-unsubstituted derivatives migrated with a unique mobility. Using the assay, we showed that some bacteria produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by Pseudomonas syringae pv. tabaci chromatographed with the properties of N-(3-oxohexanoyl)-L-homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy-forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-L-homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules.

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.

There has been much recent interest and progress in the characterization of community structure and community assembly processes through the application of phylogenetic methods. To date most focus has been on groups of taxa for which some relevant detail of their ecology is known, for which community composition is reasonably easily quantified and where the temporal scale is such that speciation is not likely to feature. Here, we explore how we might apply a molecular genetic approach to investigate community structure and assembly at broad taxonomic and geographical scales, where we have little knowledge of species ecology, where community composition is not easily quantified, and where speciation is likely to be of some importance. We explore these ideas using the class Collembola as a focal group. Gathering molecular evidence for cryptic diversity suggests that the ubiquity of many species of Collembola across the landscape may belie greater community complexity than would otherwise be assumed. However, this morphologically cryptic species-level diversity poses a challenge for attempts to characterize diversity both within and among local species assemblages. Recent developments in high throughput parallel sequencing technology, combined with mtDNA barcoding, provide an advance that can bring together the fields of phylogenetic and phylogeographic analysis to bear on this problem. Such an approach could be standardized for analyses at any geographical scale for a range of taxonomic groups to quantify the formation and composition of species assemblages.

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