Thermoregulation of N-acyl homoserine lactone-based quorum sensing in the soft rot bacterium Pectobacterium atrosepticum.

The psychrotolerant bacterium Pectobacterium atrosepticum produces four N-acyl homoserine lactones under a wide range of temperatures. Their thermoregulation differs from that of the exoenzyme production, described as being under quorum-sensing control. A mechanism involved in this thermoregulation consists of controlling N-acyl homoserine lactones synthase production at a transcriptional level.

A plasmid-borne truncated luxI homolog controls quorum-sensing systems and extracellular carbohydrate production in Methylobacterium extorquens AM1.

A cryptic plasmid of Methylobacterium extorquens AM1 was found to encode tslI, a truncated luxI homolog. tslI was shown to be expressed and to control transcription of the acyl-homoserine lactone (HSL) synthase gene msaI and thus, indirectly, acyl-HSL production. In addition, tslI was found to positively regulate extracellular polysaccharide production.

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions.

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

Evidence for a functional quorum-sensing type AI-1 system in the extremophilic bacterium Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is one of the main acidophilic chemolithotrophic bacteria involved in the bioleaching of metal sulfide ores. The bacterium-mineral interaction requires the development of biofilms, whose formation is regulated in many microorganisms by type AI-1 quorum sensing. Here, we report the existence and characterization of a functional type AI-1 quorum-sensing system in A. ferrooxidans. This microorganism produced mainly acyl-homoserine lactones (AHL) with medium and large acyl chains and different C-3 substitutions, including 3-hydroxy-C8-AHL, 3-hydroxy-C10-AHL, C12-AHL, 3-oxo-C12-AHL, 3-hydroxy-C12-AHL, C14-AHL, 3-oxo-C14-AHL, 3-hydroxy-C14-AHL, and 3-hydroxy-C16-AHL. A quorum-sensing genetic locus that includes two open reading frames, afeI and afeR, which have opposite orientations and code for proteins with high levels of similarity to members of the acyl synthase (I) and transcriptional regulator (R) protein families, respectively, was identified. Overexpression of AfeI in Escherichia coli and the associated synthesis of AHLs confirmed that AfeI is an AHL synthase. As determined by reverse transcription-PCR, the afeI and afeR genes were transcribed in A. ferrooxidans. The transcription levels of the afeI gene were higher in cells grown in sulfur and thiosulfate media than in iron-grown cells. Phosphate starvation induced an increase in the transcription levels of afeI which correlated with an increase in AHL levels. Two afe boxes which could correspond to the AfeR binding sites were identified upstream of the afeI gene. This is the first report of a functional type AI-1 quorum-sensing system in an acidophilic chemolithotrophic microorganism, and our results provide a very interesting opportunity to explore the control and regulation of biofilm formation during the bioleaching process.

Osmotic stress and phosphate limitation alter production of cell-to-cell signal molecules and rhamnolipid biosurfactant by Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, rhamnolipid production is controlled by the quorum-sensing system RhlRI, which itself depends on LasRI. These systems use cell-to-cell signal molecules: N-butyryl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3OC(12)-HSL), respectively. Whereas both HSLs were produced in M63 medium, rhamnolipid synthesis was not achieved. Phosphate limitation reduced the HSL concentrations, while allowing rhamnolipid production. Hyperosmotic shock applied during the exponential growth phase stopped the accumulation of 3OC(12)-HSL, and prevented C4-HSL and rhamnolipid production. These defects result from lower expression of genes involved in C4-HSL and rhamnolipid syntheses. The osmoprotectant glycine betaine partially restored C4-HSL and rhamnolipid production.

On-line high-performance liquid chromatography-mass spectrometric detection and quantification of N-acylhomoserine lactones, quorum sensing signal molecules, in the presence of biological matrices.

A protocol using reversed-phase liquid chromatography coupled with positive-ion electrospray ionization and ion trap mass spectrometry is described for the identification and quantification of N-acylhomoserine lactones (HSLs) in crude cell-free supernatants of bacterial cultures. The HSLs are produced by gram-negative bacteria and act as intercellular signals inducing density-dependent gene expression. Compared with the multi-step procedures previously reported, which included chemical extraction, purification and the use of Escherichia coli HSL biosensors, this on-line LC-MS-MS method is fast and detects 11 HSLs. Its speed and robustness allow the analysis of a large number of samples without loss of performance (no signal variation for a control sample after 90 chromatographic injections). The selectivity is based on the MS-MS fragment ions of the molecular [M+H]- ions and on their relative intensities. For quantification, the m/z 102 ion, specific for the lactone ring and detected with a good signal-to-noise ratio, allows low detection limits even in complex matrix samples (0.28 up to 9.3 pmol). Moreover, this method allows the quantification of 11 HSLs whatever their chemical structure, substituted or not. The protocol was applied to Vibrio vulnificus, a marine bacterium. Six HSLs were detected and quantified with relative standard deviations for repeatability of < 10%.