Abstract
Quorum sensing mediated by specific signal compounds (autoinducers) allows bacteria to monitor their cell density and enables a synchronized regulation of target gene sets. The best studied group of autoinducers are the acylhomoserine lactones (AHSLs), which are central to the regulation of virulence in many plant and animal pathogens. Variation of the acyl side chain of the AHSLs underlies the observed species specificity of this communication system. Here we show that even different strains of the plant pathogen Erwinia carotovora employ different dialects of this language and demonstrate the molecular basis for the acyl chain length specificity of distinct AHSL synthases. Under physiological concentrations, only the cognate AHSL with the "right" acyl chain is recognized as a signal that will switch on virulence genes. Mutagenesis of the AHSL synthase gene expI(SCC1) identified the changes M127T and F69L as sufficient to effectively alter ExpI(SCC1) (an N-3-oxohexanoyl-l-homoserine lactone producer) substrate specificity to that of an N-3-oxooctanoyl-l-homoserine lactone producer. Our data identify critical residues that define the size of the substrate-binding pocket of the AHSL synthase and will help in understanding and manipulating this bacterial language.
Highlights
Bacteria are able to monitor their cell density by producing and detecting specific signal compounds commonly referred to as autoinducers
After the acylhomoserine lactones (AHSLs) concentration reaches a critical threshold level corresponding to a certain quorum of bacteria, the AHSL interacts with the cognate R-protein and controls the expression of quorum sensing-regulated target genes [1, 2, 14]
In E. carotovora, the production of both carbapenem antibiotics and extracellular enzyme virulence factors is controlled by AHSLs and, requires a functional I-protein known as CarI or ExpI, respectively [1, 13, 14]
Summary
Bacterial Strains and Mutagenesis—The E. carotovora strains SCC1 and SCC3193 originate from potato field collections near the Viikki Campus of the University of Helsinki [27]. Cellulase assays were performed with 10-l supernatants of overnight grown cultures on cellulose indicator plates (carboxymethylcellulose) using Congo Red to determine cellulose digestion as described [13]. Determination of AHSL Content and Structure Elucidation—AHSLs were extracted twice with equal volumes of ethyl acetate from 2-ml E. carotovora or E. coli culture supernatants grown in LB or minimal M9 medium [29] containing 0.4% sucrose or from 20 l of macerated potato tuber tissue diluted in 500 l of water. B, minimal effective concentration (MEC) values of different AHSLs for inducing Cel activity as determined with carboxymethylcellulose plates. Their high performance liquid chromatography retention time as well as by tandem mass spectrometry analysis. Structure figures were prepared with Protein Explorer (molvis.sdsc.edu/protexpl/ frntdoor.htm), ISISDraw2.4, and Rasmol 2.5
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